Articles in PresS. Am J Physiol Renal Physiol (March 8, 2017). doi:10.1152/ajprenal.00032.

2017

1 Effect of Diuretics on Renal Tubular Transport of Calcium and Magnesium

3 R. Todd Alexander1,2,, Henrik Dimke3, #

4
1
5 Membrane Protein Disease Research Group, Department of Physiology and 2Department

6 of Pediatrics, University of Alberta, Edmonton, Canada. 3Department of Cardiovascular

7 and Renal Research, Institute of Molecular Medicine, University of Southern Denmark,

8 Odense, Denmark.

10 Word count: 10748

11 Abstract word count: 250

12 Running head: Diuretic effects on Ca2+ and Mg2+ transport

13

14 # Correspondence to:

15 Henrik Dimke

16 Department of Cardiovascular and Renal Research

17 Institute of Molecular Medicine, University of Southern Denmark

18 J.B. Winsløws Vej 21, 3th floor, 5000 Odense C, Denmark

19 Phone: +45 65508310, Fax: +45 6613 3479

20
21 E-mail: hdimke@health.sdu.dk
22

Copyright © 2017 by the American Physiological Society.

23 Abstract

24 Calcium (Ca2+) and Magnesium (Mg2+) reabsorption along the renal tubule is dependent

25 on distinct trans- and paracellular pathways. Our understanding of the molecular

26 machinery involved is increasing. Ca2+ and Mg2+ reclamation in kidney is dependent on a

27 diverse array of proteins, which are important for both forming divalent cation permeable

28 pores and channels, but also for generating the necessary driving forces for Ca2+ and

29 Mg2+ transport. Alterations in these molecular constituents lead to profound effects on

30 tubular Ca2+ and Mg2+ handling. Diuretics are used to treat a large range of clinical

31 conditions, but most commonly for the management of blood pressure and fluid balance.

32 The pharmacological targets of diuretics generally directly facilitate sodium (Na+)

33 transport, but also indirectly affect renal Ca2+ and Mg2+ handling, i.e. by establishing a

34 prerequisite electrochemical gradient. It is therefore not surprising that substantial

35 alterations in divalent cation handling can be observed following diuretic treatment. The

36 effects of diuretics on renal Ca2+ and Mg2+ handling are reviewed in the context of the

37 current understanding of basal molecular mechanisms of Ca2+ and Mg2+ transport.

38 Acetazolamide, osmotic diuretics, NHE3 inhibitors and antidiabetic SGLT blocking

39 compounds, target the proximal tubule, where paracellular Ca2+ transport predominates.

40 Loop-diuretics and ROMK inhibitors block thick ascending limb transport, a segment

41 with significant paracellular Ca2+ and Mg2+ transport. Thiazides target the distal

42 convoluted tubule, however, their effect on divalent cation transport is not limited to that

43 segment. Finally, potassium-sparing diuretics, which inhibit electrogenic Na+ transport at

44 distal sites, can also affect divalent cation transport.

45

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46 Keywords: TRPM6 and TRPV5 channels, Furosemide, Amiloride, Calciuria, Claudins.

47 Introduction

48 Diuretic are used in the management of hypertension, oedema, and a large array of other

49 diseases affected or ameliorated by altering electrolyte transport. In kidney, diuretics

50 often target transport proteins or mechanisms that are critically important for renal

51 reabsorption of sodium, chloride (NaCl) and water. Given that calcium (Ca2+) and

52 magnesium (Mg2+) transport in the kidney is dependent on electrochemical gradients

53 established by tubular NaCl transport processes, it is not surprising that diuretic treatment

54 can cause significant alterations in renal Ca2+ and Mg2+ handling. This review aims to

55 highlight the actions of diuretics in the context of our current knowledge of the molecular

56 mechanisms driving tubular Ca2+ and Mg2+ transport.

57

58 Ca2+ and Mg2+ transport in the proximal tubule

59 Approximately 55% of Ca2+, the fraction in plasma not bound to protein, is freely filtered

60 by the glomerulus into the proximal tubule (PT) (77, 79, 96). A slightly larger fraction of

61 Mg2+, approximately 70%, is not bound to plasma proteins and hence freely filterable by

62 the glomerulus (54). The majority, or 60-70% of the filtered Ca2+ load, is reabsorbed

63 along the course of the PT (67, 137, 214, 226). The Ca2+ concentration in the convoluted

64 portion of the PT accessible to micropuncture remains nearly equivalent or slightly higher

65 (1.0-1.2) to that in the ultrafiltrate (138, 226). This observation supports the generally

66 accepted premise that the majority of PT Ca2+ reabsorption occurs via a passive

67 paracellular process, driven by active solute and subsequent water reabsorption (28, 169,

68 214, 226). Proximal tubular Mg2+ reabsorption is also thought to occur via a passive

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69 paracellular process, driven by solute and water reabsorption, in a fashion similar to Ca2+.

70 However, only 10-25% of the filtered load of Mg2+ is reabsorbed from the PT, much less

71 than that reported for Ca2+ (138, 139, 193, 194). Consequently, the luminal Mg2+

72 concentration may almost double along the proximal convoluted tubule (138). This is

73 likely the consequence of greatly reduced relative permeability for Mg2+ (PMg) across this

74 segment amounting to 1 • 10-5 cm/s, in comparison to the permeability for Ca+ (PCa),

75 which has been reported to reach 8 – 27 • 10-5 cm/s (78, 119, 169).

76 In the PT, Ca2+ reabsorption closely follows Na+, suggesting that Na+ transport

77 could be a prerequisite for driving Ca2+ reabsorption across the epithelium (214, 226).

78 Na+ is the major physiological solute in the ultrafiltrate. At a molecular level, Na+

79 reabsorption from the PT occurs via a transcellular process with apical entry

80 predominantly occurring through the Na+- proton (H+) exchanger isoform 3, NHE3 (210)

81 (Figure 1). This Na+ uptake is driven by the steep Na+ gradient maintained by the Na+,

82 potassium (K+)-ATPase, which pumps 2 K+ into the cell and 3 Na+ out, thereby

83 maintaining a low cellular Na+ concentration and a steep voltage gradient across the cell

84 membrane. The epithelium of the PT has a high osmotic water permeability due to

85 abundant expression of aquaporin 1 (AQP1) in both the apical and basolateral membrane

86 (171). Thus, Na+ reabsorption via NHE3 facilitates water removal from the luminal fluid.

87 The proximal tubular epithelium is also very leaky, displaying a transepithelial resistance

88 of approximately 10 Ω · cm2 (19, 167, 213). Consequently, significant water reabsorption

89 also occurs through the paracellular shunt, although the majority moves transcellular via

90 AQP1 (208, 209) (Figure 1). Importantly, the movement of water through the paracellular

91 shunt facilitates the movement of other ions via convection in a process known as solvent

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 92 drag. This process contributes to significant paracellular Na+ reabsorption along the PT

93 (38, 168). Regardless, both Na+ and water are reabsorbed paracellularly from the PT,

94 driven by the transcellular reabsorption of Na+. Ca2+ reabsorption follows this passive

95 paracellular mechanism. Whether Ca2+ transport occurs due to water removal resulting in

96 a slightly higher electrochemical gradient favouring paracellular Ca2+ transport or via the

97 process of solvent drag has yet to be clearly delineated (Figure 1). Hence, volume

98 expansion, which reduces Na+ and water transport from the PT, also reduces Ca2+

99 reabsorption and may lead to significant urinary Ca2+ wasting. In line with this, volume

100 contraction reduces urinary Ca2+ excretion (228). However, volume status does not

101 appear to affect Mg2+ transport in the PT (186). Why Mg2+ reabsorption is not as tightly

102 coupled to Na+ reabsorption from the PT is likely due to the nature of the shunt, which is

103 discussed further below.

104 The presence of an increased fractional excretion of Ca2+ in Nhe3 null mice,

105 underlines the importance of Na+ coupled Ca2+ reabsorption from the PT (179).

106 Although specific examination of NHE3 coupled Ca2+ flux has not been measured by

107 micropuncture or microperfusion, further evidence implicating NHE3 in driving

108 paracellular Ca2+ flux is provided by work performed in a proximal tubular cell culture

109 model. When NHE3 was overexpressed, transepithelial Ca2+ flux doubled (179). This

110 effect of increased NHE3 mediated transepithelial Ca2+ flux was prevented by blocking

111 Na+/H+ exchange, through the removal of extracellular Na+. In line with paracellular Ca2+

112 flux being coupled to NHE3-dependent Na+ transport is ex vivo Ussing chamber studies

113 on intestine (210). Nhe3–deficient mice display reduced Ca2+ flux across both the

114 duodenum and cecum, highlighting the dependence of Ca2+ absorption on NHE3 activity

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115 (179, 200). Ultimately, increased renal losses and decreased intestinal absorption of Ca2+

116 led to reduced bone mineral density in Nhe3 knockout mice (179).

117 Passive paracellular Ca2+ flux requires not only a driving force, but also for the

118 tight junction to be permeable to Ca2+. The molecular identity of the tight junction

119 proteins conferring permeability to cations in the PT has yet to be clarified. The claudin

120 (CLDN) family of tight junction proteins provides the permeability characteristics of an

121 epithelium. Thus, a combination of claudins expressed in the proximal tubule likely

122 confers the leaky permeability characteristics of this nephron segment permitting Ca2+

123 flux. CLDN2, CLDN10a and CLDN17 have been identified in the proximal tubule of

124 adult animals (71, 100, 124, 134). When CLDN2 is expressed in cell culture models, it

125 consistently forms a cation selective pore (11, 81, 250). The renal expression of CLDN2

126 is restricted to the PT and early thin descending limb. Microperfusion studies on proximal

127 tubules from mice lacking the Cldn2 gene confirm a role of CLDN2 in making a cation

128 selective paracellular pore across the PT in vivo (167). Unfortunately, Ca2+ flux across the

129 proximal tubule of Cldn2 knockout mice has not been measured. However, Cldn2

130 knockout mice display hypercalciuria (167). Thus, it is very likely that CLDN2

131 contributes paracellular Ca2+ permeability across the PT. However, one must also

132 consider that Cldn2 deficient mice display altered tight junction structure and reduced PT

133 reabsorption of Na+, Cl-, and water (167). Thus, it cannot be definitively concluded that

134 Cldn2 null mice do not display urinary Ca2+ loss because of reduced solvent drag or an

135 altered Ca2+ gradient across the segment, as mentioned above, altered Na+ and water flux

136 across the PT strongly correlates with changes in PT Ca2+ transport (6, 18, 69, 229).

137 Further, it is unlikely that claudin-2 forms a Ca2+ permeable paracellular pore in isolation,

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138 as the thin descending limb has a much lower apparent Ca2+ permeability than the PT

139 (202).

140 Electroneutral bicarbonate (HCO3-) reabsorption in the early portion of the PT

141 occurs at the expense of Cl-, as illustrated in figure 1. This leads to an increased

142 concentration of Cl- in the lumen when the filtrate reaches the later portions of the PT.

143 The accumulation of Cl- in the lumen allows Cl- flux down its chemical gradient, through

144 the paracellular pathway, leaving a net lumen positive, potential difference (16). This

145 lumen positive transepithelial voltage drives Na+ reabsorption and favours passive Ca2+

146 reabsorption via the paracellular shunt (13, 16). Although the majority of Ca2+ is

147 reabsorbed via the paracellular pathway across the PT, there is evidence supporting a

148 small, yet significant, transcellular component of Ca2+ transport across both the

149 convoluted (20% of the total absorption), and straight (10%) portions of the PT (28, 203,

150 238). Finally, the tight junction proteins that confer selectivity for Ca2+ over Mg2+ in the

151 PT are completely unknown and an area requiring further study.

152

153 NHE3 inhibition/Tenapanor

154 Unlike the other nephron segments, currently there is not a direct inhibitor of apical

155 sodium entry from the PT that is employed clinically (note carbonic anhydrase inhibitors

156 indirectly prevent Na+ absorption from this segment, as discussed below). S3226, is a

157 specific NHE3 inhibitor, that has been shown to attenuate ischemia-induced renal failure

158 in an animal model (120). However, this drug has not made it to the clinic and we are

159 unaware of information on its affects on renal divalent cation handling. Given the

160 calciuria seen in Nhe3 deficient mice (180), it is not unreasonable to suggest such

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161 compounds may increase urinary Ca2+ excretion. More recently, a non-absorbable NHE3

162 inhibitor, tenapanor, has been generated and is undergoing clinical trials. NHE3 is also

163 expressed in the intestine, where it mediates significant Na+ and water reabsorption (210).

164 It is therefore not unexpected that this compound inhibits intestinal Na+ absorption and

165 lowers blood pressure in rats (135). Surprisingly, it also prevents intestinal phosphate

166 absorption and lowers plasma phosphate in patients on dialysis (25, 135). Effects on

167 divalent cation handling have not been reported for these compounds. Although given the

168 previous findings of decreased Ca2+ absorption from duodenum and cecum of NHE3 null

169 mice (180, 200), it likely also attenuates intestinal Ca2+ absorption. It is unclear whether

170 other mechanisms would compensate for this.

171

172 Osmotic diuretics

173 An osmotic diuretic is a compound that is not reabsorbed along the nephron and in

174 addition exerts an osmotic force, thereby impairing osmotic water reabsorption from the

175 tubule. This results in a diuretic effect. Mannitol is such an osmotic compound and is

176 most commonly employed as an osmotic diuretic. Similar to Ca2+, water is reabsorbed

177 from the PT, down an osmotic gradient created by the reabsorption of Na+. It is therefore

178 not surprising that infusion of D-mannitol interferes with osmotically driven Ca2+

179 reabsorption as well. Consistent with this, administration of 10-20% D-mannitol solutions

180 increases the fractional urinary excretion of Ca2+ (67, 246). Moreover, micropuncture

181 data confirms this diuretic reduces proximal Ca2+ reabsorption (246), although infusion of

182 a lower concentration of D-mannitol did not induce calciuria as evaluated by standard

183 renal clearance techniques (27), perhaps due to transcellular proximal tubular Ca2+

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184 transport in the PT (28), or compensatory pathways in the distal tubule. Mannitol

185 administration also increases urinary Mg2+ excretion (36, 181), however, whether this is

186 via reduced PT paracellular reabsorption, or an effect on the thick ascending limb (TAL),

187 where the majority of Mg2+ is reabsorbed in a paracellular fashion, is not clear.

188

189 Carbonic anhydrase inhibitors and divalent cation transport

190 Carbonic anhydrase inhibitors are a group of drugs that act by blocking the carbonic

191 anhydrase enzymes and hence the catalysis of H2CO3 to CO2 and water and vice versa.

192 Without the carbonic anhydrases, the above listed interconversion, would occur at a much

193 slower rate. They are employed for a diverse array of indications including as

194 antiglaucoma agents, diuretics, antiepileptics and in the treatment of acute altitude

195 sickness. Acetazolamide is most commonly used clinically. Acetazolamide increases

196 urinary Ca2+ excretion (15, 157, 230). There are multiple carbonic anhydrase isoforms

197 expressed in multiple tissues including red blood cells, bone as well as multiple isoforms

198 in the kidney epithelium, including the PT and the intercalated cells of the collecting duct

199 (CD)(189). Therefore, ascribing the effect of acetazolamide or other carbonic anhydrase

200 inhibitors on urinary Ca2+ excretion to specific inhibition of carbonic anhydrase in the PT

201 is not straightforward. In particular, these compounds have also been found to inhibit

202 PTH secretion in nephrectomized rats (242). However, acetazolamide exhibits a rather

203 low membrane permeability (204) and the diuretic effect in kidney is thought to be the

204 result of inhibiting H+ recycling across the apical membrane of the PT, where, luminal

205 carbonic anhydrases play a key role in converting filtered HCO3- and secreted H+ into

206 H2O and CO2. Thus, inhibition of H+ recycling, needed for Na+ influx, will impair Na+

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207 uptake into the cell, Na+ reabsorption from the PT, and consequently water reabsorption

208 from the PT (Figure 1), which ultimately will inhibit PT reabsorption of Ca2+. Consistent

209 with this, micropuncture studies on dog kidney found reduced Ca2+ reabsorption from the

210 PT after administration of acetazolamide (18). It is therefore not unreasonable to suggest

211 that reduced proximal tubular Ca2+ reabsorption is at least in part responsible for calciuria

212 induced by this drug.

213

214 Na+/glucose cotransporter inhibitors

215 Na+/glucose cotransporter type 2 (SGLT2) inhibitors are a new class of antidiabetic

216 drugs, which prevent reabsorption of glucose from the PT (Figure 1). The compounds

217 themselves are not considered diuretic agents, but their ability to increase luminal glucose

218 concentrations in the PT, can lead to a diuretic response. In normal physiology, filtered

219 glucose is almost completely reabsorbed from the PT, in a process coupled to Na+

220 reabsorption. Apical uptake occurs through the transporter SGLT2 in the convoluted

221 portion and to a lessor extent via SGLT1 in the straight PT. Basolateral efflux of glucose

222 is mediated via type 2 glucose transporters (GLUT2) (Figure 1). As a consequence,

223 reabsorption of glucose may also contribute to osmotic water removal. As such, one

224 might predict that deletion or inhibition of proximal tubular Na+ coupled glucose

225 transport, i.e. SGLT2, would lead not only to glucosuria but also Ca2+ wasting, with

226 glucose itself acting as an osmotic diuretic. Consistent with this hypothesis is the

227 presence of increased urinary Ca2+ excretion in a mutant mouse model devoid of Sglt2

228 expression (151). This animal also has increased urinary Mg2+ excretion. Furthermore, D-

229 glucose provokes significant calciuria when infused (27). To date, there is only a

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230 significant body of literature looking at the effects of canagliflozin, a SGLT2 inhibitor,

231 on Ca2+ homeostasis in patients with type 2 diabetes (reviewed in (7, 24)). There has been

232 significant interest in this area as patients treated with this drug have displayed reduced

233 bone mineral density in the hip (although not at other sites). At pharmacological doses

234 canagliflozin does not significantly elevate urinary Ca2+ or Mg2+ excretion. This might be

235 due to incomplete blockage of the transporter, compensation by SGLT1 and/or increased

236 Ca2+ absorption from the distal nephron. Although patients treated with SGLT2 inhibitors

237 display osmotic diuresis, their polyuria is significantly less and the degree of glucosuria

238 significantly lower than that observed in the knockout animal, consistent with incomplete

239 inhibition or better compensation in patients.

