Puromycin Aminonucleoside Administration Research Articles

GDF-15 Suppresses Puromycin Aminonucleoside-Induced Podocyte Injury by Reducing Endoplasmic Reticulum Stress and Glomerular Inflammation.

GDF15, also known as MIC1, is a member of the TGF-beta superfamily. Previous studies reported elevated serum levels of GDF15 in patients with kidney disorder, and its association with kidney disease progression, while other studies identified GDF15 to have protective effects. To investigate the potential protective role of GDF15 on podocytes, we first performed in vitro studies using a Gdf15-deficient podocyte cell line. The lack of GDF15 intensified puromycin aminonucleoside (PAN)-triggered endoplasmic reticulum stress and induced cell death in cultivated podocytes. This was evidenced by elevated expressions of Xbp1 and ER-associated chaperones, alongside AnnexinV/PI staining and LDH release. Additionally, we subjected mice to nephrotoxic PAN treatment. Our observations revealed a noteworthy increase in both GDF15 expression and secretion subsequent to PAN administration. Gdf15 knockout mice displayed a moderate loss of WT1+ cells (podocytes) in the glomeruli compared to wild-type controls. However, this finding could not be substantiated through digital evaluation. The parameters of kidney function, including serum BUN, creatinine, and albumin-creatinine ratio (ACR), were increased in Gdf15 knockout mice as compared to wild-type mice upon PAN treatment. This was associated with an increase in the number of glomerular macrophages, neutrophils, inflammatory cytokines, and chemokines in Gdf15-deficient mice. In summary, our findings unveil a novel renoprotective effect of GDF15 during kidney injury and inflammation by promoting podocyte survival and regulating endoplasmic reticulum stress in podocytes, and, subsequently, the infiltration of inflammatory cells via paracrine effects on surrounding glomerular cells.

IGF-1 and HGF Preconditioning of Bone Marrow Mesenchymal Stem Cells Reduced Proteinuria in Rat Focal Segmental Glomerulosclerosis Model

Background & Aim Focal Segmental Glomerulosclerosis (FSGS) is one of the leading causes of end-stage kidney disease requiring renal replacement therapy for survival. It is characterized by massive proteinuria that results in hypoalbuminemia, edema and hyperlipidemia. and is known as irreversible kidney disease that steroid-based drugs only slow down the progression. The use of Mesenchymal stem cells (MSCs) has been successful in the treatment of glomerular diseases. The biggest challenge of the MSC application is its engraftment into the site of injury and its loss due to the poor microenvironment. Preconditioning of MSCs by particular growth factors such as insulin-like growth factor-1 (IGF-1) and hepatocyte growth factor (HGF) increases their migration, immunomodulation and survival. IGF-1 and HGF have been proposed as promising therapeutic agents in recent years and are especially preferred in renal diseases with their renotrophic and nephroprotective effects. Therefore we aimed to test the effect of IGF-1 and HGF-preconditioned MSCs in a rat model of FSGS. Methods, Results & Conclusion Preconditioning of MSCs was performed by 10 ng/ml IGF-1 and 20 ng/ml HGF and designated as iMSCs. Rat (Sprague-Dawley) FSGS model was technically induced by a single-dose intraperitoneal administration of Puromycin Aminonucleoside (PAN) (150 mg/kg). There were PBS, MSC and iMSC treatment groups (n=6/gr77). The occurrence of FSGS was assessed by biochemistry (i.e. elevated urinary protein/creatinine (P/C) ratio), immunohistochemistry (i.e. decreased glomerular SYNPO protein level) and electron microscopy (i.e. presence of podocyte effacement). We applied 2 × 105 MSC or preconditioned MSC intravenously on the day of 4 following PAN administration. The effect of treatment was assessed by urinary P/C ratios in 24-hour urine which was collected on the treatment day of 9. iMSC treatment significantly decreased proteinuria (p As a result of our study, we demonstrated that iMSCs can partially reverse the glomerular damage and reduce proteinuria with regenerating glomerular filtering apparatus in the PAN model of FSGS and therefore may offer a new therapeutic strategy for human FSGS.

