Puromycin Aminonucleoside Nephropathy Research Articles

Renal protective effects of helix B surface polypeptide in rats with puromycin aminonucleoside nephropathy

Background Recent studies have reported that helix B surface polypeptide (HBSP), an erythropoietin derivative, exhibits strong tissue protective effects, independent of erythropoietic effects, in a renal ischemia-reperfusion (IR) injury model. Meanwhile, the transforming growth factor-β (TGF-β) superfamily member glial cell line-derived neurotrophic factor (GDNF) demonstrated protective effect on podocytes in vitro. Using a rat puromycin aminonucleoside nephropathy (PAN) model, this study observed the renal protective effect of HBSP and investigated its renal protective effect on podocytes and mechanism related to GDNF. Methods Rats nephropathy model was induced by injection of 60 mg/kg of PAN via the tail vein. Rats in the PAN + HBSP group were injected intraperitoneally with HBSP (8 nmol/kg) 4 h before the model was induced, followed by intraperitoneal injections of HBSP once every 24 h for 7 consecutive days. The 24-hour urinary protein level was measured once every other day, and blood and renal tissue samples were collected on the 7th day for the examination of renal function, complete blood count, renal pathological changes and the expression levels of GDNF. Results Compared with the control group, the PAN nephropathy rat model showed a large amount of urinary protein. The pathological manifestations were mainly extensive fusion and disappearance of foot processes, along with vacuolar degeneration of podocytes and their separation from the glomerular basement membrane. GDNF expression was upregulated. Compared with the PAN + vehicle group, the PAN + HBSP group showed decreased urinary protein (p < 0.05). Pathological examination revealed ameliorated glomerular injury and vacuolar degeneration of podocytes. The expression of GDNF in the PAN nephropathy group was increased, when compared with the control group. The greatest expression of GDNF observed in the PAN + HBSP group (p < 0.05). Conclusions The expression of GDNF in the kidney of PAN rat model was increased. HBSP reduced urinary protein, ameliorated pathological changes in renal podocytes, increased the expression of GDNF in the PAN rat model. HBSP is likely to exert its protective effects on podocytes through upregulation of GDNF expression.

Study on biomarkers of acteoside in treating puromycin aminonucleoside nephropathy in young rats based on non-targeted urine metabolomics technology

This study aims to reveal the endogenous metabolic characteristics of acteoside in the young rat model of purinomycin aminonucleoside nephropathy(PAN) by non-targeted urine metabolomics and decipher the potential mechanism of action. Biochemical indicators in the urine of rats from each group were determined by an automatic biochemical analyzer. The potential biomarkers and related core metabolic pathways were identified by ultra-high performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry(UHPLC-LTQ-Orbitrap MS) combined with principal component analysis(PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA). MetaboAnalyst 5.0 was used to establish the receiver operating characteristic(ROC) curve for evaluating the clinical diagnostic performance of core metabolites. The results showed that acteoside significantly decreased urinary protein-to-creatinine ratio in PAN young rats. A total of 17 differential metabolites were screened out by non-targeted urine metabolomics in PAN young rats and they were involved in phenylalanine metabolism and phenylalanine, tyrosine and tryptophan biosynthesis. Thirtten differential metabolites were screened by acteoside intervention in PAN young rats, and they were involved in phenylalanine metabolism and arginine and proline metabolism. Among them, leucylproline and acetophenone were the differential metabolites that were significantly recovered after acteoside treatment. These pathways suggest that acteoside treats PAN in young rats by regulating amino acid metabolism. The area under the curve of two core biomarkers, leucylproline and acetophenone, were both greater than 0.9. In summary, acteoside may restore amino acid metabolism by regulating endogenous differential metabolites in PAN young rats, which will help to clarify the mechanism of acteoside in treating chronic glomerulonephritis in children. The characteristic biomarkers screened out have a high diagnostic value for evaluating the treatment of chronic glomerulonephritis in children with acteoside.

