Model Of Glomerular Injury Research Articles

Podocyte-specific deletion of miR-146a increases podocyte injury and diabetic kidney disease.

Diabetic glomerular injury is a major complication of diabetes mellitus and is the leading cause of end stage renal disease (ESRD). Healthy podocytes are essential for glomerular function and health. Injury or loss of these cells results in increased proteinuria and kidney dysfunction and is a common finding in various glomerulopathies. Thus, mechanistic understanding of pathways that protect podocytes from damage are essential for development of future therapeutics. MicroRNA-146a (miR-146a) is a negative regulator of inflammation and is highly expressed in myeloid cells and podocytes. We previously reported that miR-146a levels are significantly reduced in the glomeruli of patients with diabetic nephropathy (DN). Here we report generation of mice with selective deletion of miR-146a in podocytes and use of these mice in models of glomerular injury. Induction of glomerular injury in C57BL/6 wildtype mice (WT) and podocyte-specific miR-146a knockout (Pod-miR146a–/–) animals via administration of low-dose lipopolysaccharide (LPS) or nephrotoxic serum (NTS) resulted in increased proteinuria in the knockout mice, suggesting that podocyte-expressed miR-146a protects these cells, and thus glomeruli, from damage. Furthermore, induction of hyperglycemia using streptozotocin (STZ) also resulted in an accelerated development of glomerulopathy and a rapid increase in proteinuria in the knockout animals, as compared to the WT animals, further confirming the protective role of podocyte-expressed miR-146a. We also confirmed that the direct miR-146a target, ErbB4, was significantly upregulated in the diseased glomeruli and erlotinib, an ErbB4 and EGFR inhibitor, reducedits upregulation and the proteinuria in treated animals. Primary miR146–/– podocytes from these animals also showed a basally upregulated TGFβ-Smad3 signaling in vitro. Taken together, this study shows that podocyte-specific miR-146a is imperative for protecting podocytes from glomerular damage, via modulation of ErbB4/EGFR, TGFβ, and linked downstream signaling.

Mesenchymal stromal cells (Cyto-MSC) are well-tolerated and associated with renal function improvement in patients with chronic kidney disease: an observational retrospective study

Background & Aim Background Microvascular disease and structural damage are hallmarks of chronic kidney disease (CKD). We have demonstrated previously that mesenchymal stromal cells (MSCs) could differentiate into mesangial cells in animal models of glomerular injury. We further demonstrated that MSCs promoted angiogenesis in diabetic patients with critical limb ischemia. Given the ability to improve vascularity and structure, MSCs may potentially improve renal function in CKD. Objective In this study, we evaluated the safety, tolerability and renal function of patients with CKD who had participated in MSC-related clinical trials. Methods, Results & Conclusion Methodology There were 4 clinical trials approved at the National University Malaysia in collaboration with Cytopeutics (Cyberjaya, Malaysia) which provided the mesenchymal stromal cells (Cyto-MSC) for treatment of acute stroke, heart failure, diabetes with critical limb ischemia and severe deforming osteoarthritis. All patients received 65-130 × 106 Cyto-MSC intravenously. Severity of chronic kidney disease is based on baseline estimated glomerular filtration rate (eGFR) and serum creatinine which are markers of renal dysfunction. These and other blood tests including fasting blood sugar (FBS) and Hba1c (marker of glycemic control) were measured at baseline and 12 months post infusion. Results 82 patients (53 men, 29 women; age 38-92 years) with different CKD stages (stage 3, n=65; stage 4, n=5; stage 5, n=12) were identified. 33 patients have type 2 diabetes. All patients tolerated the treatment well. There was significant improvement in serum creatinine (170±148 vs. 144±123 μmol/L; p=0.013) and eGFR values (37±17 vs. 41±18 mL/min/1.73m2; p=0.001). Patients with stage 3 CKD have the most significant eGFR value improvement (45±9 vs. 50±10 mL/min/1.73m2; p=0.006). Among diabetic patients, improvements were observed in creatinine (167±138 vs. 145±115 μmol/L, p=0.011), eGFR (37±15 vs. 41±16, p=0.05), HbA1c (7.4±2.1 vs. 6.2±1.4 %, p=0.011) and FBS (7.1±1.1 vs. 6.8±0.8 mmol/L %, p=0.021). There was also modest positive correlation between FBS with urea (p=0.001) and eGFR (p=0.016). Conclusion Cyto-MSC appears to be safe and well-tolerated by patients with moderate-severe CKD and associated with improvement in renal function particularly for moderate CKD patients. Further randomized prospective studies are warranted.