Combined use of electron microscopy and intravital imaging captures morphological and functional features of podocyte detachment.

The development of podocyte injury and albuminuria in various glomerular pathologies is still incompletely understood due to technical limitations in studying the glomerular filtration barrier (GFB) in real-time. We aimed to directly visualize the early morphological and functional changes of the GFB during the development of focal segmental glomerulosclerosis (FSGS) using a combination of transmission electron microscopy (TEM) and in vivo multiphoton microscopy (MPM) in the rat puromycin aminonucleoside (PAN) model. We hypothesized that this combined TEM + MPM experimental approach would provide a major technical improvement that would benefit our mechanistic understanding of podocyte detachment. Male Sprague-Dawley (for TEM) or Munich-Wistar-Frömter (for MPM) rats were given a single dose of 100-150mg/kg body weight PAN i.p. and were either sacrificed and the kidneys processed for TEM or surgically instrumented for in vivo MPM imaging at various times 2-14days after PAN administration. Both techniques demonstrated hypertrophy and cystic dilatations of the subpodocyte space that developed as early as 2-3days after PAN. Adhesions of the visceral epithelium to the parietal Bowman's capsule (synechiae) appeared at days 8-10. TEM provided unmatched resolution of podocyte foot process remodeling, while MPM revealed the rapid dynamics of pseudocyst filling, emptying, and rupture, as well as endothelial and podocyte injury, misdirected filtration, and podocyte shedding. Due to the complementary advantages of TEM and MPM, this combined approach can provide an unusally comprehensive and dynamic portrayal of the alterations in podocyte morphology and function during FSGS development. The results advance our understanding of the role and importance of the various cell types, hemodynamics, and mechanical forces in the development of glomerular pathology.

Smad3/Nox4-mediated mitochondrial dysfunction plays a crucial role in puromycin aminonucleoside-induced podocyte damage

Podocyte depletion due to apoptosis is the key hallmark of proteinuric kidney disease progression. Recently, several studies reported that mitochondrial (mt) dysfunction is involved in podocyte injury, while the underlying molecular mechanisms remain elusive. This study investigated the potential proximal signaling related to in vitro and in vivo mitochondrial dysfunction in a puromycin aminonucleoside (PA)-induced podocyte injury model. PA time- and dose-dependently resulted in cultured mouse podocyte damage, presenting with an increase of apoptotic cells and induction of activated caspase3/9. PA also caused mitochondrial damage and dysfunction based on the downregulation of the mtDNA level, decrease of transcriptional factors mtTfa and Nrf-1, decrease of CoxI, II and IV, and reduction of the oxygen consumption level and mitochondrial membrane potential level as well as excessive production of cellular ROS. Additionally, antioxidant MnSOD and catalase levels were decreased in mitochondrial fractions, and reduction of complex I and IV activity was also observed in PA-stimulated podocytes. Furthermore, an obvious translocation of p-Smad3 from the cytosol to nuclei and induction of mitochondrial Nox4 were detected following PA application. The PA-induced shift of cytochrome c was observed from mitochondria to the cytoplasm. Induction of Nox4 by PA administration was significantly repressed by Smad3-shRNA, while Nox4-shRNA showed no effect on PA-induced p-Smad3 activation. Notably, both Smad3 and Nox4 silencing significantly prevented the reduction of the mtDNA level, restored mitochondrial function, and decreased cellular apoptosis in PA-stimulated podocytes. A similar mitochondrial dysfunction was obtained in a PA-injected nephropathy rat, which was effectively inhibited by treatment with the antiproteinuric drug prednisone. In addition, Dab2 knockdown decreased albumin uptake and influx whereas it showed no effect on cellular apoptosis in PA-stimulated podocytes. In conclusion, our findings demonstrated that Smad3–Nox4 axis-mediated mitochondrial dysfunction is involved in PA-induced podocyte damage likely via increasing ROS generation and activating the cytochrome c–caspase9–caspase3 apoptotic signaling pathway. Dab2 may be required for the increased permeability of podocytes following injury.