#5420 A MODULAR INTRACELLULAR HIERARCHICAL-RESPONSIVE NANOCARRIER ENABLES DUAL TARGETING FOR HIGH THERAPEUTIC EFFICACY IN KIDNEY DISEASE

Abstract Background and Aims Single-loaded nanocarrier that can improve targeting capability and drug toxicity compared with free drugs demonstrates a promising strategy for disease treatment. Due to the heterogeneity of the injured cells during the disease progression, dose-induced drug resistance, and instability of nanocarriers in the blood circulation, it is challenging to achieve dual targeting to different injured cell subsets as well as reducing drug dosage but obtaining efficient treatment. In addition, developing nanocarriers is a complex and time-consuming process; there is a desire to develop a universal nanocarrier for various diseases, especially complex ones. Here, we have successfully designed a modular nanocarrier capable of co-delivery drugs. Meanwhile, the effective dosage was reduced to one-tenth of single-loaded nanocarriers. Method The dual-loaded drug-targeting nanoparticles consist of four main modules, including a chemical material, an antibody, and two drugs. First, we constructed a mesoscale spherical nanoparticle with a particle size of 400 nm from PLGA and wrapped drug A inside it. Next, the foot cell-specific antibody Nephrin is used as the target material and modified on the nanoparticle surface by the ph-sensitive short peptide. Finally, drug B is coupled to the antibody to obtain the final dual-targeting dual-drug nanoparticles. The dual-targeting dual-drug nanoparticles were injected into mice via the tail vein to evaluate their toxicity to organs and the immune system and compared with free drug. We explored the targeting and mechanisms of dual-targeting nanoparticles by organ imaging, Immunofluorescence, and other experimental methods. In mice, we established the model of acute kidney injury, puromycin aminonucleoside nephropathy (PAN), and ccRCC to compare the protective effects and mechanisms of dual targeting nanoparticles in different concentrations. Results Dual targeting nanoparticles loaded with different drugs demonstrated the ability of dual targeting of glomeruli and tubules. In treating acute kidney injury, the nanocarriers were encapsulated with rapamycin and dexamethasone acetate; for puromycin nephropathy, the drugs were changed to rapamycin and captopril; and in the treatment of kidney cancer models, gefitinib and glutathione were chosen as targeted antitumor drugs. For different diseases, the lesions were treated significantly with different drug combinations. We observed a reduction of tubular injury in the acute kidney injury model, a recovery of the foot process of podocytes in the puromycin nephropathy model, and a reduction of cancer foci in the renal cancer model. These results show that dual drug-loaded nanomaterials have an excellent ability to cope with complex situations. Conclusion Dual drug delivery system can flexibly adapt the treatment of various diseases by changing particle size, surface antibodies, and drugs.

Vitamin D Receptor/Vitamin D Response Element Directly Modulate Nestin Transcription to Ameliorate PAN-Induced Podocyte Morphological Changes

Background: The change of podocyte morphology is a pathologic feature of chronic kidney disease. Several studies have suggested that vitamin D plays a role in the protection of podocytes, but the underlying mechanism remains unclear. Methods: The effects of paricalcitol on podocyte injury were tested in a puromycin aminonucleoside (PAN)-induced rat model and cultured mouse podocytes. Proteinuria, podocyte foot process (FP) effacement, and the expression of nestin and vitamin D receptor (VDR) were evaluated. VDR-siRNA or plasmids containing VDR-shRNA were transfected into podocytes to silence VDR expression. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays were performed to verify the connection between VDR and nestin gene expression. Results: Paricalcitol significantly alleviated proteinuria and podocyte FP effacement in PAN-induced nephrosis, which was accompanied by increased VDR expression in the glomeruli. Paricalcitol also inhibited PAN-induced nestin overexpression in the glomeruli. In an in vivo study, PAN significantly inhibited VDR protein expression, stimulated nestin protein expression, and resulted in nestin filament derangement in mouse podocytes, while paricalcitol treatment abolished these effects. In contrast, downregulation of VDR resulted in derangement and overexpression of nestin. ChIP assays demonstrated the presence of a vitamin D response element (VDRE) in the nestin promoter, and paricalcitol enhanced the binding of VDR to VDRE. Furthermore, luciferase reporter assays of the nestin promoter fragment showed that paricalcitol effectively repressed nestin reporter gene expression after PAN treatment, and mutation of VDRE abolished this effect. Conclusions: Paricalcitol directly regulates nestin transcription through the interaction of VDR/VDRE, thereby preventing morphological changes of podocytes in PAN nephropathy.