240

241 Ca2+ and Mg2+ transport in the thick ascending limb of Henle’s loop

242 The TAL plays a critical role in the renal reabsorption of both Ca2+ and Mg2+.

243 Consequently alterations in transport function within the TAL can have profound effects

244 on urinary excretion of divalent cations. Detailed physiological measurements using

245 micropuncture indicate that the loop reclaims significant amounts of Ca2+ and Mg2+ (52,

246 137, 226). Reabsorption of these divalent cations is thought to occur primarily through a

247 common paracellular pore between epithelial cells in the TAL, amounting to 25% of the

248 Ca2+ and more than 60% of Mg2+, filtered by the kidney (52, 137, 226). As such,

249 paracellular transport of Ca2+ and Mg2+ appears largely in the cortical TAL, with only a

250 minor contribution from the medullary portion of the segment (53, 61, 216, 227). The

251 lumen-positive transepithelial voltage gradient is an absolute requirement for driving

252 divalent cation flux across the TAL epithelium (59, 190). This can best be appreciated

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253 when artificially reversing the transepithelial potential to a lumen negative voltage, in

254 isolated perfused cortical TAL segments. Such alterations results in reversal of fluxes,

255 with both Ca2+ and Mg2+ being secreted back into the tubular lumen (59). The lumen

256 positive diffusion potential is generated in part by the asymmetrical secretion of ions

257 across the TAL, a result of the polarized expression of specific electrolyte transport

258 proteins in the apical and basolateral membranes of the epithelial cells in this segment.

259 The Na+, K+-ATPase is situated in the basolateral membrane of the TAL cell

260 (Figure 2). The inward entry of Na+, K+, and Cl- (in the stoichiometry of 1:1:2) across the

261 apical membrane occurs via the furosemide-sensitive cotransporter, NKCC2 (93, 152,

262 219). Due to a limited amount of K+ in the tubular fluid, the uptake of monovalent ions

263 via NKCC2 is highly dependent on efficient recycling of K+ across the apical membrane.

264 Apical K+ influx is mediated by the renal outer medullary K+ (ROMK) channels (93, 220,

265 248). The two Cl- ions, transported inward via the NKCC2 cotransporter, exit the cell via

266 basolaterally located CLC-Kb channels, a process favoured by the transmembrane

267 potential difference (92, 218). These channels require the cofactor Barttin, for Cl-

268 conductance (72). The luminal recycling of each K+ coupled with the basolateral exit of

269 Cl- gives rise to the transepithelial potential difference, forming a lumen positive

270 transepithelial voltage of +5 to +10 mV across the segment (93, 94). Furthermore, the

271 transepithelial potential difference may reach values close to +30 mV in the cortical TAL

272 (91, 117, 154, 201), however, the mechanism underlying this observation remains to be

273 defined in detail.

274 The permeability of Na+ (PNa) has been reported to be as much as 2-10 times

275 higher than that of Cl- (PCl) in the TAL (34, 161), with comparatively low transepithelial

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276 resistances of 11-34 Ω · cm2 (34). Anatomically, the TAL stretches from the inner stripe

277 of outer medulla (TALISOM), trough the outer stripe of outer medulla (TALOSOM), to the

278 cortical bend encompassing the TALCTX, where it is gradually replaced by the macula

279 densa cells and cells of the distal convoluted tubule (DCT). The low water permeability

280 of the TAL enables it to function as a diluting segment. Thus the NaCl concentration of

281 the luminal fluid gradually falls along the TAL, reaching values of 30-60 mM in the

282 earliest accessible portion of the distal tubule (34). In the medullary TAL, as much as

283 50% of the Na+ is taken up via the paracellular shunt, driven by the electrochemical

284 gradient (106). Given that the TAL is responsible for reclaiming some 20-25% of NaCl

285 from the filtrate, it is not surprising that altered transport within the segment leads to

286 distorted renal handling of NaCl. This is highlighted by mutations in NKCC2, ROMK,

287 CLC-Kb, and Barttin, which underlie various types of Bartter syndrome, a disease

288 characterized by significant NaCl wasting (22, 218-220).

289 Permeation of Ca2+ and Mg2+ across the cortical TAL, driven by the lumen

290 positive transepithelial voltage gradient, occurs almost exclusively via the paracellular

291 shunt (53, 61, 216, 227). Overall, the paracellular pathway in the TAL is cation selective

292 (60, 91). These permeation characteristics are determined by specific molecular

293 interactions between claudin tight junction proteins, expressed within the segment. Two

294 main claudins, CLDN16 and CLDN19, primarily participate in forming the paracellular

295 pores, whereby Ca2+ and Mg2+ permeate the TAL epithelium. Both genes were found to

296 participate in this process via their mutation in the genetic syndrome of Familial

297 Hypomagnesemia with Hypercalciuria and Nephrocalcinosis (FHHNC)(129, 221).

298 Affected family members display severe alterations in Ca2+ and Mg2+ balance due to

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299 renal wasting of these ions. Consequently, patients suffering from this autosomal

300 recessive disorder have marked hypomagnesemia, hypercalciuria and nephrocalcinosis

301 (129, 160, 187, 221). Further implication of alterations of renal CLDN16 and CLDN19

302 expression as the main defect (12, 129, 221), is provided by a FHHNC transplant

303 recipient receiving an unaffected kidney, which normalized urinary divalent cation

304 excretion and stabilized serum Mg2+ concentrations (187).

305 Elucidation of ion selectivity conferred by individual claudins has been attempted

306 in various cell model systems. Yet, substantial variability in the effect of CLDN16 and

307 CLDN19 overexpression on permeability characteristics of cell monolayers has been

308 reported. The sources of this variability are discussed in detail elsewhere (9, 101), but

309 likely relate to the cell type and the resulting interactions between the overexpressed

310 claudins under investigation, with endogenously expressed claudins in the cell line

311 employed. Some studies suggest that CLDN16 and CLDN19 are involved in conferring

312 cation permeability of the junctional complex, with only minor effects on divalent cation

313 permeability (118). This data infers that FHHNC mutations increase Cl- backflux, thereby

314 impairing diffusion potentials in the cortical TAL and hence diminishing the lumen-

315 positive voltage gradient, leading to Ca2+ and Mg2+ wasting. However, these changes

316 should also result in NaCl wasting, which is not observed in FHHNC patients, whom

317 have normal aldosterone levels and Na+ balance (23). These observations might be

318 reconciled by very recent studies from Milatz and Himmerkus et al, which suggest that

319 there may be different transport pathways for Ca2+ and Mg2+ versus Na+ along the length

320 of the TAL (161). The authors show that CLDN10, another cation permeable claudin

321 expressed within the TAL is absent from tight junctions where CLDN16 and CLDN19

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322 are localized. Using an elegant series of experiments coupling PNa and PMg measurements

323 of single microperfused TAL tubules, with subsequent immunohistochemical staining of

324 the perfused tubule, the authors were able to elucidate the PNa and PMg of the epithelium,

325 and correlate it with the total length of the tight junction that expressed either CLDN10 or

326 CLDN16. Using this approach, permeability measurements on the TAL tubules revealed

327 a high cation selectivity of the paracellular shunt in the TALISOM, which dropped in the

328 TALOSOM/TALCTX. Furthermore, the ratio of PMg over PNa increased by more than 3-fold,

329 moving from the TALISOM to the more cortical TAL segments with the highest expression

330 of CLDN16. Concurrently, they found that TALISOM almost exclusively expresses

331 CLDN10, while CLDN16 predominates in the TALOSOM/TALCTX, ranging from 37% to

332 97% (161). These new findings support the existence of two paracellular pathways for

333 cation permeation in the TAL, where CLDN10 allows permeation of Na+, while the

334 CLDN16/CLDN19 complex permits permeation of Mg2+ and presumably Ca2+ as well.

335 This hypothesis is further supported by the observation that genetic ablation of the

336 Cldn10 gene specifically from the distal nephron, leads to an augmented lumen-positive

337 transepithelial voltage gradient, due to an alteration in permeability characteristics within

338 the paracellular shunt (31). Here the PNa over PCl is markedly decreased, thereby raising

339 the lumen-positive voltage gradient. As the short circuit current was undisturbed,

340 indicating no overall changes in transcellular transport, these findings support a role for

341 CLDN10 in forming a Na+ permeable pore (31). Furthermore, PCa and PMg increased

342 substantially over that of PNa in Cldn10 deficient mice, suggesting alterations within the

343 paracellular shunt (31). The findings of altered Ca2+ and Mg2+ permeability in the Cldn10

344 knockout mice is in part explained by a redistribution of CLDN16 and CLDN19, to the

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345 now, CLDN10 depleted junctions (161). In combination with an augmented lumen

346 positive voltage in Cldn10 deficient mice, these adaptations could form the basis for

347 enhanced reabsorption of divalent cations trough the CLDN16/19 complex (31, 161). As

348 such, Cldn10 deficient mice show increased renal divalent cation reabsorption, with

349 hypermagnesemia and nephrocalcinosis (31). Furthermore, Cldn10 deficient mice display

350 increased Cldn14 expression. This is a pore-blocking tight junction protein expressed in

351 the TAL and strongly regulated by the Ca2+-Sensing Receptor (CaSR) (31, 62), as

352 discussed below.

353 CLDN14 was originally identified in a genome-wide association study (236), The

354 investigators found a strong association between kidney stones and hypercalciuria with

355 single nucleotide polymorphisms (SNPs) located within the genomic region of the

356 CLDN14 gene (236). However, the investigators were unable to assign a direct

357 mechanistic role for Cldn14 in regulating either Ca2+ balance or stone disease.

358 Furthermore, patients with loss of function mutations in the human CLDN14 gene do not

359 have alterations in Ca2+ balance, but suffer from nonsyndromic deafness, as CLDN14 is

360 expressed in the epithelial cells located within the organ of Corti (245). Additional

361 investigations found that Cldn14 was expressed in the TAL (89). Here, regulation of the

362 tight junctional claudin was shown to be highly dependent on alterations in CaSR activity

363 (62, 89). In fact, any elevation in serum Ca2+ concentrations or by pharmacological

364 alterations of CaSR sensing using calcimimetics, greatly increased Cldn14 expression (62,

365 88, 89). Mechanistically, CLDN14 overexpression in various cell lines was found to

366 reduce both the PNa/PCl ratio as well as the permeability to Ca2+, suggesting that CLDN14

367 may act to block paracellular permeation of cations (20, 62, 89). In cells, CLDN14 seems

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368 to interact with CLDN16, but not directly with CLDN19 (89). It is therefore likely that

369 CLDN14 primarily acts by blocking divalent permeation in kidney trough the

370 CLDN16/CLDN19 complex. These findings are in line with the observations that mice

371 receiving a high Ca2+ diet have increased CLDN14 expression, but only in the cortical

372 region of the kidney, where the CLDN16/CLDN19 complex is expressed (185). Thus, the

373 increase in CLDN14 induced by hypercalcemia, may block paracellular permeation of

374 Ca2+ and thereby increase urinary excretion of Ca2+, to stabilize serum Ca2+ levels (62). A

375 recent genome-wide association study found a correlation between a SNP in the CLDN14

376 gene and the ratio of Mg2+/Ca2+ in urine (44). As such, Cldn14 deficient mice show a

377 lower fractional excretion of both Ca2+ and Mg2+, when placed on a high Ca2+ diet (89).

378 Furthermore, overexpression of the Cldn14 gene in the TAL leads to an increase in the

379 fractional excretion of both Ca2+ and Mg2+ (88).

380

381 Loop diuretics

382 Loop diuretics are a class of sulfamoylbenzoic acid derivatives that include furosemide,

383 bumetanide, torasemide, piretanide, and benzmetanide, as well as ethacrynic acid that

384 lacks a sulfonamide substituent. Among loop diuretics, furosemide is by far the most

385 commonly employed clinically. Furosemide treatment is implemented to correct edema

386 formation and may be administered in the treatment of chronic heart failure, hepatic

387 cirrhosis, renal disease, and acute pulmonary edema (74, 82, 85, 212). Furosemide is a

388 weak organic anion that is predominantly protein bound in plasma. Therefore, only a

389 minor fraction gets filtered by the kidney and the majority is delivered into the luminal

390 fluid by proximal tubular anion transporters (183). Furosemide is the diuretic of choice

17
391 when targeting the TAL and functions as an effective pharmacological inhibitor of

392 NKCC2-dependent transport, by competing for the Cl- binding site (174) (Figure 2).

393 Understandably, given the important role of the TAL in Na+ reabsorption, targeting the

394 NKCC2 transporter leads to a marked natriuretic response (69, 224). As such, patients

395 with antenatal manifestations of Bartter syndrome do not respond appropriately to

396 furosemide when tested (126).

397 Since administration of furosemide diminishes the lumen-positive transepithelial

398 voltage gradient across the TAL epithelium (35, 55, 90), by inhibiting the primary

399 mechanism generating this potential difference, it is not surprising that Ca2+ and Mg2+

400 transport in TAL is significantly inhibited by furosemide administration (59, 190). Loop

401 diuretics such as furosemide and ethacrynic acid promote hypercalciuria and

402 hypermagnesiuria in experimental animals (65, 69, 70, 141). Urinary losses of both Ca2+

403 and Mg2+ have also been reported in human subjects given loop diuretics, including

404 furosemide, torasemide and ethacrynic acid (105, 140, 198). In a double-blind placebo

405 controlled study, furosemide significantly increased urinary Ca2+ and Mg2+ excretion 3

406 hours post a single oral dose (140). However, subsequent collections for the reminder of

407 24 hours showed an overall compensatory response, with a decrease in the urinary

408 excretion of divalent cations. Similar finding were documented for urinary NaCl

409 excretion (140). In healthy volunteers, both furosemide and torasemide dose-dependently

410 increased the fractional excretion of Ca2+ and Mg2+ during 3 consecutive clearance

411 collections of 30 minutes (64). Comparison of a single dose of furosemide (40 mg) with

412 torasemide (20mg) in patients with chronic heart failure showed an increased urinary

413 excretion of Ca2+ during the first 4 hours of collection. However, only the effect of

18
414 torasemide persisted for the next 8 hours, in comparison to furosemide (205). This can be

415 explained by the longer half-life of torasemide, with both compounds acting on the same

416 tubular site, namely NKCC2-dependet transport in the TAL. Overall loss of Ca2+ and

417 Mg2+ following loop diuretic administration may depend strongly on dosing regimes.

418 Systemic Ca2+ concentrations are in general much more tightly regulated than Mg2+. This

419 may explain why hypomagnesemia occurs in some patients treated with loop diuretics,

420 while a reduction in serum Ca2+ levels are rarely reported (4, 75). However, clinical

421 studies directly comparing these side effects are lacking. The occurrence of alterations in

422 divalent cation homeostasis during prolonged treatment may therefore be related to both

423 the dosing regimen of loop diuretic as well as preexisting deficits in Ca2+ and Mg2+

424 balance.

425 Patients with Bartter’s syndrome may be hypotensive, due to NaCl wasting,

426 display hypokalemia, metabolic alkalosis, and hyperaldosteronism, along with an

427 excessive amount of prostaglandin E2 in the urine (17, 73, 156). Although the clinical

428 phenotype of Bartter syndrome may resemble prolonged administration of a loop diuretic

429 in supraphysiological dosages, variance exists between the different types of Bartter in

430 relation to divalent cation handling. The underlying mechanisms for these discrepancies

431 remain to be investigated in detail, but likely relate to the molecular components mutated,

432 keeping in mind that loop diuretics target only the NKCC2 transporter and full inhibition

433 is unlikely during normal dosing regimes. In fact, while patients with antenatal Bartter’s

434 syndrome (due to mutations in NKCC2 and ROMK) present with significant

435 hypercalciuria and secondary nephrocalcinosis, these patients do not commonly show

436 disturbed Mg2+ balance (156). However, patients suffering from classical Bartter

19
437 syndrome type III, due to mutations in the CLCNKB gene often develop

438 hypomagnesemia, but rarely hypercalciuria or secondary nephrocalcinosis. As compared

439 to the antenatal form of Bartter, the absence of Ca2+ wasting in classical Bartter patients

440 could stem from less compromised TAL transport pathways as NaCl loss in some

441 patients with CLCNKB defects is often less severe, possibly due to alternative basolateral

442 efflux mechanisms for Cl- in the TAL (92, 130). Recent evidence supports these

443 assumptions. Disruption of the mouse Clcnk2 gene encoding the CLC-K2 Cl- channel

444 (equivalent to the human CLCNKB gene encoding CLC-Kb) leads to a Bartter phenotype

445 with renal NaCl wasting and hypokalemic metabolic alkalosis. Importantly, Hennings et

446 al, found expression of the highly homologues CLC-K1 Cl- channel in the medullary

447 portion of the TAL in Clcnk2-deficient mice (108), suggesting at least some tubular NaCl

448 transport is retained in that segment. How that would affect the lumen-positive voltages

449 along the length of the TAL remains to be determined. Urinary Ca2+/creatinine ratios did

450 not differ between Clcnk2-deficient mice and wildtype littermates (108), akin to the

451 findings obtained from Bartter type III patients. Another recently developed Clcnk2-

452 deficient mouse model did not show changes in serum Mg2+ concentration, however,

453 these mice were severely volume depleted (95). Hennings et al found increased urinary

454 Mg2+/creatinine ratios in their model (108). Importantly, these mice also had altered DCT

455 morphology, with atrophy of the early portion, potentially explaining why Mg2+ wasting

456 was present (discussed in detail later).