Podocyte Expression of Membrane Transporters Involved in Puromycin Aminonucleoside-Mediated Injury

Several complex mechanisms contribute to the maintenance of the intricate ramified morphology of glomerular podocytes and to interactions with neighboring cells and the underlying basement membrane. Recently, components of small molecule transporter families have been found in the podocyte membrane, but expression and function of membrane transporters in podocytes is largely unexplored. To investigate this complex field of investigation, we used two molecules which are known substrates of membrane transporters, namely Penicillin G and Puromycin Aminonucleoside (PA).We observed that Penicillin G pre-administration prevented both in vitro and in vivo podocyte damage caused by PA, suggesting the engagement of the same membrane transporters by the two molecules. Indeed, we found that podocytes express a series of transporters which are known to be used by Penicillin G, such as members of the Organic Anion Transporter Polypeptides (OATP/Oatp) family of influx transporters, and P-glycoprotein, a member of the MultiDrug Resistance (MDR) efflux transporter family.Expression of OATP/Oatp transporters was modified by PA treatment. Similarly, in vitro PA treatment increased mRNA and protein expression of P-glycoprotein, as well as its activity, confirming the engagement of the molecule upon PA administration.In summary, we have characterized some of the small molecule transporters present at the podocyte membrane, focusing on those used by PA to enter and exit the cell. Further investigation will be needed to understand precisely the role of these transporter families in maintaining podocyte homeostasis and in the pathogenesis of podocyte injury.

Puromycin aminonucleoside modulates p130Cas of podocytes

PurposePuromycin aminonucleoside (PAN) specifically injures podocytes, leading to foot process effacement, actin cytoskeleton disorganization, and abnormal distribution of slit diaphragm proteins. p130Cas is a docking protein connecting F-actin fibers to the glomerular basement membrane (GBM) and adapter proteins in glomerular epithelial cells (GEpCs; podocytes). We investigated the changes in the p130Cas expression level in the PAN-induced pathological changes of podocytes in vitro.MethodsWe observed changes in the p130Cas expression in cultured rat GEpCs and mouse podocytes treated with various concentrations of PAN and antioxidants, including probucol, epigallocatechin gallate (EGCG), and vitamin C. The changes in the p130Cas expression level were analyzed using confocal immunofluorescence imaging, Western blotting, and polymerase chain reaction.ResultsIn the immunofluorescence study, p130Cas showed a diffuse cytoplasmic distribution with accumulation at distinct sites visible as short stripes and colocalized with P-cadherin. The fluorescences of the p130Cas protein were internalized and became granular by PAN administration in a dose-dependent manner, which had been restored by antioxidants, EGCG and vitamin C. PAN also decreased the protein and mRNA expression levels of p130Cas at high doses and in a longer exposed duration, which had been also reversed by antioxidants.ConclusionThese findings suggest that PAN modulates the quantitative and distributional changes of podocyte p130Cas through oxidative stress resulting in podocyte dysfunction.

Effects of mineralocorticoid and angiotensin II receptor blockers on proteinuria and glomerular podocyte protein expression in a model of minimal change nephrotic syndrome

