Decreased Excretion of Urinary Exosomal Aquaporin-2 in a Puromycin Aminonucleoside-Induced Nephrotic Syndrome Model.

Urinary exosomes, small extracellular vesicles present in urine, are secreted from all types of renal epithelial cells. Aquaporin-2 (AQP2), a vasopressin-regulated water channel protein, is known to be selectively excreted into the urine through exosomes (UE-AQP2), and its renal expression is decreased in nephrotic syndrome. However, it is still unclear whether excretion of UE-AQP2 is altered in nephrotic syndrome. In this study, we examined the excretion of UE-AQP2 in an experimental rat model of nephrotic syndrome induced by the administration of puromycin aminonucleoside (PAN). Rats were assigned to two groups: a control group administered saline and a PAN group given a single intraperitoneal injection of PAN (125 mg/kg) at day 0. The experiment was continued for 8 days, and samples of urine, blood, and tissue were collected on days 2, 5, and 8. The blood and urine parameters revealed that PAN induced nephrotic syndrome on days 5 and 8, and decreases in the excretion of UE-AQP2 were detected on days 2 through 8 in the PAN group. Immunohistochemistry showed that the renal expression of AQP2 was decreased on days 5 and 8. The release of exosomal marker proteins into the urine through UEs was decreased on day 5 and increased on day 8. These data suggest that UE-AQP2 is decreased in PAN-induced nephrotic syndrome and that this reflects its renal expression in the marked proteinuria phase after PAN treatment.

Usefulness of urinary biomarkers for nephrotoxicity in cynomolgus monkeys treated with gentamicin, cisplatin, and puromycin aminonucleoside.

The objective of this study was to investigate the availability of novel urinary biomarkers (BMs) such as total protein, albumin, ß2-microglobulin, clusterin, cystatin C, neutrophil gelatinase-associated lipocalin (NGAL) for the detection of acute nephrotoxicity in cynomolgus monkeys. Animals (total 9 males/3 groups) were administered gentamicin (GM) subcutaneously at 40 mg/kg for 7 days, cisplatin (CDDP) intravenously at 3 mg/kg once and puromycin aminonucleoside (PAN) intravenously at 20 mg/kg for 7 days. Two-hr urine on Days 0, 3, and 6, and 16-hr urine and blood on Days 1, 4, and 7 were collected. Novel urinary BMs and conventional clinical pathology parameters were evaluated in parallel to histopathological and electron microscopic examinations on the kidneys at termination. Urinary BMs and enzymes increased earlier than serum creatinine and blood urea nitrogen, particularly in 2-hr urine after dosing on Day 0, urinary albumin was increased in all groups and urinary NGAL with the highest magnitude of change rate among urinary BMs was observed in the GM and CDDP groups. Degeneration/necrosis and hyaline droplet of renal tubule, cellular cast and dilatation of renal tubule, and hypertrophy of podocytes were observed in the GEN, CDDP, and PAN groups, respectively. These results showed that the increases of urinary BMs reflected the agent-specific renal damages and these urinary BMs could be useful for the detection of segment-specific nephrotoxicity. Urinary albumin and NGAL are the most useful BMs to estimate glomerular and distal tubular damages, respectively, as well as proximal tubular damage in cynomolgus monkeys.

Calcineurin inhibitors cyclosporin A and tacrolimus protect against podocyte injury induced by puromycin aminonucleoside in rodent models.

