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Toxicology and Applied Pharmacology 223 (2007) 86 – 98

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Review
Glomerular nephrotoxicity of aminoglycosides
Carlos Martínez-Salgado a,⁎, Francisco J. López-Hernández a , José M. López-Novoa b
a
Unidad de Investigación, Hospital Universitario de Salamanca, Paseo San Vicente 58-182, 37007 Salamanca, Spain
b
Instituto Reina Sofía de Investigación Nefrológica, Departamento de Fisiología y Farmacología, Universidad de Salamanca, 37007 Salamanca, Spain

Received 9 February 2007; revised 17 April 2007; accepted 10 May 2007


Available online 21 May 2007

Abstract

Aminoglycoside antibiotics are the most commonly used antibiotics worldwide in the treatment of Gram-negative bacterial infections.
However, aminoglycosides induce nephrotoxicity in 10–20% of therapeutic courses. Aminoglycoside-induced nephrotoxicity is characterized by
slow rises in serum creatinine, tubular necrosis and marked decreases in glomerular filtration rate and in the ultrafiltration coefficient. Regulation
of the ultrafiltration coefficient depends on the activity of intraglomerular mesangial cells. The mechanisms responsible for tubular nephrotoxicity
of aminoglycosides have been intensively reviewed previously, but glomerular toxicity has received less attention. The purpose of this review is to
critically assess the published literature regarding the toxic mechanisms of action of aminoglycosides on renal glomeruli and mesangial cells. The
main goal of this review is to provide an actualized and mechanistic vision of pathways involved in glomerular toxic effects of aminoglycosides.
© 2007 Elsevier Inc. All rights reserved.

Keywords: Aminoglycosides; Apoptosis; Gentamicin; Glomerular toxicity; Mesangial cells; Platelet-activating factor; Proliferation

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Aminoglycosides and renal failure: tubular and glomerular involvement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Cellular uptake of aminoglycosides and stimulation of cell surface receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
Gentamicin induces mesangial cell contraction and proliferation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
Gentamicin increases cytosolic free calcium concentration in mesangial cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Potential role of CaR stimulation on aminoglycoside-induced proliferation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
CaR as a modulator of contraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Gentamicin-induced mesangial cell contraction and proliferation are mediated by platelet-activating factor . . . . . . . . . . . . . . . 90
Gentamicin induces phospholipase A2 activation and synthesis and release of eicosanoids in cultured mesangial cells . . . . . . . . . 91
Gentamicin-induced mesangial cell activation is mediated by reactive oxygen species . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Gentamicin induces apoptosis in renal glomeruli and cultured mesangial cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Jun-kinase activation and Jun–AP-1 expression can mediate the effects of gentamicin on mesangial cells . . . . . . . . . . . . . . . . 94
Changes in nitric oxide synthesis can mediate the effect of gentamicin on renal glomeruli and cultured mesangial cells . . . . . . . . 94
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

⁎ Corresponding author. Fax: +34 923 294669.


E-mail address: carlosms@usal.es (C. Martínez-Salgado).

0041-008X/$ - see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.taap.2007.05.004
C. Martínez-Salgado et al. / Toxicology and Applied Pharmacology 223 (2007) 86–98 87

Introduction stimulate mesangial cell proliferation and apoptosis with no


overt net change in total cell number.
A number of therapeutic agents have long been associated
with the development of iatrogenic renal failure. The kidney is a Aminoglycosides and renal failure: tubular and glomerular
common target for toxic xenobiotics, due to its capacity to involvement
extract and concentrate toxic substances, and to its large blood
flow share (about 20% of cardiac output). Experimental data Since their introduction into therapeutic practice in 1944,
suggest that both vascular, glomerular, and tubular targets are aminoglycoside antibiotics such as gentamicin and amikacin are
involved in drug-induced nephrotoxicity, as a result of the most commonly used antibiotics worldwide for the
mechanisms that disrupt normal cellular structures and func- treatment of Gram-negative bacterial infections. In many
tions (mitochondria, membrane integrity, etc.), induce renal cases, they have been the only effective therapeutic tools
injury through intratubular obstruction (crystal deposition), and against bacterial strains resistant to other antibiotics. Amino-
promote cellular swelling and tubular luminal occlusion glycosides are natural or semi-synthetic antibiotics with a
(through osmotic effects) (Perazella, 2003). heterocyclic structure formed by two or more aminosugars
Nephrotoxic substances damage different nephron cell types, linked by glycoside bonds to an aminocyclitol ring (Fig. 1).
although tubular epithelial cell necrosis is the phenomenon most They show a poor degree of oral absorption and hence
extensively studied. Glomerular injuries and functional altera- intravenous administration is the route usually used in patients
tions have also been described, but they are much less well with severe infections. Aminoglycosides are not metabolized
documented (Kohn et al., 2002). When some drugs (e.g. and are essentially eliminated by glomerular filtration. A
cyclosporine, cisplatin, or gentamicin) are administered to relatively large amount (about a 10%) of the intravenously
experimental animals, there is a marked fall in renal blood administered dose is accumulated in the kidney, whereas little
flow and glomerular filtration rate (GFR), with a rise in renal distribution of aminoglycosides into other tissues is observed
vascular resistance. Apparently, these effects may even occur (Nagai and Takano, 2004). However, serious complications like
without permanent gross structural changes in glomerular nephrotoxicity and ototoxicity are dose-limiting factors in the
structures and rather independently from tubular damage use of aminoglycosides. Aminoglycoside antibiotics induce a
(English et al., 1987; Bennett, 1990; Bennett et al., 1994). dose-dependent nephrotoxicity in 10–25% of therapeutic
However, the effects of aminoglycosides on glomerular structure courses, even despite rigorous monitoring of serum drug
and function have been poorly studied and, to our knowledge, concentration and adequate fluid volume control (Lauren et
there are no reviews on this topic. Thus, the purpose of the al., 1990; Kacew and Bergeron, 1990; Leehey et al., 1993).
present article is to describe the available data on the glomerular Aminoglycoside-induced nephrotoxicity is typically char-
toxic mechanisms of action of aminoglycosides, especially those acterized by tubular necrosis, without gross morphological
derived from the action of gentamicin (far and away, the most changes in glomerular structures (Rodríguez-Barbero et al.,
widely used aminoglycoside) on mesangial cells. As documen- 1997). However, chronic gentamicin treatment also causes a
ted through the text, gentamicin may (i) alter the glomerular marked decrease in GFR and alters intraglomerular dynamics,
filtration function through mesangial cell contraction, and (ii) with a marked decrease in the ultrafiltration coefficient (Kf)

Fig. 1. Chemical structure of two representative aminoglycosides: streptomycin and gentamicin.


