Phosphorylation of slit diaphragm proteins NEPHRIN and NEPH1 upon binding of HGF promotes podocyte repair.

Phosphorylation (activation) and dephosphorylation (deactivation) of the slit diaphragm proteins NEPHRIN and NEPH1 are critical for maintaining the kidney epithelial podocyte actin cytoskeleton and, therefore, proper glomerular filtration. However, the mechanisms underlying these events remain largely unknown. Here we show that NEPHRIN and NEPH1 are novel receptor proteins for hepatocyte growth factor (HGF) and can be phosphorylated independently of the mesenchymal epithelial transition receptor in a ligand-dependent fashion through engagement of their extracellular domains by HGF. Furthermore, we demonstrate SH2 domain-containing protein tyrosine phosphatase-2-dependent dephosphorylation of these proteins. To establish HGF as a ligand, purified baculovirus-expressed NEPHRIN and NEPH1 recombinant proteins were used in surface plasma resonance binding experiments. We report high-affinity interactions of NEPHRIN and NEPH1 with HGF, although NEPHRIN binding was 20-fold higher than that of NEPH1. In addition, using molecular modeling we constructed peptides that were used to map specific HGF-binding regions in the extracellular domains of NEPHRIN and NEPH1. Finally, using an in vitro model of cultured podocytes and an ex vivo model of Drosophila nephrocytes, as well as chemically induced injury models, we demonstrated that HGF-induced phosphorylation of NEPHRIN and NEPH1 is centrally involved in podocyte repair. Taken together, this is the first study demonstrating a receptor-based function for NEPHRIN and NEPH1. This has important biological and clinical implications for the repair of injured podocytes and the maintenance of podocyte integrity.

Loss of Motor Protein MYO1C Causes Rhodopsin Mislocalization and Results in Impaired Visual Function.

Unconventional myosins, linked to deafness, are also proposed to play a role in retinal cell physiology. However, their direct role in photoreceptor function remains unclear. We demonstrate that systemic loss of the unconventional myosin MYO1C in mice, specifically causes rhodopsin mislocalization, leading to impaired visual function. Electroretinogram analysis of Myo1c knockout (Myo1c-KO) mice showed a progressive loss of photoreceptor function. Immunohistochemistry and binding assays demonstrated MYO1C localization to photoreceptor inner and outer segments (OS) and identified a direct interaction of rhodopsin with MYO1C. In Myo1c-KO retinas, rhodopsin mislocalized to rod inner segments (IS) and cell bodies, while cone opsins in OS showed punctate staining. In aged mice, the histological and ultrastructural examination of the phenotype of Myo1c-KO retinas showed progressively shorter photoreceptor OS. These results demonstrate that MYO1C is important for rhodopsin localization to the photoreceptor OS, and for normal visual function.

Development of a novel cell-based assay to diagnose recurrent focal segmental glomerulosclerosis patients.

Definitive diagnosis of glomerular disease requires a kidney biopsy, an invasive procedure that may not be safe or feasible to perform in all patients. We developed a noninvasive, accurate, and economical diagnostic assay with easy commercial adaptability to detect recurrent focal segmental glomerulosclerosis (rFSGS) after kidney transplant. Since FSGS involves podocyte damage and death, our approach involved mRNA profiling of cultured podocytes treated with plasma from patients with rFSGS to identify upregulated genes involved in podocyte damage. For concept validation, three upregulated pro-apoptotic candidate genes (IL1ß, BMF, and IGFBP3) were selected, and their promoter regions were cloned into a luciferase-based reporter vector and transfected into podocytes to generate stable podocyte cell lines. Strikingly, when exposed to rFSGS patient plasma, these cell lines showed increased reporter activity; in contrast, no reporter activity was noted with plasma from patients with non-recurrent FSGS or membranous nephropathy. Area under the receiver operating characteristics curves (AUCs) for models discriminating between rFSGS and other nephropathies (non-recurrent FSGS and membranous nephropathy) and between rFSGS and non-recurrent FSGS ranged from 0.81 to 0.86, respectively. Estimated sensitivities and specificities for the diagnosis of rFSGS were greater than 80% for the IL1ß and BMF cell lines, and were slightly lower for the IGFBP3 cell line. Importantly, the novel approach outlined here for the diagnosis of rFSGS is widely applicable to the design of sensitive and specific diagnostic/prognostic assays for other glomerular diseases.