Synbindin Downregulation Participates in Slit Diaphragm Dysfunction

Introduction: Synbindin, originally identified as a neuronal cytoplasmic molecule, was found in glomeruli. The cDNA subtractive hybridization technique showed the mRNA expression of synbindin in glomeruli was downregulated in puromycin aminonucleoside (PAN) nephropathy, a mimic of minimal-change nephrotic syndrome. Methods: The expression of synbindin in podocytes was analyzed in normal rats and 2 types of rat nephrotic models, anti-nephrin antibody-induced nephropathy, a pure slit diaphragm injury model, and PAN nephropathy, by immunohistochemical analysis and RT-PCR techniques. To elucidate the function of synbindin, a gene silencing study with human cultured podocytes was performed. Results: Synbindin was mainly expressed at the slit diaphragm area of glomerular epithelial cells (podocytes). In both nephrotic models, decreased mRNA expression and the altered staining of synbindin were already detected at the early phase when proteinuria and the altered staining of nephrin, a key molecule of slit diaphragm, were not detected yet. Synbindin staining was clearly reduced when severe proteinuria was observed. When the cultured podocytes were treated with siRNA for synbindin, the cell changed to a round shape, and filamentous actin structure was clearly altered. The expression of ephrin-B1, a transmembrane protein at slit diaphragm, was clearly lowered, and synaptic vesicle-associated protein 2B (SV2B) was upregulated in the synbindin knockdown cells. Conclusion: Synbindin participates in maintaining foot processes and slit diaphragm as a downstream molecule of SV2B-mediated vesicle transport. Synbindin downregulation participates in slit diaphragm dysfunction. Synbindin can be an early marker to detect podocyte injury.

Plasminogenuria is associated with podocyte injury, edema, and kidney dysfunction in incident glomerular disease.

Urinary plasminogen/plasmin, or plasmin (ogen) uria, has been demonstrated in proteinuric patients and exposure of cultured podocytes to plasminogen results in injury via oxidative stress pathways. A causative role for plasmin (ogen) as a "second hit" in kidney disease progression has yet to have been demonstrated in vivo. Additionally, association between plasmin (ogen) uria and kidney function in glomerular diseases remains unclear. We performed comparative studies in a puromycin aminonucleoside (PAN) nephropathy rat model treated with amiloride, an inhibitor of plasminogen activation, and measured changes in plasmin (ogen) uria. In a glomerular disease biorepository cohort (n=128), we measured time-of-biopsy albuminuria, proteinuria, and plasmin (ogen) uria for correlations with kidney outcomes. In cultured human podocytes, plasminogen treatment was associated with decreased focal adhesion marker expression with rescue by amiloride. Increased glomerular plasmin (ogen) was found in PAN rats and focal segmental glomerulosclerosis (FSGS) patients. PAN nephropathy was associated with increases in plasmin (ogen) uria and proteinuria. Amiloride was protective against PAN-induced glomerular injury, reducing CD36 scavenger receptor expression and oxidative stress. In patients, we found associations between plasmin (ogen) uria and edema status as well as eGFR. Our study demonstrates a role for plasmin (ogen)-induced podocyte injury in the PAN nephropathy model, with amiloride having podocyte-protective properties. In one of the largest glomerular disease cohorts to study plasminogen, we validated previous findings while suggesting a potentially novel relationship between plasmin (ogen) uria and estimated glomerular filtration rate (eGFR). Together, these findings suggest a role for plasmin (ogen) in mediating glomerular injury and as a viable targetable biomarker for podocyte-sparing treatments.

Estrogen-related receptor-α mediates puromycin aminonucleoside-induced mesangial cell apoptosis and inflammatory injury.

Glomerular diseases are the leading cause of chronic kidney disease, and mesangial cells (MCs) have been demonstrated to be involved in the pathogenesis. Puromycin aminonucleoside (PAN) is a nephrotoxic drug that induces glomerular injury with elusive mechanisms. The present study was undertaken to investigate the role of PAN in MC apoptosis, as well as the underlying mechanism. Here we found that PAN induced MC apoptosis accompanied by declined cell viability and enhanced inflammatory response. The apoptosis was further evidenced by increments of apoptosis regulator BAX (BAX) and caspase-3 expression. In line with the apoptotic response in MCs following PAN treatment, we also found a remarkable induction of estrogen-related receptor-α (ERRα), an orphan nuclear receptor, at both mRNA and protein levels. Interestingly, ERRα silencing by an siRNA approach resulted in an attenuation of the apoptosis and inflammatory response caused by PAN. More importantly, overexpression of ERRα in MCs significantly triggered MC apoptosis in line with increased BAX and caspase-3 expression. In PAN-treated MCs, ERRα overexpression further aggravated PAN-induced apoptosis. In agreement with the in vitro study, we also observed increased ERRα expression in line with enhanced apoptotic response in renal cortex from PAN-treated rats. These data suggest a detrimental effect of ERRα on PAN-induced MC apoptosis and inflammatory response, which could help us to better understand the pathogenic mechanism of MC injury in PAN nephropathy.

Pharmacological and genetic inhibition of downstream targets of p38 MAPK in experimental nephrotic syndrome.

