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Renal physiology

Article  in  Nephrology Dialysis Transplantation · May 2012

DOI: 10.1093/ndt/gfs196

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Gilles Crambert Manoocher Soleimani

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RENAL PHYSIOLOGY
FO006 H,K-ATPASE TYPE 2 IS REQUIRED FOR RENAL
ADAPTATION TO PREGNANCY
Salhi Amel1, Gilles Crambert1 and Doucet Alain1
1
Inserm-Cordelier Research Center-Paris-France
Introduction and Aims: Renal reabsorption of K+ during dietary K+ restriction
requires the stimulation of H,K-ATPase type 2 by adrenal progesterone (Elabida et al.
2011, Kidney Int. 80, 256-262). A known physiological situation that combines both
renal K+ retention and high level of circulating progesterone is the gestation. In
human, at the end of the gestation, around 300 mmol of K+ have been accumulated,
a quantity that correspond roughly to 10 - 15 % of the total body K+ content or to
the total K+ intake of 3 day-period. Obviously, during a normal pregnancy, this
accumulation is “pumped” by the fetus and does not influence maternal plasma K+.
Our recent finding in mice that adrenal progesterone promotes K+ retention by
activating the renal HKA2 suggests that it might be involved for the
pregnancy-induced potassium retention. In this study, we, therefore, investigated
whether gestation is accompanied by a renal stimulation of HKA2 is
progesterone-dependent and could be relevant in this physiological state.
Methods: Experiments were performed on C57Bl6 wild-type (WT) and knock-out mice
for the HKA2 a subunit gene (KO). WT and KO female mice were mated with WT
males for a period of 2 nights. Females are then transferred to metabolic cages. The
metabolic studies were performed after 2 days of animal adaptation to encaging.
24h-urine samples were collected and urinary creatinine concentrations were
determined on an automatic analyzer (Konelab 20i). Urinary Na+ and K+ concentration
were determined by flame photometry. Plasma K+ concentrations were measured by tail
incision on anesthetized animal with an ABL77 pH/blood-gas analyzer (Radiometer,
Lyon, France). All animal procedures were carried out in accordance with the French
legislation for animal care and experimentation. Gene expression was investigated by
qPCR and hormone levels were measured using RIA kits.
Results: During the late part of gestation in mice, the decrease in renal K+ excretion is
accompanied by stimulation of HKA2 mRNA and activity. In this condition, kidney
function is disconnected from the dietary conditions since gravid mice increase their
food consumption (and their K+ intake) by 15%. HKA2-null mice are unable to
efficiently retain K+ during gestation compared to wild-type littermate (WT) but
maintain normal plasma K+ values (around 3.9 mM). This occurs in parallel with a
decrease of the fertility rate (86% vs. 25% of successful gestation in WT and
HKA2-null mice, respectively) and a higher mortality rate during gestation in
HKA2-null mice compared to WT. Both phenotypes are reversed when gravid
HKA2-null are given a K+ supplementation in their drinking water.
Conclusions: All together, our results provide evidence that pregnancy is a
physiological state where kidney is switched from a K+ secretive to a K+ reabsorptive
state because of stimulation of H,K- ATPase type 2. Moreover, inability to do so, have
an impact on the development of the gestation.
FO007 UNMASKING A CONSTITUTIVELY ACTIVE SALT
-ABSORBING PATHWAY IN THE KIDNEY: A NOVEL
TARGET FOR DIURETIC THERAPY
Manoocher Soleimani1, Manoocher Soleimani1, Hassane Amlal1,
Sharon Barone1, Jie Xu1 and Kamyar Zahedi1
1 afferent arterioles but not on efferent arterioles, Up4A may play a role in blood
University of Cincinnati, Cincinnati USA
Introduction and Aims: The thiazide-sensitive NaCl cotransporter NCC (SLC12A3)
and the Cl-/HCO3 - exchanger pendrin (SLC26A4) are expressed on apical
membranes of distal cortical nephron segments and mediate salt absorption, with
pendrin working in tandem with the epithelial Na channel and NCC working by
itself. Pendrin is expressed on the apical membrane of intercalated cells in late distal
convoluted tubule (DCT), connecting tubule (CNT) and the cortical collecting duct
(CCD), whereas the thiazide-sensitive NaCl cotransporter NCC is primarily
expressed on the apical membrane of DCT cells.