The Use of High-Throughput Transcriptomics to Identify Pathways with Therapeutic Significance in Podocytes.

Podocytes have a unique structure that supports glomerular filtration function, and many glomerular diseases result in loss of this structure, leading to podocyte dysfunction and ESRD (end stage renal disease). These structural and functional changes involve a complex set of molecular and cellular mechanisms that remain poorly understood. To understand the molecular signature of podocyte injury, we performed transcriptome analysis of cultured human podocytes injured either with PAN (puromycin aminonucleoside) or doxorubicin/adriamycin (ADR). The pathway analysis through DE (differential expression) and gene-enrichment analysis of the injured podocytes showed Tumor protein p53 (P53) as one of the major signaling pathways that was significantly upregulated upon podocyte injury. Accordingly, P53 expression was also up-regulated in the glomeruli of nephrotoxic serum (NTS) and ADR-injured mice. To further confirm these observations, cultured podocytes were treated with the P53 inhibitor pifithrin-α, which showed significant protection from ADR-induced actin cytoskeleton damage. In conclusion, signaling pathways that are involved in podocyte pathogenesis and can be therapeutically targeted were identified by high-throughput transcriptomic analysis of injured podocytes.

A Novel CLCN5 Mutation Associated With Focal Segmental Glomerulosclerosis and Podocyte Injury.

INTRODUCTION: Tubular dysfunction is characteristic of Dent's disease; however, focal segmental glomerulosclerosis (FSGS) can also be present. Glomerulosclerosis could be secondary to tubular injury, but it remains uncertain whether the CLCN5 gene, which encodes an endosomal chloride and/or hydrogen exchanger, plays a role in podocyte biology. Here, we implicate a role for CLCN5 in podocyte function and pathophysiology. METHODS: Whole exome capture and sequencing of the proband and 5 maternally-related family members was conducted to identify X-linked mutations associated with biopsy-proven FSGS. Human podocyte cultures were used to characterize the mutant phenotype on podocyte function. RESULTS: We identified a novel mutation (L521F) in CLCN5 in 2 members of a Hispanic family who presented with a histologic diagnosis of FSGS and low-molecular-weight proteinuria without hypercalciuria. Presence of CLCN5 was confirmed in cultured human podocytes. Podocytes transfected with the wild-type or the mutant (L521F) CLCN5 constructs showed differential localization. CLCN5 knockdown in podocytes resulted in defective transferrin endocytosis and was associated with decreased cell proliferation and increased cell migration, which are hallmarks of podocyte injury. CONCLUSIONS: The CLCN5 mutation, which causes Dent's disease, may be associated with FSGS without hyercalcuria and nepthrolithiasis. The present findings supported the hypothesis that CLCN5 participates in protein trafficking in podocytes and plays a critical role in organizing the components of the podocyte slit diaphragm to help maintain normal cell physiology and a functional filtration barrier. In addition to tubular dysfunction, mutations in CLCN5 may also lead to podocyte dysfunction, which results in a histologic picture of FSGS that may be a primary event and not a consequence of tubular damage.

Slit diaphragm protein Neph1 and its signaling: a novel therapeutic target for protection of podocytes against glomerular injury.

Podocytes are specialized epithelial cells that are critical components of the glomerular filtration barrier, and their dysfunction leads to proteinuria and renal failure. Therefore, preserving podocyte function is therapeutically significant. In this study, we identified Neph1 signaling as a therapeutic target that upon inhibition prevented podocyte damage from a glomerular injury-inducing agent puromycin aminonucleoside (PAN). To specifically inhibit Neph1 signaling, we used a protein transduction approach, where the cytoplasmic domain of Neph1 (Neph1CD) tagged with a protein transduction domain trans-activator of transcription was transduced in cultured podocytes prior to treatment with PAN. The PAN-induced Neph1 phosphorylation was significantly reduced in Neph1CD-transduced cells; in addition, these cells were resistant to PAN-induced cytoskeletal damage. The biochemical analysis using subfractionation studies showed that unlike control cells Neph1 was retained in the lipid raft fractions in the transduced cells following treatment with PAN, indicating that transduction of Neph1CD in podocytes prevented PAN-induced mislocalization of Neph1. In accordance, the immunofluorescence analysis further suggested that Neph1CD-transduced cells had increased ability to retain endogenous Neph1 at the membrane in response to PAN-induced injury. Similar results were obtained when angiotensin was used as an injury-inducing agent. Consistent with these observations, maintaining high levels of Neph1 at the membrane using a podocyte cell line overexpressing chimeric Neph1 increased the ability of podocytes to resist PAN-induced injury and PAN-induced albumin leakage. Using a zebrafish in vivo PAN and adriamycin injury models, we further demonstrated the ability of transduced Neph1CD to preserve glomerular function. Collectively, these results support the conclusion that inhibiting Neph1 signaling is therapeutically significant in preventing podocyte damage from glomerular injury.