88 C. Martínez-Salgado et al. / Toxicology and Applied Pharmacology 223 (2007) 86–98

(Baylis, 1980; Schor et al., 1981) (Fig. 2). Other drugs (i.e. gentamicin uptake needs to be established: endocytosis
cyclosporine, FK 506 or cisplatin) also induce a marked (Sandoval and Molitoris, 2004), penetration through cation
decrease in GFR, which has also been related to a marked channels (Myrdal and Steyger, 2005) and mast cell-derived
decrease in Kf (Murray et al., 1985; Pavao dos Santos et al., granule-mediated internalization have been proposed (Decorti
1991; McCauley, 1993). This reinforces a relationship between et al., 2000). Mast cell-derived, gentamicin-containing granules
GFR and Kf in the mechanism of action of toxins. Kf regulation are endocytosed by other cells in a histamine-dependent manner
is a dynamic phenomenon that depends mainly on the tone (Decorti et al., 2000), which poses an interesting link between
(degree of contraction) of intraglomerular mesangial cells by gentamicin uptake and e.g. inflamed tissues. A specific
modifying the ultrafiltration surface. Mesangial cells are transport mechanism has been identified in proximal tubule
perivascular pericytes located at the core of the glomerular epithelial cells, which accumulate aminoglycosides at higher
tuft between capillary loops (Mené et al., 1989). Mesangial tone levels than those detected in plasma (Nagai and Takano, 2004).
exerts a mechanical traction on the glomerular basal membrane It has been reported that megalin, a giant endocytic receptor
and on the endothelial capillary lining, which determines the abundantly expressed at the apical membrane of renal proximal
area of the filtration surface and Kf, and modulates glomerular tubules, plays an important role in binding and endocytosis of
vascular resistance. Consequently, most authors agree that aminoglycosides in proximal tubule cells (Nagai and Takano,
mesangial cell contraction plays a major role in the reduction of 2004). Megalin antagonists (molecules which bind megalin and
GFR, glomerular filtration surface and Kf (Mené et al., 1989; block megalin-mediated endocytosis) have been developed (e.g.
Pfeilschifter, 1989). Contraction and relaxation of mesangial cytochrome c and derived peptides), which hold promise as
cells are highly regulated by numerous vasoactive substances; prospective therapeutic agents for preventing or minimizing the
they are also altered by xenobiotics, such as certain drugs and iatrogenic tubular damage induced by gentamicin (Watanabe et
heavy metals (Mené, 2001; Rodriguez-Barbero et al., 2000). al., 2004; Nagai et al., 2006). However, megalin is not present
Mesangial cells have other functions including (a) synthesis and on mesangial cells (Teng et al., 2004). Presently, it is still
assembly of the mesangial matrix, which in turn regulates the unclear whether gentamicin accumulates in mesangial cells and,
viscoelastic and hydraulic properties of the mesangium; (b) if it does, the mechanism by which gentamicin enters these
endocytosis and processing of plasma macromolecules includ- cells.
ing immunocomplexes; and (c) release of vasoactive hormones,
growth factors and cytokines affecting renal physiology at Gentamicin induces mesangial cell contraction and
different levels, including auto and paracrine actions on proliferation
themselves (Hawkins et al., 1990).
Logically, all of these functions are potential targets for the Gentamicin stimulates mesangial cell contraction and
action of toxic substances. Notwithstanding, nephrotoxicants proliferation on primary cultures of mesangial cells (Rodrí-
have been reported to act on mesangial cells mainly through (a) guez-Barbero et al., 1995, Martínez-Salgado et al., 1997, 2000,
inducing a maintained glomerular and cellular contraction 2002, 2004). Gentamicin-induced cell proliferation appears to
(Rivas-Cabañero et al., 1997; Girardi and Elias, 1998; Potier et be (at least in vitro) cell type-specific. In fact, some authors
al., 1998; Rodriguez-Barbero et al., 2000); (b) increasing cell have described that gentamicin and other aminoglycosides
proliferation (Martínez-Salgado et al., 1997); and (c) causing (neomycin B, paromomycin, tobramycin) in vitro suppress the
cell necrosis (Barrouillet et al., 1999). Accordingly, the proliferation of human epidermal keratinocytes, supposedly by
reduction of the glomerular ultrafiltration surface induced by inhibiting tRNA processing (Tekos et al., 2003). Moreover,
glomerular contraction could also be a logical explanation for gentamicin reduced cell proliferation in an organ of Corti cell
the decrease in GFR after aminoglycoside treatment. line (Bertolaso et al., 2003) and high concentrations of
gentamicin inhibited cell proliferation in human osteoblast-
Cellular uptake of aminoglycosides and stimulation of cell like cells (Isefuku et al., 2003). The differential effect exerted by
surface receptors gentamicin on different cell types might also be dependent on
the concentration of the drug used, and also on culture and
A first issue to be discussed is whether gentamicin (and, in experimental conditions. Contractile and proliferative effects on
general, aminoglycosides) needs to enter the cell or it is able to mesangial cells have been reported for other substances with
induce all or some of its toxic effects through the activation of nephrotoxic properties (Rodriguez-Barbero et al., 2000).
specific cell surface receptors. Due to their large size and It must be born in mind that both contraction and proli-
polycationic charge, aminoglycoside antibiotics do not easily feration of mesangial cells observed in culture upon gentamicin
cross biological membranes lacking transport mechanisms. treatment occur in an in vitro artificial system bereft of many
Vestibular and auditory sensory hair cells (Aran et al., 1995; physiopathological neuroendocrine stimuli and also deprived of
Forge and Li, 2000; Steyger et al., 2003; Dai et al., 2006) of the tissue antiproliferative restrictions posed by cell–cell and cell–
inner ear accumulate gentamicin. A good correlation has been matrix interactions. Accordingly, translation of in vitro results
observed between cellular uptake of gentamicin, and tissue to the in vivo scenario must be undertaken cautiously. In ge-
localization and extent of ototoxic damage (Aran et al., 1993), neral, stimuli that in vitro induce contraction of contracting cells
suggesting that gentamicin needs to be taken in by these cells to (myocytes, mesangial cells, etc.) also induce contraction of
exert its toxic action. However the precise mechanism of those same cells in vivo. This is without detriment that their
C. Martínez-Salgado et al. / Toxicology and Applied Pharmacology 223 (2007) 86–98 89