Podocyte injury and the appearance of proteinuria are features of minimal-change disease (MCD). Cyclosporin A (CsA) and tacrolimus (FK506) has been reported to reduce proteinuria in patients with nephrotic syndrome, but mechanisms remain unknown. We, therefore, investigated the protective mechanisms of CsA and FK506 on proteinuria in a rat model of MCD induced by puromycin aminonucleoside (PAN) and in vitro cultured mouse podocytes. Our results showed that CsA and FK506 treatment decreased proteinuria via a mechanism associated to a reduction in the foot-process fusion and desmin, and a recovery of synaptopodin and podocin. In PAN-treated mouse podocytes, pre-incubation with CsA and FK506 restored the distribution of the actin cytoskeleton, increased the expression of synaptopodin and podocin, improved podocyte viability, and reduced the migrating activities of podocytes. Treatment with CsA and FK506 also inhibited PAN-induced podocytes apoptosis, which was associated with the induction of Bcl-xL and inhibition of Bax, cleaved caspase 3, and cleaved PARP expression. Further studies revealed that CsA and FK506 inhibited PAN-induced p38 and JNK signaling, thereby protecting podocytes from PAN-induced injury. In conclusion, CsA and FK506 inhibit proteinuria by protecting against PAN-induced podocyte injury, which may be associated with inhibition of the MAPK signaling pathway.

The cytoprotective role of autophagy in puromycin aminonucleoside treated human podocytes.

Autophagy is a ubiquitous catabolic process involving degradation of damaged organelles and protein aggregates. It shows cytoprotective effects in many cell types and helps to maintain cell homeostasis. In many glomerular diseases, podocyte damage leads to the disruption of the renal filtration barrier and subsequent proteinuria. Puromycin aminonucleoside (PAN) which induces podocyte apoptosis in vitro and in vivo is widely used for studying the pathophysiology of glomerular diseases. It has been shown that PAN induces autophagy in podocytes. However, the relationship between autophagy and apoptosis in PAN treated human podocytes is not known and the role of PAN-induced autophagy in podocyte survival remains unclear. Here we demonstrate that PAN induced autophagy in human podocytes prior to apoptosis which was featured with the activation of mTOR complex 1 (mTORC1). When the PAN-induced autophagy was inhibited by 3-methyladenine (3-MA) or chloroquine (CQ), podocyte apoptosis increased significantly along with the elevation of active caspase-3. Under such circumstance, the podocyte cytoskeleton was also disrupted. Collectively, our results suggested that the induced autophagy may be an early adaptive cytoprotective mechanism for podocyte survival after PAN treatment.

Transcriptome analysis of the response of cultured murine podocytes to puromycin aminonucleoside.

AIMS: Idiopathic nephrotic syndrome is known as a disease of the renal glomerular epithelial cells (podocytes). Recent advances in podocyte biology showed that podocytopathy is the culprit of nephrotic syndrome. To obtain comprehensive information about the response of podocytes to injury, we investigated the gene expression profile of podocytes in response to puromycin aminonucleoside (PAN)-induced injury. METHODS: Differentiated mouse podocyte cell line (MPC5) cells were treated with 25 microg/ml PAN for 24, 48, or 72 h. Gene expression profiles of these cells were analyzed. Real time PCR analysis was used to confirm the findings of microarray. RESULTS: Expression levels of 23 genes (differentially expressed genes, DEGs), including laminin alpha(1) and MMP3, were significantly different between PAN-treated podocytes and untreated cells. Gene ontology of DEGs indicated that their functional categories were cell adhesion, extracellular matrix (ECM) formation, and ECM degradation. Real-time PCR and indirect immunohistochemistry of PAN-treated and untreated podocytes confirmed the differential expression of DEGs. CONCLUSION: Using unbiased global gene expression profiling, we found that podocytes respond to PAN-induced injury by down-regulating the expression of genes involved in cell adhesion and extracellular matrix.

Rapid screening of glomerular slit diaphragm integrity in larval zebrafish.

Gene array-type experiments have identified large numbers of genes thought to be important for the integrity of the glomerular slit diaphragm. Confirmation of individual proteins has been limited by the expenses and time involved in generating transgenic or knockout mice for each candidate. We present a functional screening assay based on the clearance of a 70-kDa fluorescent dextran in another vertebrate system that is rapid and low in cost. In the pronephric glomerulus of larval zebrafish, we have demonstrated quantifiable loss of slit diaphragm integrity in a zebrafish model of puromycin aminonucleoside (PA) toxicity. In addition, after knockdown of CD2-associated protein (CD2AP) and podocin, two well-characterized genetic contributors to podocyte differentiation in mammals, we observed glomerular loss of serum macromolecules similar to that seen in mammalian kidneys with inborn mutations in these genes. Increased filtration of 70-kDa FITC-labeled dextran correlates with effacement of podocyte foot processes in ultrastructural analysis. These findings document the value of the zebrafish model in genomics and pharmacological screening applications.