Several proteins constituting the slit diaphragm are considered important for maintaining capillary wall permselectivity. Early intervention with blockers of angiotensin II receptors (AR) and mineralocorticoid receptors (MR) is effective against proteinuria in models of chronic hypertensive and protein-induced renal damage. However, the effects of AR and/or MR blockers in a model of acute nephrotic syndrome remain unknown. The effects of AR and MR blockers were examined in puromycin aminonucleoside (PAN)-treated rats. Six week old male Sprague-Dawley (SD) rats were injected with PAN or vehicle and assigned to groups as follows: vehicle (group C); PAN (group P); PAN followed 3 days later by administration of the MR blocker, eplerenone (group MR), and by the AR blocker, losartan (group AR). Blood pressure and urinary protein excretion were measured and all rats were killed for immunohistochemical investigation on day 14 after PAN administration. Blood pressure did not change throughout the study period. Proteinuria was decreased in groups MR and AR compared with group P (on day 14 after PAN administration, respectively; group P vs AR, P < 0.01; group P vs MR, P < 0.05). Nephrin, podocin and podocalyxin staining was preserved in the glomeruli of groups MR and AR compared with group P. The MR and AR blockers decreased proteinuria in the acute model of nephrotic syndrome with preserved expression of glomerular podocyte protein independently of blood pressure.

The renal clearance of dextran sulfate decreases in puromycin aminonucleoside-induced glomerulosclerosis: A puzzle observation

Background Puromycin aminonucleoside-induced nephrosis is characterized by increased renal excretion of plasma proteins. We employed this experimental model to study the urinary clearance of dextran sulfate. Methods The dextran sulfate eliminated by the urine was determined using a metachromatic assay. Polysaccharide fragments were analyzed by chromatographic and electrophoretic procedures. Disaccharide composition of the glomerular heparan sulfate was assessed using digestion with specific lyases. Results In normal rats dextran sulfate is partially degraded to lower molecular weight fragments and only then eliminated by the urine. Surprisingly, in puromycin aminonucleoside-induced glomerulosclerosis the molecular size of the fragments of dextran sulfate found in the urine is the same or even lower than in control animals in spite of the marked proteinuria. Furthermore, urinary excretion of dextran sulfate decreases in the experimentally induced nephrosis. This observation cannot be totally attributed to a reduced number of physiologically active nephrons since the glomerular filtration rate decreases ∼ 32% after puromycin aminonucleoside administration while the urinary excretion of 8 kDa-dextran sulfate decreases 3-fold. The glomerular heparan sulfate shows reduced sulfation when compared with normal animals. Possibly puromycin aminonucleoside decreases the activity of kidney endoglycosidases, which reduce the molecular size of the sulfated polysaccharide, leading to a decrease in its renal clearance. Reduced sulfation of the glomerular heparan sulfate in the puromycin aminonucleoside-induced nephrosis does not alter the size of the dextran sulfate eliminated by the kidney, as suggested for protein. Conclusions Each pathological process induces a particular modification in the kidney, which in turn can affect the renal selectivity to specific macromolecules in different ways.

In vivo detection of intrinsic reactive oxygen species using acyl-protected hydroxylamine in puromycin nephrosis

Intrinsic reactive oxygen species (ROS) in a rat model of human minimal change nephropathy were detected directly using an in vivo electron paramagnetic resonance (EPR) method with 1-acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine (ACP) in real time. The nephrosis was induced by the intravenous administration of 75 mg/kg of puromycin aminonucleoside (PAN). It was found that ROS in the kidney were increased 1 h after the administration of PAN. This increased oxidative stress declined at 24 h and returned to a normal level 3 days after PAN administration. This is the first non-invasive in vivo detection and quantification of specific ROS in an experimental nephrosis model.

Effect of a novel free radical scavenger, edaravone, on puromycin aminonucleoside induced nephrosis in rats