action can be counterbalanced or nullified by relaxing stimuli cium influxes from the external medium and calcium release
working in vivo in the opposite direction or by opposing stimuli from internal stores (Rodríguez-Barbero et al., 1995; Martinez-
derived from the action of the contracting factor on neighboring Salgado et al., 2000). Gentamicin-induced mesangial cell
or distant tissues (e.g. release of vasoactive substances or mod- contraction and proliferation are reduced by the calcium
ulation of nervous activity). However, the case of proliferation channel blocker verapamil and by the endoplasmic reticulum
is somewhat more complicated. Many stimuli are capable of calcium release blocker TMB-8 (Rodríguez-Barbero et al.,
inducing proliferation, apoptosis or other effects depending on 1995; Martinez-Salgado et al., 2000), thus suggesting that the
the cellular scenario given at a certain time in a particular cell. It increased cytosolic free calcium also plays a major role in the
is the case, e.g., of transforming growth factor-β, Fas stimul- contractile and proliferative effects of this antibiotic.
ation or tumor necrosis factor-α. As such, the proliferative effect A hypothetical scenario can be proposed for gentamicin-
induced by gentamicin both in vitro and in vivo might respond induced calcium mobilization as follows. Acting on CaR, gen-
to the same effect or, alternatively, to different phenomena. tamicin (in general, aminoglycosides) induces an inositol-3-
Hypothetically, it might be argued that the direct proliferative phosphate-mediated calcium release from internal stores (likely
effect of gentamicin in vitro might become blocked in vivo by the sarcoplasmic reticulum), which results in an increased Ca2+i.
an antiproliferative phenotype induced in mesangial cells by A part of the Ca2+ released is taken back by internal stores,
their entourage. However, the proliferative effect of gentamicin whereas an undetermined fraction escapes out of the cell (as
on mesangial cells has also been observed in vivo (Martínez- now the outward concentration gradient favors diffusion and
Salgado et al., 2004). According to this argument, the increased facilitated transport). After undetermined (one or more) cycles
proliferation detected in vivo could be interpreted, for example, of stimulation, because not all the Ca2+ released returns to the
as a repair response derived from, e.g., a direct or indirect internal stores, their growingly lower concentration of calcium
damage caused by gentamicin to the mesangial compartment or reaches a threshold that triggers a rather well described
adjacent structures. These questions bear a great interest when signaling pathway that opens cell surface calcium channels an
knowledge of the mechanism of action of toxic substances (at allows refilling of intracellular stores, a process termed capa-
the tissue, cellular or molecular levels) is intended to be used in citative Ca2+ entry (Putney, 1986). Capacitative Ca2+ entry has
the development of therapeutic strategies. been conceptually separated from voltage-operated Ca2+ influx,
An important challenge is, thus, to unravel the cell signaling sensitive to calcium antagonists, e.g. verapamil. Still, in some
pathways involved in contraction and proliferation induced by excitable cells, depletion of intracellular Ca2+ stores activates
aminoglycosides, both in vitro and in vivo. Mechanistic studies capacitative Ca2+ entry and concomitantly depolarizes the cell
carried out in vitro with cultured cells reduce enormously the membrane, thus activating voltage-operated Ca2+ influx (Mora-
amount of inter-related variables existing in vivo. However, les et al., 2004), and capacitative Ca2+ entry may be compa-
such a simplification mandates further corroboration in vivo. ratively low with respect to voltage-operated Ca2+ influx
Interestingly, cell contraction and proliferation appear to be (Morales et al., 2004). This could provide a theoretical
interlinked events that share a logical pathophysiological explanation for the verapamil-sensitivity of gentamicin effects.
connection, not only in mesangial cells but also more generally
in contracting cells, and maybe that is why thus they also share Potential role of CaR stimulation on
some signaling pathways. For instance, many stimuli and cell aminoglycoside-induced proliferation
signaling events known to lead to contraction (of contracting
cells) have also been reported to stimulate proliferation, either McLarnon et al. (2002) showed that several aminoglycoside
through the tension generated (which induces tissue remodel- antibiotics (gentamicin, tobramycin, neomycin) are capable of
ing, involving sometimes tissue hypertrophy by cell prolifera- activating the calcium-sensing receptor (CaR), a cell membrane,
tion), or directly in a tension-independent manner. As described G-protein-coupled receptor (of the glutamate/γ-aminobutyric
in the next sections, mediators involved in gentamicin-induced acid B/pheromone receptors family), which is sensitive to
contraction and proliferation include increased cytosolic free extracellular calcium concentration (Ca2+o) (Brown et al., 1993;
calcium, phospholipase A2 activation, platelet-activating factor Brown and MacLeod, 2001). A cationic agonist (including
and eicosanoids release, increased production of reactive polyamine aminoglycosides) specific binding region is located
oxygen species and alterations in nitric oxide synthesis. within the extracellular domain (Hammerland et al., 1999).
Renal cells express CaR (Chattopadhyay et al., 1996), which
Gentamicin increases cytosolic free calcium concentration mediates intracellular calcium mobilization and phospholipase
in mesangial cells C-dependent Erk activation in response to aminoglycoside
antibiotics. Thus, it might be thought that CaR could play a
Early findings showed the ability of calcium channel block- role in their nephrotoxicity (Ward et al., 2002). More re-
ers to inhibit mesangial cell contraction and proliferation cently, these authors showed that aminoglycosides induce
induced by different substances (Shultz and Raij, 1990; Mon- both acute (an increase in proliferation rate) and chronic (in-
tero et al., 1995), indicating that an increase in cytosolic free duction of apoptosis) changes in tubular cells. They have also
calcium (Ca2+i) concentration might be necessary for the demonstrated that the proximal tubular CaR likely contributes
induction of both processes in these cells. Specifically, genta- to the signaling underlying tubule cell apoptosis (Ward et al.,
micin increases Ca2+i; indeed, gentamicin activates both cal- 2005).
90 C. Martínez-Salgado et al. / Toxicology and Applied Pharmacology 223 (2007) 86–98