Increased cyclosporine bioavailability induced by experimental nephrotic syndrome in rats.

Components of whole blood and plasma are highly altered during the presentation of nephrotic syndrome. The present study was aimed to explore the influence of nephrotic syndrome on the pharmacokinetics of cyclosporine (CsA) (10 mg/kg) administered i.v. to control or puromycin-induced nephrotic rats (P-NS). We found an increase in CsA bioavailability in the nephrotic group compared with controls. The area under the curve of blood CsA versus time (AUCiv) increased from 27.7 +/- 5.3 to 60.6 +/- 13.8 mug.h.mL-1 in control and P-NS rats, respectively. The AUCiv augmentation was positively correlated with cholesterol levels. On the other hand, the total body clearance was significantly lower (0.38 +/- 0.06 vs. 0.17 +/- 0.03 L.(kg body mass)-1.h-1) and the volume of distribution at steady state (3.70 +/- 0.52 vs. 2.85 +/- 0.32 L/kg) was significantly smaller in nephrotic rats as compared with control. These pharmacokinetic changes lead to a longer terminal half-life of CsA in P-NS rats (11.8 +/- 1.6 vs. 6.9 +/- 0.91 h). We conclude that the physiopathologic changes induced by the nephrotic syndrome in P-NS animals result in a significant increase in CsA blood exposure by both the decrease in drug distribution and the reduction in elimination rate of CsA.

Morphologic, molecular and hemodynamic cardiac alterations in a model of nephropathy induced by puromycin aminonucleoside.

INTRODUCTION: Proteinuria and decreased glomerular filtration rate are assuming increased importance in defining cardiovascular risk in chronic renal insufficiency. The aim of this work was to study morphologic, molecular and hemodynamic cardiac alterations in an animal model of proteinuria and renal insufficiency induced by puromycin aminonucleoside (PAN). METHODS: Normotensive rats (n = 14) were injected with PAN (150 mg/kg, i.p.) or with vehicle. Blood pressure was measured daily and the animals were placed in metabolic cages for evaluation of urinary excretion of sodium, protein and creatinine. Fourteen days after PAN administration left ventricular hemodynamics were evaluated through a pressure tip micromanometer and heart morphology was examined. Transmural samples of left ventricle were then taken for mRNA quantification of SERCA2a, phospholamban (PLB), insulin-like growth factor 1 (IGF-1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). RESULTS: The animals treated with PAN presented a decrease in creatinine clearance (14th day: 2.24 +/- 0.32 vs. 4.51 +/- 1.08 ml/min) and an increase in proteinuria (14th day: 51.0 +/- 9.0 vs. 3.8 +/- 0.7 mg/mg creatinine), without changes in systolic (14th day: 151 +/- 7 vs. 141 +/- 6 mmHg) or diastolic blood pressure (14th day: 85 +/- 7 vs. 86 +/- 3 mmHg), These alterations were accompanied by cardiac atrophy with decreased left ventricular contractility. A reduction in the SERCA2a/PLB mRNA ratio was observed without significant alteration in the expression of IGF-1 in the left ventricle. CONCLUSIONS: PAN-induced nephropathy is accompanied by cardiac atrophy, left ventricular dysfunction and alterations in the expression of genes involved in myocardial calcium kinetics. These findings were not accompanied by increases in blood pressure and may contribute to our understanding of the increased cardiovascular risk in chronic renal insufficiency.

Peroxisome proliferator-activated receptor-gamma agonist is protective in podocyte injury-associated sclerosis.

