Puromycin Aminonucleoside Nephrosis Research Articles

Pharmacological Melanocortin 5 Receptor Activation Attenuates Glomerular Injury and Proteinuria in Rats with Puromycin Aminonucleoside Nephrosis

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Differential Expression of Renal and Hepatic PCSK9 During Development of Hypercholesterolemia in the Puromycin Aminonucleoside Nephrosis Rat Model of Nephrotic Syndrome

Differential Expression of Renal and Hepatic PCSK9 During Development of Hypercholesterolemia in the Puromycin Aminonucleoside Nephrosis Rat Model of Nephrotic Syndrome

Pharmacological Melanocortin 5 Receptor Activation Attenuates Glomerular Injury and Proteinuria in Rats With Puromycin Aminonucleoside Nephrosis.

Clinical evidence indicates that the melanocortin peptide ACTH is effective in inducing remission of nephrotic glomerulopathies like minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS), including those resistant to steroids. This suggests that a steroid-independent melancortinergic mechanism may contribute. However, the type of melanocortin receptor (MCR) that conveys this beneficial effect as well as the underlying mechanisms remain controversial. Burgeoning evidence suggests that MC5R is expressed in glomeruli and may be involved in glomerular pathobiology. This study aims to test the effectiveness of a novel highly selective MC5R agonist (MC5R-A) in puromycin aminonucleoside (PAN) nephrosis. Upon PAN injury, rats developed evident proteinuria on day 5, denoting an established nephrotic glomerulopathy. Following vehicle treatment, proteinuria continued to persist on day 14 with prominent histologic signs of podocytopathy, marked by ultrastructural glomerular lesions, including extensive podocyte foot process effacement. Concomitantly, there was loss of podocyte homeostatic markers, such as synaptopodin and podocin, and de novo expression of the podocyte injury marker desmin. Treatment with MC5R-A attenuated urine protein excretion and mitigated the loss of podocyte marker proteins, resulting in improved podocyte ultrastructural changes. In vitro in cultured podocytes, MC5R-A prevented the PAN-induced disruption of actin cytoskeleton integrity and apoptosis. MC5R-A treatment in PAN-injured podocytes also reinstated inhibitory phosphorylation and thus averted hyperactivity of GSK3β, a convergent point of multiple podocytopathic pathways. Collectively, pharmacologic activation of MC5R by using the highly selective small-molecule agonist is likely a promising therapeutic strategy to improve proteinuria and glomerular injury in protenuric nephropathies.

Rodent models to study sodium retention in experimental nephrotic syndrome.

Sodium retention and edema are hallmarks of nephrotic syndrome (NS). Different experimental rodent models have been established for simulating NS, however, not all of them feature sodium retention which requires proteinuria to exceed a certain threshold. In rats, puromycin aminonucleoside nephrosis (PAN) is a classic NS model introduced in 1955 that was adopted as doxorubicin-induced nephropathy (DIN) in 129S1/SvImJ mice. In recent years, mice with inducible podocin deletion (Nphs2Δipod ) or podocyte apoptosis (POD-ATTAC) have been developed. In these models, sodium retention is thought to be caused by activation of the epithelial sodium channel (ENaC) in the distal nephron through aberrantly filtered serine proteases or proteasuria. Strikingly, rodent NS models follow an identical chronological time course after the development of proteinuria featuring sodium retention within days and spontaneous reversal thereafter. In DIN and Nphs2Δipod mice, inhibition of ENaC by amiloride or urinary serine protease activity by aprotinin prevents sodium retention, opening up new and promising therapeutic approaches that could be translated into the treatment of nephrotic patients. However, the essential serine protease(s) responsible for ENaC activation is (are) still unknown. With the use of nephrotic rodent models, there is the possibility that this (these) will be identified in the future. This review summarizes the various rodent models used to study experimental nephrotic syndrome and the insights gained from these models with regard to the pathophysiology of sodium retention.