Mesangial cells and mesangial cell lines also express CaR of CaR (lacking exon 5) were detected in these cells by RT–
(Ward et al., 2002; Kwak et al., 2005). Activation of CaR in PCR, it was proposed that expression of CaR alternative
mesangial cells (by increasing Ca2+o) provokes an elevation of splicing variants might determine the affinity or the response to
Ca2+i and inositol-3-phosphate concentration, likely mediated different CaR agonists. This provides new grounds (to be
by phospholipase C activation, which results in an increased cell explored) for the explanation of different roles of CaR
proliferation rate. Interestingly, inhibition of CaR with the stimulation and inhibition in different tissues, under physiolo-
antagonists NPS 2390 prevents Ca2+o-induced proliferation gical or pathological circumstances.
(Kwak et al., 2005). Still, stimulation of aortic vascular smooth Putting all these data together, it is probable that gentamicin-
muscle cells with high Ca2+o induces an elevation of Ca2+i and induced contraction and proliferation of mesangial cells be
cell proliferation, which are inhibited by the CaR antagonist mediated, at least partially, by CaR stimulation. From a purely
NPS 2390 (Smajilovic et al., 2006). These effects are inositol-3- speculative perspective it is also possible that CaR-mediated
phosphate production-independent and mitogen extracellular signaling, also signals open directly cell membrane calcium
kinase-1-dependent (Smajilovic et al., 2006). channels. Furthermore, scenarios involving CaR activation can
be hypothesized for explaining the dependency of gentamicin
CaR as a modulator of contraction action on verapamil-inhibited targets (likely cell surface
calcium channels). For example, it might be argued that
The participation of CaR in mesangial cell contraction has gentamicin, in a CaR-independent manner, directly or indirectly
not been studied yet. It is noteworthy though, that the contractile activate calcium entry. All these possibilities need to be further
effect of aminoglycosides on mesangial cells appears to be cell explored, as they potentially offer additional targets for thera-
type-specific, since they induce relaxation of other smooth peutic action. For example, the effect of CaR antagonists should
muscle containing tissues (see below in this section). This opens be tested in animal models of gentamicin-induced renal damage.
up new mechanistic and teleological questions. In fact, They might prove helpful against aminoglycoside-induced ne-
aminoglycosides have long been demonstrated to prevent in phrotoxicity provided that, as commented above, contraction,
vitro vascular Ca2+ mobilization (Goodman and Adams, 1976) proliferation and apoptosis of renal cells finally proved to be
and the contraction of dissected vessels (including renal veins deleterious effects of these drugs, rather than repair responses
Hester, 1983) stimulated with a variety of contracting stimuli, as elicited by the kidneys in an effort to respond to other toxic
well as to relax precontracted arteries (Goodman and Adams, actions of aminoglycosides.
1976; Hester, 1983; Gergawy et al., 1998; Wickman et al.,
2001) and myogenic tone (Ohanian et al., 2005). However, Gentamicin-induced mesangial cell contraction and
contraction and relaxation of blood vessels is the result of an proliferation are mediated by platelet-activating factor
integrated effect primed by direct action of the prospective
substance on the contracting cell type (vascular smooth muscle Platelet-activating factor (PAF: 1-O-alkyl-2-acetyl-sn-gly-
cells in the lamina media), plus indirect stimulation derived cero-3-phosphocholine) is a potent lipid mediator that exerts
from the endothelium. CaR has been found to be expressed in biological responses at subnanomolar levels. PAF is rapidly
vascular tissues, including vascular smooth muscle (Smajilovic produced and secreted by mesangial cells in culture in response
et al., 2006) and endothelial cells (Ziegelstein et al., 2006). It to various stimuli, including the calcium ionophore A23187,
might be argued that gentamicin exerts a contracting effect on lipopolysaccharide, tumor necrosis factor-α, interleukin-1β,
vascular smooth muscle cells, but a stronger endothelium- activated complement, and angiotensin II (Schlondorff et al.,
dependent relaxation, with a net result being vasodilatation. 1986; Neuwirth et al., 1989; Camussi et al., 1992). PAF induces
However, gentamicin-induced vasorelaxation has even been an increase in Ca2+i in mesangial cells, in a phospholipase C-
shown to occur in endothelium-denuded cerebral artery rings, and inositol-3-phosphate-dependent manner (Kester et al.,
indicating that the relaxant effect is an endothelium-indepen- 1992). PAF is able to induce isolated glomeruli and mesangial
dent, direct effect on vascular smooth muscle cells, at least in cell contraction, mesangial cell proliferation and other effects in
these arteries (Gergawy et al., 1998). A direct relaxing action of the kidney (Arriba et al., 1987; López-Farré et al., 1991; López-
gentamicin on smooth muscle cells is reinforced by the fact that Novoa, 1999; Montero et al., 1993; Schlondorff and Neuwirth,
aminoglycosides (not only gentamicin) induce relaxation of 1986). The increase in Ca2+i induced by gentamicin (as above
other smooth muscle bearing tissues, such as the myometrium described) is inhibited by the PAF-antagonist BN-52021
(Ocal et al., 2004) and vas deferens (Papaioannidou et al., (Rodríguez-Barbero et al., 1995). Gentamicin-induced mesan-
1988), and of ventricular myocytes (Belus and White, 2002). gial cell contraction and proliferation were also reduced by BN-
Yet, a contribution of CaR-mediated, endothelium dependent 52021 and by other two structurally different PAF receptor
relaxation cannot be excluded. In fact, Ziegelstein et al. (2006) antagonists: alprazolam and BB-823. Moreover, gentamicin
demonstrated that stimulation of human aortic endothelial cells stimulated the synthesis and release of PAF in cultured rat
with the CaR agonist spermine elicited an inositol-3-phosphate- mesangial cells (Rodríguez-Barbero et al., 1995).
mediated, ryanodine-sensitive increase in Ca2+i and nitric oxide PAF production was also increased in glomeruli from rats
(NO) production that were inhibited by CaR RNA interference. treated with gentamicin (Rodríguez-Barbero et al., 1995). It
None of these effects was elicited by other CaR agonists (i.e. should be noted that in vivo treatment with PAF antagonists
high Ca2+o, neomycin or gadolinium). Because splicing variants ameliorated renal dysfunction and the decrease in Kf induced by
C. Martínez-Salgado et al. / Toxicology and Applied Pharmacology 223 (2007) 86–98 91