We have previously observed increased expression of peroxisome proliferator-activated receptor gamma (PPARgamma) in podocytes in both rat and human sclerotic conditions. The aim of the present study was to investigate whether activation of PPARgamma can attenuate podocyte injury-associated glomerulosclerosis in vivo. Puromycin aminonucleoside nephropathy was induced in Sprague-Dawley rats. The animals then either received no further treatment (control group (CONT)); or the PPARgamma agonist, pioglitazone (Pio) starting at week 0 (P0); or Pio starting at week 6 (P6), with sacrifice at week 12. At week 12, urinary protein excretion and systolic blood pressure were similar in the three groups. Glomerular filtration rate and glomerulosclerosis were decreased in CONT and P0 at week 12, but preserved in P6 rats. PPARgamma expression in CONT at 12 weeks was increased in podocytes and in mesangial WT-1 cells in segmentally sclerotic glomeruli, with less Wilms' tumor 1 (WT-1) staining. In P6 rats, mesangial WT-1 staining was lessened, but podocyte staining was strongly accentuated. Delayed treatment with Pio partially restored podocyte staining and tended to decrease the ratio of proliferating cell nuclear antigen-positive to apoptotic cells in glomeruli. Both treatment groups showed significantly reduced infiltrating glomerular macrophages and plasminogen activator inhibitor-1 mRNA expression in cortex, with no change in transforming growth factor-beta1 and tissue inhibitor of metalloproteinase-1 mRNA. Pio also decreased renal cortical angiopoietin-like protein 4 expression to almost 20% of CONT group, associated with increased vascular endothelial-derived growth factor expression in glomeruli. We conclude that treatment with PPARgamma agonist has protective effects on progression of glomerulosclerosis.

Novel expression of sodium/myo-inositol co-transporter in podocytes in puromycin aminonucleoside nephrosis.

BACKGROUND: How podocytes respond to injury is poorly understood, although podocyte injury in the glomerulus has been proposed as the crucial mechanism in the pathogenesis of proteinuria and focal segmental glomerulosclerosis. An increase in sodium/myo-inositol co-transporter (SMIT) transcripts, an osmoprotective gene, has been demonstrated in a variety of brain injury models. In the present study, we investigated SMIT expression in podocytes in experimental nephrosis. METHODS: Two types of nephrosis were induced in rats: puromycin aminonucleoside (PAN) nephrosis and monoclonal antibody (mAb) 5-1-6 nephropathy. Podocyte injury was morphologically distinct in the former type of nephrosis and limited to a minimum in the latter. SMIT expression in isolated glomeruli was estimated by ribonuclease protection assay. Localization of SMIT-expressing cells in glomeruli was examined by in situ hybridization. RESULTS: SMIT transcripts in glomeruli increased conspicuously in the nephrotic stage of PAN nephrosis, whereas the transcripts in cortices and medullae did not show significant changes. In situ hybridization revealed that podocytes were predominant cells expressing SMIT in the glomerulus. Significant increase of SMIT mRNA in the glomeruli was detected before the onset of massive proteinuria. In contrast, up-regulation of SMIT expression was not observed in mAb 5-1-6 nephropathy, whose urinary protein levels were comparable with those in the nephrotic stage of PAN nephrosis. CONCLUSIONS: These findings suggest that SMIT expression in podocytes is not provoked by an effect of massive proteinuria but by extensive cellular injury.

Retinoids as a potential treatment for experimental puromycin-induced nephrosis.

1 Puromycin aminonucleoside (PAN)-induced nephrosis is a model of human minimal change disease. In rats, PAN induces nephrotic-range proteinuria, renal epithelial cell (podocyte) damage, infiltration of mononuclear leukocytes, and apoptosis of several renal cell types. 2 Retinoic acid (RA) modulates a wide range of biological processes, such as inflammation and apoptosis. Since renal damage by PAN is characterized by inflammatory infiltration and epithelial cell death, the effect of treatment with all-trans RA (tRA) was examined in the PAN nephrosis model and in the cultured differentiated podocyte. 3 Treatment with tRA 4 days after PAN injection did not inhibit the proteinuria peak but reversed it significantly. However, treatment with tRA both before and 2 days after the injection of PAN protected the glomerular epithelial cells, diminishing the cellular edema and diffuseness of the foot process effacement. Preservation of the podocyte architecture correlated with the inhibition of proteinuria. The anti-inflammatory effect of tRA was evidenced by the inhibition of PAN-induced interstitial mononuclear cell infiltration and the decreased renal expression of two molecules involved in monocyte infiltration: fibronectin and monocyte chemoattractant protein-1. TUNEL assays showed that tRA inhibited the PAN-induced apoptosis of cultured differentiated mouse podocytes. 4 We conclude that tRA treatment may prevent proteinuria by protecting the podocytes from injury and diminishing the interstitial mononuclear infiltrate in the model of PAN nephrosis. Retinoids are a potential new treatment for kidney diseases characterized by proteinuria and mononuclear cell infiltration.