Delayed Treatment with a Novel Highly Selective Small-Molecule Agonist of MC5R Attenuates Podocyte Injury and Proteinuria in Puromycin Aminonucleoside Nephrosis

Delayed Treatment with a Novel Highly Selective Small-Molecule Agonist of MC5R Attenuates Podocyte Injury and Proteinuria in Puromycin Aminonucleoside Nephrosis

Urinary ERdj3 and mesencephalic astrocyte-derived neutrophic factor identify endoplasmic reticulum stress in glomerular disease

Podocyte injury and endoplasmic reticulum (ER) stress have been implicated in the pathogenesis of various glomerular diseases. ERdj3 (DNAJB11) and mesencephalic astrocyte-derived neurotrophic factor (MANF) are ER chaperones lacking the KDEL motif, and may be secreted extracellularly. Since podocytes reside in the urinary space, we examined if podocyte injury is associated with secretion of KDEL-free ER chaperones from these cells into the urine, and if chaperones in the urine reflect ER stress in glomerulonephritis. In cultured podocytes, ER stress increased ERdj3 and MANF intracellularly and in culture medium, whereas GRP94 (KDEL chaperone) increased only intracellularly. ERdj3 and MANF secretion was blocked by the secretory trafficking inhibitor, brefeldin A. Urinary ERdj3 and MANF increased in rats injected with tunicamycin (in the absence of proteinuria). After induction of passive Heymann nephritis (PHN) and puromycin aminonucleoside nephrosis (PAN), there was an increase in glomerular ER stress, and appearance of ERdj3 and MANF in the urine, coinciding with the onset of proteinuria. Rats with PHN were treated with the chemical chaperone, 4-phenyl butyrate (PBA), starting at the time of disease induction, or after disease was established. In both protocols, 4-PBA reduced proteinuria and urinary ER chaperone secretion, compared with PHN rats treated with saline (control). In conclusion, urinary ERdj3 and MANF reflect glomerular ER stress. 4-PBA protected against complement-mediated podocyte injury and the therapeutic response could be monitored by urinary ERdj3 and MANF.

Association of Proteinuria with Urinary Concentration Defect in Puromycin Aminonucleoside Nephrosis.

BackgroundPuromycin aminonucleoside (PA) can induce nephrotic syndrome in rats, and proteinuria is an important mediator of tubulointerstitial injury in glomerulopathy. We assumed that glomerular proteinuria may affect tubular function, such as urinary concentration, and investigated whether a urinary concentration defect is associated with proteinuria in puromycin aminonucleoside nephrosis (PAN). We also investigated the defect response to enalapril.MethodsGlomerular proteinuria was induced by a single intraperitoneal injection of PA (150mg/kg BW) in male Sprague-Dawley rats. In a half of these rats, enalapril (35mg/kg BW) was administered daily in a food mixture for two weeks. After the animal experiment, kidneys were harvested for immunoblot analysis and histopathologic examination.ResultsCompared with the control group, PA-treated rats had severe proteinuria, polyuria, and a lower urine osmolality. PA treatment induced remarkable tubulointerstitial injury and significant reductions in protein abundances of aquaporin-1 and Na-K-2Cl co-transporter type 2 (NKCC2). Proteinuria significantly correlated with osteopontin expression in the kidney and inversely correlated with renal expression of aquaporin-1, aquaporin-2, and NKCC2. The degree of tubulointerstitial injury significantly correlated with proteinuria, urine output, and osteopontin expression and inversely correlated with urine osmolality and renal expression of aquaporin-1, aquaporin-2, and NKCC2. No significant differences in parameters were found between PA-treated rats with and without enalapril.ConclusionIn PAN, glomerular proteinuria was associated with tubulointerstitial injury and water diuresis. Downregulation of aquaporin-1 and NKCC2 can impair countercurrent multiplication and cause a urinary concentration defect in PAN.