Alternatively spliced isoforms of WT1 control podocyte-specific gene expression

The Wilms' tumor suppressor WT1 is a key regulator of podocyte function that is mutated in Denys-Drash and Frasier syndromes. Here we have used an integrative approach employing ChIP, exon array, and genetic analyses in mice to address general and isoform-specific functions of WT1 in podocyte differentiation. Analysis of ChIP-Seq data showed that almost half of the podocyte-specific genes are direct targets of WT1. Bioinformatic analysis further identified coactivator FOXC1-binding sites in proximity to WT1-bound regions, thus supporting coordinated action of these transcription factors in regulating podocyte-specific genes. Transcriptional profiling of mice lacking the WT1 alternative splice isoform (+KTS) had a more restrictive set of genes whose expression depends on these alternatively spliced isoforms. One of these genes encodes the membrane-associated guanylate kinase MAGI2, a protein that localizes to the base of the slit diaphragm. Using functional analysis in mice, we further show that MAGI2α is essential for proper localization of nephrin and the assembly of the slit diaphragm complex. Finally, a dramatic reduction of MAGI2 was found in an LPS mouse model of glomerular injury and in genetic cases of human disease. Thus, our study highlights the central role of WT1 in podocyte differentiation, identifies that WT1 has a central role in podocyte differentiation, and identifies MAGI2α as the crucial isoform in slit diaphragm assembly, suggesting a causative role of this gene in the etiology of glomerular disorders.

Anti-inflammatory role of DPP-4 inhibitors in a nondiabetic model of glomerular injury.

Dipeptidyl peptidase (DPP)-4 is an enzyme that cleaves and inactivates incretin hormones capable of stimulating insulin secretion from pancreatic β-cells. DPP-4 inhibitors are now widely used for the treatment of type 2 diabetes. Experimental studies have suggested a renoprotective role of DPP-4 inhibitors in various models of diabetic kidney disease, which may be independent of lowering blood glucose levels. In the present study, we examined the effect of DPP-4 inhibitors in the rat Thy-1 glomerulonephritis model, a nondiabetic glomerular injury model. Rats were injected with OX-7 (1.2 mg/kg iv) and treated with the DPP-4 inhibitor alogliptin (20 mg·kg(-1)·day(-1)) or vehicle for 7 days orally by gavage. Alogliptin significantly reduced the number of CD68-positive inflammatory macrophages in the kidney, which was associated with a nonsignificant tendency to ameliorate glomerular injury and reduce proteinuria. Another DPP-4 inhibitor, anagliptin (300 mg·kg(-1)·day(-1) mixed with food) and a glucagon-like peptide-1 receptor agonist, exendin-4 (10 mg/kg sc), similarly reduced CD68-positive macrophage infiltration to the kidney. Furthermore, ex vivo transmigration assays using peritoneal macrophages revealed that exendin-4, but not alogliptin, dose dependently reduced monocyte chemotactic protein-1-stimulated macrophage infiltration. These data suggest that DPP-4 inhibitors reduced macrophage infiltration directly via glucagon-like peptide-1-dependent signaling in the rat Thy-1 nephritis model and indicate that the control of inflammation by DPP-4 inhibitors is useful for the treatment of nondiabetic kidney disease models.

CD40 Generation 2.5 Antisense Oligonucleotide Treatment Attenuates Doxorubicin-induced Nephropathy and Kidney Inflammation.

