Expression of renal aquaporins 1, 2, and 3 in a rat model of cisplatin-induced polyuria

Expression of renal aquaporins 1, 2, and 3 in a rat model of cisplatin-induced polyuria.BackgroundCisplatin (CP)-induced polyuria in rats is attributed to decreased medullary hypertonicity and/or an end-organ resistance to vasopressin. However, the roles of renal aquaporins (AQPs) have not yet been explored.MethodsMale Sprague-Dawley rats (230 to 245 g) received either a single injection of CP (5 mg/kg, N = 4) or saline (N = 4) intraperitoneally five days before sacrifice. Urine, blood, and kidney samples were analyzed.ResultsPlatinum accumulated in the cortex and outer medulla of CP-treated rats (39.05 ± 7.50 and 36.48 ± 12.44 μg/g vs. 2.52 ± 0.43 and 1.87 ± 0.84 μg/g dry tissue in controls, respectively). Histologically, tubular damage and decreased AQP1 immunolabeling were detected in the S3 segment of proximal tubules. CP treatment caused 4.4- and 4.8-fold increases, respectively, in blood urea nitrogen and urine volume, and a 4.4-fold decrease in urine osmolality. Immunoblots showed that AQP2 and AQP3 were significantly reduced to 33 ± 10% (P < 0.001) and 69 ± 11% (P < 0.05), respectively, in the inner medulla of CP-treated rats. Immunocytochemical analysis showed a decrease in AQP2 labeling in the inner medulla of CP-treated rats. Northern hybridization revealed a 33 ± 11% (P < 0.002) decrease in AQP2 mRNA expression in the inner medulla of CP-treated rats. AQP1 protein expression levels were modestly (67 ± 7%, P = 0.057) and significantly (53 ± 13%, P < 0.007) decreased in outer and inner medullae, respectively, of CP-treated rats.ConclusionsCP-induced polyuria in rats is associated with a significant decrease in the expression of collecting duct (AQP2 and AQP3) and proximal nephron and microvascular (AQP1) water channels in the inner medulla

Annexin A4 Reduces Water and Proton Permeability of Model Membranes but Does Not Alter Aquaporin 2–mediated Water Transport in Isolated Endosomes

Annexin A4 (Anx4) belongs to a ubiquitous family of Ca2+-dependent membrane-binding proteins thought to be involved in membrane trafficking and membrane organization within cells. Anx4 localizes to the apical region in epithelia; however, its physiological role is unclear. We show that Anx4 exhibited binding to liposomes (phosphatidylcholine:phosphatidylserine, 1:1) in the presence of Ca2+ and binding was reversible with EDTA. Anx4 binding resulted in liposome aggregation and a reduction in membrane water permeability of 29% (P < 0.001) at 25°C. These effects were not seen in the presence of Ca2+ or Anx4 alone and were reversible with EDTA. Measurements of membrane fluidity made by monitoring fluorescence anisotropy of 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD-HPC) demonstrated that Anx4 binding rigidified the outer leaflet of the bilayer (P < 0.001), thus providing a molecular explanation for the inhibition of water flux. To determine whether Anx4 would produce similar effects on physiological membranes we constructed liposomes which recapitulated the lipid composition of the inner leaflet of the MDCK apical membrane. These membranes exhibited reductions to water permeability upon Anx4 binding (19.5% at 25°C, 31% at 37°C; P < 0.01 and P < 0.001, respectively) and to proton permeability (15% at 25°C, 19.5% at 37°C; P < 0.05). Since our in vitro experiments indicated an effect on membrane permeability, we examined localization of Anx4 in the kidney collecting duct, a region of the nephron responsible for concentrating urine through water reabsorbtion. Anx4 was shown to colocalize apically with aquaporin 2 (AQP2) in collecting duct epithelia. To test for the existence of a functional interaction between Anx4 and AQP2 we isolated AQP2-containing endosomes and exposed them to Anx4/Ca2+. Water flux rates were unchanged, indicating Anx4 does not directly regulate AQP2. We conclude that Anx4 can alter the physical properties of membranes by associating with them and regulate passive membrane permeability to water and protons. These properties represent important new functions for Anx4

Polyanionic compounds as protectants against aminoglycoside-induced nephrotoxicity : biochemical and morphological studies