SUN-190 GFB-887, a small molecule inhibitor of TRPC5, protects against podocyte injury and attenuates proteinuria in models of FSGS

Focal segmental glomerulosclerosis (FSGS) is a rare podocytopathy with a high likelihood of progression to end stage renal disease. The Ca2+-permeable transient receptor potential canonical 5 (TRPC5) channel is a critical mediator of proteinuria in FSGS. Activation of the TRPC5 pathway in podocytes culminates in activation of Rac1, which is the primary driver of proteinuria in many forms of familial and sporadic FSGS. Inhibition of TRPC5 channel activity has been shown to protect against proteinuria and podocyte loss in AT1R transgenic and Dahl salt-sensitive rats. We performed high throughput screening of a structurally diverse compound collection followed by lead optimization for the development of several potent TRPC5 inhibitors. We characterized TRPC5 inhibitors in vitrousing a mouse podocyte cell line and podocyte injury models derived from human inducible pluripotent stem cells (hiPSC), and in two in vivomodels of FSGS, the unilaterally nephrectomized deoxycorticosterone acetate (DOCA)-salt rat model and the puromycin aminonucleoside nephrosis (PAN) rat model Here we report the identification of GFB-887, a potent, selective and orally bioavailable small molecule inhibitor of TRPC5. Potency and selectivity across TRP channels were determined using FLIPR based assays and electrophysiology. We show that TRPC5 inhibition protects against loss of stress fibers induced by protamine sulfate, a known activator of TRPC5, and restores stress fibers in podocytes after knockdown of synaptopodin. We further show that inhibition of TRPC5 suppresses pathogenic podocyte motility in a scratch assay. Finally, we demonstrate that inhibition of TRPC5 attenuates proteinuria in hypertension-induced FSGS in DOCA-salt rat model without altering blood pressure, and in non-hypertensive FSGS in the PAN rat model suggesting direct glomerular protection in vivo. We have identified GFB-887 as a new selective small molecule inhibitor of TRPC5 for the treatment of proteinuria in FSGS

TRPC6 Inactivation Does Not Protect Against Diabetic Kidney Disease in Streptozotocin (STZ)‐treated Sprague‐Dawley Rats

Glomerular TRPC6 channels are up‐regulated in rodent models of diabetes mellitus, but the role of TRPC6 in driving progression of experimental diabetic nephropathy is controversial. We recently used CRISPR/Cas9 methods to generate Sprague‐Dawley rats with a deletion in exon 2 of the Trpc6 gene that renders the channels non‐functional [1]. In addition, we observed that animals lacking functional TRPC6 were protected in the chronic puromycin aminonucleoside (PAN) model of acquired FSGS [1]. In the present study, 300‐gram adult Trpc6del/del rats and their wild‐type Trpc6wt/wt littermates were examined in the STZ model of type‐1 diabetes using protocols approved by the University of Houston IACUC committee. In our experiments, diabetes was induced by a single intraperitoneal injection of STZ, while controls received vehicle. Animals were not treated with insulin. STZ‐treated rats with severe hyperglycemia (>450 mg/dl) by 5 days after treatment were used for analysis and were monitored for the next 10 weeks. At 4 and 10 weeks after STZ treatment, compared to vehicle‐treated controls, STZ‐treated rats had severe hyperglycemia (745 ± 144 mg/dl, mean ± SD at 10 weeks). They also had elevated plasma triglycerides and total cholesterol, and failed to gain weight. STZ‐treated rats also exhibited markedly increased urine albumin excretion, increased blood urea nitrogen (BUN), and increased plasma creatinine and decreased creatinine clearance, indicating marked declines in renal function in diabetic rats. Two‐way ANOVA analyses of these datasets revealed no differences between Trpc6wt/wt and Trpc6del/del rats in any of these parameters. There was no time in the course of the experiment where we obtained functional data suggesting a protective effect of TRPC6 inactivation on blood glucose, or kidney function in STZ diabetes. Histological analysis carried out 10 weeks after STZ treatment revealed marked glomerular and tubulointerstitial disease in STZ‐treated rats. Mesangial expansion was the prominent feature in glomeruli of STZ‐treated animals, and glomeruli were less disrupted than we previously observed in the chronic PAN model [1]. Blind semi‐quantitative analysis of glomerular histology revealed a significant effect of STZ but no protective effect of TRPC6 inactivation on glomerular pathology. In summary, TRPC6 inactivation does not prevent declines in renal function and appears to have no protective effect in the STZ model of type‐1 diabetes in Sprague‐Dawley rats. This is in marked contrast to our previous work in chronic PAN nephrosis, where TRPC6 inactivation preserves renal function and histology.Support or Funding InformationSupported by NIH grant R01‐DK104708.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Morphological Processes of Foot Process Effacement in Puromycin Aminonucleoside Nephrosis Revealed by FIB/SEM Tomography.