The role of the renin-angiotensin system in cholesterol and puromycin mediated renal injury.

BACKGROUND: Puromycin aminonucleoside (PAN) nephropathy is a widely studied model of glomerular sclerosis (GS) in the rat, and cholesterol feeding exacerbates the injury induced by PAN. The importance of the interaction of angiotensin II (Ang II) with the AT2 receptor is unclear. We investigated the role of the renin-angiotensin system, particularly with regard to AT1 and AT2 receptor dynamics, in PAN and cholesterol-mediated GS. METHODS: Sprague-Dawley rats were given a 4% cholesterol diet (group II), subcutaneous PAN (group III), or a 4% cholesterol diet and PAN (group IV) and compared with a control group given PAN vehicle (group I). After 16 weeks, kidneys were harvested and tissue Ang II concentration, angiotensin-converting enzyme (ACE) activity, and ACE, AT1, and AT2 mRNA levels were determined. RESULTS: Compared with control rats, proteinuria was significantly higher in groups II to IV. Kidney ACE activity and ACE mRNA levels in groups III and IV were 2- and 3-fold higher than in groups I and II, respectively. Kidney Ang II concentration also was increased in the experimental groups. Whereas kidney AT1 mRNA was significantly lower in groups III and IV, kidney AT2 mRNA was significantly increased in groups II to IV. CONCLUSION: In these experimental models of GS, there is significant activation of the tissue-based renin-angiotensin system. Puromycin with and without cholesterol decreased the AT1 receptor mRNA and increased the AT2 receptor mRNA. Up-regulation of AT2 receptors may be important in ameliorating the proliferative effects of Ang II, which presumably occur through the AT1 receptor.

Plasma and urinary endothelin-1 in focal segmental glomerulosclerosis.

The kidney is an important site of endothelin-1 (ET-1) production and is particularly susceptible to ET-1 action. Infusion of ET-1 in rats induces both functional and morphological alterations in the kidneys. Increased plasma level of ET-1 has been reported in patients with chronic renal failure. However, there are still no reports on the plasma and urinary ET-1 levels in patients with focal segmental glomerulosclerosis (FSGS). In the present study, we have measured the plasma concentration and urinary excretion rate of ET-1 in 15 patients with nephrotic syndrome due to FSGS, and observed the serial changes of plasma and urinary ET-1 in nephrotic rats with FSGS, induced by repeated injection with puromycin aminonucleoside (PAN). ET-1 was measured with radioimmunoassay. The results showed that plasma ET-1 concentration in FSGS patients was significantly higher than in normal controls (P < 0.05), and that urinary ET-1 excretion rate was also significantly higher in FSGS patients than in normal controls (P < 0.01). In FSGS patients, the plasma and urinary ET-1 was significantly correlated (P < 0.05), and the urinary ET-1 excretion rate was significantly correlated with the amount of proteinuria (P < 0.05) and the glomerular sclerosing score (P < 0.01). In the ten rats with PAN-induced FSGS, serial examination showed a significant increase in plasma ET-1 after 8 weeks of injections, while the urinary ET-1 excretion rate showed a biphasic increase that showed a peak after 4 to 6 weeks. The same changes in plasma and urinary ET-1 levels were not observed in control rats injected with normal saline at the same frequency. Our results suggest that ET-1 may be involved in the pathogenesis of FSGS in both humans and rats.