457

458 ROMK inhibitors

20
459 The recent development of inhibitors of ROMK allows targeting of NKCC2 dependent

460 NaCl transport, by inhibiting K+ recycling in the TAL (Figure 2). Furthermore, ROMK is

461 also expressed in more distal portions of the renal tubule, where it allows K+ secretion.

462 Here the electrochemical gradient for K+ secretion is strongly dependent on the

463 electrogenic uptake of Na+, through the epithelial Na+ channel, ENaC (Figure 3).

464 Compensatory increases in Na+ transport via ENaC occur during inhibition of TAL

465 transport, which leads to depolarization of the cell, and ROMK-dependent K+ secretion.

466 Therefore, blocking the ROMK channel would allow inhibition of both Na+ and K+

467 transport in the TAL, along with substantial inhibition of K+ secretion in more distal

468 parts, potentially minimizing urinary K+ losses. In fact, pharmacological inhibition of

469 ROMK leads to significant natriuresis and diuresis, effectively lowering blood pressure,

470 without substantial effects on urinary K+ excretion (103). Administration of ROMK

471 inhibitors to dogs for 7 days, did not alter serum levels of either Ca2+ or Mg2+ (103).

472 However, a single dose elicited an initial increase in urinary Ca2+ and Mg2+ excretion,

473 when measured initially or after 7 days, of treatment, paralleling the loss of Cl- (103). In

474 spontaneously hypertensive rats, urinary Ca2+ dose-dependently increased after ROMK

475 inhibitor administration for 4 days (103). Furthermore, high dose ROMK inhibition

476 increased urinary excretion of Ca2+ in Dahl salt-sensitive rats on a high NaCl diet.

477 However, since dietary NaCl supplementation results in markedly increased urinary

478 divalent cation excretion due to inhibition of paracellular transport in kidney, it is difficult

479 to assess the direct effect of ROMK inhibition on Ca2+ or Mg2+ transport in this

480 experimental setting (251). Overall, it seems that ROMK inhibitors target the TAL in a

481 similar manner as furosemide, thereby dissipating the transepithelial voltage gradient,

21
482 which is of upmost importance for driving the paracellular reabsorption of Ca2+ or Mg2+

483 across the epithelium and this likely contributes to loss of divalent cations.

484

485 Transcellular transport of Ca2+ and Mg2+ in the distal convolution and collecting

486 system

487 The distal convolution is a microanatomical division of the renal tubule that encompasses

488 several segments of the nephron and collecting system. These include mainly the distal

489 convoluted tubule (DCT), the connecting tubule (CNT), as well as the initial superficial

490 portion of the cortical collecting ducts (CD) (132). Besides differences in the structural

491 appearance of cells, the distal convolution can be identified by the specific expression of

492 key transport proteins (Figure 3). Cells in the DCT express the thiazide-sensitive NaCl

493 cotransporter, NCC (176). The DCT segment can be further subdivided into a DCT1 and

494 DCT2. The latter segment is identified by the coexpression of NCC and ENaC (37, 149).

495 ENaC expression is also observed throughout the remainder of the collecting system.

496 Furthermore, the vasopressin regulated water channel Aquaporin 2 (AQP2) is expressed

497 exclusively in the CNT and CD segments of the tubule (170). In addition to the TAL,

498 ROMK is expressed in the distal nephron and plays a role in K+ secretion in the CNT and

499 CD (128, 143, 159) (Figure 3). Along the length of the distal convolution, intercalated

500 cells appear, with highest abundance in the CNT and CD. These cells are involved in

501 acid-base handling and also facilitate electroneutral NaCl reabsorption (99, 144). Species

502 differences are observed in the arrangement of the above-mentioned segments within the

503 distal convolution. As such, the subsegmentation of the DCT as well as the histological

504 division of the individual segments and appearance of various transport proteins, differ

22
505 between mice, rat, rabbit and man (147, 149). For instance, the DCT2 segment is

506 pronounced in mouse and rat, shortened in human and absent in rabbit (147). Given the

507 markedly different expression pattern of transporters along the distal convolution, with

508 predominantly electroneutral NaCl transport occurring in the early distal convolution

509 transitioning towards an electrogenic Na+ uptake in the late convolution, it is not

510 surprising that the transepithelial voltage gradually decreases from between 0 and -5 mV

511 in the DCT to -40 mV in the CNT (84, 153, 195, 223, 247). The transepithelial voltage

512 gradient across the CNT is much larger than in the cortical CD, due to a comparatively

513 higher transport of Na+ and K+ in the CNT segment. Consequently, the transepithelial

514 voltage in the CCD may drop to -15 mV (123, 127, 175, 231).

515 The cells lining the distal convolution give rise to a high resistance epithelium,

516 with values reported of 150 Ω · cm2 in the early distal convolution, and increasing by

517 almost 6-fold in the cortical CD (107, 153). Although expression of CLDN16 and

518 CLDN19 extends into the DCT, suggesting possible paracellular transport of Ca2+ and

519 Mg2+ (124, 129, 221) in this segment (not depicted in figure 3), divalent cation transport

520 across the distal convolution is thought to occur via a transcellular route. Active transport

521 of Ca2+ and Mg2+ has been described in the distal convolution, with unidirectional

522 divalent transport occurring in the presence of lumen negative transepithelial voltages

523 (137, 192). As estimated by classical micropuncture studies, approximately 3-7% of the

524 Ca2+ filtered by the kidney is reabsorbed in the distal convolution (5, 137, 139). Similar

525 physiological measurements suggest that 5-6% of filtered Mg2+ is reabsorbed in the distal

526 convolution (14, 33, 139).

23
527 The transport machinery driving active transcellular Ca2+ transport in the distal

528 convolution is well defined. It consists of the Transient Receptor Potential Vanilloid 5

529 (TRPV5) channel located on the apical membrane, which facilitates the uptake of Ca2+

530 from the filtrate (109, 110) (Figure 3). In mouse, TRPV5 is expressed in the DCT2

531 segment as well as the CNT and the initial portion of the CD (114, 147, 149). Expression

532 and localization of TRPV5 in the human kidney has not been determined. In mouse,

533 TRPV5 localizes with Calbindin-D28K, which may function as an intracellular Ca2+ buffer

534 and aid in shuttling Ca2+ from apical to basolateral aspects of the cell (131). Extrusion of

535 Ca2+ from the cell into renal interstitium, is driven by the Na2+/Ca2+ exchanger type 1

536 (NCX1) and the plasma membrane Ca2+-ATPase proteins (PMCA1 and PMCA4) (8, 197,

537 239). Both NCX and PMCA4 are highly abundant in the DCT2 and CNT region of the

538 mouse, colocalizing with TRPV5, however weaker expression of the basolateral Ca2+

539 extrusion protein have also been noted in DCT (not depicted in figure 3), although no

540 apical Ca2+ channel has been documented in the segment (8, 149). Targeted transgenic

541 strategies have been implemented to understand the contribution of these genes to overall

542 Ca2+ transport within the distal convolution. Mice with a targeted deletion of Trpv5, show

543 the most pronounced phenotype and display massive renal Ca2+ wasting, osteopenia, and

544 compensatory hyperabsorption from the intestine (112). Further, Ca2+ concentration was

545 determined in relation to tubular fluid K+ along the length of the superficial distal

546 convolution accessible to micropuncture in Trpv5-deficient mice. The K+ concentration

547 increases in luminal fluid along the length of the distal convolution, due to water removal

548 from the tubular fluid in combination with the onset of electrogenic Na+ transport along

549 the tubule. Thus, in basal states, the luminal K+ concentration is a good marker for

24
550 distance along the distal convolution. In wildtype mice, fractional Ca2+ delivery markedly

551 decreases concomitantly with increased luminal K+ concentration, suggesting that

552 significant reabsorption of Ca2+ takes place along the length of the distal convolution, in

553 line with the expression pattern of TRPV5. In mice lacking Trpv5, this relationship

554 between Ca2+ delivery and luminal K+ concentration was no longer apparent, with a

555 markedly elevated fractional Ca2+ delivery found along the distal convolution, indicative

556 of significant wasting within that segment (112). In line with these findings, a rare

557 missense variant in TRPV5 correlates with recurrent kidney stones (177), however no

558 loss-of-function mutations have been described in humans.

559 Transport of Mg2+ in the distal convolution is predicted to occur via the divalent

560 cation selective Transient Receptor Potential Melastin 6 channel (TRPM6) (241). The

561 role of TRPM6 was determined via elucidation of the causative gene defective in the

562 autosomal recessive disorder of Hypomagnesemia with Secondary Hypocalcemia (HSH).

563 These patients display a severe Mg2+ intestinal absorptive defect, as TRPM6 is expressed

564 in the colonic enterocyte (162, 206, 243). A role for TRPM6 in renal reabsorption of

565 Mg2+ is supported by the observation that HSH patients display an elevated fraction

566 excretion of Mg2+ even in the presence of hypomagnesemia (206, 243). Given a low

567 serum Mg2+ concentration, failure to sufficiently lower urinary fractional Mg2+ excretion

568 is consistent with renal wasting (4). Furthermore, when HSH patients are subjected to

569 intravenous Mg2+ loading, the urinary Mg2+ leak becomes apparent (243). Detailed

570 micropuncture studies utilizing early and late puncture sites from the same distal

571 convolution, demonstrate that Mg2+ transport dominates in the early portion of the

572 convolution, likely the DCT (14). Immunohistochemical localization studies identified

25
573 TRPM6 in the apical membrane of cells in the DCT (241), consistent with the

574 micropuncture data reported above. Very recently, Chubanov, Gudermann and colleagues

575 performed a series of elegant experiments utilizing various targeted transgenic strategies

576 in order to understand the contribution of Trpm6 to intestinal and renal Mg2+ transport

577 (41). The selective intestinal deletion of Trpm6 resulted in a marked phenotype of severe

578 Mg2+ depletion, due to inefficient Mg2+ uptake from the intestine. However, specific

579 deletion of Trpm6 from renal epithelial cells, using the Ksp-Cre model did not cause a

580 change in serum levels nor the renal excretion of Mg2+, suggesting that, at least in mouse,

581 TRPM6 is not the only apical entry mechanism for Mg2+ reabsorption from the distal

582 convolution (41). It still remains to be clarified whether the structurally similar channel

583 homologue TRPM7 also participates in apical uptake of Mg2+ from the DCT and hence in

584 renal Mg2+ transport (42, 63, 145, 207, 241). Several genes localizing to the distal

585 convolution cause genetic syndromes of hypomagnesemia (51, 63). These gene defects

586 suggest that the resting membrane potential of the DCT cell is critical for Mg2+ transport

587 in kidney (26, 51, 63, 87, 158). Consistent with this, the intracellular Mg2+ concentration

588 ranges between 0.2 and 1.0 mM (98, 191), while that in the luminal fluid reaching the

589 DCT is estimated to be 0.2 - 0.7 mM (49). Therefore, the membrane potential of the DCT

590 cell likely determines the apical uptake of Mg2+ into the DCT cell (43, 104). Aside from

591 TRPM6, the molecular transport machinery involved in transepithelial Mg2+ transport in

592 the distal convolution remains poorly defined.

593 Ca2+ and Mg2+ transport appears largely restricted to the distal convolution,

594 predominantly the DCT and CNT segments depending on the ion. Little transport of Ca2+

595 and Mg2+ is expected to occur within the remainder of the CD, although some studies

26
596 found evidence of small yet measurable flux. Micropuncture studies, using the lumen K+

597 concentrations for orientation along the distal convolution, found that the fractional

598 delivery of Ca2+ is very small towards the end of the distal convolution, suggesting

599 negligible transport of Ca2+ in downstream segments (112, 150, 173). Consistent with

600 this, Loffing et al, found that NCX1 and TRPV5 expression ends in the CNT and is not

601 detectable in the CCD (149). Conversely, mice expressing green fluorescent protein after

602 the Trpv5 promoter demonstrate expression of the transgene in cortical CD as well (113).

603 In rabbit, microperfusion of isolated cortical CD did not reveal significant Ca2+ flux (30,

604 217). However, other studies report significant permeability for Ca2+ in the

605 microperfused rabbit cortical CD (115, 121). Microcatheterization studies of rat inner

606 medullary CD found a small, albeit significant decrease in Ca2+ over inulin ratio over the

607 length of the inner medullary CD (21). Thus, the contribution of the CD to overall Ca2+

608 transport appears to be minor, but physiologically relevant Ca2+ transport across the

609 segment cannot be excluded.

610 Mg2+ transport in the CD appears to be absent. Microperfusion experiments on

611 rabbit cortical CD could not find significant Mg2+ flux (217). Furthermore, the

612 microcatherization studies outlined above, failed to find significant absorption of Mg2+

613 along the inner medullary CD (21).

614

615 Thiazide-type diuretics

616 The class of thiazide-type diuretics includes a range of compounds with a

617 benzothiadiazine structure e.g. hydrochlorothiazide, methyclothiazide, cyclothiazide,

618 bendroflumethiazide. Furthermore, chlorthalidone, metolazone, and indapamide are

27
619 thiazide-like diuretics, which do not share the common benzothiadiazine structure.

620 However all the above-mentioned compounds block NaCl transport in the DCT, by

621 inhibiting the NaCl cotransporter, NCC (Figure 3)(195, 237, 240). The mechanism of

622 action for metolazone has been suggested to result from interaction with the Cl- binding

623 site of NCC, thereby blocking transport (237). However, a subsequent study found that

624 the Cl- binding site and the binding site of metolazone might reside in different locations

625 within the cotranporter (164). Furthermore, thiazides likely also target another transport

626 process in intercalated cells, which contributes to electroneutral NaCl reabsorption (144).

627 In addition to direct diuretic actions on the DCT, effects of thiazides and the thiazide-like

628 diuretic indapamide on vascular function have been noted in experimental animals and

629 humans (reviewed in detail by (68, 234)). Acutely, hydrochlorothiazide can elicit

630 endothelium-dependent vasodilation, which may contribute to the blood pressure

631 lowering effect. However, since thiazides do not seem to affect blood pressure in

632 normotensive patients, it is questionable whether these effects are clinically relevant (68).

633 Indapamide can also elicit a decrease in total peripheral resistance, by acting on the

634 vasculature. Mechanistically, this has been described to result from reductions in

635 membranous Ca2+ influx in the smooth muscle cell, leading to a reduction in vascular

636 reactivity in response to vasoconstrictors (163). Interestingly, indapamide seems to exert

637 its antihypertensive effect in the absence of a strong diuretic activity at standard dosing

638 schemes of 2.5 mg daily, used for the treatment of hypertension (2, 50, 165). Similar

639 findings can be recapitulated in hypertensive patients with severe renal impairment and

640 hence almost no renal function (average creatinine clearance of 16 ml/min) (97),

28
641 suggesting that the blood pressure lowering effect of indapamide may in part result from

642 reducing total peripheral resistance.

643 Among this class of diuretics, hydrochlorothiazide is the most commonly

644 prescribed. The Joint National Committee for the detection, evaluation, and treatment of

645 high blood pressure, recommend thiazide diuretics as first line treatment or in

646 combination with other antihypertensive agents for patients with stage I hypertension

647 (uncomplicated) and stage II hypertension, respectively (39). Furthermore, thiazides are

648 available at a low cost making them important compounds in the treatment of

649 hypertension worldwide. In addition to lowering blood pressure by eliciting renal NaCl

650 loss, thiazides also promote hypocalciuria and may lead to hypercalcemia in some

651 patients (40, 244). Due to these properties, thiazides are frequently used in the treatment

652 of kidney stone disease by reducing urinary Ca2+ and to alleviate hypocalcemia by

653 increasing renal Ca2+ reabsorption (136, 178). Furthermore, chronic thiazide treatment

654 can cause hypomagnesemia (133, 182), likely due to renal wasting (140). More

655 pronounced perturbations in divalent cation balance can be observed in individuals with

656 Gitelman syndrome. These patients have a defect in the SLC12A3 gene encoding the

657 thiazide-sensitive NaCl cotransporter, NCC (86, 222). Loss of function mutations in

658 SLC12A3 leads to NaCl wasting, hypokalemia, metabolic acidosis, hypomagnesemia and

659 hypocalciuria (86).