Foot process effacement is one of the pathologic indicators of podocyte injury. However, the morphologic changes associated with it remain unclear. To clarify the developmental process, we analyzed puromycin nephrotic podocytes reconstructed from serial focused-ion beam/scanning electron microscopy (FIB/SEM) images. Intact podocytes consisted of four subcellular compartments: cell body, primary process, ridge-like prominence (RLP), and foot process. The RLP, a longitudinal protrusion from the basal surface of the cell body and primary process, served as an adhesive apparatus for the cell body and primary process to attach to the glomerular basement membrane. Foot processes protruded from both sides of the RLP. In puromycin nephrotic podocytes, foot process effacement occurred in two ways: by type-1 retraction, where the foot processes retracted while maintaining their rounded tips; or type-2 retraction, where they narrowed across their entire lengths, tapering toward the tips. Puromycin nephrotic podocytes also exhibited several alterations associated with foot process effacement, such as deformation of the cell body, retraction of RLPs, and cytoplasmic fragmentation. Finally, podocytes were reorganized into a broad, flattened shape. The three-dimensional reconstruction of podocytes by serial FIB/SEM images revealed the morphologic changes involved in foot process effacement in greater detail than previously described.

Expression of Cathepsin L and Its Intrinsic Inhibitors in Glomeruli of Rats With Puromycin Aminonucleoside Nephrosis.

Cathepsin L, a lysosomal cysteine proteinase, may have a key role in various biological and disease processes by intracellular and extracellular degradation of proteins. We examined the levels of cathepsin L and its intrinsic inhibitors in glomeruli of rats with puromycin aminonucleoside (PAN) nephrosis. In contrast to the weak levels of cathepsin L in normal glomeruli, on days 4 and 8, strong immunostaining was detected in almost all podocytes when proteinuria and pathological changes of the podocytes developed. Cathepsin L was reduced after day 28, but remained in a focal and segmental manner. Cystatin β, an intracellular inhibitor, was not detected in podocytes. However, cystatin C, an extracellular inhibitor, was detected in podocytes after day 4, coincident with cathepsin L. Cystatin C levels were gradually reduced but sustained in many podocytes on day 28, while cystatin C was not detected in podocytes sustained cathepsin L. These results demonstrated that cathepsin L levels are not always accompanied by the levels of its inhibitors in podocytes of PAN nephrosis, suggesting a potential role of cathepsin L in podocyte injury, which is a critical process for the development and progression of tuft adhesion and sclerosis.

In glomerular cells of puromycin aminonucleoside nephrosis rats both phosphorylated and total STAT3 levels increased during proteinuria

Recent studies showed that JAK/STAT pathway plays role in glomerular damages. The fact that STAT3 could be activated also by oxidative stress make Puromycin Aminonucleoside (PAN) Nephrosis model very appropriate for examination of STAT3 expression changes in glomerular pathology. Along with a control group, three PAN groups sacrificed on different days were formed by the i.p. injection of PAN for 5 consecutive days. Throughout the experiment, 24-hour-urines were collected on specific days and proteinuria levels were monitored. At the end of the experiments, tissue specimens were stained immunohistochemically for both total and phosphorylated STAT3 and evaluated subjectively. They were also examined ultrastructurally in transmission electron microscope. The proteinuria levels did not increase significantly on 5th day but showed a dramatic increase on 10th and 15th days. On 20th and 25th days, urinary protein levels gradually decreased. Ultrastructural examinations showed glomerular damages such as significant decrease in slit pore number, a significant gradual increase in glomerular basement membrane thickness and podocyte hypertrophy on 5th and 15th days; besides significant increase in mesangial matrix. The first significant increases in phosphorylated and total STAT3 levels occurred in 5th day and 15th day groups respectively. These increases diminished in 25th day group. Regarding all the findings, it was deduced that STAT3 is one of the active factors in glomerular pathologies.