Polymorphism in glutathione S-transferase P1 is associated with susceptibility to Plasmodium vivax malaria compared to P. falciparum and upregulates the GST level during malarial infection.

Glutathione S-transferase P1 (GSTP1) is a member of the GST superfamily, which has well-established multiple roles in various infectious and parasitic diseases. The genetic regulation of GSTP1 has been extensively studied. Thus, its biological significance and disease association prompted us to investigate the role of GSTP1 polymorphisms in Plasmodium-mediated pathogenesis in infected humans. The genotypic distribution of Ile105Val in Plasmodium vivax infection was observed to be significant and strongly associated (OR=4.5) with the progression of pathology, whereas in P. falciparum infection no significant association was observed compared to healthy subjects. Interestingly, we observed significant elevation of GST in vivax infection, with both genotypes Ile105Val and Val105Val, compared to healthy subjects, whereas in P. falciparum infection we found marginally elevated GST levels of mutated genotypes but significantly depleted compared to healthy subjects. Further, during vivax and falciparum infection overall significant elevations of glutathione, glutathione peroxidase, and GST levels were observed. Expression of both GSTP1 mRNA and protein was significantly upregulated during vivax infection compared to falciparum infection and both were significantly upregulated compared to the levels in healthy subjects as well. These studies suggest that GSTP1 polymorphism is involved in the pathogenesis of malaria and it may serve as a valuable molecular marker, possessing a promising rationale for diagnostic potential in assessing disease progression during clinical malaria.

CYP1A1, CYP1A2 and CYBA gene polymorphisms associated with oxidative stress in COPD.

BACKGROUND: The genetic susceptibility to chronic obstructive pulmonary disease (COPD) depends on detoxification and antioxidant enzymes, which detoxify cigarette smoke reactive components that, otherwise, generate oxidative stress. METHODS: In a case-control study of 346 subjects with and without COPD, we examined the polymorphisms 462Ile/Val, 3801T/C of CYP1A1, -3860G/A of CYP1A2 and -930A/G, 242C/T of CYBA individually or in combination and their contribution to oxidative stress markers by measuring malondialdehyde (MDA), catalase (CAT), glutathione (GSH) and glutathione peroxidase (GPx). RESULTS: COPD patients had significantly increased MDA concentration (p<0.001) and decreased CAT activity, GSH concentration, GPx activity (p< or =0.01). The patients were over-represented by the alleles 462Val, 3801C of CYP1A1 and -930G, 242C of CYBA (p<0.001, p=0.003, p=0.030 and p=0.031, respectively) and consequently the haplotypes of same alleles i.e. 462Val:3801C, 462Val:3801T and -930G:242C (p=0.048, p=0.016 and p=0.039, respectively). Similarly, CYP1A1 and CYP1A2 haplotypes, 462Val:3860G and 462Val:3801T:3860G were significantly over-represented (p=0.001 and p=0.003), respectively in patients. The same alleles-associated genotype-combinations between genes were more prevalent in patients. Of note, the genotypes, 462Ile/Val+Val/Val, 3801TC+CC of CYP1A1 and -930AG+GG of CYBA associated with increased MDA concentration (p=0.018, p=0.045 and p=0.017, respectively), decreased CAT activity (p<0.0001, p=0.080 and p<0.0001, respectively) and GSH concentration (p<0.0001, p=0.0002 and p=0.011, respectively) in patients. CONCLUSION: The identified alleles, its haplotypes and the genotype-combination along with increased oxidative stress, signify the importance in susceptibility to COPD.