Garlic ameliorates hyperlipidemia in chronic aminonucleoside nephrosis.

Nephrotic syndrome (NS) is characterized by proteinuria, oxidative stress and endogenous hyperlipidemia. Hyperlipidemia and oxidative stress may be involved in coronary heart disease and the progression of renal damage in these patients. Garlic has been suggested to be beneficial in various disease states. Some of the beneficial effects of garlic may be secondary to its hypolipidemic and antioxidant properties. Therefore, the effect of a 2% garlic diet on acute and chronic experimental NS induced by puromycin aminonucleoside (PAN) was studied in this work. Acute NS was induced by a single injection of PAN to rats which were sacrificed 10 days later. Chronic NS was induced by repeated injections of PAN to rats which were sacrificed 84 days after the first injection. Garlic treatment was unable to modify proteinuria in either acute or chronic NS, and hypercholesterolemia and hypertriglyceridemia in acute NS. However, garlic treatment diminished significantly total-cholesterol, LDL-cholesterol and triglycerides, but not HDL-cholesterol in chronic NS. Garlic induced no change in the percentage of sclerotic glomeruli in chronic NS and a significative decrease on the percentage of sclerotic area of these glomeruli (33 +/- 3% in NS+Garlic group vs. 47 +/- 4% in NS group, p = 0.0126). The enhanced in vivo renal H2O2 production and the diminished renal Cu, Zn-SOD and catalase activities in acute NS, and the decreased renal catalase activity in chronic NS were not prevented by garlic treatment. These data indicate that garlic treatment ameliorates hyperlipidemia and renal damage in chronic NS which is unrelated to proteinuria or antioxidant enzymes.

MR imaging of intrarenal macrophage infiltration in an experimental model of nephrotic syndrome.

The objective of this study was to use MR imaging to detect macrophage infiltration of the kidney after injection of ultrasmall superparamagnetic iron oxide (USPIO) particles in a rat model of experimental nephropathy. Ninety micromol of USPIO were injected intravenously in 10 rats with nephropathy secondary to intravenous injection of 5 mg of puromycin aminonucleoside (PAN), and in 10 control rats. The signal intensity was measured in each kidney compartment before and 24 h after injection of the contrast agent. FLASH sequences were performed on a spectrometer operating at 4.7 T. MR findings were compared with histological data. Twenty-four hours after injection of USPIO, a significant decrease (P < 0.0001) was observed in signal intensity in each kidney compartment in the PAN group. There was no variation in the control group. In the diseased kidneys, histological data revealed the presence of macrophages with iron oxide particles within their cytoplasm and lysosomes. Using USPIO, MR imaging can evidence infiltration of the rat kidney by macrophages.

Failure of antioxidant therapy to attenuate interstitial disease in rats with reversible nephrotic syndrome.

The present two studies were designed to determine whether oxidized LDL contributes to the tubulointerstitial changes seen in rats during the acute phase of acute puromycin aminonucleoside nephrosis (PAN). In the single-dose study, rats were given one injection of puromycin aminonucleoside (PA; 15 mg/100 g body wt) and killed 1, 2, or 3 wk thereafter. The four animal groups were saline controls, PAN controls, PAN plus probucol, and PAN plus lovastatin. This study showed that the addition of probucol significantly reduced the mean levels of serum cholesterol and renal lipid-peroxidation products, an effect not seen with lovastatin therapy. Compared with saline controls, PAN controls had a significant increase in total kidney collagen (7.9 +/- 1.2 versus 5.9 +/- 0.6 mg/kidney at 3 wk). Neither probucol nor lovastatin therapy attenuated the interstitial inflammation or fibrosis. In the multidose study, rats were given the same initial PA dose and were uninephrectomized on day 12. They were killed on day 35 after two smaller PA doses were given on days 16 and 23. Animal groups were saline controls, PAN controls, PAN plus probucol, and PAN plus vitamin E. Hepatic lipid-peroxidation products were significantly lower in the probucol-treated, but not in the vitamin E-treated, PAN groups when compared with the PAN controls. Neither probucol nor vitamin E prevented the increase in total kidney collagen that was observed in the PAN control group (7.4 +/- 0.7, 10.1 +/- 2.6, and 9.3 +/- 1.8 mg of collagen/kidney, respectively, versus 5.4 +/- 0.5 mg/kidney for the saline controls). Renal cortical mRNA levels for matrix-encoding genes and protease inhibitors were similar in the three nephrotic groups. Transforming growth factor-beta1 mRNA levels were highly variable within each group and not significantly different at day 35, but showed a significant positive correlation with the degree of albuminuria (r = 0.70). The present results demonstrate that the treatment of acutely nephrotic rats with antioxidant therapy did not attenuate interstitial inflammation or fibrosis. We speculate that other factors, possibly a consequence of proteinuria itself, are the predominant pathogenetic mediators of the tubulointerstitial damage in acute nephrotic syndrome.