Preclinical and clinical data suggest CD40 activation contributes to renal inflammation and injury. We sought to test whether upregulation of CD40 in the kidney is a causative factor of renal pathology and if reduction of renal CD40 expression, using antisense oligonucleotides (ASOs) targeting CD40, would be beneficial in mouse models of glomerular injury and unilateral ureter obstruction. Administration of a Generation 2.5 CD40 ASO reduced CD40 mRNA and protein levels 75–90% in the kidney. CD40 ASO treatment mitigated functional, transcriptional, and pathological endpoints of doxorubicin-induced nephropathy. Experiments using an activating CD40 antibody revealed CD40 is primed in kidneys following doxorubicin injury or unilateral ureter obstruction and CD40 ASO treatment blunted CD40-dependent renal inflammation. Suborgan fractionation and imaging studies demonstrated CD40 in glomeruli before and after doxorubicin administration that becomes highly enriched within interstitial and glomerular foci following CD40 activation. Such foci were also sites of ASO distribution and activity and may be predominately comprised from myeloid cells as bone marrow CD40 deficiency sharply attenuated CD40 antibody responses. These studies suggest an important role of interstitial renal and/or glomerular CD40 to augment kidney injury and inflammation and demonstrate that ASO treatment could be an effective therapy in such disorders.

Podocyte-associated talin1 is critical for glomerular filtration barrier maintenance

Podocytes are specialized actin-rich epithelial cells that line the kidney glomerular filtration barrier. The interface between the podocyte and the glomerular basement membrane requires integrins, and defects in either α3 or β1 integrin, or the α3β1 ligand laminin result in nephrotic syndrome in murine models. The large cytoskeletal protein talin1 is not only pivotal for integrin activation, but also directly links integrins to the actin cytoskeleton. Here, we found that mice lacking talin1 specifically in podocytes display severe proteinuria, foot process effacement, and kidney failure. Loss of talin1 in podocytes caused only a modest reduction in β1 integrin activation, podocyte cell adhesion, and cell spreading; however, the actin cytoskeleton of podocytes was profoundly altered by the loss of talin1. Evaluation of murine models of glomerular injury and patients with nephrotic syndrome revealed that calpain-induced talin1 cleavage in podocytes might promote pathogenesis of nephrotic syndrome. Furthermore, pharmacologic inhibition of calpain activity following glomerular injury substantially reduced talin1 cleavage, albuminuria, and foot process effacement. Collectively, these findings indicate that podocyte talin1 is critical for maintaining the integrity of the glomerular filtration barrier and provide insight into the pathogenesis of nephrotic syndrome.

Background Strain and the Differential Susceptibility of Podocyte-Specific Deletion of Myh9 on Murine Models of Experimental Glomerulosclerosis and HIV Nephropathy

We previously reported that podocyte-specific deletion of Myh9 (conventional myosin heavy chain 2A) in C57BL/6 mice does not cause spontaneous kidney disease but instead results in a predisposition to glomerulosclerosis in response to a second model of glomerular injury. In contrast, other investigators reported that podocyte-specific deletion of Myh9 (PodΔMyh9) resulted in spontaneous glomerulosclerosis in mice on a mixed background, suggesting that the glomerulosclerosis is dependent on background strain. In order to elucidate the cause of this strain dependent effect Podocin::Cre and Myh9flox alleles were backcrossed to mouse strain FVB/N, which is highly susceptible to glomerulosclerosis, with the aim of intercrossing susceptible FVB/N and resistant C57BL/6 mice in subsequent congenic analyses. However, after backcrossing mice to FVB/N and aging mice to 28 weeks, we found no evidence of glomerular disease in PodΔMyh9 mice vs control littermates (urine MAC ratio all p>0.05). We also tested C57BL/6 PodΔMyh9 mice for a predisposition to injury from models other than Adriamycin including HIV nephropathy (HIVAN), puromycin nephropathy, and sheep nephrotoxic serum. In the Tg26 model of HIVAN, we found that podocyte-specific deletion of Myh9 resulted in a modest hypersensitivity in adults compared to Tg26+ control littermates (urine MAC ratio, p<0.05 or less). In contrast, we found that PodΔMyh9 mice were not predisposed to injury in response to other injury models including puromycin nephropathy and sheep nephrotoxic serum. While the mechanism of injury in these models is not fully understood, we conclude that PodΔMyh9 results in a variable susceptibility to glomerulosclerosis in response to different models of glomerular injury. In addition, based on the lack of a spontaneous phenotype of glomerulosclerosis in both C57BL/6 and FVB/N mice, we propose that Myh9 is not absolutely required in adult podocytes.