Ischemic injury to kidney induces glomerular podocyte effacement and dissociation of slit diaphragm proteins Neph1 and ZO-1.

Glomerular injury is often characterized by the effacement of podocytes, loss of slit diaphragms, and proteinuria. Renal ischemia or the loss of blood flow to the kidneys has been widely associated with tubular and endothelial injury but rarely has been shown to induce podocyte damage and disruption of the slit diaphragm. In this study, we have used an in vivo rat ischemic model to demonstrate that renal ischemia induces podocyte effacement with loss of slit diaphragm and proteinuria. Biochemical analysis of the ischemic glomerulus shows that ischemia induces rapid loss of interaction between slit diaphragm junctional proteins Neph1 and ZO-1. To further understand the effect of ischemia on molecular interactions between slit diaphragm proteins, a cell culture model was employed to study the binding between Neph1 and ZO-1. Under physiologic conditions, Neph1 co-localized with ZO-1 at cell-cell contacts in cultured human podocytes. Induction of injury by ATP depletion resulted in rapid loss of Neph1 and ZO-1 binding and redistribution of Neph1 and ZO-1 proteins from cell membrane to the cytoplasm. Recovery resulted in increased Neph1 tyrosine phosphorylation, restoring Neph1 and ZO-1 binding and their localization at the cell membrane. We further demonstrate that tyrosine phosphorylation of Neph1 mediated by Fyn results in significantly increased Neph1 and ZO-1 binding, suggesting a critical role for Neph1 tyrosine phosphorylation in reorganizing the Neph1-ZO-1 complex. This study documents that renal ischemia induces dynamic changes in the molecular interactions between slit diaphragm proteins, leading to podocyte damage and proteinuria.

COX2 and p53 risk-alleles coexist in COPD.

BACKGROUND: Cigarette smoke stimulates airway epithelial cells to release pro-inflammatory cytokines which influence various inflammation-related genes, including COX2, whereas p53 expression is known to alter in such a condition. Since both the genes share several common physiological functions including inflammation and oxidative stress, we investigated within gene and gene-gene interactions towards susceptibility to the disease. METHOD: In a prospective gene-association study we conducted PCR-RFLP for genotyping the COX2 -765G/C and 8473T/C and p53 72Pro/Arg polymorphisms in 229 COPD patients and 147 healthy controls. RESULTS: The -765GC+CC genotypes of COX2 and Pro/Pro+Pro/Arg genotypes of p53 were prevalent in patients with significant odds ratio, 2.05 and 2.30, respectively (p=0.001; p=0.009, respectively), as a consequence, the -765C and 72Pro alleles were prevalent (p

Correlation of oxidative status with BMI and lung function in COPD.

OBJECTIVES: The imbalance in oxidative status together with nutrition depletion and low body weight play a vital role in the pathogenesis and severity of chronic obstructive pulmonary disease (COPD). The study was undertaken to ascertain if a relationship existed between oxidative status and BMI in COPD. In addition, association of oxidative status and BMI with lung function of the disease was also examined. MATERIALS AND METHODS: In 202 COPD patients and 136 healthy controls plasma lipid peroxidation (LPO), reduced glutathione (GSH), glutathione peroxidase (GPx), catalase (CAT) activities, BMI and FEV(1)% predicted were looked for interactions. RESULTS: The patients had increased LPO (p=0.006) and decreased antioxidants (GSH, p=0.005; GPx, p=0.035 and CAT, p=0.008, respectively). Of note are the correlations of oxidative stress markers with BMI and FEV(1)% predicted in the patients. LPO inversely and GSH, GPx, and CAT positively correlated with both BMI (p=0.007, p<0.001, p=0.045 and p=0.009, respectively), and FEV(1)% of predicted (LPO, p=0.001; GSH, p<0.001; GPx, p=0.043 and CAT, p<0.001) in the patients. Further, a positive correlation existed between BMI and FEV(1)% predicted (p=0.016) in COPD. CONCLUSION: The intimate relationship of oxidative status with BMI and lung function, and the direct correlation between BMI and FEV(1) may potentiate severity of the disease.