Structure-activity relationship for two lipoxygenase inhibitors and their potential for inducing nephrotic syndrome.

In a study of structure-activity relationship with drug-induced nephropathy two lipoxygenase inhibitors, the N-hydroxyurea derivative 70C ((E)-N-{3-[3-(4-fluorophenoxy) phenyl]-1-(R, S)-methylprop-2-enyl}-N-hydroxyurea) and the N-hydroxamic acid analogue 360C ((E)-N-{3-[3-(4-fluorophenoxy) phenyl]-1-(R, S)-methylprop-2-enyl}-N-hydroxamic acid), were administered to rats. 70C and 360C were dosed to female Wistar rats at 100 mg/kg po daily for 7 days. Another group of rats was given a single intravenous bolus dose of puromycin aminonucleoside (PAN) at 100 mg/kg. Urine samples were collected from all groups during the study and plasma samples were collected after 7 days. Kidneys were excised and fixed for examination by electron microscopy. 70C- and PAN-treated groups both showed early changes in the glomeruli, in which the visceral cells appeared enlarged and showed varying degrees of foot process loss. This foot process loss was associated with decreases in total plasma protein and albumin and increases in the plasma cholesterol, triglycerides, creatinine, and urea were recorded. Marked proteinuria was observed in both the 70C and PAN groups. The foot process loss together with increased proteinuria, hypoalbuminemia, hypercholesterolemia, and lipemia are all characteristic of the human condition, Minimal Change Nephrotic Syndrome. All the biochemical and morphological investigations showed that 360C-treated rats were similar to the control group, suggesting that the hydroxyurea moiety of 70C is responsible, either directly or indirectly, for the induction of the nephrotic syndrome seen in rats.

Reduced renal medullary water channel expression in puromycin aminonucleoside--induced nephrotic syndrome.

The aquaporins are molecular water channels that mediate transcellular water transport across water-permeable epithelia. To investigate the cause of the concentrating defect in the nephrotic syndrome, immunoblotting using membrane fractions from inner medulla was utilized to assess the level of expression of four aquaporin water channels in vehicle-treated versus puromycin aminonucleoside (PAN)-treated rats. Scanning electron microscopy demonstrating loss of glomerular foot processes and measurements of urinary protein excretion confirmed the efficacy of the PAN treatment. In rats receiving PAN, there was an increase in plasma vasopressin, without a change in plasma sodium concentration. Inner medullary tissue hypertonicity was sustained in PAN-treated rats while the urinary osmolality was low, pointing to defective osmotic equilibration across the collecting ducts in PAN-nephrosis. Among collecting duct aquaporins, there was an 87% decrease in aquaporin-2 expression and a 70% decrease in aquaporin-3 expression in the inner medulla, whereas aquaporin-4 expression was unaltered. Transmission electron microscopy of the inner medullary collecting ducts of PAN-treated rats showed normal-appearing cells. Thus, PAN-nephrosis is associated with an extensive downregulation of collecting duct water channel expression despite increased circulating vasopressin, providing an explanation for the concentrating defect associated with the nephrotic syndrome.