gentamicin (Pavao dos Santos et al., 1991; Bagnis et al., 1996; Increased release of PAF can also play a role in gentamicin-
Rodríguez-Barbero et al., 1997). All these data suggest that mediated increase in eicosanoids production. PAF stimulates
increased PAF synthesis and release plays a major role thromboxane A2 and prostaglandin E2 production by cultured rat
mediating the glomerular effects of gentamicin. This is not mesangial cells, being PAF stimulation of thromboxane B2
surprising as it has been reported that PAF mediates mesangial several-fold greater than that of prostaglandin E2 (Arribas-
cell contraction and other renal effects of substances involved in Gomez et al., 1995). This fact is in agreement with the studies
the genesis of acute renal failure such as endothelin (López- showing that gentamicin induced a higher increase in thrombox-
Farré et al., 1991; López-Novoa, 1999). ane B2 than in prostaglandin E2 production (Martínez-Salgado
According to these results, it seems plausible that gentamicin et al., 1997). PAF can also activate PLA2 (Schlondorff and
induces the release of PAF, which then acts in an auto- and Neuwirth, 1986) and increase (i) cytosolic free calcium (Kester
paracrine manner on its cell surface receptors to induce an et al., 1987; Olivera et al., 1994) and (ii) arachidonic acid
elevation of Ca2+i, contraction and proliferation (Fig. 3). availability by activating phospholipase C and diacylglycerol
However, it can be also hypothesized that the gentamicin- lipase or phospholipase D (Shukla, 1991) in mesangial cells.
induced increase in cytosolic calcium stimulates PAF synthesis PAF-induced PLA2 activation, with the subsequent increase in
secondary to an activation of phospholipase A2 (PLA2), as later PAF and thromboxane B2 production (Kester et al., 1992),
described. represents a mechanism of positive feedback by which a small
increase in cytosolic free calcium can induce a small activation
Gentamicin induces phospholipase A2 activation and of PLA2 and a small synthesis of PAF, which in turn can increase
synthesis and release of eicosanoids in cultured mesangial its own synthesis, induce further activation of PLA2 and
cells probably other phospholipases, and thus amplify the contractile
and proliferative effect of gentamicin on mesangial cells.
Gentamicin also induces a release of arachidonic acid and According to the data presented so far, PAF bears the
increases the production of prostaglandin E2 and thromboxane capacity to induce Ca2+ mobilization and thus calcium-
B2 by cultured mesangial cells (Martínez-Salgado et al., 1997). dependent PLA2 activation and arachidonic acid production in
Gentamicin-induced arachidonic acid release could be second- mesangial cells. Because, as described in the previous section,
ary to the activation of PLA2 or diacylglycerol lipase. Genta- distinct PAF antagonists inhibit gentamicin-induced Ca2+i
micin-induced arachidonic acid release was markedly inhibited elevation, it should be concluded that PAF activation precedes
by the PLA2 inhibitor aristolochic acid. In addition, PLA2 was calcium mobilization. On the other hand, it must be considered
markedly activated by the calcium ionophore A-23187, whereas that, in excitable cells, an elevation of Ca2+i is a sufficient event
verapamil, in addition to inhibiting gentamicin-induced cell to trigger contraction, and it has also been related to pro-
contraction and proliferation, also inhibited gentamicin-induced liferation (Berridge, 1995). As such, it is difficult to explain how
increases in thromboxane B2 and prostaglandin E2 synthesis PLA2 inhibition (downstream of calcium mobilization) might
(Martínez-Salgado et al., 1997). All these data suggest that at affect mesangial cell contraction and proliferation, as demon-
least a part of gentamicin-induced arachidonic acid release is strated by our studies using PLA2 inhibitors. An explanation
mediated by calcium-dependent PLA2 activation. Interestingly, must then incorporate a calcium-independent PLA2 activation
the same study also demonstrated that incubation of mesangial step before Ca2+ mobilization, located right after PAF activation
cells with the specific PLA2 inhibitors aristolochic acid and or previous to it. Both PAF (Schlondorff and Neuwirth, 1986)
quinacrine significantly inhibited gentamicin induced mesan- and CaR (Kifor et al., 1997) (hypothetically located upstream of
gial cell contraction and completely prevented gentamicin- PAF activation) have been demonstrated to activate PLA2,
induced mesangial cell proliferation. although it is not clear whether they activate a calcium-
independent PLA2 directly coupled to their respective mem-
brane receptors. All these data together suggest a major role for
PLA2 activation and a subsequent increase in PAF and
thromboxane A2 production, in gentamicin-induced mesangial
cell contraction and proliferation, although the precise sequence
of events of the signaling messengers involved is until unclear.
Fig. 3 shows the possible role of intracellular free calcium,
PLA2 and PAF in gentamicin-induced mesangial cell activation,
and therefore, in GFR.