660 Thiazide treatment can cause a robust decrease in the urinary excretion of Ca2+ (32,

661 122). This effect is independent of parathyroid hormone, as similar findings are observed

662 in patients with hypo- and hyperparathyroidism (32). A hypocalciuric effect has also been

663 reported when analyzing 24-hour urinary Ca2+ excretion following a single dose of

29
664 thiazide to healthy postmenopausal women and volunteers (32, 140). However, when

665 urinary Ca2+ excretion was measured more frequently following a single oral dose, the

666 hypocalciuric effect was only observed in the urine collection obtained 12-24 hours post-

667 administration, even though effects on NaCl and fluid excretion were evident earlier

668 (140). Similar effects as seen in humans were reported on divalent cation balance in

669 rodents following chronic thiazide treatment and in mouse models of Gitelman syndrome

670 (142, 150, 172, 211, 249). However, acute administration of thiazides following a bolus

671 injection to animals yielded conflicting results, with respect to the onset of thiazide-

672 induced hypocalciuria, relative to the timing of thiazide-induced NaCl losses (142, 173,

673 249).

674 The mechanisms leading to hypocalciuria after thiazide treatment or in Gitelman

675 patients is still not fully delineated and may depend on alterations in both proximal and

676 distal transport mechanisms as reviewed in detail by Reilly and Huang (196). The

677 hypocalciuric effect from alterations in proximal transport likely depends on volume

678 contraction, following administration of thiazides. Specifically it is thought that renal

679 losses of NaCl due to NCC inhibition augment Na+ reabsorption in the PT, in order to

680 normalize extracellular circulating volume. Since, Ca2+ transport in the PT occurs in

681 parallel with that of Na+, Na+ hyperabsorption leads to increased Ca2+ transport and hence

682 hypocalciuria. Substantial experimental evidence supports this. For instance, when

683 healthy volunteers had their NaCl losses replaced, the hypocalciuric effect of thiazide

684 administration was absent (32). Detailed physiological measurements using

685 micropuncture in wildtype mice revealed an increased PT reabsorption of Na+, fluid, and

686 Ca2+ following administration of thiazides for 6 days, clearly supporting a contribution of

30
687 this pathway to thiazide-induced hypocalciuria (173). Similar findings localizing the

688 hypocalciuric effect to the PT have been obtained from micropuncture studies in mice

689 lacking the Slc12a3 gene (150). Use of NaCl depletion to alter volume status resulted in a

690 robust reduction in Ca2+ excretion in wildtype mice, while such dietary manipulations did

691 not affect Ca2+ excretion in Slc12a3–deficient mice (211). These findings are supportive

692 of an already reduced extracellular volume and hence increased PT transport in mice

693 lacking the Slc12a3 gene. Micropuncture studies conducted on mouse kidney following

694 chronic thiazide administration did not reveal an additional distal reabsorptive effect.

695 However, the fractional Ca2+ delivery to distal puncture sites was measured in relation to

696 K+ concentration. This is potentially problematic as NCC inhibition alters K+

697 reabsorption in the distal nephron, by inducing a shift towards electrogenic ENaC-

698 dependent Na+ transport and concomitant K+ secretion (233). Nonetheless, the thiazide-

699 dependent hypocalciuric effect was still visible in Trpv5-deficient mice, suggesting an

700 important contribution of the PT pathway to the mechanism of thiazide-induced Ca2+

701 retention (173).

702 Indapamide also reduces urinary Ca2+ excretion in a number of trials, including in

703 stone-formers (10, 29, 50, 155). In line with a dual mechanism of action for this

704 compound, the blood pressure lowering effect obtained by acting on both the vasculature

705 and by inhibiting NaCl transport in the DCT, could both lead to activation of the renin-

706 angiotensin-aldosterone system (50) and hence stimulate Na+ transport in the PT (102).

707 Indapamide may thus promote hypocalciuria by stimulating PT reabsorption, similar to

708 the other thiazide-type diuretics, although the mechanisms of action might partly differ.

31
709 A distal mechanism contributing to thiazide-induced hypocalciuria has also been

710 suggested. Some studies found an additional effect of thiazides on Ca2+ transport in the

711 distal convolution (46, 76, 142, 196). Consistent with this, increased expression of genes

712 involved in transcellular Ca2+ transport in the distal convolution have been seen after

713 thiazide administration (e.g. TRPV5 and Calbindin-28K), but only when salt

714 supplementation (NaCl and KCl) was given, suggesting that the distal effect may be

715 apparent only when extracellular volume contraction is prevented (142). However, in the

716 same study, salt supplementation alone had similar effects. As such, decreased PT

717 transport in response to volume expansion, which could be envisioned to elicit an

718 increase in the distal transport machinery, to compensate for PT losses of Ca2+.

719 Chronic thiazide treatment in hypertensive subjects results in lower than normal

720 serum Mg2+ concentrations, although the difference compared to patients not receiving

721 diuretics is relatively subtle (133). As such, the appearance of hypomagnesemia was

722 reported to be uncommon in this population of thiazide treated patients.

723 Hypomagnesemia is more commonly observed in the elderly treated with thiazide-

724 diuretics (182). Furthermore, hypomagnesemia remains a hallmark feature of Gitelman

725 syndrome (86). The reason for these differences may relate to incomplete inhibition of

726 the cotransporter during thiazide treatment and preexisting deficits in Mg2+ stores, which

727 may tend to be more prevalent in the elderly. As such, hypertensive patients receiving the

728 highest dose of thiazides were more prone to developing hypomagnesemia, than patients

729 receiving lower doses (133). Unlike Ca2+, renal Mg2+ wasting is evident within 3 hours

730 following thiazide-administration to healthy volunteers (140). The effect of chronic

731 thiazide administration or Gitelman syndrome on renal Mg2+ handling as understood

32
732 from animal models are complicated somewhat by the substantial alterations that occur in

733 the morphology of the distal convolution (148, 150, 173, 211, 249). Both mice and rats

734 treated with thiazides and transgenic models of Gitelman syndrome display marked

735 atrophy of the early DCT1 segment and compensatory increases in the DCT2 and CNT

736 (148, 150, 249). Other experimental models using thiazide administration in mice, found

737 no evidence of significant alterations in DCT volume (142, 173). These findings are

738 likely dependent on dosage and duration of treatment (148). Since transcellular Mg2+

739 transport within the distal convolution is primarily localized to the DCT, it is not

740 surprising that atropy of the early portion of the segment i.e. DCT1 promotes Mg2+

741 wasting, possibly by downreagulation of TRPM6. However, one study reported

742 downregulation of TRPM6 in the absence of changes in morphology of the distal

743 convolution (173). It is also interesting to note that indapamide does not seem to affect

744 the urinary excretion of Mg2+ at therapuetical dosages used in the treatment of

745 hypertension (1.5-2.5 mg), as does the other thiazide-type diuretics. Electrolyte

746 measurement in a 24 hour urine collection followin a single dose of 2.5 mg indapamide

747 showed increased urinary NaCl excretion, reduced urinary Ca2+ excretion, but the total

748 amount of Mg2+ excreted in the urine was not altered (199). In patients with stone

749 disease, 6 or 18 months of treatment with indapamide, 1.5 mg sustained release

750 formulations, reduced urinary Ca2+, but did not affect urinary Mg2+ excretion (10).

751 Similarly, no effect could be documented on serum Mg2+ levels (10, 232). Based on the

752 limited amount of data, it is difficult to draw firm conclusions on this, however the

753 avaliable studies do suggest that indapamide may have a Mg2+-sparing effect, by virtue of

754 its mechanism of action, namely by acting on both the vasculature and the DCT to lower

33
755 blood pressure. As such, a comparatively lower inhibiton of NCC activity, may allow a

756 lesser inhibiton of TRPM6 function, either by preventing atrophy of the DCT or

757 amiliorating direct downregulation of the channel. In conclusion, additional studies are

758 needed to get a clearer understanding of the mechanisms leading to atrophy of the DCT

759 as well as direct inhibition of Mg2+ transport within the distal convolution conferred by

760 thiazide-type diuretics.

761

762 K+-sparing diuretics

763 K+-sparing diuretics block either ENaC (which is expressed in the DCT2, CNT and CD),

764 or the mineralocorticoid receptor (MR), which has a slightly broader expression pattern

765 along the epithelium of the distal tubule) (3, 37, 149) (Figure 3). MR deletion in a subset

766 of tubular epithelial cells, does not alter NCC expression, while it has marked effects on

767 ENaC and Na+/K+-ATPase expression in the collecting system (48), suggesting that the

768 primary effect for both types of diuretics on electrolyte transport is the blockade of

769 electrogenic ENaC-mediated Na+ reabsorption in the distal convolution and CD. Such

770 alterations yield concomitant reductions in tubular K+-secretion. The compounds directly

771 blocking ENaC, amiloride and triamterene, as well as the MR antagonists spironolactone

772 and eplerenone, are the most commonly used drugs in this category. K+-sparing diuretics

773 are weak diuretics often used in combination with other diuretics to lower blood pressure

774 or to prevent fluid accumulation in edematous conditions such as congestive heart failure

775 ((1, 184)). Their K+-sparing ability is important to minimize the risk of hypokalemia,

776 either when combined with other diuretics or alone in conditions with frank hypokalemia.

34
777 Both classes of K+-sparing diuretics alter renal Ca2+ and Mg2+ transport.

778 Administration of triamterene to healthy subjects resulted in an initial Ca2+ loss, which

779 after 6 hours changed to a decrease in urinary Ca2+ excretion (105). In patients with

780 essential hypertension, spironolactone reduces urinary Ca2+ excretion (83). Other studies

781 found an increase in urinary Ca2+ excretion following spironolactone administration,

782 however as summarized by Prati et al, at least some formulations of spironolactone and

783 amiloride contain significant amounts of Ca2+ (188). Evidence from experimental animals

784 suggests that K+-sparing diuretics have a Ca2+ retaining effect. Both triamterene and

785 amiloride lower the Ca2+/Na+ clearance ratio in dogs, although the effect was much lower

786 than that for thiazides (47). However, only amiloride directly altered the fractional

787 excretion of Ca2+ in this experimental series. In rats, Costanzo observed a reduced

788 fractional excretion of Ca2+ after administration of amiloride (45). Using micropuncture,

789 addition of amiloride to the perfusate augmented reabsorption of Ca2+ from the late

790 portion of the distal convolution, yet both fluid and Na+ reabsorption was inhibited (45).

791 No such effect was present in the early portion, suggesting a direct effect on distal Ca2+

792 transport machinery. The authors note that the increase in Ca2+ reclamation was tightly

793 correlated to the degree of inhibition of Na+ absorption. Similarly, administration of

794 amiloride to rats treated with furosemide, reduced the fractional excretion of Ca2+,

795 although this was not seen in the absence of furosemide (57). For spironolactone, no Ca2+

796 sparing effect has been described in experimental animals. Thus K+-sparing diuretics may

797 exert a modest effect, if any, upon Ca2+ transport in kidney. The mechanism behind this

798 remains to be fully delineated, but may relate to hyperpolarization of the cell during

799 inhibition of ENaC. In mouse DCT cells, amiloride blocks Na+ uptake, hyperpolarizes the

35
800 membrane potential and increases Ca2+ uptake by 39% (80). Furthermore, TRPV5

801 displays hyperpolarization-dependent activation (111), suggesting that the effect of K+-

802 sparing diuretics on Ca2+ transport may occur via increased TRPV5 mediated Ca2+

803 absorption. An additional proximal effect could be envisioned if these compounds reduce

804 extracellular volume, as they may also relieve the inhibition of PT transport during

805 volume expanded states and thereby enhance paracellular Ca2+ reabsorption in the PT, as

806 occurs for thiazides.

807 An effect of K+-sparing diuretics on urinary excretion of Mg2+ has been

808 documented in both humans and animals. Hypertensive patients treated with triamterene

809 display higher serum Mg2+ levels than untreated patients (133). Furthermore, in healthy

810 subjects, oral administration of triamterene lead to Mg2+ retention, already after the first

811 hour, an effect that lasted for the entire study period of 10 hours (105). In contrast,

812 another study conducted on healthy subjects did not find acute changes in urinary Mg2+

813 excretion following a single oral dose of amiloride (125). Patients suffering form primary

814 aldosteronism have normal serum Mg2+ concentrations, but display a higher urinary Mg2+

815 excretion (116). Horton & Biglieri found that adrenalectomy or spironolactone treatment

816 reduced urinary Mg2+ excretion, although it was unclear whether the effect of

817 spironolactone relates to reductions in extracellular fluid volume (116). In healthy

818 volunteers, spironolactone treatment for 5 days decreased urinary Mg2+ excretion (225).

819 Another spironolactone-like compound (Canrenoate potassium) also has Mg2+ sparing

820 properties in liver cirrhosis patients (146). A comparison of amiloride and spironolactone

821 in thiazide-treated healthy subjects, only found a positive effect of amiloride on serum

822 Mg2+ concentrations (166).

36
823 In experimental animals, Devane and Ryan found that both amiloride and

824 triamterene reduced urinary excretion of Mg2+ in saline loaded rats. Furthermore, the

825 authors found that amiloride inhibited urinary excretion of Mg2+, even when

826 coadministered with furosemide (56, 57). In another study, the highest dose of amiloride

827 elicited the greatest reduction in the fractional excretion of Mg2+ from furosemide

828 infusion, while no alteration in hematocrit was seen (56), suggesting that the effect was

829 independent of changes in volume status. In another study, the authors found no change

830 in glomerular filtration or serum Mg2+ concentration, during three 30 minute urine

831 collections in clearance experiments in rats undergoing amiloride infusion (58).

832 Importantly, in this study, they were able to control for extracellular volume changes by

833 fluid and electrolyte infusion, which probably yielded an even larger extracellular volume

834 by the end of the study period. Under these conditions, amiloride was still able to reduce

835 urinary fractional excretion of Mg2+ (58). Few animal studies have examined the effect of

836 spironolactone on Mg2+ balance, while studies addressing the direct effect on renal Mg2+

837 handling is scarce. In dogs with degenerative mitral valve disease, spironolactone

838 stimulated a small yet significant increase in serum Mg2+ concentrations after 20 weeks

839 (235). Injection of spironolactone for 5 days in rabbits also resulted in a higher serum

840 Mg2+ concentration (215). Overall, based on these studies, the underlying mechanism for

841 Mg2+ retention induced by K+-sparing diuretics seems to occur independent of changes in

842 extracellular volume. An often cited explanation, is the fact that ENaC blockade

843 hyperpolarizes the cell by inhibiting Na+ permeability of the apical membrane (66),

844 thereby increasing the driving force for Mg2+ entry into the cells of the early distal

845 convolution. Indeed, several gene defects highlight the sensitivity of Mg2+ transport in the

37
846 DCT to changes in resting membrane potential (26, 51, 63, 87, 158). Structurally, the

847 localization of Mg2+ transport along the DCT and the presence of ENaC in only the

848 DCT2 segment of mice (Figure 3), could account for such an effect in this later segment,

849 since various cell types are intermingled along this portion of the nephron (149).

850 However, the DCT2 is very small in humans (147) and it is therefore hard to envision

851 that inhibition of ENaC activity in this more distal portion, would amplify Mg2+ uptake in

852 the early distal convolution. Nevertheless, to date, very little information has been

853 published about the precise localization of the transcellular Mg2+ transport machinery in

854 human kidney. In conclusion, more studies are required to further delineate the potential

855 effects of K+-sparing diuretics on renal tubular transport of Ca2+ and Mg2+.

856

857 Summary

858 Many of the proteins involved in tubular transport of Ca2+ and Mg2+ have been identified

859 and studied within the last decades. These findings have contributed significantly to our

860 understanding of the molecular mechanisms mediating Ca2+ and Mg2+ transport within

861 the kidney. In contrast, diuretics have long been known to affect Ca2+ and Mg2+ balance,

862 by changing divalent cation handling in the kidney. The mechanism(s) whereby this

863 occurs has been detailed for some, but not all diuretics. However, an increased

864 understanding of the molecular transport pathways permitting Ca2+ and Mg2+

865 reabsorption from the renal tubule aid in clarifying these effects, although our

866 understanding is far from complete. Ongoing studies complemented with targeted

867 transgenic models, to allow specific deletions of transporters that are targeted by

38
868 diuretics, within select tubular epithelial cells, will be critical to further our understanding

869 of this topic.

870

871
872
873

39
874 Acknowledgements

875 The laboratory of H. Dimke is funded by Fabrikant Vilhelm Pedersen og Hustrus

876 Mindelegat (on recommendation from the Novo Nordisk Foundation), the Novo Nordisk

877 Foundation, the Carlsberg Foundation, the A.P. Møller Foundation, the Beckett

878 Foundation, and the Lundbeck Foundation. Dr. R. Todd Alexander is the Canada

879 Research Chair in Renal Tubular Epithelial Transport Physiology and an Alberta

880 Innovates Health Solutions Clinical Investigator. The laboratory of R. Todd Alexander is

881 funded by grants from the Natural Sciences and Engineering Research Council (NSERC)

882 of Canada and the Canadian Institute of Health Research (CIHR).

883

884

40
885 Figure legends

886 Figure 1

887 Schematic representation of the PT epithelial cell. Please note that this model represents

888 the convoluted S1 and S2 PT segments, where HCO3- reabsorption predominates. NHE3,

889 Na+/H+ exchanger 3; AQP1, Aquaporin 1; CA, Carbonic anhydrase; CLDN2, Claudin-2;

890 NBCe1, Electrogenic Na+, HCO3- cotransporter 1; SGLT2, Na+/glucose cotransporter

891 type 2; GLUT2, Glucose transporter type 2. Transepithelial potential differences across

892 the convoluted portion of the PT are obtained from (16).

893

894 Figure 2

895 Schematic model outlining the molecular transport proteins in the TAL. NKCC2, the

896 furosemide-sensitive Na+, K+, 2Cl- cotransporter; ROMK, renal outer medullary K+

897 channel; CLC-Kb, Cl- channel Kb; CLC-Ka, Cl- channel Ka, BSND, Barttin; CLDN10-

898 19, Claudin 10-19. The transepithelial voltages are listed. In the TAL, the transepithelial

899 potential difference may be +5 to +10 mV (93, 94), although the transepithelial voltage

900 can increase to values approximating +30 mV in the cortical protion of the segment as

901 outlined in the top of the figure (91, 117, 201).