Nie X, Chanley MA, Pengal R, Thomas DB, Agrawal S, Smoyer WE. Pharmacological and genetic inhibition of downstream targets of p38 MAPK in experimental nephrotic syndrome. Am J Physiol Renal Physiol 314: F602-F613, 2018. First published November 29, 2017; doi: 10.1152/ajprenal.00207.2017 .-The p38 MAPK pathway plays a crucial role in various glomerulopathies, with activation being associated with disease and inhibition being associated with disease amelioration. We hypothesized that the downstream targets of p38 MAPK, MAPK-activated protein kinase 2 and/or 3 (MK2 and/or MK3), play an important role in mediating injury in experimental nephrotic syndrome via their actions on their downstream substrates heat shock protein B1 (HSPB1) and cyclooxygenase-2 (COX-2). To test this hypothesis, the effects of both pharmacological and genetic inhibition of MK2 and MK3 were examined in mouse adriamycin (ADR) and rat puromycin aminonucleoside (PAN) nephropathy models. MK2-/-, MK3-/-, and MK2-/-MK3-/- mice were generated in the Sv129 background and subjected to ADR-induced nephropathy. MK2 and MK3 protein expression was completely abrogated in the respective knockout genotypes, and massive proteinuria and renal histopathological changes developed after ADR treatment. Furthermore, renal cortical HSPB1 was induced in all four genotypes by day 21, but HSPB1 was activated only in the wild-type and MK3-/- mice. Expression of the stress proteins HSPB8 and glucose-regulated protein 78(GRP78) remained unaltered across all genotypes. Finally, while MK2 and/or MK3-knockout downregulated the proinflammatory enzyme COX-2, ADR significantly induced renal cortical COX-2 only in MK2-/- mice. Additionally, pharmacological MK2 inhibition with PF-318 during PAN-induced nephropathy did not result in significant proteinuria reduction in rats. Together, these data suggest that while the inhibition of MK2 and/or MK3 regulates the renal stress response, our currently available approaches are not yet able to safely and effectively reduce proteinuria in experimental nephrotic syndrome and that other p38MAPK downstream targets should also be considered to improve the future treatment of glomerular disease.

Original Research: Potential of urinary nephrin as a biomarker reflecting podocyte dysfunction in various kidney disease models.

Urinary nephrin is a potential non-invasive biomarker of disease. To date, however, most studies of urinary nephrin have been conducted in animal models of diabetic nephropathy, and correlations between urinary nephrin-to-creatinine ratio and other parameters have yet to be evaluated in animal models or patients of kidney disease with podocyte dysfunction. We hypothesized that urinary nephrin-to-creatinine ratio can be up-regulated and is negatively correlated with renal nephrin mRNA levels in animal models of kidney disease, and that increased urinary nephrin-to-creatinine ratio levels are attenuated following administration of glucocorticoids. In the present study, renal nephrin mRNA, urinary nephrin-to-creatinine ratio, urinary protein-to-creatinine ratio, and creatinine clearance ratio were measured in animal models of adriamycin nephropathy, puromycin aminonucleoside nephropathy, anti-glomerular basement membrane glomerulonephritis, and 5/6 nephrectomy. The effects of prednisolone on urinary nephrin-to-creatinine ratio and other parameters in puromycin aminonucleoside (single injection) nephropathy rats were also investigated. In all models tested, urinary nephrin-to-creatinine ratio and urinary protein-to-creatinine ratio increased, while renal nephrin mRNA and creatinine clearance ratio decreased. Urinary nephrin-to-creatinine ratio exhibited a significant negative correlation with renal nephrin mRNA in almost all models, as well as a significant positive correlation with urinary protein-to-creatinine ratio and a significant negative correlation with creatinine clearance ratio. Urinary protein-to-creatinine ratio exhibited a significant negative correlation with renal nephrin mRNA. Following the administration of prednisolone to puromycin aminonucleoside (single injection) nephropathy rats, urinary nephrin-to-creatinine ratio was significantly suppressed and exhibited a significant positive correlation with urinary protein-to-creatinine ratio. In addition, the decrease in number of glomerular Wilms tumor antigen-1-positive cells was attenuated, and urinary nephrin-to-creatinine ratio exhibited a significant negative correlation in these cells. In conclusion, these results suggest that urinary nephrin-to-creatinine ratio level is a useful and reliable biomarker for predicting the amelioration of podocyte dysfunction by candidate drugs in various kidney disease models with podocyte dysfunction. This suggestion will also be validated in a clinical setting in future studies.