Single deletion of pendrin or NCC does not cause salt wasting or excessive diuresis
under basal conditions. Kidney functions, including sodium and chloride excretion,
urine output, and blood urea nitrogen (BUN) levels in mutant mice are comparable
to wild type animals. Both pendrin KO and NCC KO mice, however, show signs of
volume depletion or develop hypotension during salt restriction. These findings have
led investigators to conclude that pendrin and NCC are predominantly active during
salt depletion (and or in response to increased aldosterone levels), and their
contribution to salt reabsorption at baseline conditions is minimal.
© The Author 2012. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com
Nephrology Dialysis Transplantation 27 (Supplement 2): ii3–ii4, 2012
doi:10.1093/ndt/gfs196
Aims: We hypothesized that NCC and pendrin may compensate for loss of the other
under basal conditions; thereby masking the role that each plays in salt reabsorption.
Methods: To test our hypothesis, we generated double-knockout of pendrin and
NaCl cotransporter mice by crossing animals with single deletion for NCC and
pendrin. Balanced studies were performed in double pendrin/NCC KO, single NCC
or pendrin KO and wild type (WT) mice.
Results: The double Pendrin/NCC KO mice displayed sharp increases in urine
output (by ∼400% in 24 hr urine volume, p<0.0001 Vs. single KOs or WT) and salt
wasting (by ∼100% increase in 24 hrs sodium and chloride excretion, p<0.001 Vs.
single KOs or WT mice) under basal conditions. As a result, animals developed
profound dehydration (as shown by ∼7 fold increase in the expression of renin in
the kidney, p<0.0001 Vs. WT or single KO mice), renal failure (∼5 fold increase in
BUN, p<0.0001 Vs. WT or single KO mice) and metabolic alkalosis (serum
bicarbonate of 33.3 mEq/L in double KO mice Vs. 23.1 in WT animals, p<0.01)
without hypokalemia (serum K of 4.2 and 4.6 in double KO and WT, respectively,
p>0.05). All metabolic abnormalities (renin expression, dehydration, renal failure and
metabolic alkalosis) were corrected by ∼90% after 6 days of high salt (7%) liquid diet
intake.
Conclusions: We propose that the combined inhibition of pendrin and NCC can
provide a novel and strong diuretic regimen without causing hypokalemia for
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patients with fluid overload, including patients with congestive heart failure,
nephrotic syndrome, diuretic resistance and generalized edema. We further suggest a
revision on the role of active salt reabsorbing pathways in the distal tubule is
warranted.
FO008 UP4A AFFECTING THE GLOMERULAR FILTRATION RATE
Zidek Walter1, Vera Jankowski2 and Jankowski Joachim3
1
Charité, Berlin, Garmany, 2Charitè, 3Charite, Berlin, Germany
Introduction and Aims: It was described that Uridine adenosine tetraphosphate
(Up4A) released from endothelial cells after stimulation with mechanical stress was a
strong vasoconstrictor. In this study, we isolated and identified Up4A from kidney
tissue, and we characterized the essential varying effects of Up4A on the afferent
arterioles and efferent arterioles.
Methods: Porcine and human kidney tissue was fractionated by
size-exclusion-chromatography, affinity-chromatography and reverse
phase-chromatography. In fractions purified to homogeneity, Up4A was identified by
matrix assisted laser desorption/ionisation mass- spectrometry (MALDI-TOF-MS),
MALDI-LIFT-fragment-mass-spectrometry (MALDI- TOF-TOF-MS), retention-time
comparison, and enzymatic cleavage analysis. We analysed the release of Up4A from
cultivated renal proximal tubule cells after stimulation of protein kinase C with
oleoyl-2-acetyl-sn-glycerol (OAG).