902

903 Figure 3

904 Schematic representation of the cells lining the distal convolution. The epithelial cells

905 representing the DCT1, DCT2 and CNT are depicted. NCC, thiazide-sensitive NaCl

906 cotransporter; ROMK, renal outer medullary K+ channel; CLC-Kb, Cl- channel Kb;

907 ENaC, epithelial Na+ channel; TRPM6, transient receptor potential Melastin 6 Mg2+

41
908 channel; TRPV5, transient receptor potential Vanilloid 5 Ca2+ channel; NCX1, Na2+/Ca2+

909 exchanger type 1; PMCA, Plasma membrane Ca2+-ATPase proteins (PMCA1 and

910 PMCA4); MR, Mineralocorticoid receptor. The transepithelial voltage decreases from

911 about 0 to -5 mV in the DCT towards -40 mV in the CNT (84, 153, 195, 223, 247).

912 PMCA4 and NCX1 are highly abundant in the DCT2 and CNT region of the mouse,

913 colocalizing with TRPV5, however weaker expression of the basolateral Ca2+ extrusion

914 protein have also been noted in DCT (not displayed in figure) (8, 149). Although the MR

915 is expressed in the DCT, no effect on NCC has been documented, when the MR is

916 deleted (48). Spironolactone may also inhibit MR in the DCT1 cell – with unknown

917 consequences for Ca2+ and Mg2+ transport (not displayed in figure).

918
919
920
921
922
923
924
925

42
926 References
927
928 1. Consensus recommendations for the management of chronic heart failure. On
929 behalf of the membership of the advisory council to improve outcomes
930 nationwide in heart failure. Am J Cardiol 83: 1A-38A, 1999.
931 2. Acchiardo SR, and Skoutakis VA. Clinical efficacy, safety, and
932 pharmacokinetics of indapamide in renal impairment. Am Heart J 106: 237-244,
933 1983.
934 3. Ackermann D, Gresko N, Carrel M, Loffing-Cueni D, Habermehl D, Gomez-
935 Sanchez C, Rossier BC, and Loffing J. In vivo nuclear translocation of
936 mineralocorticoid and glucocorticoid receptors in rat kidney: differential effect of
937 corticosteroids along the distal tubule. American journal of physiology 299:
938 F1473-1485, 2010.
939 4. Agus ZS. Hypomagnesemia. J Am Soc Nephrol 10: 1616-1622, 1999.
940 5. Agus ZS, Chiu PJ, and Goldberg M. Regulation of urinary calcium excretion in
941 the rat. The American journal of physiology 232: F545-549, 1977.
942 6. Agus ZS, Gardner LB, Beck LH, and Goldberg M. Effects of parathyroid
943 hormone on renal tubular reabsorption of calcium, sodium, and phosphate. The
944 American journal of physiology 224: 1143-1148, 1973.
945 7. Alba M, Xie J, Fung A, and Desai M. The effects of canagliflozin, a sodium
946 glucose co-transporter 2 inhibitor, on mineral metabolism and bone in patients
947 with type 2 diabetes mellitus. Curr Med Res Opin 32: 1375-1385, 2016.
948 8. Alexander RT, Beggs MR, Zamani R, Marcussen N, Frische S, and Dimke H.
949 Ultrastructural and immunohistochemical localization of plasma membrane Ca2+-
950 ATPase 4 in Ca2+-transporting epithelia. American journal of physiology 309:
951 F604-616, 2015.
952 9. Alexander RT, Rievaj J, and Dimke H. Paracellular calcium transport across
953 renal and intestinal epithelia. Biochem Cell Biol 92: 467-480, 2014.
954 10. Alonso D, Pieras E, Piza P, Grases F, and Prieto RM. Effects of short and
955 long-term indapamide treatments on urinary calcium excretion in patients with
956 calcium oxalate dihydrate urinary stone disease: a pilot study. Scand J Urol
957 Nephrol 46: 97-101, 2012.
958 11. Amasheh S, Meiri N, Gitter AH, Schoneberg T, Mankertz J, Schulzke JD,
959 and Fromm M. Claudin-2 expression induces cation-selective channels in tight
960 junctions of epithelial cells. Journal of cell science 115: 4969-4976, 2002.
961 12. Angelow S, El-Husseini R, Kanzawa SA, and Yu AS. Renal localization and
962 function of the tight junction protein, claudin-19. American journal of physiology
963 293: F166-177, 2007.
964 13. Aronson PS. Role of ion exchangers in mediating NaCl transport in the proximal
965 tubule. Kidney international 49: 1665-1670, 1996.
966 14. Bailly C, Roinel N, and Amiel C. Stimulation by glucagon and PTH of Ca and
967 Mg reabsorption in the superficial distal tubule of the rat kidney. Pflugers Arch
968 403: 28-34, 1985.
969 15. Barker ES, Elkinton JR, and Clark JK. Studies of the renal excretion of
970 magnesium in man. The Journal of clinical investigation 38: 1733-1745, 1959.

43
 971 16. Barratt LJ, Rector FC, Jr., Kokko JP, and Seldin DW. Factors governing the
972 transepithelial potential difference across the proximal tubule of the rat kidney.
973 The Journal of clinical investigation 53: 454-464, 1974.
974 17. Bartter FC, Pronove P, Gill JR, Jr., and Maccardle RC. Hyperplasia of the
975 juxtaglomerular complex with hyperaldosteronism and hypokalemic alkalosis. A
976 new syndrome. The American journal of medicine 33: 811-828, 1962.
977 18. Beck LH, and Goldberg M. Effects of acetazolamide and parathyroidectomy on
978 renal transport of sodium, calcium, and phosphate. The American journal of
979 physiology 224: 1136-1142, 1973.
980 19. Bello-Reuss E. Cell membranes and paracellular resistances in isolated renal
981 proximal tubules from rabbit and Ambystoma. The Journal of physiology 370: 25-
982 38, 1986.
983 20. Ben-Yosef T, Belyantseva IA, Saunders TL, Hughes ED, Kawamoto K, Van
984 Itallie CM, Beyer LA, Halsey K, Gardner DJ, Wilcox ER, Rasmussen J,
985 Anderson JM, Dolan DF, Forge A, Raphael Y, Camper SA, and Friedman
986 TB. Claudin 14 knockout mice, a model for autosomal recessive deafness
987 DFNB29, are deaf due to cochlear hair cell degeneration. Human molecular
988 genetics 12: 2049-2061, 2003.
989 21. Bengele HH, Alexander EA, and Lechene CP. Calcium and magnesium
990 transport along the inner medullary collecting duct of the rat. The American
991 journal of physiology 239: F24-29, 1980.
992 22. Birkenhager R, Otto E, Schurmann MJ, Vollmer M, Ruf EM, Maier-Lutz I,
993 Beekmann F, Fekete A, Omran H, Feldmann D, Milford DV, Jeck N, Konrad
994 M, Landau D, Knoers NV, Antignac C, Sudbrak R, Kispert A, and
995 Hildebrandt F. Mutation of BSND causes Bartter syndrome with sensorineural
996 deafness and kidney failure. Nature genetics 29: 310-314, 2001.
997 23. Blanchard A, Jeunemaitre X, Coudol P, Dechaux M, Froissart M, May A,
998 Demontis R, Fournier A, Paillard M, and Houillier P. Paracellin-1 is critical
999 for magnesium and calcium reabsorption in the human thick ascending limb of
1000 Henle. Kidney international 59: 2206-2215, 2001.
1001 24. Blevins TC, and Farooki A. Bone effects of canagliflozin, a sodium glucose co-
1002 transporter 2 inhibitor, in patients with type 2 diabetes mellitus. Postgrad Med 1-
1003 10, 2016.
1004 25. Block GA, Rosenbaum DP, Leonsson-Zachrisson M, Astrand M, Johansson
1005 S, Knutsson M, Langkilde AM, and Chertow GM. Effect of Tenapanor on
1006 Serum Phosphate in Patients Receiving Hemodialysis. J Am Soc Nephrol 2017.
1007 26. Bockenhauer D, Feather S, Stanescu HC, Bandulik S, Zdebik AA, Reichold
1008 M, Tobin J, Lieberer E, Sterner C, Landoure G, Arora R, Sirimanna T,
1009 Thompson D, Cross JH, van't Hoff W, Al Masri O, Tullus K, Yeung S,
1010 Anikster Y, Klootwijk E, Hubank M, Dillon MJ, Heitzmann D, Arcos-Burgos
1011 M, Knepper MA, Dobbie A, Gahl WA, Warth R, Sheridan E, and Kleta R.
1012 Epilepsy, ataxia, sensorineural deafness, tubulopathy, and KCNJ10 mutations.
1013 The New England journal of medicine 360: 1960-1970, 2009.
1014 27. Boland PS, and Garland HO. Effects of D-glucose, L-glucose and D-mannitol
1015 on renal calcium handling and general renal function in the rat. Exp Physiol 78:
1016 165-174, 1993.

44
1017 28. Bomsztyk K, George JP, and Wright FS. Effects of luminal fluid anions on
1018 calcium transport by proximal tubule. The American journal of physiology 246:
1019 F600-608, 1984.
1020 29. Borghi L, Elia G, Trapassi MR, Melloni E, Amato F, Barbarese F, and
1021 Novarini A. Acute effect of indapamide on urine calcium excretion in
1022 nephrolithiasis and human essential hypertension. Pharmacology 36: 348-355,
1023 1988.
1024 30. Bourdeau JE, and Hellstrom-Stein RJ. Voltage-dependent calcium movement
1025 across the cortical collecting duct. The American journal of physiology 242: F285-
1026 292, 1982.
1027 31. Breiderhoff T, Himmerkus N, Stuiver M, Mutig K, Will C, Meij IC,
1028 Bachmann S, Bleich M, Willnow TE, and Muller D. Deletion of claudin-10
1029 (Cldn10) in the thick ascending limb impairs paracellular sodium permeability
1030 and leads to hypermagnesemia and nephrocalcinosis. Proceedings of the National
1031 Academy of Sciences of the United States of America 109: 14241-14246, 2012.
1032 32. Brickman AS, Massry SG, and Coburn JW. changes in serum and urinary
1033 calcium during treatment with hydrochlorothiazide: studies on mechanisms. The
1034 Journal of clinical investigation 51: 945-954, 1972.
1035 33. Brunette MG, Vigneault N, and Carriere S. Micropuncture study of
1036 magnesium transport along the nephron in the young rat. The American journal of
1037 physiology 227: 891-896, 1974.
1038 34. Burg M, and Good D. Sodium chloride coupled transport in mammalian
1039 nephrons. Annual review of physiology 45: 533-547, 1983.
1040 35. Burg MB. Tubular chloride transport and the mode of action of some diuretics.
1041 Kidney international 9: 189-197, 1976.
1042 36. Calverley RK, Jenkins LC, and Griffiths J. A clinical study of serum
1043 magnesium concentrations during anaesthesia and cardiopulmonary bypass. Can
1044 Anaesth Soc J 20: 499-518, 1973.
1045 37. Campean V, Kricke J, Ellison D, Luft FC, and Bachmann S. Localization of
1046 thiazide-sensitive Na(+)-Cl(-) cotransport and associated gene products in mouse
1047 DCT. American journal of physiology 281: F1028-1035, 2001.
1048 38. Chantrelle BM, Cogan MG, and Rector FC, Jr. Active and passive
1049 components of NaCl absorption in the proximal convoluted tubule of the rat
1050 kidney. Miner Electrolyte Metab 11: 209-214, 1985.
1051 39. Chobanian AV, Bakris GL, Black HR, Cushman WC, Green LA, Izzo JL,
1052 Jr., Jones DW, Materson BJ, Oparil S, Wright JT, Jr., and Roccella EJ. The
1053 Seventh Report of the Joint National Committee on Prevention, Detection,
1054 Evaluation, and Treatment of High Blood Pressure: the JNC 7 report. Jama 289:
1055 2560-2572, 2003.
1056 40. Christensson T, Hellstrom K, and Wengle B. Hypercalcemia and primary
1057 hyperparathyroidism. Prevalence in patients receiving thiazides as detected in a
1058 health screen. Arch Intern Med 137: 1138-1142, 1977.
1059 41. Chubanov V, Ferioli S, Wisnowsky A, Simmons DG, Leitzinger C, Einer C,
1060 Jonas W, Shymkiv Y, Bartsch H, Braun A, Akdogan B, Mittermeier L, Sytik
1061 L, Torben F, Jurinovic V, van der Vorst EP, Weber C, Yildirim OA, Sotlar
1062 K, Schurmann A, Zierler S, Zischka H, Ryazanov AG, and Gudermann T.

45
1063 Epithelial magnesium transport by TRPM6 is essential for prenatal development
1064 and adult survival. eLife 5: 2016.
1065 42. Chubanov V, Waldegger S, Mederos y Schnitzler M, Vitzthum H, Sassen
1066 MC, Seyberth HW, Konrad M, and Gudermann T. Disruption of
1067 TRPM6/TRPM7 complex formation by a mutation in the TRPM6 gene causes
1068 hypomagnesemia with secondary hypocalcemia. Proceedings of the National
1069 Academy of Sciences of the United States of America 101: 2894-2899, 2004.
1070 43. Cohen B, Giebisch G, Hansen LL, Teuscher U, and Wiederholt M.
1071 Relationship between peritubular membrane potential and net fluid reabsorption
1072 in the distal renal tubule of Amphiuma. The Journal of physiology 348: 115-134,
1073 1984.
1074 44. Corre T, Olinger E, Harris SE, Traglia M, Ulivi S, Lenarduzzi S, Belge H,
1075 Youhanna S, Tokonami N, Bonny O, Houillier P, Polasek O, Deary IJ, Starr
1076 JM, Toniolo D, Gasparini P, Vollenweider P, Hayward C, Bochud M, and
1077 Devuyst O. Common variants in CLDN14 are associated with differential
1078 excretion of magnesium over calcium in urine. Pflugers Arch 469: 91-103, 2017.
1079 45. Costanzo LS. Comparison of calcium and sodium transport in early and late rat
1080 distal tubules: effect of amiloride. The American journal of physiology 246: F937-
1081 945, 1984.
1082 46. Costanzo LS, and Weiner IM. On the hypocalciuric action of chlorothiazide.
1083 The Journal of clinical investigation 54: 628-637, 1974.
1084 47. Costanzo LS, and Weiner IM. Relationship between clearances of Ca and Na:
1085 effect of distal diuretics and PTH. The American journal of physiology 230: 67-
1086 73, 1976.
1087 48. Czogalla J, Vohra T, Penton D, Kirschmann M, Craigie E, and Loffing J. The
1088 mineralocorticoid receptor (MR) regulates ENaC but not NCC in mice with
1089 random MR deletion. Pflugers Arch 468: 849-858, 2016.
1090 49. Dai LJ, Ritchie G, Kerstan D, Kang HS, Cole DE, and Quamme GA.
1091 Magnesium transport in the renal distal convoluted tubule. Physiological reviews
1092 81: 51-84, 2001.
1093 50. Danielsen H, Pedersen EB, and Spencer ES. Effect of indapamide on the renin-
1094 aldosterone system, and urinary excretion of potassium and calcium in essential
1095 hypertension. Br J Clin Pharmacol 18: 229-231, 1984.
1096 51. de Baaij JH, Hoenderop JG, and Bindels RJ. Magnesium in man: implications
1097 for health and disease. Physiological reviews 95: 1-46, 2015.
1098 52. de Rouffignac C, Corman B, and Roinel N. Stimulation by antidiuretic
1099 hormone of electrolyte tubular reabsorption in rat kidney. The American journal
1100 of physiology 244: F156-164, 1983.
1101 53. De Rouffignac C, Di Stefano A, Wittner M, Roinel N, and Elalouf JM.
1102 Consequences of differential effects of ADH and other peptide hormones on thick
1103 ascending limb of mammalian kidney. The American journal of physiology 260:
1104 R1023-1035, 1991.
1105 54. de Rouffignac C, and Quamme G. Renal magnesium handling and its hormonal
1106 control. Physiological reviews 74: 305-322, 1994.