Renal tubular angiogenic dysregulation in anti-Thy1.1 glomerulonephritis

Peritubular vascular changes and hypoxia after glomerular injury may explain subsequent tubulointerstitial injury and fibrosis. Several studies suggested that the expected tubulointerstitial angiogenic response is actively suppressed in this setting. The mechanism of this aberrant response has not been clearly identified. We used a common model of glomerular injury in rats to assess vascular changes and to identify potential factors associated with this aberrant response. Anti-Thy1.1 antibody administration (1 or 4 weekly doses) led to a dose-dependent renal damage characterized by elevated urea and tubulointerstitial fibrosis as assessed by Picro-Sirius Red staining. We quantified peritubular capillaries using CD31 and CD34 immunohistochemistry and showed that tubular angiogenic dysregulation was associated with peritubular capillary rarefaction. Using laser capture microdissection, we demonstrated an early induction of fibrogenic and angiogenic factors in the glomeruli and a subsequent dysregulated angiogenic response in the tubulointerstitial compartment. Proximal tubules of anti-Thy1.1-treated animals had increased pigment epithelial-derived factor (PEDF) expression by immunohistochemistry. Protein taken by laser capture microdissection also showed that PEDF was upregulated. Temporally associated with PEDF expression was a transient downregulation of tubular hypoxia-inducible factor (HIF)1α. In a human proximal tubular cell culture, we show that PEDF downregulates HIF1α protein and gene expression in cells exposed to 1% oxygen. In anti-Thy1.1 glomerulonephritis, there is aberrent tubular angiogenesis associated with glomerular injury and tubulointersititial fibrosis. We showed that PEDF may be involved by downregulating HIF1α. Further work is needed to elucidate the mechanism of PEDF upregulation and action in the tubules.

Phosphorylation status of nephrin in human membranous nephropathy

We recently reported that nephrin, a major slit-diaphragm protein, is phosphorylated at Y1204 and Y1228 in normal human glomeruli and that phosphorylation decreased significantly in minimal-change nephrosis. These results indicate that phosphorylation of nephrin is important for maintenance of normal podocyte morphology and function. On the other hand, phosphorylation of nephrin was reportedly increased in certain animal models of glomerular injury. We performed immunofluorescent and immunoelectron staining of phosphorylated nephrin in human kidney biopsy specimens of membranous nephropathy (MN) to investigate whether phosphorylation of nephrin was altered in human MN and whether it correlated with MN staging. Although aberrant localization of phosphorylated nephrin was detected using immunoelectron microscopy in stage I MN, a decrease in the immunofluorescent intensity of phosphorylated nephrin was not observed in stage I, and only a slight decrease was seen in stages II, III, and IV compared with controls. No significant correlation between nephrin phosphorylation and proteinuria was observed. Nephrin phosphorylation was not significantly decreased in the early stage of MN.

Podocyte antigens, dendritic cells and T cells contribute to renal injury in newly developed mouse models of glomerulonephritis

Correspondence and offprint requests to: Matthias Mack; E-mail: matthias.mack@klinik.uni-regensburg.de ∗Comment on Bao L, Haas M, Pippin J et al. Focal and segmental glomerulosclerosis induced in mice lacking decay-accelerating factor in T cells. J Clin Invest 2009; 119: 1264–1274; and Heymann F, Meyer-Schwesinger C, Hamilton-Williams EE et al. Kidney dendritic cell activation is required for progression of renal disease in a mouse model of glomerular injury. J Clin Invest 2009; 119: 1286–1297.