Trpc6 inactivation confers protection in a model of severe nephrosis in rats

Mutations in canonical transient receptor potential-6 (TRPC6) channels give rise to rare familial forms of focal and segmental glomerulosclerosis (FSGS). Here we examined a possible role for TRPC6 in the progression of chronic puromycin aminonucleoside (PAN) nephrosis in Sprague-Dawley rats, a classic model of acquired nephrotic syndromes. We used CRISPR/Cas9 technology to delete a 239-bp region within exon 2 of the Trpc6 gene (Trpc6del allele). Trpc6del/del rats expressed detectable Trpc6 transcripts missing exon 2, and TRPC6 proteins could be detected by immunoblot of renal cortex. However, the abundance of Trpc6 transcripts and TRPC6 protein in renal cortex was much lower than in Trpc6wt/wt littermates, and functional TRPC6 channels could not be detected in whole-cell recordings from glomerular cells cultured from Trpc6del/del animals, possibly because of disruption of ankyrin repeats 1 and 2. During the chronic phase of PAN nephrosis, Trpc6del/del rats had reduced urine albumin excretion, reduced serum cholesterol and triglycerides, and improved azotemia compared to wild-type Trpc6wt/wt littermates. Glomerulosclerosis was severe during chronic PAN nephrosis in Trpc6wt/wt rats but was markedly reduced in Trpc6del/del littermates. Trpc6del/del animals also had less severe tubulointerstitial fibrosis as assessed by several biochemical and histological analyses, as well as reduced foot process effacement and glomerular basement thickening compared to Trpc6wtt/wt controls. None of the manipulations in this study affected the abundance of TRPC5 channels in renal cortex. TRPC3 was increased in PAN nephrosis and in Trpc6del/del rats. These data support a role for TRPC6 channels in driving an acquired form of secondary FSGS.Key messagesWe examined aminonucleoside nephrosis in rats with wild type and inactivated TRPC6.TRPC6 channels were inactivated by CRISPR/Cas9 editing of the Trpc6 gene.TRPC6 inactivation reduced albuminuria in the chronic but not the acute phase.TRPC6 inactivation reduced glomerulosclerosis and ultrastructural changes.TRPC6 inactivation also reduced interstitial changes and renal fibrosis.

CRISPR/Cas9 Deletion of a Portion of Exon 2 in the Trpc6 Gene Produces a Hypomorphic Variant that Confers Protection in the Chronic Puromycin Aminonucleoside Nephrosis Model in Sprague‐Dawley Rats

Mutations in TRPC6 channels give rise to rare familial forms of focal and segmental glomerulosclerosis (FSGS). Here we examined the role of TRPC6 channels in the chronic puromycin aminonucleoside (PAN) model of acquired FSGS in Sprague‐Dawley rats, using protocols approved by the University of Houston IACUC. In our disease model, animals received two i.p. injections of PAN at 30 day intervals, a protocol that has long been known to induce albuminuria and FSGS lesions in this species. To test our central hypothesis, we used CRISPR/Cas9 methods to delete a 239‐bp fragment in Exon 2 of Trpc6, which resulted in a frame shift that we expected to introduce numerous premature stop codons downstream of the deletion. However as a result of a recently described process known as exon skipping, short Trpc6 transcripts missing Exon 2 were detected by RT‐PCR in Trpc6del/del rats, albeit at considerably lower levels in renal cortex compared to the normal full‐length transcripts seen in Trpc6wt/wt controls. The Exon 2 deletion encodes a portion of an ankyrin‐repeat domain in the N‐terminal of the channel subunit. The function of that domain in the channel protein is not known. Using two different antibodies directed at different motifs downstream of the deletion, we detected very low levels of TRPC6 protein in immunoblots of the renal cortex of Trpc6del/del rats, whereas signal was robust in Trpc6wt/wt controls with both antibodies. In addition, we did not observe ATP‐activated cationic currents mediated by TRPC6 in whole‐cell recordings from glomerular cells resembling podocytes cultured from Trpc6del/del rats, but these currents were readily seen in Trpc6wt/wt controls. Kidney disease in chronic PAN nephrosis (measured 60 days after the first PAN injection) was markedly decreased in Trpc6del/del rats compared to Trpc6wt/wt littermates. This conclusion is based on reduced 24‐hr albumin excretion, reduced serum cholesterol and triglycerides, and improved blood urea nitrogen in Trpc6del/del rats. Trpc6del/del rats also had reduced glomerulosclerosis, less severe tubulointerstitial disease based on biochemical and histological measures, and reduced foot process effacement and glomerular basement thickening in transmission EM compared to Trpc6wt/wt rats. Protection in Trpc6del/del rats was robust but not complete. Thus, most of the quantifiable parameters of disease severity were reduced by about 50% in Trpc6del/del rats compared to wild‐type littermates. We did not discern any difference between renal function or glomerular structure in saline‐treated Trpc6del/del and Trpc6wt/wt rats. We also observed that glomerular TRPC3 abundance in renal cortex was increased in Trpc6del/del rats, although TRPC3 abundance did not increase further in chronic PAN nephrosis. None of the manipulations in this study affected the abundance of TRPC5 channels in renal cortex. These results support TRPC6 family channels as therapeutic targets for acquired forms of FSGS.Support or Funding InformationSupported by NIH grant RO1‐DK104708This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Renoprotective effect of topiroxostat via antioxidant activity in puromycin aminonucleoside nephrosis rats.