46
1107 55. Deschenes G, Wittner M, Stefano AD, Jounier S, and Doucet A. Collecting
1108 Duct Is a Site of Sodium Retention in PAN Nephrosis: A Rationale for Amiloride
1109 Therapy. J Am Soc Nephrol 12: 598-601, 2001.
1110 56. Devane J, and Ryan MP. Dose-dependent reduction in renal magnesium
1111 clearance by amiloride during frusemide-induced diuresis in rats. Br J Pharmacol
1112 80: 421-428, 1983.
1113 57. Devane J, and Ryan MP. The effects of amiloride and triamterene on urinary
1114 magnesium excretion in conscious saline-loaded rats. Br J Pharmacol 72: 285-
1115 289, 1981.
1116 58. Devane J, and Ryan MP. Evidence for a magnesium-sparing action by amiloride
1117 during renal clearance studies in rats. Br J Pharmacol 79: 891-896, 1983.
1118 59. Di Stefano A, Roinel N, de Rouffignac C, and Wittner M. Transepithelial Ca2+
1119 and Mg2+ transport in the cortical thick ascending limb of Henle's loop of the
1120 mouse is a voltage-dependent process. Ren Physiol Biochem 16: 157-166, 1993.
1121 60. Di Stefano A, Wittner M, Gebler B, and Greger R. Increased Ca++ or mg++
1122 concentration reduces relative tight-junction permeability to Na+ in the cortical
1123 thick ascending limb of Henle's loop of rabbit kidney. Ren Physiol Biochem 11:
1124 70-79, 1988.
1125 61. Di Stefano A, Wittner M, Nitschke R, Braitsch R, Greger R, Bailly C, Amiel
1126 C, Roinel N, and de Rouffignac C. Effects of parathyroid hormone and
1127 calcitonin on Na+, Cl-, K+, Mg2+ and Ca2+ transport in cortical and medullary
1128 thick ascending limbs of mouse kidney. Pflugers Arch 417: 161-167, 1990.
1129 62. Dimke H, Desai P, Borovac J, Lau A, Pan W, and Alexander RT. Activation
1130 of the Ca2+-Sensing Receptor Increases Renal Claudin-14 Expression and
1131 Urinary Ca2+ Excretion. Am J Physiol Renal Physiol 2013.
1132 63. Dimke H, Hoenderop JG, and Bindels RJ. Hereditary tubular transport
1133 disorders: implications for renal handling of Ca2+ and Mg2+. Clin Sci (Lond)
1134 118: 1-18, 2010.
1135 64. Dodion L, Ambroes Y, and Lameire N. A comparison of the pharmacokinetics
1136 and diuretic effects of two loop diuretics, torasemide and furosemide, in normal
1137 volunteers. Eur J Clin Pharmacol 31 Suppl: 21-27, 1986.
1138 65. Duarte CG. Effects of ethacrynic acid and furosemide on urinary calcium,
1139 phosphate and magnesium. Metabolism 17: 867-876, 1968.
1140 66. Duarte CG, Chomety F, and Giebisch G. Effect of amiloride, ouabain, and
1141 furosemide on distal tubular function in the rat. The American journal of
1142 physiology 221: 632-640, 1971.
1143 67. Duarte CG, and Watson JF. Calcium reabsorption in proximal tubule of the dog
1144 nephron. The American journal of physiology 212: 1355-1360, 1967.
1145 68. Duarte JD, and Cooper-DeHoff RM. Mechanisms for blood pressure lowering
1146 and metabolic effects of thiazide and thiazide-like diuretics. Expert Rev
1147 Cardiovasc Ther 8: 793-802, 2010.
1148 69. Edwards BR, Baer PG, Sutton RA, and Dirks JH. Micropuncture study of
1149 diuretic effects on sodium and calcium reabsorption in the dog nephron. The
1150 Journal of clinical investigation 52: 2418-2427, 1973.
1151 70. Eknoyan G, Suki WN, and Martinez-Maldonado M. Effect of diuretics on
1152 urinary excretion of phosphate, calcium, and magnesium in

47
1153 thyroparathyroidectomized dogs. The Journal of laboratory and clinical medicine
1154 76: 257-266, 1970.
1155 71. Enck AH, Berger UV, and Yu AS. Claudin-2 is selectively expressed in
1156 proximal nephron in mouse kidney. American journal of physiology 281: F966-
1157 974, 2001.
1158 72. Estevez R, Boettger T, Stein V, Birkenhager R, Otto E, Hildebrandt F, and
1159 Jentsch TJ. Barttin is a Cl- channel beta-subunit crucial for renal Cl- reabsorption
1160 and inner ear K+ secretion. Nature 414: 558-561, 2001.
1161 73. Fanconi A, Schachenmann G, Nussli R, and Prader A. Chronic hypokalaemia
1162 with growth retardation, normotensive hyperrenin-hyperaldosteronism ("Bartter's
1163 syndrome"), and hypercalciuria. Report of two cases with emphasis on natural
1164 history and on catch-up growth during treatment. Helv Paediatr Acta 26: 144-163,
1165 1971.
1166 74. Felker GM, Lee KL, Bull DA, Redfield MM, Stevenson LW, Goldsmith SR,
1167 LeWinter MM, Deswal A, Rouleau JL, Ofili EO, Anstrom KJ, Hernandez
1168 AF, McNulty SE, Velazquez EJ, Kfoury AG, Chen HH, Givertz MM,
1169 Semigran MJ, Bart BA, Mascette AM, Braunwald E, O'Connor CM, and
1170 Network NHFCR. Diuretic strategies in patients with acute decompensated heart
1171 failure. The New England journal of medicine 364: 797-805, 2011.
1172 75. Fiaccadori F, Pedretti G, Pasetti G, Pizzaferri P, and Elia G. Torasemide
1173 versus furosemide in cirrhosis: a long-term, double-blind, randomized clinical
1174 study. Clin Investig 71: 579-584, 1993.
1175 76. Friedman PA. Codependence of renal calcium and sodium transport. Annu Rev
1176 Physiol 60: 179-197, 1998.
1177 77. Friedman PA. Renal Calcium Transport: Sites and Insights. News Physiol Sci 3:
1178 17-20, 1988.
1179 78. Friedman PA, Figueiredo JF, Maack T, and Windhager EE. Sodium-calcium
1180 interactions in the renal proximal convoluted tubule of the rabbit. The American
1181 journal of physiology 240: F558-568, 1981.
1182 79. Friedman PA, and Gesek FA. Cellular calcium transport in renal epithelia:
1183 measurement, mechanisms, and regulation. Physiological reviews 75: 429-471,
1184 1995.
1185 80. Friedman PA, and Gesek FA. Stimulation of calcium transport by amiloride in
1186 mouse distal convoluted tubule cells. Kidney international 48: 1427-1434, 1995.
1187 81. Furuse M, Furuse K, Sasaki H, and Tsukita S. Conversion of zonulae
1188 occludentes from tight to leaky strand type by introducing claudin-2 into Madin-
1189 Darby canine kidney I cells. The Journal of cell biology 153: 263-272, 2001.
1190 82. Gandhi SK, Powers JC, Nomeir AM, Fowle K, Kitzman DW, Rankin KM,
1191 and Little WC. The pathogenesis of acute pulmonary edema associated with
1192 hypertension. The New England journal of medicine 344: 17-22, 2001.
1193 83. Garca Puig J, Miranda ME, Mateos F, Herrero E, Lavilla P, and Gil A.
1194 Hydrochlorothiazide versus spironolactone: long-term metabolic modifications in
1195 patients with essential hypertension. J Clin Pharmacol 31: 455-461, 1991.
1196 84. Giebisch G, Malnic G, Klose RM, and Windhager EE. Effect of ionic
1197 substitutions on distal potential differences in rat kidney. The American journal of
1198 physiology 211: 560-568, 1966.

48
1199 85. Gines P, Cardenas A, Arroyo V, and Rodes J. Management of cirrhosis and
1200 ascites. The New England journal of medicine 350: 1646-1654, 2004.
1201 86. Gitelman HJ, Graham JB, and Welt LG. A new familial disorder characterized
1202 by hypokalemia and hypomagnesemia. Trans Assoc Am Physicians 79: 221-235,
1203 1966.
1204 87. Glaudemans B, van der Wijst J, Scola RH, Lorenzoni PJ, Heister A, van der
1205 Kemp AW, Knoers NV, Hoenderop JG, and Bindels RJ. A missense mutation
1206 in the Kv1.1 voltage-gated potassium channel-encoding gene KCNA1 is linked to
1207 human autosomal dominant hypomagnesemia. The Journal of clinical
1208 investigation 119: 936-942, 2009.
1209 88. Gong Y, and Hou J. Claudin-14 Underlies Ca++-Sensing Receptor-Mediated
1210 Ca++ Metabolism via NFAT-microRNA-Based Mechanisms. J Am Soc Nephrol
1211 2013.
1212 89. Gong Y, Renigunta V, Himmerkus N, Zhang J, Renigunta A, Bleich M, and
1213 Hou J. Claudin-14 regulates renal Ca(++) transport in response to CaSR
1214 signalling via a novel microRNA pathway. The EMBO journal 2012.
1215 90. Good DW, Knepper MA, and Burg MB. Ammonia and bicarbonate transport
1216 by thick ascending limb of rat kidney. American journal of physiology 247: F35-
1217 44, 1984.
1218 91. Greger R. Cation selectivity of the isolated perfused cortical thick ascending limb
1219 of Henle's loop of rabbit kidney. Pflugers Arch 390: 30-37, 1981.
1220 92. Greger R, and Schlatter E. Properties of the basolateral membrane of the
1221 cortical thick ascending limb of Henle's loop of rabbit kidney. A model for
1222 secondary active chloride transport. Pflugers Arch 396: 325-334, 1983.
1223 93. Greger R, and Schlatter E. Properties of the lumen membrane of the cortical
1224 thick ascending limb of Henle's loop of rabbit kidney. Pflugers Arch 396: 315-
1225 324, 1983.
1226 94. Greger R, and Velazquez H. The cortical thick ascending limb and early distal
1227 convoluted tubule in the urinary concentrating mechanism. Kidney international
1228 31: 590-596, 1987.
1229 95. Grill A, Schiessl IM, Gess B, Fremter K, Hammer A, and Castrop H. Salt-
1230 losing nephropathy in mice with a null mutation of the Clcnk2 gene. Acta Physiol
1231 (Oxf) 218: 198-211, 2016.
1232 96. Grimellec CL, Poujeol P, and Rouffignia C. 3H-inulin and electrolyte
1233 concentrations in Bowman's capsule in rat kidney. Comparison with artificial
1234 ultrafiltration. Pflugers Arch 354: 117-131, 1975.
1235 97. Grimm M, Weidmann P, Meier A, Keusch G, Ziegler W, Gluck Z, and
1236 Beretta-Piccoli C. Correction of altered noradrenaline reactivity in essential
1237 hypertension by indapamide. Br Heart J 46: 404-409, 1981.
1238 98. Grubbs RD. Intracellular magnesium and magnesium buffering. Biometals 15:
1239 251-259, 2002.
1240 99. Gueutin V, Vallet M, Jayat M, Peti-Peterdi J, Corniere N, Leviel F, Sohet F,
1241 Wagner CA, Eladari D, and Chambrey R. Renal beta-intercalated cells
1242 maintain body fluid and electrolyte balance. The Journal of clinical investigation
1243 123: 4219-4231, 2013.

49
1244 100. Gunzel D, Stuiver M, Kausalya PJ, Haisch L, Krug SM, Rosenthal R, Meij
1245 IC, Hunziker W, Fromm M, and Muller D. Claudin-10 exists in six
1246 alternatively spliced isoforms that exhibit distinct localization and function.
1247 Journal of cell science 122: 1507-1517, 2009.
1248 101. Gunzel D, and Yu AS. Function and regulation of claudins in the thick ascending
1249 limb of Henle. Pflugers Arch 2008.
1250 102. Gurley SB, Riquier-Brison AD, Schnermann J, Sparks MA, Allen AM, Haase
1251 VH, Snouwaert JN, Le TH, McDonough AA, Koller BH, and Coffman TM.
1252 AT1A angiotensin receptors in the renal proximal tubule regulate blood pressure.
1253 Cell Metab 13: 469-475, 2011.
1254 103. Hampton C, Zhou X, Priest BT, Pai LY, Felix JP, Thomas-Fowlkes B, Liu J,
1255 Kohler M, Xiao J, Corona A, Price O, Gill C, Shah K, Rasa C, Tong V,
1256 Owens K, Ormes J, Tang H, Roy S, Sullivan KA, Metzger JM, Alonso-
1257 Galicia M, Kaczorowski GJ, Pasternak A, and Garcia ML. The Renal Outer
1258 Medullary Potassium Channel Inhibitor, MK-7145, Lowers Blood Pressure, and
1259 Manifests Features of Bartter's Syndrome Type II Phenotype. J Pharmacol Exp
1260 Ther 359: 194-206, 2016.
1261 104. Hansen LL, Schilling AR, and Wiederholt M. Effect of calcium, furosemide
1262 and chlorothiazide on net volume reabsorption and basolateral membrane
1263 potential of the distal tubule. Pflugers Arch 389: 121-126, 1981.
1264 105. Hanze S, and Seyberth H. [Studies of the effect of the diuretics furosemide,
1265 ethacrynic acid and triamterene on renal magnesium and calcium excretion]. Klin
1266 Wochenschr 45: 313-314, 1967.
1267 106. Hebert SC, and Andreoli TE. Ionic conductance pathways in the mouse
1268 medullary thick ascending limb of Henle. The paracellular pathway and
1269 electrogenic Cl- absorption. The Journal of general physiology 87: 567-590, 1986.
1270 107. Helman SI, Grantham JJ, and Burg MB. Effect of vasopressin on electrical
1271 resistance of renal cortical collecting tubules. The American journal of physiology
1272 220: 1825-1832, 1971.
1273 108. Hennings JC, Andrini O, Picard N, Paulais M, Huebner AK, Cayuqueo IK,
1274 Bignon Y, Keck M, Corniere N, Bohm D, Jentsch TJ, Chambrey R, Teulon J,
1275 Hubner CA, and Eladari D. The ClC-K2 Chloride Channel Is Critical for Salt
1276 Handling in the Distal Nephron. J Am Soc Nephrol 2016.
1277 109. Hoenderop JG, Hartog A, Stuiver M, Doucet A, Willems PH, and Bindels RJ.
1278 Localization of the epithelial Ca(2+) channel in rabbit kidney and intestine. J Am
1279 Soc Nephrol 11: 1171-1178, 2000.
1280 110. Hoenderop JG, van der Kemp AW, Hartog A, van de Graaf SF, van Os CH,
1281 Willems PH, and Bindels RJ. Molecular identification of the apical Ca2+
1282 channel in 1, 25-dihydroxyvitamin D3-responsive epithelia. The Journal of
1283 biological chemistry 274: 8375-8378, 1999.
1284 111. Hoenderop JG, van der Kemp AW, Hartog A, van Os CH, Willems PH, and
1285 Bindels RJ. The epithelial calcium channel, ECaC, is activated by
1286 hyperpolarization and regulated by cytosolic calcium. Biochemical and
1287 biophysical research communications 261: 488-492, 1999.
1288 112. Hoenderop JG, van Leeuwen JP, van der Eerden BC, Kersten FF, van der
1289 Kemp AW, Merillat AM, Waarsing JH, Rossier BC, Vallon V, Hummler E,

50
1290 and Bindels RJ. Renal Ca2+ wasting, hyperabsorption, and reduced bone
1291 thickness in mice lacking TRPV5. The Journal of clinical investigation 112:
1292 1906-1914, 2003.
1293 113. Hofmeister MV, Fenton RA, and Praetorius J. Fluorescence isolation of mouse
1294 late distal convoluted tubules and connecting tubules: effects of vasopressin and
1295 vitamin D3 on Ca2+ signaling. American journal of physiology 296: F194-203,
1296 2009.
1297 114. Hofmeister MV, Fenton RA, and Praetorius J. Fluorescence-isolation of mouse
1298 late distal convoluted tubules and connecting tubules: effects of vasopressin and
1299 vitamin D3 on Ca2+ signaling. American journal of physiology 2008.
1300 115. Holt WF, and Lechene C. ADH-PGE2 interactions in cortical collecting tubule.
1301 II. inhibition of Ca and P reabsorption. The American journal of physiology 241:
1302 F461-467, 1981.
1303 116. Horton R, and Biglieri EG. Effect of aldosterone on the metabolism of
1304 magnesium. The Journal of clinical endocrinology and metabolism 22: 1187-
1305 1192, 1962.
1306 117. Hou J, Paul DL, and Goodenough DA. Paracellin-1 and the modulation of ion
1307 selectivity of tight junctions. Journal of cell science 118: 5109-5118, 2005.
1308 118. Hou J, Renigunta A, Konrad M, Gomes AS, Schneeberger EE, Paul DL,
1309 Waldegger S, and Goodenough DA. Claudin-16 and claudin-19 interact and
1310 form a cation-selective tight junction complex. The Journal of clinical
1311 investigation 118: 619-628, 2008.
1312 119. Houillier P. Mechanisms and regulation of renal magnesium transport. Annu Rev
1313 Physiol 76: 411-430, 2014.
1314 120. Hropot M, Juretschke HP, Langer KH, and Schwark JR. S3226, a novel
1315 NHE3 inhibitor, attenuates ischemia-induced acute renal failure in rats. Kidney
1316 international 60: 2283-2289, 2001.
1317 121. Imai M. Effects of parathyroid hormone and N6,O2'-dibutyryl cyclic AMP on
1318 Ca2+ transport across the rabbit distal nephron segments perfused in vitro.
1319 Pflugers Arch 390: 145-151, 1981.
1320 122. Jorgensen FS. Effect of thiazide diuretics upon calcium metabolism. Dan Med
1321 Bull 23: 223-230, 1976.
1322 123. Kaufman JS, and Hamburger RJ. Potassium transport in the connecting tubule.
1323 Miner Electrolyte Metab 22: 242-247, 1996.
1324 124. Kiuchi-Saishin Y, Gotoh S, Furuse M, Takasuga A, Tano Y, and Tsukita S.
1325 Differential expression patterns of claudins, tight junction membrane proteins, in
1326 mouse nephron segments. J Am Soc Nephrol 13: 875-886, 2002.
1327 125. Knauf H, Reuter K, and Mutschler E. Limitation on the use of amiloride in
1328 early renal failure. Eur J Clin Pharmacol 28: 61-66, 1985.
1329 126. Kockerling A, Reinalter SC, and Seyberth HW. Impaired response to
1330 furosemide in hyperprostaglandin E syndrome: evidence for a tubular defect in the
1331 loop of Henle. The Journal of pediatrics 129: 519-528, 1996.
1332 127. Koeppen BM, Biagi BA, and Giebisch GH. Intracellular microelectrode
1333 characterization of the rabbit cortical collecting duct. The American journal of
1334 physiology 244: F35-47, 1983.