Kidney dendritic cell activation is required for progression of renal disease in a mouse model of glomerular injury

Kidney dendritic cell activation is required for progression of renal disease in a mouse model of glomerular injury

Kidney dendritic cell activation is required for progression of renal disease in a mouse model of glomerular injury

The progression of kidney disease to renal failure correlates with infiltration of mononuclear immune cells into the tubulointerstitium. These infiltrates contain macrophages, DCs, and T cells, but the role of each cell type in disease progression is unclear. To investigate the underlying immune mechanisms, we generated transgenic mice that selectively expressed the model antigens ovalbumin and hen egg lysozyme in glomerular podocytes (NOH mice). Coinjection of ovalbumin-specific transgenic CD8+ CTLs and CD4+ Th cells into NOH mice resulted in periglomerular mononuclear infiltrates and inflammation of parietal epithelial cells, similar to lesions frequently observed in human chronic glomerulonephritis. Repetitive T cell injections aggravated infiltration and caused progression to structural and functional kidney damage after 4 weeks. Mechanistic analysis revealed that DCs in renal lymph nodes constitutively cross-presented ovalbumin and activated CTLs. These CTLs released further ovalbumin for CTL activation in the lymph nodes and for simultaneous presentation to Th cells by distinct DC subsets residing in the kidney tubulointerstitium. Crosstalk between tubulointerstitial DCs and Th cells resulted in intrarenal cytokine and chemokine production and in recruitment of more CTLs, monocyte-derived DCs, and macrophages. The importance of DCs was established by the fact that DC depletion rapidly resolved established kidney immunopathology. These findings demonstrate that glomerular antigen-specific CTLs and Th cells can jointly induce renal immunopathology and identify kidney DCs as a mechanistic link between glomerular injury and the progression of kidney disease.

Podocyte Repopulation Contributes to Regression of Glomerular Injury Induced by Ace Inhibition

Angiotensin-converting enzyme (ACE) inhibition induces glomerular repair in the Munich Wistar Frömter (MWF) rat, a model of spontaneous glomerular injury. In this study, we investigated whether this effect is related to changes in glomerular cell number, particularly of podocytes, which are progressively lost with age. MWF rats with advanced nephropathy were studied at both 40 weeks and after 20 weeks of observation either with or without treatment with the ACE inhibitor lisinopril. Forty-week-old Wistar rats were used as controls. In untreated MWF rats, proteinuria, hypertension, glomerulosclerosis, and renal function worsened, while lisinopril induced regression of both functional and structural changes. Despite glomerular hypercellularity in untreated MWF rats, the number of endothelial cells per glomerulus did not change, and podocyte number even decreased. ACE inhibition halted the progressive increase in glomerular cell number and enhanced endothelial cell volume density. Surprisingly, lisinopril not only halted age-related podocyte loss but also increased the number of glomerular podocytes above baseline, which was associated with an increased number of proliferating Wilms tumor 1-positive cells, loss of cyclin-dependent kinase inhibitor p27 expression, and increased number of parietal podocytes. These data indicate that ACE inhibition restructures glomerular capillary, primarily by restoring the podocyte population in this model of glomerular injury. Increased parietal podocyte number in lisinopril-treated MWF rats suggests that the remodeling of Bowman's capsule epithelial cells contributes to this effect.

Interferon-β

Experimental studies to test the latest cytokine (or antibodies to it) in models of glomerular injury appear on an almost industrial scale. Most show modest benefits without providing serious mechanistic insights or the confidence that they will ever become clinical therapies. The article by