Recent studies indicate that excessive production of oxidants plays a role in the pathogenesis of glomerular injury leading to proteinuria in patients with minimal-change nephrotic syndrome (MCNS). The novel free radical scavenger, edaravone (EDA), which was recently developed in Japan, is currently used in patients with stroke. We studied whether this new agent would be beneficial in patients with MCNS by its antioxidant activity and examined its effect on proteinuria in nephrosis induced by puromycin-aminonucleoside (PAN) in rats. Nineteen Wistar-Kyoto rats injected with PAN were assigned to four groups: group 1, without EDA (n=4); group 2, concomitant EDA injection from 1 day prior to PAN administration (n=5); group 3, concomitant EDA injection from 1 day after PAN administration (n=5); group 4, concomitant EDA injection from 3 days after PAN administration (n=5). Daily urinary excretions of protein and 8-hydroxy-2'-deoxyguanosine (8-OHdG), a new sensitive marker of oxidative DNA damage in vivo, were measured in each group from the 1st to the 30th day after PAN injection. In group 1 proteinuria developed from the 5th day and reached the peak level on the 9th day. In groups 2, 3, and 4 proteinuria did not appear until the 6th day. The excretions in urinary protein and 8-OHdG were significantly lower in groups 2, 3, and 4 than group 1 on days 5, 9, and 25. In conclusion, EDA could delay and ameliorate the urinary protein excretion in accordance with the urinary 8-OHdG excretion in PAN-induced nephrosis.

Downregulation of nitric oxide synthase in nephrotic syndrome: role of proteinuria

Blood pressure is frequently elevated, blood volume is usually normal or increased and plasma renin and aldosterone are usually low in nephrotic syndrome (NS). These observations challenge the conventional view attributing sodium retention in NS to a hypoalbuminemia-induced intravascular volume contraction. Given the pivotal role of nitric oxide (NO) in regulation of renal sodium (Na) handling, vascular resistance and sympathetic activity, we considered that Na retention and hypertension in NS may be associated with impaired NO system. Urinary excretion of Na and NO metabolites (NOx), as well as immunodetectable endothelial (eNOS), inducible (iNOS) and neuronal (nNOS) NO synthases were determined in rats with puromycin aminonucleoside (PAN)-induced NS, rats with protein overload proteinuria, Nagase rats (NAR) with inherited analbuminemia, iNOS inhibitor (aminoguanidine)-treated rats, prenephrotic PAN-treated and placebo-treated control rats. The NS group showed marked proteinuria, hypoalbuminemia, decreased fractional excretion of Na (FE Na), reduced urinary NOx excretion, and severe reduction of iNOS and nNOS protein abundance in the kidney. Similar results were found in rats with protein overload proteinuria in which proteinuria was present without hypoalbuminemia. In contrast, despite extreme hypoalbuminemia, NAR showed normal FE Na, increased urinary NOx excretion and upregulations of iNOS and nNOS protein abundance in the kidney. Administration of aminoguanidine for 3 weeks lowered FE Na in normal rats to levels approximating those found in the NS group. Animals studied 2 days after PAN administration (wherein proteinuria was absent) showed no abnormality. Thus, chronic PAN-induced NS results in downregulation of kidney iNOS and nNOS, which can contribute to the reduction of FE Na by augmenting renal tubular Na reabsorption, and preglomerular vasoconstriction. Findings in the NAR, which had profound hypoalbuminemia without proteinuria, and in rats with protein overload proteinuria, which had proteinuria without hypoalbuminemia, point to proteinuria as the primary mediator of kidney iNOS and nNOS deficiency and impaired Na excretion in PAN-induced NS.

Identification by an EPR technique of decreased mitochondrial reducing activity in puromycin aminonucleoside-induced nephrosis.

The temporal changes in the electron paramagnetic resonance (EPR) signal intensities of a nitroxide radical, 4-hydroxy 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), in the kidney in rat puromycin aminonucleoside (PAN) nephrosis were investigated in vivo and in vitro. The rats of the PAN nephrosis group received intraperitoneal injections of PAN at 75 mg/kg body weight while those of control group received saline. The in vivo renal half-lives of TEMPOL were calculated from the decay curve of EPR signal intensities after the intravenous injection of the TEMPOL solution. The mitochondrial half-lives were obtained from the decay curve of the EPR signals after mixing the mitochondrial fraction of the kidney and TEMPOL solution. The in vivo half-lives of TEMPOL of the kidney from 7 to 14 d after PAN administration were significantly longer than those of the controls. The mitochondrial half-lives of TEMPOL on the 9th day after the PAN administration prolonged remarkably compared to the controls (378 +/- 69 vs. 676 +/- 183 s, p <.01). These findings indicate that the in vivo and mitochondrial reducing activity in PAN treated rats decreased markedly, because the half-life of TEMPOL in the kidney reflects the renal reducing activity.