51
1335 128. Kohda Y, Ding W, Phan E, Housini I, Wang J, Star RA, and Huang CL.
1336 Localization of the ROMK potassium channel to the apical membrane of distal
1337 nephron in rat kidney. Kidney international 54: 1214-1223, 1998.
1338 129. Konrad M, Schaller A, Seelow D, Pandey AV, Waldegger S, Lesslauer A,
1339 Vitzthum H, Suzuki Y, Luk JM, Becker C, Schlingmann KP, Schmid M,
1340 Rodriguez-Soriano J, Ariceta G, Cano F, Enriquez R, Juppner H,
1341 Bakkaloglu SA, Hediger MA, Gallati S, Neuhauss SC, Nurnberg P, and
1342 Weber S. Mutations in the tight-junction gene claudin 19 (CLDN19) are
1343 associated with renal magnesium wasting, renal failure, and severe ocular
1344 involvement. American journal of human genetics 79: 949-957, 2006.
1345 130. Konrad M, Vollmer M, Lemmink HH, van den Heuvel LP, Jeck N, Vargas-
1346 Poussou R, Lakings A, Ruf R, Deschenes G, Antignac C, Guay-Woodford L,
1347 Knoers NV, Seyberth HW, Feldmann D, and Hildebrandt F. Mutations in the
1348 chloride channel gene CLCNKB as a cause of classic Bartter syndrome. J Am Soc
1349 Nephrol 11: 1449-1459, 2000.
1350 131. Koster HP, Hartog A, Van Os CH, and Bindels RJ. Calbindin-D28K facilitates
1351 cytosolic calcium diffusion without interfering with calcium signaling. Cell
1352 calcium 18: 187-196, 1995.
1353 132. Kriz W, and Bankir L. A standard nomenclature for structures of the kidney.
1354 The Renal Commission of the International Union of Physiological Sciences
1355 (IUPS). Kidney international 33: 1-7, 1988.
1356 133. Kroenke K, Wood DR, and Hanley JF. The value of serum magnesium
1357 determination in hypertensive patients receiving diuretics. Arch Intern Med 147:
1358 1553-1556, 1987.
1359 134. Krug SM, Gunzel D, Conrad MP, Rosenthal R, Fromm A, Amasheh S,
1360 Schulzke JD, and Fromm M. Claudin-17 forms tight junction channels with
1361 distinct anion selectivity. Cellular and molecular life sciences : CMLS 69: 2765-
1362 2778, 2012.
1363 135. Labonte ED, Carreras CW, Leadbetter MR, Kozuka K, Kohler J, Koo-
1364 McCoy S, He L, Dy E, Black D, Zhong Z, Langsetmo I, Spencer AG, Bell N,
1365 Deshpande D, Navre M, Lewis JG, Jacobs JW, and Charmot D.
1366 Gastrointestinal Inhibition of Sodium-Hydrogen Exchanger 3 Reduces
1367 Phosphorus Absorption and Protects against Vascular Calcification in CKD. J Am
1368 Soc Nephrol 26: 1138-1149, 2015.
1369 136. Laerum E, and Larsen S. Thiazide prophylaxis of urolithiasis. A double-blind
1370 study in general practice. Acta medica Scandinavica 215: 383-389, 1984.
1371 137. Lassiter WE, Gottschalk CW, and Mylle M. Micropuncture study of renal
1372 tubular reabsorption of calcium in normal rodents. The American journal of
1373 physiology 204: 771-775, 1963.
1374 138. Le Grimellec C. Micropuncture study along the proximal convoluted tubule.
1375 Electrolyte reabsorption in first convolutions. Pflugers Arch 354: 133-150, 1975.
1376 139. Le Grimellec C, Roinel N, and Morel F. Simultaneous Mg, Ca, P,K,Na and Cl
1377 analysis in rat tubular fluid. I. During perfusion of either inulin or ferrocyanide.
1378 Pflugers Arch 340: 181-196, 1973.

52
1379 140. Leary WP, Reyes AJ, Wynne RD, and van der Byl K. Renal excretory actions
1380 of furosemide, of hydrochlorothiazide and of the vasodilator flosequinan in
1381 healthy subjects. J Int Med Res 18: 120-141, 1990.
1382 141. Lee CT, Chen HC, Lai LW, Yong KC, and Lien YH. Effects of furosemide on
1383 renal calcium handling. American journal of physiology 293: F1231-1237, 2007.
1384 142. Lee CT, Shang S, Lai LW, Yong KC, and Lien YH. Effect of thiazide on renal
1385 gene expression of apical calcium channels and calbindins. American journal of
1386 physiology 287: F1164-1170, 2004.
1387 143. Lee WS, and Hebert SC. ROMK inwardly rectifying ATP-sensitive K+ channel.
1388 I. Expression in rat distal nephron segments. The American journal of physiology
1389 268: F1124-1131, 1995.
1390 144. Leviel F, Hubner CA, Houillier P, Morla L, El Moghrabi S, Brideau G,
1391 Hassan H, Parker MD, Kurth I, Kougioumtzes A, Sinning A, Pech V,
1392 Riemondy KA, Miller RL, Hummler E, Shull GE, Aronson PS, Doucet A,
1393 Wall SM, Chambrey R, and Eladari D. The Na+-dependent chloride-
1394 bicarbonate exchanger SLC4A8 mediates an electroneutral Na+ reabsorption
1395 process in the renal cortical collecting ducts of mice. The Journal of clinical
1396 investigation 120: 1627-1635, 2010.
1397 145. Li M, Jiang J, and Yue L. Functional characterization of homo- and heteromeric
1398 channel kinases TRPM6 and TRPM7. J Gen Physiol 127: 525-537, 2006.
1399 146. Lim P, and Jacob E. Magnesium-saving property of an aldosterone antagonist in
1400 the treatment of oedema of liver cirrhosis. Br Med J 1: 755-756, 1978.
1401 147. Loffing J, and Kaissling B. Sodium and calcium transport pathways along the
1402 mammalian distal nephron: from rabbit to human. American journal of physiology
1403 284: F628-643, 2003.
1404 148. Loffing J, Loffing-Cueni D, Hegyi I, Kaplan MR, Hebert SC, Le Hir M, and
1405 Kaissling B. Thiazide treatment of rats provokes apoptosis in distal tubule cells.
1406 Kidney international 50: 1180-1190, 1996.
1407 149. Loffing J, Loffing-Cueni D, Valderrabano V, Klausli L, Hebert SC, Rossier
1408 BC, Hoenderop JG, Bindels RJ, and Kaissling B. Distribution of transcellular
1409 calcium and sodium transport pathways along mouse distal nephron. American
1410 journal of physiology 281: F1021-1027, 2001.
1411 150. Loffing J, Vallon V, Loffing-Cueni D, Aregger F, Richter K, Pietri L, Bloch-
1412 Faure M, Hoenderop JG, Shull GE, Meneton P, and Kaissling B. Altered
1413 renal distal tubule structure and renal Na(+) and Ca(2+) handling in a mouse
1414 model for Gitelman's syndrome. J Am Soc Nephrol 15: 2276-2288, 2004.
1415 151. Ly JP, Onay T, Sison K, Sivaskandarajah G, Sabbisetti V, Li L, Bonventre
1416 JV, Flenniken A, Paragas N, Barasch JM, Adamson SL, Osborne L, Rossant
1417 J, Schnermann J, and Quaggin SE. The Sweet Pee model for Sglt2 mutation. J
1418 Am Soc Nephrol 22: 113-123, 2011.
1419 152. Lytle C, Xu JC, Biemesderfer D, and Forbush B, 3rd. Distribution and
1420 diversity of Na-K-Cl cotransport proteins: a study with monoclonal antibodies.
1421 The American journal of physiology 269: C1496-1505, 1995.
1422 153. Malnic G, and Giebisch G. Some electrical properties of distal tubular
1423 epithelium in the rat. The American journal of physiology 223: 797-808, 1972.

53
1424 154. Mandon B, Siga E, Roinel N, and de Rouffignac C. Ca2+, Mg2+ and K+
1425 transport in the cortical and medullary thick ascending limb of the rat nephron:
1426 influence of transepithelial voltage. Pflugers Arch 424: 558-560, 1993.
1427 155. Martins MC, Meyers AM, Whalley NA, Margolius LP, and Buys ME.
1428 Indapamide (Natrilix): the agent of choice in the treatment of recurrent renal
1429 calculi associated with idiopathic hypercalciuria. Br J Urol 78: 176-180, 1996.
1430 156. McCredie DA, Rotenberg E, and Williams AL. Hypercalciuria in potassium-
1431 losing nephropathy: a variant of Bartter's syndrome. Aust Paediatr J 10: 286-295,
1432 1974.
1433 157. McIntosh HW, Seraglia M, Uhlemann I, and Kore R. Effect of Acetazolamide
1434 and Triple Sulfonamide on Citrate and Calcium Excretion. Can Med Assoc J 89:
1435 1332-1333, 1963.
1436 158. Meij IC, Koenderink JB, van Bokhoven H, Assink KF, Groenestege WT, de
1437 Pont JJ, Bindels RJ, Monnens LA, van den Heuvel LP, and Knoers NV.
1438 Dominant isolated renal magnesium loss is caused by misrouting of the
1439 Na(+),K(+)-ATPase gamma-subunit. Nature genetics 26: 265-266, 2000.
1440 159. Mennitt PA, Wade JB, Ecelbarger CA, Palmer LG, and Frindt G.
1441 Localization of ROMK channels in the rat kidney. J Am Soc Nephrol 8: 1823-
1442 1830, 1997.
1443 160. Michelis MF, Drash AL, Linarelli LG, De Rubertis FR, and Davis BB.
1444 Decreased bicarbonate threshold and renal magnesium wasting in a sibship with
1445 distal renal tubular acidosis. (Evaluation of the pathophysiological role of
1446 parathyroid hormone). Metabolism 21: 905-920, 1972.
1447 161. Milatz S, Himmerkus N, Wulfmeyer VC, Drewell H, Mutig K, Hou J,
1448 Breiderhoff T, Muller D, Fromm M, Bleich M, and Gunzel D. Mosaic
1449 expression of claudins in thick ascending limbs of Henle results in spatial
1450 separation of paracellular Na+ and Mg2+ transport. Proceedings of the National
1451 Academy of Sciences of the United States of America 2016.
1452 162. Milla PJ, Aggett PJ, Wolff OH, and Harries JT. Studies in primary
1453 hypomagnesaemia: evidence for defective carrier-mediated small intestinal
1454 transport of magnesium. Gut 20: 1028-1033, 1979.
1455 163. Mironneau J, Savineau JP, and Mironneau C. Compared effects of
1456 indapamide, hydrochlorothiazide and chlorthalidone on electrical and mechanical
1457 activities in vascular smooth muscle. Eur J Pharmacol 75: 109-113, 1981.
1458 164. Moreno E, Cristobal PS, Rivera M, Vazquez N, Bobadilla NA, and Gamba
1459 G. Affinity-defining domains in the Na-Cl cotransporter: a different location for
1460 Cl- and thiazide binding. The Journal of biological chemistry 281: 17266-17275,
1461 2006.
1462 165. Morledge JH. Clinical efficacy and safety of indapamide in essential
1463 hypertension. Am Heart J 106: 229-232, 1983.
1464 166. Murdoch DL, Forrest G, Davies DL, and McInnes GT. A comparison of the
1465 potassium and magnesium-sparing properties of amiloride and spironolactone in
1466 diuretic-treated normal subjects. Br J Clin Pharmacol 35: 373-378, 1993.
1467 167. Muto S, Hata M, Taniguchi J, Tsuruoka S, Moriwaki K, Saitou M, Furuse K,
1468 Sasaki H, Fujimura A, Imai M, Kusano E, Tsukita S, and Furuse M. Claudin-
1469 2-deficient mice are defective in the leaky and cation-selective paracellular

54
1470 permeability properties of renal proximal tubules. Proceedings of the National
1471 Academy of Sciences of the United States of America 107: 8011-8016, 2010.
1472 168. Neumann KH, and Rector FC, Jr. Mechanism of NaCl and water reabsorption
1473 in the proximal convoluted tubule of rat kidney. The Journal of clinical
1474 investigation 58: 1110-1118, 1976.
1475 169. Ng RC, Rouse D, and Suki WN. Calcium transport in the rabbit superficial
1476 proximal convoluted tubule. The Journal of clinical investigation 74: 834-842,
1477 1984.
1478 170. Nielsen S, DiGiovanni SR, Christensen EI, Knepper MA, and Harris HW.
1479 Cellular and subcellular immunolocalization of vasopressin-regulated water
1480 channel in rat kidney. Proceedings of the National Academy of Sciences of the
1481 United States of America 90: 11663-11667, 1993.
1482 171. Nielsen S, Smith BL, Christensen EI, Knepper MA, and Agre P. CHIP28
1483 water channels are localized in constitutively water-permeable segments of the
1484 nephron. J Cell Biol 120: 371-383, 1993.
1485 172. Nijenhuis T, Hoenderop JG, Loffing J, van der Kemp AW, van Os CH, and
1486 Bindels RJ. Thiazide-induced hypocalciuria is accompanied by a decreased
1487 expression of Ca2+ transport proteins in kidney. Kidney international 64: 555-
1488 564, 2003.
1489 173. Nijenhuis T, Vallon V, van der Kemp AW, Loffing J, Hoenderop JG, and
1490 Bindels RJ. Enhanced passive Ca2+ reabsorption and reduced Mg2+ channel
1491 abundance explains thiazide-induced hypocalciuria and hypomagnesemia. The
1492 Journal of clinical investigation 115: 1651-1658, 2005.
1493 174. O'Grady SM, Palfrey HC, and Field M. Characteristics and functions of Na-K-
1494 Cl cotransport in epithelial tissues. The American journal of physiology 253:
1495 C177-192, 1987.
1496 175. O'Neil RG, and Sansom SC. Electrophysiological properties of cellular and
1497 paracellular conductive pathways of the rabbit cortical collecting duct. J Membr
1498 Biol 82: 281-295, 1984.
1499 176. Obermuller N, Bernstein P, Velazquez H, Reilly R, Moser D, Ellison DH, and
1500 Bachmann S. Expression of the thiazide-sensitive Na-Cl cotransporter in rat and
1501 human kidney. The American journal of physiology 269: F900-910, 1995.
1502 177. Oddsson A, Sulem P, Helgason H, Edvardsson VO, Thorleifsson G,
1503 Sveinbjornsson G, Haraldsdottir E, Eyjolfsson GI, Sigurdardottir O,
1504 Olafsson I, Masson G, Holm H, Gudbjartsson DF, Thorsteinsdottir U,
1505 Indridason OS, Palsson R, and Stefansson K. Common and rare variants
1506 associated with kidney stones and biochemical traits. Nature communications 6:
1507 7975, 2015.
1508 178. Ohkawa M, Tokunaga S, Nakashima T, Orito M, and Hisazumi H. Thiazide
1509 treatment for calcium urolithiasis in patients with idiopathic hypercalciuria. Br J
1510 Urol 69: 571-576, 1992.
1511 179. Pan W, Borovac J, Spicer Z, Hoenderop JG, Bindels RJ, Shull GE, Doschak
1512 MR, Cordat E, and Alexander RT. The Epithelial Sodium-Proton Exchanger,
1513 Nhe3, Is Necessary for Renal and Intestinal Calcium (Re)Absorption. American
1514 journal of physiology 2012.