Topiroxostat is a novel inhibitor of xanthine oxidase, and is postulated to exert a renoprotective effect. Puromycin aminonucleoside nephrosis (PAN) is a rat model of minimal change nephrotic syndrome. In this study, we examined whether topiroxostat ameliorates the kidney injury in PAN rats that was induced by a single intraperitoneal injection of PA (100 mg/kg body weight). Rats were divided into four groups: control rats, PAN rats, control rats treated with topiroxostat (1.0 mg/kg/day), and PAN rats treated with topiroxostat. Topiroxostat significantly reduced the amount of uric acid in the kidney cortex, while serum UA concentration remained unaffected by this treatment. Urinary protein excretion decreased significantly on day 10 in PAN rats upon topiroxostat treatment. Podocyte injury in PAN rats, as indicated by the reduction in WT‐1‐positive cell numbers and podocin immunoreactivity and foot process effacement, was partially yet significantly alleviated with topiroxostat treatment. In the kidney cortex, the increase in oxidative stress markers such as nitrotyrosine and 8‐hydroxy‐2‐deoxyguanosine (8‐OHdG) and the enhanced expressions of xanthine oxidase and NADPH oxidase 4 (NOX4) in PAN rats were significantly ameliorated by topiroxostat. Using cultured podocytes NOX4 expression was upregulated by adding 12 mg/dL UA into the culture medium. These results suggest that topiroxostat ameliorates proteinuria and kidney injury in PAN rats by lowering oxidative stress and tissue UA concentration. The renoprotective effects of topiroxostat could be attributed to its potential to inhibit xanthine oxidase and NOX4 in concert with suppression of intracellular UA production.

Adrenomedullin ameliorates podocyte injury induced by puromycin aminonucleoside in vitro and in vivo through modulation of Rho GTPases.

Podocyte injury is a key event in proteinuric kidney disease and eventually glomerular scarring. While adrenomedullin (AM), a potent vasodilatory peptide, has been reported to confer renoprotection in several experimental models of kidney diseases, its effect on injured podocytes and the related mechanism is still largely unknown. Employing Western blotting analysis, immunoprecipitation and immunofluorescence, we investigated the effects of AM on the expressions of podocyte cytoskeletal proteins and Rho-family small GTPases (Rho GTPases) in puromycin aminonucleoside (PAN)-induced podocyte injury, both in cultured podocytes and in PAN nephrosis rats. Urinary protein excretion and the morphologic changes of kidney in PAN nephrosis rats were evaluated. Glutathione-S-transferase pull-down assay was applied for Rho GTPases activity. PAN induced massive albuminuria and morphologic injury, which were significantly mitigated by AM administration. AM significantly antagonized not only the PAN-decreased expressions of synaptopodin, nephrin, CD2AP and podocin, but also the PAN-disrupted interactions between synaptopodin-RhoA, nephrin-CD2AP, and CD2AP-Rac1-cortactin. These effects of AM in cultured podocytes were mostly significantly blocked by H89, a PKA inhibitor. AM dramatically upregulated the PAN-induced Rho GTPases protein expressions and their activities. PAN increased the expressions of endogenous AM and its receptor RAMP2 which was furthermore upregulated by AM administration. AM alleviated podocyte injury induced by PAN both in cell culture and in PAN nephrosis. The beneficial effects of AM on podocytes can be attributable to direct modulation of podocyte cytoskeletal proteins and Rho GTPases, mainly via a PKA-dependent pathway.