Lack of a role for inducible nitric oxide synthase in an experimental model of nephrotic syndrome

Puromycin aminonucleoside (PAN) administration in rats produces an experimental model of nephrotic syndrome characterized by glomerular epithelial cell injury and proteinuria. The purpose of this study was to examine the role of nitric oxide (NO) in this model of minimal change glomerular disease. Aminoguanidine (AG) was used to inhibit inducible nitric oxide synthase (iNOS). Sprague–Dawley rats were divided into Control (N = 9), PAN (N = 14), AG (N = 2), and PAN + AG (N = 12) treatment groups. Control animals received saline (i.v.), PAN animals received PAN (75 mg/kg, i.v.), and PAN + AG animals received PAN plus AG (50 mg/kg, i.p., twice daily). AG animals received a saline injection (i.v.) on day 0 in the place of PAN and then AG on the same schedule as the PAN + AG group. Animals were kept in metabolic cages, and urinary protein excretion and nitrite (NO 2 −) excretion were measured daily. PAN administration increased urinary NO 2 − excretion by day 2, and levels remained elevated through day 7. AG prevented this PAN-induced increase in urinary NO 2 − excretion. Plasma nitrate (NO 3 −) and NO 2 − (NOx) concentrations were also increased in the PAN and PAN + AG groups. iNOS protein expression was not detected in either the glomeruli or the cortex at day 7. Proteinuria developed in PAN animals on day 4 and increased steadily through day 7. PAN + AG animals showed a pattern similar to that of the PAN group. These results indicated that in contrast to models of proliferative glomerulonephritis, NO formation during PAN-induced nephrotic syndrome is increased but does not participate in the development of glomerular injury as measured by proteinuria.

An integrated proteomic approach to studying glomerular nephrotoxicity.

A single dose of puromycin aminonucleoside (PAN) given parenterally to rats induces ultrastructural glomerular changes and a nephrotic syndrome similar in many respects to human minimal change nephropathy. The exact aetiologies of both the human and the experimental syndromes are unknown, and are probably multifactorial. However, among the observed consequences in humans and rats is increased plasma protein excretion in urine, beginning in the latter typically 3-6 days after PAN administration. In view of this, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has been used to profile urinary proteins during PAN-induced nephrotoxicity and subsequent recovery in the rat. In addition, urinary high performance liquid chromatography (HPLC) profiles and high resolution proton nuclear magnetic resonance (NMR) spectroscopy has been utilised to simultaneously detect toxin-induced changes in the relative concentrations of a number of metabolites. The proteomic approach, in conjunction with these other techniques, has the potential to provide significantly more mechanistic information than is provided readily by traditional clinical chemistry.

An integrated proteomic approach to studying glomerular nephrotoxicity

A single dose of puromycin aminonucleoside (PAN) given parenterally to rats induces ultrastructural glomerular changes and a nephrotic syndrome similar in many respects to human minimal change nephropathy. The exact aetiologies of both the human and the experimental syndromes are unknown, and are probably multifactorial. However, among the observed consequences in humans and rats is increased plasma protein excretion in urine, beginning in the latter typically 3-6 days after PAN administration. In view of this, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has been used to profile urinary proteins during PAN-induced nephrotoxicity and subsequent recovery in the rat. In addition, urinary high performance liquid chromatography (HPLC) profiles and high resolution proton nuclear magnetic resonance (NMR) spectroscopy has been utilised to simultaneously detect toxin-induced changes in the relative concentrations of a number of metabolites. The proteomic approach, in conjunction with these other techniques, has the potential to provide significantly more mechanistic information than is provided readily by traditional clinical chemistry.