Interferon-β Reduces Proteinuria in Experimental Glomerulonephritis

Interferon-beta (IFN-beta) is a multifunctional cytokine with immunomodulatory properties. We examined the effect of IFN-beta in three separate rat models of glomerular injury and in cultured human glomerular endothelial cells and podocytes. In nephrotoxic nephritis in WKY rats, recombinant rat IFN-beta started either at induction or after establishment of disease significantly reduced 24-h proteinuria by up to 73% and 51%, respectively, but did not affect serum creatinine. There was a slight reduction in numbers of glomerular macrophages, but no difference in glomerular or tubulointerstitial scarring. In Thy-1 nephritis in Lewis rats, IFN-beta started at induction of disease reduced proteinuria by up to 66% with no effect on numbers of glomerular macrophages, but a reduced number of proliferating cells. In puromycin nephropathy in Wistar rats, IFN-beta started at induction of disease reduced proteinuria by up to 93%, but had no effect on glomerular histology. In cultured cells, human IFN-beta-1a had a dramatic effect on barrier properties, increasing electrical resistance across monolayers of either glomerular endothelial cells or podocytes and decreasing trans-monolayer passage of albumin. In conclusion, these results show that IFN-beta reduces proteinuria in three different rat models of glomerular injury and that its anti-proteinuric action may result from direct effects on cells that comprise the glomerular filtration barrier. These data indicate that IFN-beta may have potential as a therapeutic agent in proteinuric renal disease.

Effects of reactive oxygen species on in vitro filtration of water and albumin across glomerular basement membrane

Most of the interest in the glomerular basement membrane (GBM) stems from the observation that it undergoes morphological changes in renal disease. Studies on persistent proteinuria in experimental animal models have shown that the permeability properties of the GBM have been altered as a result of protein degradation and cross-linking of type IV collagen via its NC1 domains promoted by reactive oxygen species (ROS) and extrusion of tubular cell contents. We used the in vitro ultrafiltration technique to assess permeability properties of bare isolated GBM films to water and albumin in the Munich Wistar Fromter rat model of glomerular injury. Hydraulic permeability for water and albumin solutions and albumin fractional clearances were measured for rats treated with lisinopril [an angiotensin converting enzyme (ACE) inhibitor] and were compared with those measured for rats treated with dimethylthiouria (an ROS scavenger) and their control groups, at four pressure levels (50, 100, 200, and 300 mmHg). The ACE inhibitors and ROS scavengers treatment regimens for studied rats in addition to significantly lowering their systolic blood pressure and urinary protein excretion values to normal levels, have significantly increased their in vitro hydraulic and Darcy permeability, which is a measure of the intrinsic hydraulic conductance of the GBM, either in the absence or presence of albumin; in comparison with control animals. We believe that these favorable effects may derive from ROS scavenging beneficial effects that preserve the GBM protein structure by reducing entactin and laminin degradation and type IV collagen cross-linking.

Lack of Integrin α1β1 Leads to Severe Glomerulosclerosis after Glomerular Injury

Severity of fibrosis after injury is determined by the nature of the injury and host genetic susceptibility. Metabolism of collagen, the major component of fibrotic lesions, is, in part, regulated by integrins. Using a model of glomerular injury by adriamycin, which induces reactive oxygen species (ROS) production, we demonstrated that integrin alpha1-null mice develop more severe glomerulosclerosis than wild-type mice. Moreover, primary alpha1-null mesangial cells produce more ROS both at baseline and after adriamycin treatment. Increased ROS synthesis leads to decreased cell proliferation and increased glomerular collagen IV accumulation that is reversed by antioxidants both in vivo and in vitro. Thus, we have identified integrin alpha1beta1 as a modulator of glomerulosclerosis. In addition, we showed a novel pathway where integrin alpha1beta1 modulates ROS production, which in turn controls collagen turnover and ultimately fibrosis. Because integrin alpha1beta1 is expressed in many cell types this may represent a generalized mechanism of controlling matrix accumulation, which has implications for numerous diseases characterized by fibrosis.

Glomerular expression of the ATP-sensitive P2X7 receptor in diabetic and hypertensive rat models