Antioxidative Stress Effects of Salvia przewalskii Extract in Experimentally Injured Podocytes

Background: Oxidative stress is a leading cause of puromycin aminonucleoside (PAN)-induced nephrosis. As the inhibition of oxidative stress may improve injury of podocyte, we aimed at examining the effect of total phenolic acid extract of Salvia przewalskii (SPE) on PAN-induced oxidative stress in vivo and in vitro. Methods: Seventy-two male Sprague-Dawley rats were randomly assigned into 6 groups (n = 12), PAN alone, tacrolimus (TAC), SPE (50, 100 and 200 mg/kg) and normal control group. Salvianolic acid B (SalB, 5.52%) and rosmarinic acid (RA, 31.58%) were isolated from SPE. The intensities of 8-oxo-2'-deoxyguanosine (8-OHdG) were evaluated by immunofluorescence. In vitro, the podocytes were assigned into groups of control, PAN alone, TAC (1 μg/ml), SPE (158, 316 μg/ml), SalB (8.5, 17 μg/ml) and RA (25, 50 μg/ml). The intracellular reactive oxygen species (ROS) production and cell apoptosis rate were measured by flow cytometry. Form factor and aspect ratio were calculated to assess mitochondrial morphology. Results: In vivo, PAN increased the intensity of 8-OHdG in the renal tissue in the PAN group (p < 0.05). The high-dose SPE reduced 8-OHdG significantly at levels comparable to TAC alone (p > 0.05) on day 15. The intracellular ROS production, podocytes apoptosis rate and mitochondrial fragmentation increased significantly following PAN exposure in podocytes (p < 0.05). Treatment with high-dose SalB significantly ameliorated the increase in the expression of ROS and revised the structure of mitochondria. The percentage of apoptotic cells was decreased compared with the PAN group after SPE, SalB, RA, and TAC treatment for 24 h (p < 0.05). Conclusion: These findings suggest that high-dose SPE significantly attenuated 8-OHdG in PAN nephrosis. Antioxidative stress effects of high-dose SPE, SalB against PAN-stimulated cultured podocyte via mechanisms include suppression of ROS expression and mitochondria fission. In addition, SPE, SalB and RA can suppress PAN-induced apoptosis.

Inducible Expression of Claudin-1 in Glomerular Podocytes Generates Aberrant Tight Junctions and Proteinuria through Slit Diaphragm Destabilization.

The tight junction (TJ) has a key role in regulating paracellular permeability to water and solutes in the kidney. However, the functional role of the TJ in the glomerular podocyte is unclear. In diabetic nephropathy, the gene expression of claudins, in particular claudin-1, is markedly upregulated in the podocyte, accompanied by a tighter filtration slit and the appearance of TJ-like structures between the foot processes. However, there is no definitive evidence to show slit diaphragm (SD) to TJ transition in vivo Here, we report the generation of a claudin-1 transgenic mouse model with doxycycline-inducible transgene expression specifically in the glomerular podocyte. We found that induction of claudin-1 gene expression in mature podocytes caused profound proteinuria, and with deep-etching freeze-fracture electron microscopy, we resolved the ultrastructural change in the claudin-1-induced SD-TJ transition. Notably, immunolabeling of kidney proteins revealed that claudin-1 induction destabilized the SD protein complex in podocytes, with significantly reduced expression and altered localization of nephrin and podocin proteins. Mechanistically, claudin-1 interacted with both nephrin and podocin through cis- and trans-associations in cultured cells. Furthermore, the rat puromycin aminonucleoside nephrosis model, previously suspected of undergoing SD-TJ transition, exhibited upregulated expression levels of claudin-1 mRNA and protein in podocytes. Together, our data attest to the novel concept that claudins and the TJ have essential roles in podocyte pathophysiology and that claudin interactions with SD components may facilitate SD-TJ transition that appears to be common to many nephrotic conditions.