A vital role for myosin-9 in puromycin aminonucleoside-induced podocyte injury by affecting actin cytoskeleton

ABSTRACTPodocyte injury is an early pathological change of many kidney diseases. In particular, the actin cytoskeleton plays an important role in maintaining the normal function of podocytes. Disruption of the actin cytoskeleton is a feature of podocyte injury in proteinuric nephropathies. Recent studies showed that myosin-9 was localized in the podocyte foot processes and was necessary in maintaining podocyte structural homeostasis. However, it is unclear whether myosin-9 maintains podocyte structure by affecting actin cytoskleton. Here, the role of myosin-9 in puromycin aminonucleoside (PAN)-induced podocyte injury was explored both in vitro and in vivo. In cultured mouse podocytes (MPC5), it was determined that PAN downregulated myosin-9 expression, disrupted the actin cytoskeleton and reduced the adhesion ability. Reduced myosin-9 expression by siRNA precipitated podocyte cytoskeletal damage and accelerated PAN-induced podocyte detachment. Overexpression of myosin-9 protected against PAN-induced podocyte detachment. Furthermore, administration of an antioxidant Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP) inhibited PAN-induced podocyte cytoskeletal damage and podocyte detachment by restoring the expression of myosin-9. In the rat PAN nephropathy model, MnTBAP could also attenuate PAN-induced reduction of myosin-9 and podocyte loss. Taken together, these findings pinpointed that oxidative stress contributed to PAN-induced podocyte injury through the repression of a cytoskeletal protein myosin-9, which provided novel insights into a potential target for the treatment of podocyte injury-associated glomerulopathies.

Immunohistochemical Analyses of Serum Proteins in Puromycin Aminonucleoside Nephropathy of Living Rat Kidneys by Cryotechniques

Immunohistochemical Analyses of Serum Proteins in Puromycin Aminonucleoside Nephropathy of Living Rat Kidneys by Cryotechniques

Alteration in the podoplanin-ezrin-cytoskeleton linkage is an important initiation event of the podocyte injury in puromycin aminonucleoside nephropathy, a mimic of minimal change nephrotic syndrome.

Podoplanin was identified as a protein associated with the transformation of arborized foot processes of glomerular epithelial cells (podocytes) to flat feet. However, the function of podoplanin in the podocyte is not yet fully clarified. In this study, we analyzed the molecular nature of podoplanin, and its expression in rat nephrotic models and patients with minimal change nephrotic syndrome (MCNS). We demonstrated here that podoplanin has two forms: one contains abundant sialic acid and the other a lesser amount of sialic acid. Podoplanin bound ezrin to interact with the cytoskeleton. The silencing of podoplanin in cultured podocytes caused a change in the cell shape and the distribution of ezrin and actin. The expression of podoplanin was clearly reduced before the onset of proteinuria in puromycin aminonucleoside (PAN) nephropathy, a mimic of MCNS, and the decrease in the expression of podoplanin became more evident at the proteinuric stage. Podoplanin was detected in normal urine samples, and the amount of urinary podoplanin markedly increased on day 1 of PAN nephropathy. Urinary ezrin was also detected. The amount of the phosphorylated ezrin was reduced, while the amount of the podoplanin-interacting ezrin increased. The podoplanin expression was reduced in a patient with active-phase MCNS. It is conceivable that the alteration of the podoplanin-ezrin-cytoskeleton linkage is an important event of the podocyte injury in MCNS.

Quantitative analysis of markers of podocyte injury in the rat puromycin aminonucleoside nephropathy model

Podocytes are an essential component of the renal glomerular filtration barrier, their injury playing an early and important role in progressive renal dysfunction. This makes quantification of podocyte marker immunoreactivity important for early detection of glomerular histopathological changes. Here we have specifically applied a state-of-the-art automated computational method of glomerulus recognition, which we have recently developed, to study quantitatively podocyte markers in a model with selective podocyte injury, namely the rat puromycin aminonucleoside (PAN) nephropathy model. We also retrospectively investigated mRNA expression levels of these markers in glomeruli which were isolated from the same formalin-fixed, paraffin-embedded kidney samples by laser microdissection. Among the examined podocyte markers, the immunopositive area and mRNA expression level of both podoplanin and synaptopodin were decreased in PAN glomeruli. The immunopositive area of podocin showed a slight decrease in PAN glomeruli, while its mRNA level showed no change. We have also identified a novel podocyte injury marker β-enolase, which was increased exclusively by podocytes in PAN glomeruli, similarly to another widely used marker, desmin. Thus, we have shown the specific application of a state-of-the-art computational method and retrospective mRNA expression analysis to quantitatively study the changes of various podocyte markers. The proposed methods will open new avenues for quantitative elucidation of renal glomerular histopathology.