The molecular identification and characterization of the adenosine triphosphate (ATP)-sensitive family of P2 receptors is comparatively new. There are two main subgroups, each with several subtypes and widespread tissue distribution, including the kidney. A unique member of the P2X subgroup of P2 receptors is the ATP-gated ion channel P2X(7), which on activation can cause cell blebbing, cytokine release, and cell death by necrosis or apoptosis. We report expression of this receptor in normal rat kidney and in two chronic models of glomerular injury: streptozotocin-induced (STZ) diabetes and ren-2 transgenic (TGR) hypertension. At different time points in these models, we used a polyclonal antibody to the P2X(7) receptor and immunohistochemistry to determine its expression and distribution. We also used Western blotting and real-time polymerase chain reaction (PCR) to detect changes in P2X(7) receptor protein and mRNA expression, respectively. We found only low-level glomerular immuno-staining for the P2X(7) receptor in normal rat kidney, but intense P2X(7) receptor immunostaining of glomeruli in kidneys from diabetic animals at 6 and 9 weeks, and in hypertensive animals at 12 weeks. In diabetic animals, real-time PCR demonstrated a approximately tenfold increase in glomerular P2X(7) receptor mRNA relative to control, and Western blotting confirmed an increase in protein. Immunohistochemistry and immunoelectron microscopy showed staining of glomerular podocytes, which was both intracellular and at the plasma membrane. We conclude that the P2X(7) receptor is not expressed appreciably under normal conditions, but that following glomerular injury it is significantly up-regulated, mainly in podocytes, though also in endothelial and mesangial cells, of animals with STZ-induced diabetes mellitus or TGR hypertension. Although the exact function and regulation of this receptor remain unclear, its association with inflammatory cytokine release and cell death suggests that increased expression might be involved in the pathogenesis of glomerular cell injury or repair.

Lipoic acid as a rescue agent for adriamycin-induced hyperlipidemic nephropathy in rats

Adriamycin [ADR] is a cell independent model of glomerular injury. Hyperlipidemia associated with nephrotic syndrome may play a role in the deterioration of renal function. The study was carried out with adult male albino rats of Wistar strain comprising six rats in each group. Rats were fed with a standard rat pellet diet and were given access to water ad libitum. Group I rats served as controls (0.5 ml/day/rat i.p). Group II rats received ADR (1 mg/kg body weight/day) intravenously through the tail vein. Group III rats were given lipoic acid [LA] (35 mg/kg body weight/day) intraperitoneally. Group IV rats received LA 24 hours prior to the administration of ADR. ADR administered rats showed a significant increase ( p < 0.05) in serum lipids and lipoprotein fractions. However, the kidney of ADR administered rat showed a decline ( p < 0.05) in the content of phospholipids and free fatty acids. Significant ( p < 0.05) alterations were also observed in the activities of lipid metabolizing enzymes Pre-treatment with LA reverted the levels of lipid profiles and serum lipoproteins to near normal. The activities of lipid metabolizing enzymes also showed considerable changes on LA pre-treatment. This study highlights the antilipemic action of LA and its role as a protective agent for hyperlipidemia, associated with ADR induced nephrotic syndrome.

Cytochrome P450 2B1 mediates oxidant injury in puromycin-induced nephrotic syndrome

Reactive oxygen metabolites (ROM) are important mediators of puromycin aminonucleoside (PAN) induced minimal change nephrotic syndrome (NS) in rats. We have recently shown that cytochrome P450 (CYP) is a significant source of catalytic iron in this model of glomerular injury. The current study was designed to identify the CYP isozyme(s) in the rat glomeruli and explore the role of the specific isozyme(s) in PAN-induced minimal change NS. NS was induced in rats by a single intravenous injection of PAN. Animals were sacrificed at different time points for variety of biochemical assays including Western blot, immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). Ultrastructural histochemistry was utilized to study hydrogen peroxide (H2O2) generation in the kidney. Several CYP isozymes were tested and CYP2B1 was localized exclusively in the rat glomeruli but not in the tubules. Treatment with PAN resulted in the generation of H2O2 in the glomerular basement membrane with significant loss of CYP2B1 content accompanied by a marked increase in the catalytic iron. CYP2B1 inhibitors cimetidine and piperine significantly reduced H2O2 generation, and prevented the loss of CYP2B1 content and the increase in the catalytic iron. CYP2B1 inhibitors also provided significant protection against PAN induced proteinuria. The induction of heme oxygenase and ferritin also was observed in the glomeruli in PAN-treated rats. Both cimetidine and piperine reduced the up-regulation of these proteins. Our data indicate that CYP2B1 plays an important role in PAN induced NS by serving as a site for ROM generation and a significant source of catalytic iron.