Results: Up4A was identified in renal tissue, and the effect of Up4A on the vascular
tone of isolated perfused afferent arterioles and efferent arterioles was tested.
Stimulation of tubule cells with OAG increased the release-rate of Up4A from tubule
cells about ten fold. Up4A acts as a strong vasoconstrictive mediator on afferent
arterioles, but has no significant effect on the tone of efferent arterioles, suggesting a
functional role of Up4A as an autocrine hormone for glomerular perfusion. Because
of the predominant effect of the Up4A on afferent arterioles, we assume that Up4A
may decrease glomerular perfusion, intraglomerular pressure, and hence glomerular
filtration rate. The release of Up4A from renal tubular cells may be an additional
mechanism whereby tubular cells could affect renal perfusion. Up4A release may
further contribute to renal vascular autoregulation mechanism.
Conclusions: In Conclusion, Up4A occurs in renal tissue and has marked effects on
pressure regulation and in renal hemodynamics.
FO009 RENAL UPTAKE OF 99MTC-DMSA IS DEPENDENT ON
ENDOCYTOSIS BY THE MEGALIN/CUBILIN RECEPTOR
COMPLEX
Kathrin Weyer1, Rikke Nielsen2, Erik Christensen2, Michael Rehling3 and
Henrik Birn2
1
Department of Biomedicine, Aarhus University, 2Department of Biomedicine,
Aarhus University, Aarhus, Denmark, 32department of Clinical Physiology and
Nuclear Medicine, Aarhus University Hospital, Skejby, Aarhus, Denmark
Introduction and Aims: Dimercaptosuccinic acid (DMSA) labelled with
technetium-99m (99mTc) is the major renal imaging agent used in diagnosis of renal
parenchymal disorders. 99mTc-DMSA is highly accumulated in the kidney cortex,
but despite its extensive clinical use, the mechanism for renal targeting of the tracer
Abstracts
remains elusive. Two routes of 99mTc-DMSA kidney uptake have been proposed; the
basolateral uptake from plasma and the endocytic uptake from the ultrafiltrate.
Megalin and cubilin are co-operating receptors that mediate the endocytic uptake of
proteins from the glomerular ultrafiltrate by the proximal tubule epithelial cells in
the kidney. Here, we used megalin/cubilin-deficient mice to determine whether
receptor-mediated endocytosis from the ultrafiltrate is responsible for the renal
uptake of 99mTc-DMSA tracer.
Methods: Control mice or conditional megalin/cubilin-deficient mice were i.v.
injected with 0.5 MBq of the tracer 99mTc-DMSA or 99mTc-MAG3
(mercaptoacetyltriglycine). Six hours post-injection, samples of plasma, urine, and
whole kidneys were collected and analysed on a gamma counter. Whole-body
autoradiographs of the mice were made using a gamma-camera. The size of excreted
99mTc-DMSA-bound peptides was estimated by fractionation of urine samples by
ultracentrifugation or separation by SDS-PAGE followed by autoradiography.
Results: The absence of the megalin/cubilin receptor complex had a dramatic effect
on the renal uptake of 99mTc-DMSA, where no activity could be detected in the
kidneys of megalin/cubilin-deficient mice in whole-body autoradiographs (Figure 1).
Compared to control mice, the renal uptake of 99mTc-DMSA was found to be
reduced to 11.4% (± 2.5%, n=7) in the megalin/cubilin-deficient mice, which
correlates to a megalin/cubilin-deficiency of about 90% in the kidneys of these mice.
The lack of renal uptake was accompanied by a corresponding increase in the urinary
excretion of 99mTc-DMSA by the megalin/cubilin- deficient mice. In contrast, the
urinary excretion of 99mTc-MAG3 was similar to control mice, suggesting that the
basolateral uptake of the proximal tubule cells is not disturbed by the deficiency of
megalin and cubilin. Size-dependent separation of the urine from
megalin/cubilin-deficient mice by ultracentrifugation or SDS-PAGE, further
99m
FO009 Figure 1. Megalin/cubilin-dependent renal uptake of Tc -DMSA.
ii | Abstracts
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Nephrology Dialysis Transplantation
demonstrated that an increased amount of 99mTc-DMSA was excreted in a
protein-bound form.