Angiotensin II type 1 receptor blockade ameliorates proteinuria in puromycin aminonucleoside nephropathy by inhibiting the reduction of NEPH1 and nephrin.

The precise pathogenic mechanism and role of angiotensin II (Ang II) action in the development of proteinuria in minimal change nephrotic syndrome (MCNS) is uncertain. The glomerular expressions of the slit diaphragm (SD) molecules nephrin, podocin and NEPH1 in rat puromycin aminonucleoside (PAN) nephropathy, a mimic of MCNS, were analyzed. The effects of Ang II receptor blockade (ARB) (irbesartan 15 mg/kg body weight/day) on proteinuria and on the expression of the SD molecules were analyzed. mRNA expressions of nephrin, podocin and NEPH1 were decreased to an undetectable level at 1 h. The staining of these SD molecules shifted to a discontinuous pattern, and their intensity was reduced. NEPH1 staining was reduced to an undetectable level on day 10. ARB treatment ameliorated the peak value of proteinuria (237.6 ± 97.0 vs. 359.0 ± 63.3 mg/day, p < 0.05), and prevented the decrease in the mRNA expression of the SD molecules (nephrin 66.96 %, podocin 60.40 %, NEPH1 77.87 % of normal level). The immunofluorescence staining of NEPH1 was restored by ARB. ARB treatment enhanced the expression of NEPH1 of normal rats. Dysfunction of the SD molecules including NEPH1 is a crucial initiation event of PAN nephropathy. ARB treatment ameliorates proteinuria in PAN nephropathy by inhibiting the reduction of NEPH1 and nephrin. Ang II action regulates the expression of NEPH1 and nephrin in not only the pathological but also physiological state.

Expression of a novel stress-inducible protein, sestrin 2, in rat glomerular parietal epithelial cells.

Sestrin 2, initially identified as a p53 target protein, accumulates in cells exposed to stress and inhibits mammalian target of rapamycin (mTOR) signaling. In normal rat kidneys, sestrin 2 was selectively expressed in parietal epithelial cells (PECs), identified by the marker protein gene product 9.5. In adriamycin nephropathy, sestrin 2 expression decreased in PECs on day 14, together with increased expression of phosphorylated S6 ribosomal protein (P-S6RP), a downstream target of mTOR. Sestrin 2 expression was markedly decreased on day 42, coinciding with glomerulosclerosis and severe periglomerular fibrosis. In puromycin aminonucleoside nephropathy, decreased sestrin 2 expression, increased P-S6RP expression, and periglomerular fibrosis were observed on day 9, when massive proteinuria developed. These changes were transient and nearly normalized by day 28. In crescentic glomerulonephritis, sestrin 2 expression was not detected in cellular crescents, whereas P-S6RP increased. In conditionally immortalized cultured PECs, the forced downregulation of sestrin 2 by short hairpin RNA resulted in increased expression of P-S6RP and increased apoptosis. These data suggest that sestrin 2 is involved in PEC homeostasis by regulating the activity of mTOR. In addition, sestrin 2 could be a novel marker of PECs, and decreased expression of sestrin 2 might be a marker of PEC injury.

Therapeutic target for nephrotic syndrome: Identification of novel slit diaphragm associated molecules.

The slit diaphragm bridging the neighboring foot processes functions as a final barrier of glomerular capillary wall for preventing the leak of plasma proteins into primary urine. It is now accepted that the dysfunction of the sit diaphragm contributes to the development of proteinuria in several glomerular diseases. Nephrin, a gene product of NPHS1, a gene for a congenital nephrotic syndrome of Finnish type, constitutes an extracellular domain of the slit diaphragm. Podocin was identified as a gene product of NPHS2, a gene for a familial steroid-resistant nephrotic syndrome of French. Podocin binds the cytoplasmic domain of nephrin. After then, CD2 associated protein, NEPH1 and transient receptor potential-6 were also found as crucial molecules of the slit diaphragm. In order to explore other novel molecules contributing to the development of proteinuria, we performed a subtraction hybridization assay with a normal rat glomerular RNA and a glomerular RNA of rats with a puromycin aminonucleoside nephropathy, a mimic of a human minimal change type nephrotic syndrome. Then we have found that synaptic vesicle protein 2B, ephrin-B1 and neurexin were already downregulated at the early stage of puromycin aminonucleoside nephropathy, and that these molecules were localized close to nephrin. It is conceivable that these molecules are the slit diaphragm associated molecules, which participate in the regulation of the barrier function. These molecules could be targets to establish a novel therapy for nephrotic syndrome.