Gentamicin-induced mesangial cell activation is mediated


by reactive oxygen species

Previous in vivo and in vitro studies strongly suggested the


Fig. 2. Schematic diagram showing the major mechanisms leading to glo-
merular filtration rate reduction by gentamicin. GFR: glomerular filtration rate; mediation of reactive oxygen species (ROS) in the tubular and
Kf: ultrafiltration coefficient; RBF: renal blood flow; RVR: renal vascular glomerular effects of gentamicin. In vivo, ROS have been
resistance. identified as mediators of proximal tubular necrosis and acute
92 C. Martínez-Salgado et al. / Toxicology and Applied Pharmacology 223 (2007) 86–98

Fig. 3. Suggested roles of increased cytosolic free calcium, activation of phospholipase A2, platelet-activating factor, thromboxane A2 and prostaglandin A2 on
gentamicin-induced reduction of ultrafiltration coefficient and glomerular filtration rate. [Ca2+]i: intracellular calcium concentration; DAG: diacyl glycerol; GFR:
glomerular filtration rate; Kf: ultrafiltration coefficient; PAF: platelet-activating factor; PGE2: prostaglandin E2; PLA2: phospholipase A2; PLC: phospholipase C;
ROS: reactive oxygen species; TXA2: thromboxane A2. –: inhibition.

renal failure caused by gentamicin (Du and Yang, 1994), and of 2002). Another possible origin of O2− could be the activation of
the increased renal susceptibility to gentamicin observed in PLA2 (Pfeilschifter and Huwiler, 1997). In this sense, it should
obstructive jaundice (Tajiri et al., 1995). Moreover, ROS be noted that gentamicin activates a calcium-dependent PLA2
scavengers have proved to be beneficial at reducing the (Martínez-Salgado et al., 1997). There is increasing evidence
development of renal damage induced by the administration suggesting that ROS may be second messengers involved in
of endotoxin and gentamicin (Zurovsky and Haber, 1995). signal transduction for cell proliferation in mesangial cells (Ali,
Specifically, (i) administration of antioxidants such as super- 1995), rather than toxic effectors (Suzuki et al., 1997; Schulze-
oxide dismutase (SOD) or dimethyl-thiourea prevented the Osthoff et al., 1997). Martínez-Salgado et al. (2002) show
gentamicin-induced GFR reduction (Nakajima et al., 1994; enough evidence that the amount of ROS produced by genta-
Zurovsky and Haber, 1995; Ali, 1995), (ii) treatment with SOD micin does not affect the cell membrane structure, as gentamicin
in gentamicin-treated rats is associated to an increase in renal did not induce any detectable change in lipid peroxidation or in
blood flow, and therefore, superoxide anion (O2−) is proposed to membrane fluidity, either in the outer layer or in the hydro-
be involved in gentamicin-induced renal vasoconstriction phobic core of the plasma membrane. Still, cytotoxic effects
(Nakajima et al., 1994). Thus, an enhanced production of associated to an oxidative stress derived from an exaggerated
ROS has been consistently demonstrated to be involved in the production of ROS upon treatment with gentamicin cannot be
development of gentamicin-induced acute renal failure. discarded. In fact, (a) an oxidative stress generated by addition
A role for ROS, either as physiological second messengers or of xanthine plus xanthine oxidase to the culture medium caused
as inducers of oxidative stress (or both), is increasingly more apoptosis of mesangial cells (Martínez-Salgado et al., 2004);
evident in mesangial cell activation and damage. Duque et al. and (b) gentamicin-induced mesangial cell apoptosis was
(1992) showed that H2O2 was able to induce mesangial cell inhibited by addition of the ROS scavengers SOD and catalase
contraction in vitro, as well as an increase in preglomerular to the culture medium (Martínez-Salgado et al., 2004, and see
resistance in vivo. The involvement of ROS on mesangial cell below).
contraction and proliferation induced by gentamicin has been It has also been reported that administration of trans-
further demonstrated in vitro; in fact, gentamicin induces an resveratrol, a potent inhibitor of ROS production with strong
increase in O2− production and SOD activity; also, gentamicin- antioxidant activity and an inhibitor of lipid peroxidation (Baur
induced mesangial cell contraction and proliferation are and Sinclair, 2006), markedly reduced the decrease in
inhibited by the intracellular ROS scavengers SOD and catalase glomerular filtration rate and renal blood flow induced by a
(Martínez-Salgado et al., 2002) and by the antioxidant flavonoid nephrotoxic dose of gentamicin in rats (Morales et al., 2002).
trans-resveratrol (Morales et al., 2005). In these cells, gen- This protective effect of trans-resveratrol on the impairment of
tamicin-induced production of O2− seems to be due, at least in renal function induced by gentamicin was associated with its
part, to NADP(H) oxidase activity (Martínez-Salgado et al., ability to prevent the increase in lipid peroxidation induced by
C. Martínez-Salgado et al. / Toxicology and Applied Pharmacology 223 (2007) 86–98 93