The doctoral work presented here, in our view, contributed not only to decipher the mechanism of protection afforded by polyaspartic acid but also to the in depth understanding of the basic mechanism of aminoglycoside-induced nephrotoxicity itself. It also resulted in certain incidental discoveries and opened new vistas for further research. These are summarised below. 1 Contribution to the understanding of the mechanism of protection afforded by polyaspartic acid 1.1. Points proved beyond doubt : (a) Apart from polyaspartic acid, in vitro a variety of other polyanions could bind gentamicin and displace it from negatively-charged phospholipid layers. This displacement, at least for the polyanionic peptide tested, was associated with a restoration of the activity of gentamicin-inhibited lysosomal phospholipase A1. (b) We have also disproved the hypothesis of Williams on the nature of the interference seen with polyaspartic acid in the membrane binding of gentamicin and questioned her theory that membrane binding is the key event in the toxicity of these drugs. (c) We demonstrated that the protection afforded by different polyanionic peptides varied enormously depending upon the nature of the peptides? Thus, poly-L-Gly could not protect despite the fact that its in vitro behaviour was very similar to poly-L-Asp. The reason(s) for this could be many, including the sensitivity or resistance of the polyanions for lysosomal hydrolases (which we could demonstrate in vitro) or the different rates of transport and accumulation of these polyanions in lysosomes of renal cortex (that we are yet to investigate) or others. (d) Co-oxydextran despite its other toxic effects reduced the gentamicin-induced accumulation of phospholipids in the renal cortex of rats, without decreasing the drug accumulation levels. (e) Experiments with NH4Cl disclosed that raising the intralysosomal pH results in phospholipidosis and may aggravate the phosphlipidosis induced by gentamicin. (f) Experiments with leupetin disclosed that gentamicin-induced phospholipidosis can be partly blocked by leupeptin by an unknown mechanism. And in the presence of a proteolytic inhibitor like leupeptin, poly-L-Glu exhibited protective effect against gentamicin-induced phospholipidosis. 1.2 Points not proved or assumed : (a) We have not demonstrated in vivo that poly-L-Asp or other polyanions bind gentamicin and displace it from the negatively-charged phospholipids and thereby restore the activity of the gentamicin-inhibed lysosomal phospholipases. We assumed all these by extrapolating the in vitro findings made under the conditions existing in vivo. (b) We have not demonstrated the in vivo rates of degradation of polyanionic peptides in lysosomes of rat kidney and assumed that their in vitro degradation rates by liver lysosomal extracts are comparable to those in vivo rates in kidney cortex. (c) We have demonstrated that poly-L-Asp and poly-L-Glu are taken up and accumulated in the lysosomes of kidney cortex to the same extent. Based on the preliminary results and the similar biophysical properties of these peptides, we assumed that there may not be large differences in theirs rates of uptake and accumulation by kidney. (d) We have not demonstrated that poly-L-Glu is not hydrolysed preferentially by plasma proteinases or brush-border peptidases. We simply bypassed these factors and reproduced the observations made in animal models in cell culture models. 1.3 Incidental discoveries (a) The nephrotoxic potential of poly-D-Glu, a polyanionic peptide. Several aspects of this finding constitute interesting research problems for the future. (b) Toxicity induced by co-oxydextran is also an interesting aspect that may probably unravel the various modes by which kidney tissue reacts to toxic insults. (c) Raising the intralysosomal pH by NH4Cl induces phospholipidosis in cultured cells and aggravates the phospholipidosis induced by gentamicin. (d) Leupeptin, an inhibitor of cathepsin B, stimulated the activity of acid sphingomyelinase in cultured fibroblasts and partly protects against the gentamicin-induced lysosomal phospholipidosis. 1.4 Prospectives : (a) All the points that were not proved or assumed (section 1.2) constitute the research problems for the immediate future. (b) Nephrotoxicity of poly-D-Glu can be pursued further to include histochemical or autoradiographic or subcellular fractionation studies. (c) Work on leupeptin may open new vistas for research. 2 Contributions to the understanding of the aminoglycoside nephrotoxicity : 2.1 Strengthening of the ‘cascade’ The work presented here strengthened the cascade proposed by Laurent and Tulkens (1987). Conversely, the cascade can be a good model to explain the results obtained here on the mechanism of protection afforded by polyaspartic acid. Several points in this regard have already been discussed. 2.2 Disproving Williams’ hypothesis: As already discussed the work also disproves the Williams’ hypothesis on the mechanism of protection afforded by polyaspartic acid. It also questions her theory that renal membrane binding is the key event in the toxicity of aminoglycosides. 2.3 Other theories of toxicity : This work also puts the other theories on the neprhotoxicity of aminoglycosides in an inconvenient position, especially in view of the facts (i) that polyaspartic acid did not change the intracellular drug disposition as shown by Lallay and Tulkens (1989), (ii) polyaspartic acid, which blocked the development of lysosomal phospholipidosis, also prevented the cell necrosis, (iii) poly-L-glutamic acid which did not block the development of lysosomal phospholipidosis also could not prevent cell necrosis and (iv) in all these cases, whether protected or not, morphologically the alterations were limited to lysosomes only and no other subcellular oranelles exhibited abnormalitiesThèse de doctorat en sciences biomédicales -- UCL, 199

Modulation of the in vitro activity of lysosomal phospholipase A1 by membrane lipids

Lysosomal phospholipases play a critical role for degradation of cellular membranes after their lysosomal segregation. We investigated the regulation of lysosomal phospholipase A1 by cholesterol, phosphatidylethanolamine, and negatively-charged lipids in correlation with changes of biophysical properties of the membranes induced by these lipids. Lysosomal phospholipase A1 activity was determined towards phosphatidylcholine included in liposomes of variable composition using a whole-soluble lysosomal fraction of rat liver as enzymatic source. Phospholipase A1 activity was then related to membrane fluidity, lipid phase organization and membrane potential as determined by fluorescence depolarization of DPH, 31P NMR and capillary electrophoresis. Phospholipase A1 activity was markedly enhanced when the amount of negatively-charged lipids included in the vesicles was increased from 10 to around 30% of total phospholipids and the intensity of this effect depended on the nature of the acidic lipids used (ganglioside GM1<phosphatidylinositol approximately phosphatidylserine approximately phosphatidylglycerol approximately phosphatidylpropanol<phosphatidic acid). For liposomes containing phosphatidylinositol, this increase of activity was not modified by the presence of phosphatidylethanolamine and enhanced by cholesterol only when the phosphatidylinositol content was lower than 18%. Our results, therefore show that both the surface-negative charge and the nature of the acidic lipid included in bilayers modulate the activity of phospholipase A1 towards phosphatidylcholine, while the change in lipid hydration or in fluidity of membrane are less critical. These observations may have physiological implications with respect to the rate of degradation of cellular membranes after their lysosomal segregation