Effect of clofibrate on fatty acid metabolism in the kidney of puromycin-induced nephrotic rats.

Proteinuria plays an essential role in the progression of tubulointerstitial damage, which causes end-stage renal disease. An increased load of fatty acids bound to albumin reabsorbed into proximal tubular epithelial cells (PTECs) contributes to tubulointerstitial damage. Fibrates, agonists of peroxisome proliferator-activated receptor α (PPARα), have renoprotective effects against proteinuria whereas the effects of these compounds on fatty acid metabolism in the kidney are still unknown. Therefore, the present study examined whether the renoprotective effects of clofibrate were associated with improvement of fatty acid metabolism in puromycin aminonucleoside (PAN)-induced nephrotic rats. Rats were allocated to the control, PAN or clofibrate-treated PAN group. Biochemical parameters, renal injury and changes in fatty acid metabolism were studied on day14. PAN increased proteinuria, lipid accumulation in PTECs, excretions of N-acetyl-β-D-glucosaminidase (NAG) and 8-hydroxydeoxyguanosine (8OHdG) and the area of caspase 3-positive tubular cells. It decreased renal expressions of medium-chain acyl-CoA dehydrogenase (MCAD), cytochrome P450 (CYP)4A, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and estrogen-related receptor α (ERRα) without change of the expression of PPARα. Clofibrate reduced proteinuria, lipid accumulation, NAG excretion and the area of caspase 3-positive tubular cells. However, albumin excretion was not reduced and 8OHdG excretion was increased. Clofibrate minimized changes in MCAD, CYP4A, PGC-1α and ERRα expressions with increased PPARα, very long-chain acyl-CoA dehydrogenase (VLCAD) and long-chain acyl-CoA dehydrogenase (LCAD) expressions. Clofibrate is protective against renal lipotoxicity in PAN nephrosis. This study indicates that clofibrate has renoprotective effects through maintaining fatty acid metabolism in the kidney of PAN-induced nephrotic rats.

Semiautomated quantitative image analysis of glomerular immunohistochemistry markers desmin, vimentin, podocin, synaptopodin and WT-1 in acute and chronic rat kidney disease models.

Five different glomerular immunohistochemistry markers were evaluated and compared in four different acute and chronic rat kidney disease models. Progression of glomerular or podocyte damage was shown in the puromycin aminonucleoside nephrosis (PAN) and Zucker fatty/spontaneously hypertensive heart failure F1 hybrid (ZSF1) rat model. Progression and prevention of glomerular damage was demonstrated in the Zucker diabetic fatty (ZDF) and Dahl salt-sensitive (Dahl SS) rat. Immunohistochemistry was performed for desmin, vimentin, podocin, synaptopodin and Wilms tumor protein-1 (WT-1), and evaluation of glomerular immunohistochemistry markers was done by semiautomated quantitative image analysis. We found desmin and WT-1 as the most sensitive markers for podocyte damage in both acute and chronic glomerular damage followed by vimentin, podocin and synaptopodin. We were able to demonstrate that early podocyte damage as shown by increased desmin and vimentin staining together with either a phenotypic podocyte change or podocyte loss (reduced numbers of WT-1-stained podocytes) drives the progression of glomerular damage. This is followed by a reduction in podocyte-specific proteins such as podocin and synaptopodin. Our report describes the different sensitivity of glomerular or podocyte markers and gives future guidance for the selection of the most sensitive markers for efficacy testing of new drugs as well as for the selection of tissue-based toxicity markers for glomerular or podocyte injury. In addition to functional clinical chemistry markers, desmin and WT-1 immunohistochemistry offers reliable and valuable data on the morphologic state of podocytes.