ROS or by inhibiting O2− generation by NADP(H) oxidase, tructive uropathy (Truong et al., 1996) and in a subtotal-
prospectively preventing mesangial cell contraction, as these in nephrectomy model of chronic renal failure (Thomas et al.,
vitro results indicate (Morales et al., 2005), or cytotoxic 1998). Glomerular proliferation and apoptosis have been shown
damage. Thus, from the above discussed data it can be in several glomerulosclerotic lesions (Sugiyama et al., 1996;
suggested that an increased ROS production might play a Shimizu et al., 1995; Tamura et al., 2000) and in glomeruli of
major role in aminoglycoside-induced renal mesangial cell uninephrectomized spontaneously hypertensive rats (Rodri-
contraction and proliferation, as it is schematized in Fig. 4. guez-López et al., 1998). These facts might indicate that either
proliferation or apoptosis (or both) might be (at least in some
Gentamicin induces apoptosis in renal glomeruli and pathophysiological circumstances) the consequence of tissue
cultured mesangial cells homeostasis as a response to an insult, rather than a direct effect
of the insult on target cells (as argued in previous sections).
As described in previous sections, gentamicin activates The case of gentamicin, though, becomes complicated by the
mesangial cell proliferation in vitro (Rodríguez-Barbero et al., dual direct effect (proliferation and apoptosis) exerted by this
1995; Martínez-Salgado et al., 1997, 2000, 2002, 2004). aminoglycoside on cultured mesangial cells (Martínez-Salgado
Therefore, it was hypothesized that gentamicin could induce et al., 2004). A closer look indicates, notwithstanding, that in
mesangial cell proliferation and simultaneous cell apoptosis, the experimental conditions and gentamicin dose used in this
thus maintaining glomerular cell number within normal limits, study, proliferation was stronger than apoptosis, and thus a net
after a gentamicin-induced glomerular insult. However, there increase in cell number was observed upon gentamicin treat-
were no studies reporting increases in the number of mesangial ment. Yet, it might be speculated that growing doses of genta-
cells in patients or animals treated with gentamicin. The few micin would induce further cytotoxicity (i.e. apoptosis), so that
studies dealing with the apoptotic effect of gentamicin in the at a certain dose (the equilibrium dose) proliferation might equal
kidney are restricted to the tubular epithelium, mainly to apoptosis. With even higher doses, gentamicin-induced apop-
proximal cells (Bledsoe et al., 2006). Martínez-Salgado et al. tosis would outweigh gentamicin-induced proliferation, and a
(2004) indicated that in vivo treatment of rats with gentamicin net cytotoxic effect would be observed. These experiments have
induced a simultaneous increase in glomerular (mainly not been performed yet. However, it is practically impossible
mesangial) cell proliferation and apoptosis without apparent that the in vivo studies carried out by multiple researchers have
changes in the number of glomerular cells. However, used a dose of gentamicin that results in the equilibrium
simultaneous proliferation and apoptosis are not specific of concentration in the glomerular entourage, so that all of them
gentamicin-induced damage. In fact, both are observed in many have observed no alteration in glomerular cell number as a
etiologically different models of renal damage. Concomitant result of gentamicin administration. As a result, a purely spe-
proliferation and apoptosis have been also described in tubular, culative explanation according to present-day knowledge would
interstitial and glomerular cells in a model of chronic obs- be that gentamicin would not cause a direct proliferative effect
in vivo on mesangial cells (for the reasons commented in
previous sections). On the contrary, gentamicin would cause a
dose-dependent apoptosis on mesangial cells, which would
trigger a homeostatic, repair, proliferative response.
In the last years, it has been shown that gentamicin causes
apoptosis in renal tubular cell lines with successive alteration of
the permeability of lysosomes, triggering of the mitochondrial
pathway, and activation of caspase-3 (Servais et al., 2005),
which correlates with a strong toxic effect consistently seen in
vivo at the tubular level. Incidentally, one of the reasons for
gentamicin to be a milder apoptosis inducer in mesangial cells
(correlating with the lesser effect observed in vivo, completely
offset by proliferation in the mesangial compartment) may
actually be a lower uptake by these cells, rendering an
insufficient concentration at the intracellular, apoptosis-trigger-
ing targets, for the same extracellular concentration of the drug.
Alternatively, it might be thought that the uptake is similar in
both cell types but, due to pharmacokinetic reasons, the
bioavailability of the drug would be different in the vicinity
of both compartments. Gentamicin-associated cytotoxic effects
in mesangial cells can also be proposed to involve CaR
Fig. 4. Putative pathways of gentamicin-induced mesangial cell proliferation activation, as it has been proposed for tubular cells (Ward et al.,
and apoptosis through the increased reactive oxygen species production,
cytosolic free calcium and AP-1 activation. AP-1: activating protein-1; [Ca2+]i: 2002). In such a case, several scenarios open up: (i) the decisive
intracellular calcium concentration; ROS: reactive oxygen species; SOD: determinant of cytotoxicity is the concentration of the amino-
superoxide dismutase. –: inhibition. glycoside in the surroundings of the CaR (not its cellular up-
94 C. Martínez-Salgado et al. / Toxicology and Applied Pharmacology 223 (2007) 86–98