Differential expressions of miRNAs in kidney in puromycin aminonucleoside nephropathy model and intervened effects of Leizhi capsule

To observe the differential expression characteristics of microRNAs (miRNAs) in renal tissues in puromycin aminonucleoside (PAN) nephritic model, and its relationship with key structural molecules of slid diaphragm (SD) nephrin and podocin and expression of skeleton protein synaptopodin; and to explore the in vivo mechanisms of Leizhi capsule (LZC) for ameliorating the expressions of nephrin, podocin and synaptopodin and reducing proteins by regulating the modal rat renal tissues miRNAs. Fifty male Wistar rats were randomly divided into five groups: the control group (A), the model group (B), the LZC-treated group (C), the multi-glycoside of Tripterygium wilfordii (GTW)-treated group (D) and the valsartan-treated group (E). Apart from group A, all of rats in the remaining groups are injected with PAN (100 mg x kg(-1)) through jugular veins to establish the PAN nephropathy model. On the 2nd day after PAN nephropathy model was established, group C was orally administered with LZC (5 mL x kg(-1) x d(-1)) in group C, group DGTW (10 mL x kg(-1) x d(-1)), and E group valsartan (7.5 mL x kg(-1) x d(-1)), while groups A and B were intervened with physiological saline, for 10 days. Body weight and 24 h urinary protein ration (Upro) in all rats were measured at day 0, 3, and 9. All rats were sacrificed at day 11 after the establishment of the model, and their blood and renal tissues were collected to observe such blood biochemical indicators including albumin (Alb), serum creatinine (Scr), blood urea nitrogen (BUN) and glomerular ultrastructure (podocyte foot process form) and expressions of dicer enzyme, nephrin, podocin and synaptopodin in renal tissues. Meanwhile, the differential expressional characteristics of miRNAs in renal cortex were analyzed by biochip assay. Additionally, the differential expressional volumes of rno-miR-23a, rno-miR-300-3p, rno-miR-24 and rno-miR-30c were measured by real-time PCR. Proteinuria, renal dysfunction, hypoproteinemia and podocyte foot process fusion were investigated in model rats induced by PAN. In renal tissues of PAN nephropathy model rats, dicer enzyme affected the expressions of nephrin, podocin and synaptopodin in podocytes, up-regulated the expressions of rno-miR-23a and rno-miR-300-3p, and down-regulated the expressions of rno-miR-24 and rno-miR-30c. The miRNAs with differential expressions included rno-miR-24, rno-miR-30c, rno-miR-23a and rno-miR-300-3p. LZC could improve the general state, proteinuria, serum BUN and podocyte foot process fusion of PAN nephropathy model rats, reduced the expressions of dicer enzyme, increased the expressions of nephrin, podocin and synaptopodin in podocytes, weakened the up-regulated rno-miR-23a and rno-miR-300-3p, and strengthened the down-regulated rno-miR-24 and rno-miR-30c in renal tissues. PAN in vivo impacts the expressions of miRNAs in renal tissues, intervenes the expressions of nephrin, podocin and synaptopodin in podocytes, damages podocyte structures and functions and generates proteinuria by means of differential expression of dicer enayme/miRNAs. LZC can reduce proteinuria in PAN nephropathy model rats. Its mechanism may intervene dicer enayme/miRNAs differential expression, regulate nephrin, podocin and synaptopodin in podocytes and improve podocyte structures and functions.

Systemic administration of naked plasmid encoding HGF attenuates puromycin aminonucleoside-induced damage of murine glomerular podocytes

Podocyte injury is considered to play important roles in the pathogenesis of human glomerular disease. There is accumulating evidence suggesting that hepatocyte growth factor (HGF) elicits preventive activity for glomerular cells in animal models of chronic renal diseases. In this study, we demonstrated that delivery of a naked plasmid vector encoding the human HGF gene into mice by a hydrodynamic-based in vivo gene transfection approach markedly reduced proteinuria and attenuated podocyte injury in a mouse model induced by puromycin aminonucleoside (PAN) injection. Systemic administration by rapid injection via the tail vein of a naked plasmid containing HGF cDNA driven under a cytomegalovirus promoter (pCMV-HGF) produced a remarkable level of human HGF protein in the circulation. Tissue distribution studies suggested that the kidney expressed a high level of the HGF transgene. Meanwhile, compared with tubules and interstitium, a higher level of exogenous HGF protein was detected in the glomeruli. Administration of pCMV-HGF dramatically abated the urine albumin excretion and podocyte injury in PAN nephropathy in mice. Exogenous expression of HGF produced evidently beneficial effects, leading to restoration of Wilms' tumor-1 (WT1) and α-actinin-4 expression and attenuation of ultrastructural damage of the podocytes. In vitro, HGF not only restored WT1 and α-actinin-4 expression but also inhibited albumin leakage of podocytes incubated with PAN in a Transwell culture chamber. These results suggest that HGF might provide a novel strategy for amelioration of podocyte injury.