Conclusions: Our data provide evidence that 99mTc-DMSA is reabsorbed from the
glomerular ultrafiltrate via specific, receptor-mediated endocytosis by the megalin/
cubilin receptor complex. Our findings further suggest that this uptake may depend
on the binding of 99mTc-DMSA to plasma proteins, followed by glomerular
filtration and endocytosis by proximal tubular cells. 99mTc-DMSA scintigraphy can
therefore be applied to evaluate proximal tubule endocytic function in patients.
Whole-body gamma camera scintigraphy showed no uptake in the kidneys of
megalin/cubilin-deficient mice, whereas evidence of accumulation of the tracer in the
bladder was seen.
FO010 EXOSOME ISOLATION, COUNT AND CHARACTERIZATION
FROM NORMAL URINE
Veronica Dimuccio1, Andrea Ranghino2, Giovanni Camussi2 and
Benedetta Bussolati2
1
Department of Internal Medicine, University of Torino, 2Dept of Internal
Medicine, Torino, Italy
Introduction and Aims: Exosomes are microvescicles secreted from various types of
activated cells. In the kidney, exosomes are mainly released into urine by cells of the
nephron. Recent studies demonstrated that exosomes express surface receptors, as
well as selected patterns of mRNA and microRNA of the cell of origin. Since it is a
readily available fluid, urine may be regarded as the ideal source of information on
renal conditions. In particular, exosomes appear to bet the perfect mirror of the
Downloaded from http://ndt.oxfordjournals.org/ by guest on October 11, 2016
pathological conditions of the different segments of the nephron. In this perspective,
membrane proteins and RNA content of exosomes could be useful markers of renal
pathology. The purpose of the present study is the set up of a protocol for exosomes
isolation, and the quantification and analysis of surface markers and micro-RNA
(miRNA) content.
Methods: Exosomes were isolated from a pool of urine from healthy subjects. A total
of four protocols of exosome isolation were tested in this study, based on 1)
ultracentrifugation (100.000g at 4°C for 1h); 2) nanomembrane concentrator Amicon
(100k); 3) nanomembrane concentrator Vivaspin 500 (Sartorius); 4) or denaturation
of Tamm-Horsfall Protein (THP) with DTT (200mg/ml) followed by
ultracentrifugation. Exosome quantification was performed with Bradford for protein
content, or with Nanosigh count. mRNA was extracted using mirVana kit (Ambion)
and miRNA analysis was performed using quantitative RT-PCR. As exosomes are
smaller than the lower limit of sensitivity of the cytofluorimetric, cytofluorimetric
analysis was performed after adsorption of isolated vesicles on 4 μm aldehyde–
sulphate latex beads.
Results: The protein concentration tested with a Bradford assay only showed a
very low exosomes concentration for protocol n.2. Nanosight analysis showed
high concentration of exosomes in samples obtained using protocols n.1-2 (4.7 ×
108 and 3,5 × 108 exosomes/ml) while other two were very poor. RNA extracted
from exosomes from protocol n.1-2 showed the presence of a panel of 20
miRNAs. A citofluorimetric assay, set up with latex-beads, was able to identify
the presence of the classical exosome markers (CD63, CD24, CD9) as well as of
markers of the renal cell of origin (Aquaporins, integrins, membrane
transporters).
Conclusions: In the present study we identified a protocol based on
ultracentrifugation as the most suitable to obtain exosomes from urine. Exosome
count using the Nanosight analysis was more reliable than protein quantification
possibly due to a contamination by urinary proteins. In addition, we identified
in exosomes from normal urine surface markers involved in the process of
exocytosis and in renal cell physiology using a latex-beads based FACS analysis
and a panel of miRNAs possibly relevant for renal cell functions. These findings
can be a valid starting point for the further development of studies in a wide
variety of renal pathologies.
Volume 27 | Supplement 2 | May 2012