take), and then one should invoke a different availability in the The time-course of AP-1 expression in glomeruli of rats treated
tubular and mesangial compartments; (ii) gentamicin uptake is with gentamicin is similar to the expression of proliferating cell
necessary for the activation of other co-stimulatory signaling nuclear antigen showed previously (Martínez-Salgado et al.,
pathways required for apoptosis to occur upon CaR stimulation. 2004), suggesting that Jun/AP-1 may be involved in mesangial
Experimental data also suggest that O2− is involved in cell proliferation or apoptosis inhibition. It can also be
gentamicin-induced mesangial apoptosis, because the O2− donor suspected that Jun/AP-1 participates in the transcription of
system xanthine plus xanthine-oxidase enhanced the number of genes involved in mesangial cell proliferation in vitro, since the
apoptotic cells in a degree quantitatively similar to that induced lowest AP-1 expression was found in quiescent cells, and AP-1
by gentamicin; moreover, gentamicin-induced apoptosis is expression increased when mesangial cells were incubated with
inhibited by incubation with SOD plus catalase, a ROS sca- 10% FCS, which induces the maximal proliferative state. The
venger system (Martínez-Salgado et al., 2004), as commented time-course of AP-1 expression is also similar to the expression
in a previous section. There is increasing evidence showing that of the pro-apoptotic protein Bax previously described (Martí-
pro-oxidant stimuli elicit an apoptotic response in mesangial nez-Salgado et al., 2004).
cells (Choi et al., 2000; Ishikawa and Kitamura, 2000). In The possible relationships between increased cytosolic cal-
addition, glomerular and mesangial apoptosis induced by tumor cium, ROS production and JNK/AP-1 activation are schema-
necrosis factor-α and interleukin-1α is mediated by ROS tized in Fig. 5.
(Bohler et al., 2000). In agreement with this hypothesis, O2− has
been recognized as a selective mediator for tumor necrosis Changes in nitric oxide synthesis can mediate the effect of
factor-α-induced apoptosis of rat mesangial cells (Moreno- gentamicin on renal glomeruli and cultured mesangial cells
Manzano et al., 2000). Gentamicin-induced, oxidant-mediated
apoptosis in mesangial cells is also characterized by an early Nitric oxide (NO) plays a major role in the local regulation of
elevation in the levels of the pro-apoptotic protein Bax and a renal cortical blood flow in the rat and may also influence
delayed increase in the survival-promoting protein Bcl-2 intraglomerular dynamics. Rivas-Cabañero et al. (1994)
(Martínez-Salgado et al., 2004). An up-regulation of Bcl-2 demonstrated an enhanced NO formation in glomeruli of
expression has also been observed in an ovary cell line in gentamicin-treated rats. This increased NO production seems to
response to geneticin, an aminoglycoside rather similar to partially protect the kidney against the gentamicin-induced
gentamicin (Tey et al., 2000). Bcl-2 localizes at the sites of ROS reduction in GFR (Rivas-Cabañero et al., 1997). NO production
generation and is recognized to prevent oxidative damage. is also increased in cultured mesangial cells incubated with
Thus, Bcl-2 overexpression was described to have a protective gentamicin (Rivas-Cabañero et al., 1997). Recently, Martínez-
role against oxidant-induced apoptosis in a neural cell line Salgado et al. (2005) showed increases in the expression of both
(Zhong et al., 1993) and in mesangial cells (Sandau and Brune, inducible (iNOS) and constitutive (cNOS) NO synthase
2000). Bcl-2 itself can function as an antioxidant to prevent isoforms in glomeruli from rats treated with gentamicin.
apoptosis (Park and Hockenbery, 1996; Korsmeyer et al., 1995). cNOS is expressed constitutively in glomeruli from untreated
From these data we can suggest that the increased Bax/Bcl-2 animals, whereas iNOS expression was induced as a result of
ratio plays a major role in gentamicin-induced mesangial cell
apoptosis.

Jun-kinase activation and Jun–AP-1 expression can


mediate the effects of gentamicin on mesangial cells

The AP-1 transcription factor, mainly composed of Jun, Fos


and ATF protein homo- and heterodimers, regulates processes
such as proliferation, differentiation, apoptosis and transforma-
tion (Hess et al., 2004). The mechanism of post-translational
control is most extensively documented in the case of mitogen-
and cellular-stress-induced hyperphosphorylation and, in parti-
cular, activation of Jun through the Jun N-terminal kinase (JNK)
cascade (Hess et al., 2004). It has been recently observed that
gentamicin stimulates AP-1 expression both in vitro and in vivo
and that gentamicin induces JNK activation in cultured
mesangial cells, which was reduced by co-incubation with the
ROS scavengers SOD and catalase (Martínez-Salgado et al.,
2005); thus, ROS seems to mediate the JNK activation induced Fig. 5. Schematic diagram showing the suggested roles of increased reactive
by gentamicin treatment. This study also shows that gentami- oxygen species production, increased cytosolic free calcium, increased nitric
oxide synthesis and AP-1 activation on mesangial cell proliferation and apop-
cin-induced JNK activation was reduced by the calcium channel tosis. AP-1: activating protein-1; [Ca2+]i: intracellular calcium concentration;
blocker verapamil, suggesting that increases in intracellular JNK: Jun kinase; NO: nitric oxide; PCNA: proliferating cell nuclear antigen;
calcium also mediates JNK activation induced by gentamicin. ROS: reactive oxygen species; SOD: superoxide dismutase. –: inhibition.
C. Martínez-Salgado et al. / Toxicology and Applied Pharmacology 223 (2007) 86–98 95

gentamicin treatment. The increase in NO synthase expression synthesis, JNK and Jun–AP-1 activation and alterations in the
may be the responsible of the increases in NO production ratio Bcl-2/Bax. The changes in all these intracellular
previously shown (Rivas-Cabañero et al., 1994, 1997). More- mediators seem to play also a major role in the contractile,
over, increased iNOS activity can also be responsible for proliferative and apoptotic effects induced by gentamicin in
increased ROS production observed after gentamicin treatment renal glomeruli and cultured mesangial cells. A better
(Martínez-Salgado et al., 2002, 2004). knowledge of the mechanisms involved in renal glomerular
NO shows an unusual divergence of action, working as a damage induced by aminoglycosides help to design ther-
physiologic signaling molecule, a protector of cell function or as apeutical strategies to prevent aminoglycoside-induced renal
a toxic mediator (Valdivielso and Blantz, 2002). NO-mediated failure.
cellular damage occurs by several mechanisms including un-
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