Suppressed Activities of Cathepsins and Metalloproteinases in the Chronic Model of Puromycin Aminonucleoside Nephrosis

Glomerulosclerosis and tubulointerstitial fibrosis are the hallmarks of chronic renal diseases. In the present study, we have investigated the potential involvement of various proteinases in these alterations in the model of puromycin aminonucleoside (PAN) nephrosis. Two groups of male Wistar rats were given either three or seven injections of PAN (2.0 mg/100 g body weight) over a 4– and 12–week period, respectively. The two control groups received saline injections. Activities of cathepsins (B, H and L) were determined in isolated glomeruli and proximal tubules. Moreover, collagenaselike and gelatinaselike activities were analyzed in isolated glomeruli. Three weeks after weekly PAN injection, the rats developed heavy proteinuria (140.8±22.0 vs. 13.5±3.29 mg/day; p<0.001), and at week 11 protein excretion reached 606.6±23.00 vs. 22.8±1.5 mg/day. Renal morphology revealed minimal glomerular mesangial changes at the 4th week after PAN administration. At the 12th week a marked mesangial matrix accumulation as well as severe tubulointerstitial infiltration and fibrosis associated with tubular dilation and atrophy were observed. Glomerular cathepsins B, H, and L and gelatinaselike activities decreased at the 4th week after the first PAN injection and remained at this low level throughout the entire study period. Glomerular collagenaselike activity decreased at the 4th week (p<0.05) and was still mildly lower than that of the control group at the 12th week, but without significance. In the isolated proximal tubules, the activities of cathepsins B, H, and L showed the same pattern of decreases as those found in the glomeruli over the whole experimental period. Taken together, our data in the model of chronic PAN nephrosis suggest that the suppressed activities of cathepsins as well as the decreased gelatinase– and collagenaselike activities participate in the accumulation of extracellular matrix and thereby may contribute to the development of glomerulosclerosis and tubulointerstitial fibrosis.

Pituitary–ovarian dysfunction in rats with induced nephrotic syndrome

The reproductive hormonal profile was evaluated in female rats with experimental nephrotic syndrome induced with a single subcutaneous dose of puromycin aminonucleoside (PAN). Serum concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), progesterone (P4), testosterone and 17 beta-estradiol (E2) were determined sequentially in control and experimental groups on days 1, 3, 7 and 10 after PAN administration. Prolactin levels were also assessed on day 10. In both groups, vaginal smears were taken daily throughout the study to evaluate cyclic histological changes. At the end of the experiment the histological appearance of the ovaries was evaluated by light microscopy. Nephrotic rats had a rapid loss of the estrous cycle starting on day 4, which set them at diestrus. At the same time the hormonal evaluation indicated a gradual decrease in E2, LH and P4 concentrations, starting from days 3, 7 and 10, respectively. No significant changes were noted in FSH or testosterone values. Besides, on day 10, prolactin concentrations remained unmodified. Even though most hormonal levels were found low on day 10, all values except E2 (undetectable) corresponded to the interval reported for the diestrus phase. Likewise, histological examination of ovarian tissue from nephrotic rats showed a considerable increase in the number of atretic follicles. These findings indicate that female rats with nephrotic syndrome develop an important endocrine dysfunction that probably involves steroidogenic tissues (ovary and/or adrenal gland), and suggest the existence of a hypothalamic-hypophyseal impairment.