55
1515 180. Pan W, Borovac J, Spicer Z, Hoenderop JG, Bindels RJ, Shull GE, Doschak
1516 MR, Cordat E, and Alexander RT. The epithelial sodium/proton exchanger,
1517 NHE3, is necessary for renal and intestinal calcium (re)absorption. American
1518 journal of physiology 302: F943-956, 2012.
1519 181. Parfitt AM. The acute effects of mersalyl, chlorothiazide and mannitol on the
1520 renal excretion of calcium and other ions in man. Clin Sci 36: 267-282, 1969.
1521 182. Petri M, Cumber P, Grimes L, Treby D, Bryant R, Rawlins D, and Ising H.
1522 The metabolic effects of thiazide therapy in the elderly: a population study. Age
1523 Ageing 15: 151-155, 1986.
1524 183. Pichette V, and du Souich P. Role of the kidneys in the metabolism of
1525 furosemide: its inhibition by probenecid. J Am Soc Nephrol 7: 345-349, 1996.
1526 184. Pitt B, Zannad F, Remme WJ, Cody R, Castaigne A, Perez A, Palensky J,
1527 and Wittes J. The effect of spironolactone on morbidity and mortality in patients
1528 with severe heart failure. Randomized Aldactone Evaluation Study Investigators.
1529 The New England journal of medicine 341: 709-717, 1999.
1530 185. Plain A, Wulfmeyer VC, Milatz S, Klietz A, Hou J, Bleich M, and
1531 Himmerkus N. Corticomedullary difference in the effects of dietary Ca(2)(+) on
1532 tight junction properties in thick ascending limbs of Henle's loop. Pflugers Arch
1533 468: 293-303, 2016.
1534 186. Poujeol P, Chabardes D, Roinel N, and De Rouffignac C. Influence of
1535 extracellular fluid volume expansion on magnesium, calcium and phosphate
1536 handling along the rat nephron. Pflugers Arch 365: 203-211, 1976.
1537 187. Praga M, Vara J, Gonzalez-Parra E, Andres A, Alamo C, Araque A, Ortiz A,
1538 and Rodicio JL. Familial hypomagnesemia with hypercalciuria and
1539 nephrocalcinosis. Kidney international 47: 1419-1425, 1995.
1540 188. Prati RC, Alfrey AC, and Hull AR. Spironolactone-induced hypercalciuria. J
1541 Lab Clin Med 80: 224-230, 1972.
1542 189. Purkerson JM, and Schwartz GJ. The role of carbonic anhydrases in renal
1543 physiology. Kidney international 71: 103-115, 2007.
1544 190. Quamme GA. Effect of furosemide on calcium and magnesium transport in the
1545 rat nephron. The American journal of physiology 241: F340-347, 1981.
1546 191. Quamme GA, and Dai LJ. Presence of a novel influx pathway for Mg2+ in
1547 MDCK cells. The American journal of physiology 259: C521-525, 1990.
1548 192. Quamme GA, and Dirks JH. Intraluminal and contraluminal magnesium on
1549 magnesium and calcium transfer in the rat nephron. The American journal of
1550 physiology 238: F187-198, 1980.
1551 193. Quamme GA, and Smith CM. Magnesium transport in the proximal straight
1552 tubule of the rabbit. The American journal of physiology 246: F544-550, 1984.
1553 194. Quamme GA, Wong NL, Dirks JH, Roinel N, De Rouffignac C, and Morel F.
1554 Magnesium handling in the dog kidney: a micropuncture study. Pflugers Arch
1555 377: 95-99, 1978.
1556 195. Reilly RF, and Ellison DH. Mammalian Distal Tubule: Physiology,
1557 Pathophysiology, and Molecular Anatomy. Physiol Rev 80: 277-313, 2000.
1558 196. Reilly RF, and Huang CL. The mechanism of hypocalciuria with NaCl
1559 cotransporter inhibition. Nat Rev Nephrol 7: 669-674, 2011.

56
1560 197. Reilly RF, Shugrue CA, Lattanzi D, and Biemesderfer D. Immunolocalization
1561 of the Na+/Ca2+ exchanger in rabbit kidney. The American journal of physiology
1562 265: F327-332, 1993.
1563 198. Reyes AJ. Effects of diuretics on outputs and flows of urine and urinary solutes in
1564 healthy subjects. Drugs 41 Suppl 3: 35-59, 1991.
1565 199. Reyes AJ, Leary WP, and Van der Byl K. Urinary magnesium output after a
1566 single dose of indapamide in healthy adults. S Afr Med J 64: 820-822, 1983.
1567 200. Rievaj J, Pan W, Cordat E, and Alexander RT. The Na(+)/H(+) exchanger
1568 isoform 3 is required for active paracellular and transcellular Ca(2)(+) transport
1569 across murine cecum. Am J Physiol Gastrointest Liver Physiol 305: G303-313,
1570 2013.
1571 201. Rocha AS, and Kokko JP. Sodium chloride and water transport in the medullary
1572 thick ascending limb of Henle. Evidence for active chloride transport. The Journal
1573 of clinical investigation 52: 612-623, 1973.
1574 202. Rocha AS, Magaldi JB, and Kokko JP. Calcium and phosphate transport in
1575 isolated segments of rabbit Henle's loop. The Journal of clinical investigation 59:
1576 975-983, 1977.
1577 203. Rouse D, Ng RC, and Suki WN. Calcium transport in the pars recta and thin
1578 descending limb of Henle of the rabbit, perfused in vitro. The Journal of clinical
1579 investigation 65: 37-42, 1980.
1580 204. Saarikoski J, and Kaila K. Simultaneous measurement of intracellular and
1581 extracellular carbonic anhydrase activity in intact muscle fibres. Pflugers Arch
1582 421: 357-363, 1992.
1583 205. Scheen AJ, Vancrombreucq JC, Delarge J, and Luyckx AS. Diuretic activity
1584 of torasemide and furosemide in chronic heart failure: a comparative double blind
1585 cross-over study. Eur J Clin Pharmacol 31 Suppl: 35-42, 1986.
1586 206. Schlingmann KP, Weber S, Peters M, Niemann Nejsum L, Vitzthum H,
1587 Klingel K, Kratz M, Haddad E, Ristoff E, Dinour D, Syrrou M, Nielsen S,
1588 Sassen M, Waldegger S, Seyberth HW, and Konrad M. Hypomagnesemia with
1589 secondary hypocalcemia is caused by mutations in TRPM6, a new member of the
1590 TRPM gene family. Nature genetics 31: 166-170, 2002.
1591 207. Schmitz C, Dorovkov MV, Zhao X, Davenport BJ, Ryazanov AG, and
1592 Perraud AL. The channel kinases TRPM6 and TRPM7 are functionally
1593 nonredundant. The Journal of biological chemistry 280: 37763-37771, 2005.
1594 208. Schnermann J, Chou CL, Ma T, Traynor T, Knepper MA, and Verkman AS.
1595 Defective proximal tubular fluid reabsorption in transgenic aquaporin-1 null mice.
1596 Proceedings of the National Academy of Sciences of the United States of America
1597 95: 9660-9664, 1998.
1598 209. Schnermann J, Huang Y, and Mizel D. Fluid reabsorption in proximal
1599 convoluted tubules of mice with gene deletions of claudin-2 and/or aquaporin1.
1600 American journal of physiology 305: F1352-1364, 2013.
1601 210. Schultheis PJ, Clarke LL, Meneton P, Miller ML, Soleimani M, Gawenis LR,
1602 Riddle TM, Duffy JJ, Doetschman T, Wang T, Giebisch G, Aronson PS,
1603 Lorenz JN, and Shull GE. Renal and intestinal absorptive defects in mice
1604 lacking the NHE3 Na+/H+ exchanger. Nature genetics 19: 282-285, 1998.

57
1605 211. Schultheis PJ, Lorenz JN, Meneton P, Nieman ML, Riddle TM, Flagella M,
1606 Duffy JJ, Doetschman T, Miller ML, and Shull GE. Phenotype resembling
1607 Gitelman's syndrome in mice lacking the apical Na+-Cl- cotransporter of the
1608 distal convoluted tubule. The Journal of biological chemistry 273: 29150-29155,
1609 1998.
1610 212. Seedat YK. High-dose furosemide (Lasix) in renal insufficiency. S Afr Med J 46:
1611 1371-1374, 1972.
1612 213. Seely JF. Variation in electrical resistance along length of rat proximal
1613 convoluted tubule. The American journal of physiology 225: 48-57, 1973.
1614 214. Seldin DW. Renal handling of calcium. Nephron 81 Suppl 1: 2-7, 1999.
1615 215. Shaheen NM, T; Siddiqui, M, Aslam Siddiqui; Haleem, MA. Effect Of
1616 Repeated Treatment With Low Doses Of Spironolactone On Electrolytes And
1617 Osmolality. Pakistan Journal of Pharmacology 22: 23-28, 2005.
1618 216. Shareghi GR, and Agus ZS. Magnesium transport in the cortical thick ascending
1619 limb of Henle's loop of the rabbit. The Journal of clinical investigation 69: 759-
1620 769, 1982.
1621 217. Shareghi GR, and Agus ZS. Phosphate transport in the light segment of the
1622 rabbit cortical collecting tubule. The American journal of physiology 242: F379-
1623 384, 1982.
1624 218. Simon DB, Bindra RS, Mansfield TA, Nelson-Williams C, Mendonca E,
1625 Stone R, Schurman S, Nayir A, Alpay H, Bakkaloglu A, Rodriguez-Soriano
1626 J, Morales JM, Sanjad SA, Taylor CM, Pilz D, Brem A, Trachtman H,
1627 Griswold W, Richard GA, John E, and Lifton RP. Mutations in the chloride
1628 channel gene, CLCNKB, cause Bartter's syndrome type III. Nature genetics 17:
1629 171-178, 1997.
1630 219. Simon DB, Karet FE, Hamdan JM, DiPietro A, Sanjad SA, and Lifton RP.
1631 Bartter's syndrome, hypokalaemic alkalosis with hypercalciuria, is caused by
1632 mutations in the Na-K-2Cl cotransporter NKCC2. Nature genetics 13: 183-188,
1633 1996.
1634 220. Simon DB, Karet FE, Rodriguez-Soriano J, Hamdan JH, DiPietro A,
1635 Trachtman H, Sanjad SA, and Lifton RP. Genetic heterogeneity of Bartter's
1636 syndrome revealed by mutations in the K+ channel, ROMK. Nature genetics 14:
1637 152-156, 1996.
1638 221. Simon DB, Lu Y, Choate KA, Velazquez H, Al-Sabban E, Praga M, Casari
1639 G, Bettinelli A, Colussi G, Rodriguez-Soriano J, McCredie D, Milford D,
1640 Sanjad S, and Lifton RP. Paracellin-1, a renal tight junction protein required for
1641 paracellular Mg2+ resorption. Science (New York, NY 285: 103-106, 1999.
1642 222. Simon DB, Nelson-Williams C, Bia MJ, Ellison D, Karet FE, Molina AM,
1643 Vaara I, Iwata F, Cushner HM, Koolen M, Gainza FJ, Gitleman HJ, and
1644 Lifton RP. Gitelman's variant of Bartter's syndrome, inherited hypokalaemic
1645 alkalosis, is caused by mutations in the thiazide-sensitive Na-Cl cotransporter.
1646 Nature genetics 12: 24-30, 1996.
1647 223. Solomon S. Transtubular potential differences of rat kidney. J Cell Physiol 49:
1648 351-365, 1957.

58
1649 224. Stanton BA, and Kaissling B. Adaptation of distal tubule and collecting duct to
1650 increased Na delivery. II. Na+ and K+ transport. The American journal of
1651 physiology 255: F1269-1275, 1988.
1652 225. Stergiou GS, Mayopoulou-Symvoulidou D, and Mountokalakis TD.
1653 Attenuation by spironolactone of the magnesiuric effect of acute frusemide
1654 administration in patients with liver cirrhosis and ascites. Miner Electrolyte Metab
1655 19: 86-90, 1993.
1656 226. Suki WN. Calcium transport in the nephron. The American journal of physiology
1657 237: F1-6, 1979.
1658 227. Suki WN, Rouse D, Ng RC, and Kokko JP. Calcium transport in the thick
1659 ascending limb of Henle. Heterogeneity of function in the medullary and cortical
1660 segments. The Journal of clinical investigation 66: 1004-1009, 1980.
1661 228. Suki WN, Schwettmann RS, Rector FC, Jr., and Seldin DW. Effect of chronic
1662 mineralocorticoid administration on calcium excretion in the rat. The American
1663 journal of physiology 215: 71-74, 1968.
1664 229. Sutton RA, and Dirks JH. The renal excretion of calcium: a review of
1665 micropuncture data. Canadian journal of physiology and pharmacology 53: 979-
1666 988, 1975.
1667 230. Sutton RA, and Walker VR. Responses to hydrochlorothiazide and
1668 acetazolamide in patients with calcium stones. Evidence suggesting a defect in
1669 renal tubular function. The New England journal of medicine 302: 709-713, 1980.
1670 231. Taniguchi J, and Imai M. Flow-dependent activation of maxi K+ channels in
1671 apical membrane of rabbit connecting tubule. J Membr Biol 164: 35-45, 1998.
1672 232. Taylor DR, Constable J, Sonnekus M, and Milne FJ. Effect of indapamide on
1673 serum and red cell cations, with and without magnesium supplementation, in
1674 subjects with mild hypertension. S Afr Med J 74: 273-276, 1988.
1675 233. Terker AS, Zhang C, McCormick JA, Lazelle RA, Zhang C, Meermeier NP,
1676 Siler DA, Park HJ, Fu Y, Cohen DM, Weinstein AM, Wang WH, Yang CL,
1677 and Ellison DH. Potassium modulates electrolyte balance and blood pressure
1678 through effects on distal cell voltage and chloride. Cell Metab 21: 39-50, 2015.
1679 234. Thomas JR. A review of 10 years of experience with indapamide as an
1680 antihypertensive agent. Hypertension 7: II152-156, 1985.
1681 235. Thomason JD, Rockwell JE, Fallaw TK, and Calvert CA. Influence of
1682 combined angiotensin-converting enzyme inhibitors and spironolactone on serum
1683 K+, Mg 2+, and Na+ concentrations in small dogs with degenerative mitral valve
1684 disease. J Vet Cardiol 9: 103-108, 2007.
1685 236. Thorleifsson G, Holm H, Edvardsson V, Walters GB, Styrkarsdottir U,
1686 Gudbjartsson DF, Sulem P, Halldorsson BV, de Vegt F, d'Ancona FC, den
1687 Heijer M, Franzson L, Christiansen C, Alexandersen P, Rafnar T,
1688 Kristjansson K, Sigurdsson G, Kiemeney LA, Bodvarsson M, Indridason OS,
1689 Palsson R, Kong A, Thorsteinsdottir U, and Stefansson K. Sequence variants
1690 in the CLDN14 gene associate with kidney stones and bone mineral density.
1691 Nature genetics 41: 926-930, 2009.
1692 237. Tran JM, Farrell MA, and Fanestil DD. Effect of ions on binding of the
1693 thiazide-type diuretic metolazone to kidney membrane. The American journal of
1694 physiology 258: F908-915, 1990.

59
1695 238. Ullrich KJ, Rumrich G, and Kloss S. Active Ca2+ reabsorption in the proximal
1696 tubule of the rat kidney. Dependence on sodium- and buffer transport. Pflugers
1697 Arch 364: 223-228, 1976.
1698 239. van der Hagen EA, Lavrijsen M, van Zeeland F, Praetorius J, Bonny O,
1699 Bindels RJ, and Hoenderop JG. Coordinated regulation of TRPV5-mediated Ca
1700 transport in primary distal convolution cultures. Pflugers Arch 2014.
1701 240. Velazquez H, and Wright FS. Effects of diuretic drugs on Na, Cl, and K
1702 transport by rat renal distal tubule. The American journal of physiology 250:
1703 F1013-1023, 1986.
1704 241. Voets T, Nilius B, Hoefs S, van der Kemp AW, Droogmans G, Bindels RJ,
1705 and Hoenderop JG. TRPM6 forms the Mg2+ influx channel involved in
1706 intestinal and renal Mg2+ absorption. The Journal of biological chemistry 279:
1707 19-25, 2004.
1708 242. Waite LC. Carbonic anhydrase inhibitors, parathyroid hormone and calcium
1709 metabolism. Endocrinology 91: 1160-1165, 1972.
1710 243. Walder RY, Landau D, Meyer P, Shalev H, Tsolia M, Borochowitz Z,
1711 Boettger MB, Beck GE, Englehardt RK, Carmi R, and Sheffield VC.
1712 Mutation of TRPM6 causes familial hypomagnesemia with secondary
1713 hypocalcemia. Nature genetics 31: 171-174, 2002.
1714 244. Wermers RA, Kearns AE, Jenkins GD, and Melton LJ, 3rd. Incidence and
1715 clinical spectrum of thiazide-associated hypercalcemia. The American journal of
1716 medicine 120: 911 e919-915, 2007.
1717 245. Wilcox ER, Burton QL, Naz S, Riazuddin S, Smith TN, Ploplis B,
1718 Belyantseva I, Ben-Yosef T, Liburd NA, Morell RJ, Kachar B, Wu DK,
1719 Griffith AJ, Riazuddin S, and Friedman TB. Mutations in the gene encoding
1720 tight junction claudin-14 cause autosomal recessive deafness DFNB29. Cell 104:
1721 165-172, 2001.
1722 246. Wong NL, Quamme GA, Sutton RA, and Dirks JH. Effects of mannitol on
1723 water and electrolyte transport in the dog kidney. J Lab Clin Med 94: 683-692,
1724 1979.
1725 247. Wright FS. Increasing magnitude of electrical potential along the renal distal
1726 tubule. The American journal of physiology 220: 624-638, 1971.
1727 248. Xu JZ, Hall AE, Peterson LN, Bienkowski MJ, Eessalu TE, and Hebert SC.
1728 Localization of the ROMK protein on apical membranes of rat kidney nephron
1729 segments. The American journal of physiology 273: F739-748, 1997.
1730 249. Yang SS, Lo YF, Yu IS, Lin SW, Chang TH, Hsu YJ, Chao TK, Sytwu HK,
1731 Uchida S, Sasaki S, and Lin SH. Generation and analysis of the thiazide-
1732 sensitive Na+ -Cl- cotransporter (Ncc/Slc12a3) Ser707X knockin mouse as a
1733 model of Gitelman syndrome. Hum Mutat 31: 1304-1315, 2010.
1734 250. Yu AS, Cheng MH, Angelow S, Gunzel D, Kanzawa SA, Schneeberger EE,
1735 Fromm M, and Coalson RD. Molecular basis for cation selectivity in claudin-2-
1736 based paracellular pores: identification of an electrostatic interaction site. The
1737 Journal of general physiology 133: 111-127, 2009.
1738 251. Zhou X, Forrest MJ, Sharif-Rodriguez W, Forrest G, Szeto D, Urosevic-Price
1739 O, Zhu Y, Stevenson AS, Zhou Y, Stribling S, Dajee M, Walsh SP, Pasternak
1740 A, and Sullivan KA. Chronic Inhibition of Renal Outer Medullary Potassium

60
1741 Channel Not Only Prevented but Also Reversed Development of Hypertension
1742 and End-Organ Damage in Dahl Salt-Sensitive Rats. Hypertension 2016.
1743

61