Role of smooth muscle protein SM22α in glomerular epithelial cell injury

Podocytes are considered terminally differentiated cells in the mature kidney under normal conditions. In the face of injury, podocytes may proceed along several possible pathways, including dedifferentiation and proliferation, persistent cell cycle arrest, hypertrophy, apoptosis, or necrosis. There is mounting evidence that transdifferentiation into a dysregulated phenotype may also be a potential cell fate. We have previously reported that the transcript of SM22α, an actin-binding protein considered one of the earliest markers of smooth muscle differentiation, is upregulated nearly 70-fold in glomeruli of rats with passive Heymann nephritis (PHN). In contrast, the SM22α transcript is absent in normal adult rat glomeruli. The purpose of this study was to define SM22α's expression during kidney development and its role in glomerular diseases characterized by podocyte injury and proteinuria. During glomerulogenesis and podocyte differentiation, SM22α was expressed in glomeruli. This expression disappeared with glomerular maturation. Along with SM22α induction in PHN, confirmed at both mRNA and protein levels, SM22α was also induced across a broad range of proteinuric diseases, including experimental animal models (puromycin aminonucleoside nephropathy, adriamycin nephropathy, passive nephrotoxic nephritis, and diet-induced obesity) and human diseases (collapsing glomerulopathy, diabetic nephropathy, classic focal segmental glomerulosclerosis, IgA nephropathy, minimal-change disease, membranous nephropathy, and membranoproliferative glomerulonephritis). Crescentic glomerulonephritis was induced in SM22α +/+ and SM22α -/- mice by intraperitoneal injection of sheep anti-rabbit glomeruli antibody 12.5 mg/20 g body wt × 2 doses (n = 12-15/group), with mice euthanized at 7 and 14 days. Compared with SM22α -/- mice, SM22α +/+ mice demonstrated worse disease by histopathological parameters. In addition, there was greater apoptosis (cleaved caspase-3 immunostaining), fewer podocytes (Wilms' tumor-1 immunostaining), and less proliferation (Ki-67 immunostaining) in diseased SM22α +/+ mice. Furthermore, there was decreased activation of Erk1/2 in diseased SM22α +/+ mice. We conclude that the de novo expression of SM22α in glomerular epithelial cells affects the course of crescentic glomerulonephritis.

The Immunosuppressive Drug Mizoribine Directly Prevents Podocyte Injury in Puromycin Aminonucleoside Nephrosis

Background/Aims: Mizoribine (MZR) is an imidazole nucleoside used as a therapeutic immunosuppressive agent. Though a previous report showed that MZR ameliorates proteinuria in puromycin aminonucleoside (PAN) nephropathy, the effect of MZR on podocytes has not been clarified. In this study, we determined whether MZR directly prevents PAN-induced podocyte injury. Methods: Rats were intravenously injected once on day 0 with 100 mg/kg of PAN and received daily subcutaneous injections of MZR at a dose of 10 mg/kg from days 0 to 14. Cultured podocytes were pretreated with 50 µg/ml of MZR and then treated with 30 µg/ml of PAN. Results: In rat PAN nephrosis, treatment with MZR from days 0 to 14 almost completely inhibited proteinuria. Immunofluorescence staining of nephrin was diminished, showing a discontinuous pattern in saline-treated PAN rats. In contrast, MZR treatment resulted in maintenance of a normal linear pattern. In cultured podocytes exposed to PAN, the percentages of viable cells were significantly increased with MZR treatment. The protective effect of MZR on PAN-induced podocyte injury was independent of inosine 5′-monophosphate dehydrogenase that is a known target enzyme of MZR as an immunosuppressant. MZR reduced PAN-induced integrin-linked kinase activation (ILK) and phosphorylation of glycogen synthase kinase-3β (GSK3β) in vivo and in vitro. Conclusion: MZR directly prevents PAN-induced podocyte injury, possibly by affecting signaling cascades involving ILK and GSK3β.