Messenger RNA Expression for Growth Factors in Glomeruli from Focal Glomerular Sclerosis

Focal glomerular sclerosis was induced in rats by chronic injections of puromycin aminonucleoside (PAN) on Days 0, 27, 34, and 41 and by unilateral nephrectomy on Day 22. Rats were sacrificed on Days 0, 8, and 20 (acute phase) and on Days 48, 60, and 80 (sclerotic phase). The percentage of sclerosing glomeruli was 16.6% on Day 48 and increased significantly to 72.8% on Day 80. We examined glomerular mRNA levels for proliferating cell nuclear antigen (PCNA), platelet-derived growth factor (PDGF)-A and B chains, transforming growth factor (TGF)-β, epidermal growth factor (EGF), insulin-like growth factor (IGF)-I, and basic fibroblast growth factor (bFGF) on Days 0, 8, 20, 48, 60, and 80. Although these growth factor mRNA levels showed little change in glomeruli until Day 20, all growth factor mRNA levels increased in glomeruli during the sclerotic phase of PAN nephrosis as glomerular sclerosis progressed. On Day 80, mRNA levels for PCNA, PDGF-A and B chains, TGF-β, EGF, IGF-I, and bFGF increased 12-, 10-, 12-, 15-, 2-, 2-, and 8-fold, respectively, in the glomeruli of PAN-treated rats with marked glomerular sclerosis when compared with control rats. Unilateral nephrectomy without PAN administration did not cause glomerular sclerosis up to Day 80 and mRNA levels for PCNA, PDGF-A and -B chains, TGF-β, EGF, IGF-I, and bFGF in this group were almost the same as those in the normal sham-operated group. These data suggest that changes in growth factor mRNA levels in glomeruli may contribute to the development of PAN-induced glomerular sclerosis.

Rat glomerular epithelial cells in culture express characteristics of parietal, not visceral, epithelium.

Glomerular epithelial cells (GEC) in culture are derived from intact isolated glomeruli. Although there is general agreement about distinguishing GEC from mesangial and endothelial cells, there is still uncertainty regarding the visceral versus parietal origin of cultured GEC. If these cells are to provide a useful model system, it is necessary to establish well-defined cell populations. The purpose of this study was to evaluate this important issue by comparing the characteristics of cultured GEC with glomerular epithelium from rat kidney sections. By electron microscopy, GEC were polygonal, with cilia and desmosomes between cells, similar to parietal cells in situ. Because intermediate filaments are specifically expressed in differentiated cells in the kidney, the expression of intermediate filaments in cultured GEC were compared with those of intact glomeruli. Cultured GEC are positive for cytokeratin and negative for vimentin and desmin, identical to parietal cells in situ. In contrast, podocytes are positive for vimentin and desmin and negative for cytokeratin. In vivo, podocytes express gp330 and puromycin-aminonucleoside (PAN) susceptibility, which are used as markers for cultured GEC. Immunoperoxidase staining of rat kidney sections with monoclonal anti-gp330 demonstrated gp330 localization to the cell surface and coated pits of the parietal cells, similar to its localization in podocytes. The presence of gp330 in cultured GEC was confirmed by immunoblot. PAN administration to rats induced vacuolization and detachment from the basement membrane in the parietal cells of Bowman's capsule, similar to the cytotoxicity observed in podocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclosporin reduces proteinuria in rats with aminonucleoside nephrosis.

The effect of cyclosporin (CS) was assessed in Sprague-Dawley rats with puromycin aminonucleoside (PA) nephrosis induced by a single intraperitoneal injection of PA. Three groups of rats were injected intraperitoneally with CS (10 mg/kg body weight) daily, beginning 1 day before PA administration, or 5 or 10 days after PA administration, for 10 days. CS significantly reduced proteinuria in rats with PA nephrosis in comparison with untreated nephrotic controls. After discontinuation of the CS treatment, proteinuria gradually increased, reaching values similar to those in control nephrotic rats. CS pretreatment did not prevent the induction of PA-induced nephrotic syndrome. Light microscopy and assessment of anionic sites in the glomerular basement membrane revealed no differences between normal rats, nephrotic controls, and CS-treated rats. These results show that CS can reduce proteinuria in PA nephrosis, but cannot ameliorate the glomerular changes.