Genetic polymorphisms of GSTP1 and mEPHX correlate with oxidative stress markers and lung function in COPD.

The genetic susceptibility to COPD might depend on variations in detoxification enzymes that activate and detoxify cigarette smoke products, which otherwise generate oxidative stress causing pathogenesis. In a case-control study of 202 COPD patients and 136 normals, we examined the association of polymorphisms I105V, A114V of GSTP1 and Y113H, H139R of mEPHX individually or in combination with disease and their contribution to oxidative stress markers such as MDA, GSH, GPx and airflow obstruction. Patients were over-represented by the alleles 105V, 114V of GSTP1 and 113H, 139H of mEPHX (chi(2)=10.63, p=0.001, chi(2)=13.92, p<0.001, chi(2)=13.02, p<0.001 and chi(2)=4.48, p=0.034, respectively) and the haplotypes of same alleles i.e. 105V-114V and 113H-139H (chi(2)=14.58, p<0.001 and chi(2)=23.14, p<0.001). Moreover, there was marked over-representation of combination of genotypes, I105I+A114A of GSTP1 (53% vs. 36%) in controls; whereas, the combinations with 105V/114V alleles (64% vs. 47%) of GSTP1 (OR=1.99; 95% CI=1.28-3.09; p=0.002) and the homozygotes H113H+H139H (27% vs.10%) of mEPHX (OR=3.26; 95% CI=1.73-6.15; p=0.0001) in patients. Patients had significantly elevated MDA level (p<0.001) and decreased GSH level (p<0.001) and GPx activity (p=0.035), respectively. Of note, the genotypes, I105V/V105V, A114V/V114V of GSTP1 and Y113H/H113H of mEPHX associated with increased MDA level (p=0.04, p=0.03 and p=0.003), decreased GSH level (p=0.019, p=0.007 and p=0.0006) and lower FEV1 (p=0.23, p=0.037 and p=0.029), respectively, in patients; so was the correlation of these biomarkers and lung function with the combinations of the genotypes. In conclusion, 105V/114V alleles of GSTP1 and 113H/139H alleles of mEPHX and the combination of genotypes with same alleles associated with imbalanced oxidative stress and lung function in patients, signifying the importance in the disease.

Association of CYP2E1 and NAT2 gene polymorphisms with chronic obstructive pulmonary disease.

BACKGROUND: Detoxification genes are potential candidates in the susceptibility of patients with chronic obstructive pulmonary disease. Polymorphisms in these genes alter the metabolism of xenobiotics such as present in cigarette smoke. METHODS: We conducted a case-control study to investigate total 9 polymorphisms of CYP2E1, CYP2D6 and NAT2 genes by PCR-RFLP. RESULTS: The -1053C/T and -1293G/C promoter polymorphisms of CYP2E1 were found to be in complete linkage disequilibrium (LD) (D'=1.00, r(2)=1.0, p<0.0001), whereas -1293G/C and 7632T/A polymorphisms of the same gene were also in significant LD (D'=0.5183, r(2)=1.0, p=0.01) in patients. The patients over-represented the -1293GC+CC genotypes of -1293G/C polymorphism of CYP2E1 (p=0.03) and NAT2*4/7, NAT2()5/6, NAT2*5/7, NAT2*6/6 and NAT2*6/7 genotypes of NAT2 (p=0.01, p=0.039, p=0.01, p=0.032, p=0.006, respectively), resulting in to higher frequency of -1293C (OR=7.02, 95% CI=1.63-30.15, p=0.002), NAT2*6 (OR=1.90, 95% CI=1.27-2.83, p=0.001) and NAT2*7 (OR=2.91, 95% CI=1.65-5.12, p=0.0001) alleles. The 7632T/A and 9893C/G polymorphisms of CYP2E1 and 1934G/A polymorphism of CYP2D6 did not associate with the disease (p>0.05). The haplotypes -1293G:9893C and -1293G:7632T:9893C were under-represented (p<0.001), whereas haplotypes -1293C:7632T, -1293C:9893C, -1293C:9893G and -1293C:7632T:9893C of the 4 CYP2E1 polymorphisms were over-represented in patients (p<0.05). CONCLUSION: The CYP2E1 and NAT2 variants associated with COPD.