cytochrome-c-t and Proteinuria

Podocyte damage exerts a key role in proteinuria. We have demonstrated that calcineurin-binding protein 1 (Cabin1) upregulated during podocyte injury, yet its function in podocyte is still unclear.. We established 5/6 nephrectomized rats and angiotensin II (AngII)-injured podocyte, as well as knocked down Cabin1 with siRNA in cultured podocytes. Rats were killed at 4 or 8 weeks after 5/6 nephrectomy. The localization of podocyte cytoskeleton was detected after immunofluorescence staining. Podocyte mitochondrial morphology was observed under electron microscopy. Podocyte mitochondrial transmembrane potential (MMP) was measured with MitoCapture kit. Cabin1 and cytochrome c protein expression were detected by western blot.. Massive proteinuria, as well as obvious segmental glomerular sclerosis, was found in rats at 8 weeks after nephrectomy, accompanied with the disruption of synaptopodin. Moreover, mitochondria changed from large and ellipsoid shape to the small, long, and irregular shape in rats at 4 weeks after operation. At 8 weeks, mitochondria were swollen and cristae were remarkably dissolved. Compared to sham-operated rats, Cabin1 protein expression was obviously upregulated in rats at 8 weeks. AngII induced the decrease in MMP, as well as the overexpression of Cabin1 and cytochrome c protein in podocytes. Knocking down Cabin1 induced the disruption of F-actin and overexpression of cytochrome c (1.81 ± 0.21 in siRNA group vs. 0.86 ± 0.11 in negative control group).. Knocking down Cabin1 induces the disruption of cytoskeleton and mitochondrial dysfunction in podocyte. Cabin1 could be a crucial factor in podocyte damage.

Topics: Actins; Angiotensin II; Animals; Apoptosis Regulatory Proteins; Cells, Cultured; Cytochromes c; Cytoskeleton; Gene Knockdown Techniques; Membrane Potentials; Mitochondria; Nephrectomy; Podocytes; Proteinuria; Rats; RNA, Small Interfering; Up-Regulation; Vasoconstrictor Agents

To investigate the effects of Huaiqihuang Granules (, HQH), a mixture of Chinese herbs including Trametes robiniophila Murr, Fructus Lycii and Polygonatum sibiricum, on adriamycininduced nephropathy (ADRN) in rats and its underlying mechanisms.. Rats with ADRN were divided into four groups: the sham group, the model group (distilled water), the low-dose HQH-treated (2 g/kg) group, and the high-dose HQH-treated (4 g/kg) group. Body weight and 24-h urinary protein (Upro) were checked every week. After 5-week intervention, at the end of the study, the rats were sacrificed and blood samples were collected for examination of biochemical parameters, including glomerular morphological makers, podocyte shape, cellular apoptosis, expressions of nephrin, inflammatory and apoptosis markers.. HQH ameliorated the rat's general status, proteinuria, renal morphological appearance and glomerulosclerosis. The decreased expression of nephrin in ADRN rats was increased by HQH, as well as the impaired podocyte foot process fusion. Cytosolic levels of p65 and inhibitor of nuclear factor κBα (IκBα) were decreased in ADRN rats, and recovered by the treatment of HQH. Consistently, the induced expression of tumor necrosis factor α (TNF-α), phosphorylated nuclear factor κB p65 (p-NFκB p65) and IκBα in ADRN were markedly suppressed by HQH. In addition, induction of Bax, cleaved caspase-3 and cytochrome C in ADRN rats were suppressed by HQH, indicating the amelioration of apoptosis.. HQH could ameliorate renal impairments in ADRN rats by increasing nephrin expression, inhibiting NF-κB signaling pathway via the down-regulation of p-NF-κB p65 and p-IκBα, and suppression of glomerular and tubular apoptosis.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Body Weight; Caspase 3; Chromatography, High Pressure Liquid; Cytochromes c; Doxorubicin; Drugs, Chinese Herbal; Kidney; Kidney Diseases; Kidney Glomerulus; Kidney Tubules; Male; Membrane Proteins; NF-kappa B; NF-KappaB Inhibitor alpha; Organ Size; Proteinuria; Rats, Sprague-Dawley; Signal Transduction; Transcription Factor RelA; Tumor Necrosis Factor-alpha

Angiotensin (Ang)II is involved in induction of proteinuria, renal injury, and apoptosis and thus a major contributor to the development of chronic kidney disease. Podocytes are of major importance for the pathogenesis of several kidney diseases. Decrease of podoplanin (PDPN) in podocytes and podocyte loss has been associated with the development of proteinuria. Little is known about the regulation and biological function of PDPN in podocytes and its role in AngII-mediated kidney damage. Here, we determined the influence of AngII on the expression of PDPN, microRNA (miRNA)-29b and miRNA-497 in human podocytes. Further, we analyzed the impact of small interfering RNA-mediated downregulation of PDPN on AngII-induced apoptosis and viability. Moreover, we characterized the role of miRNA-29b and miRNA-497 in expression regulation of PDPN.. Cell viability and apoptosis were determined by functional assays. Expression analyses were done via Real-Time PCR and western blot analyses. Dual luciferase assay was performed to characterize miRNA-mediated expression control.. AngII increased the expression of miRNA-29b and reduced PDPN. Small interfering RNA-mediated downregulation of PDPN increased proapoptotic caspase-3 activation and cytochrome C translocation, whereas cell viability and Akt phosphorylation were reduced in AngII-stimulated podocytes. In contrast to miRNA-497, transfection of cells with miRNA-29b mimics significantly decreased PDPN. Cotransfection of cells with miRNA-29b and a dual luciferase reporter vector decreased the luciferase activity compared with controls.. These data demonstrate the posttranscriptional control of PDPN expression by miRNA-29b and support a role of PDPN as an antiapoptotic prosurvival factor in AngII-induced injury of human podocytes.

Topics: Angiotensin II; Apoptosis; Caspase 3; Cell Survival; Cells, Cultured; Cytochromes c; Down-Regulation; Gene Expression Regulation; Humans; Membrane Glycoproteins; MicroRNAs; Phosphorylation; Podocytes; Proteinuria; Proto-Oncogene Proteins c-akt; RNA, Small Interfering; Transfection; Vasoconstrictor Agents

Proteinuria may contribute to progressive renal damage by inducing tubulointerstitial inflammation, fibrosis, and tubular cell injury and death, but the mechanisms underlying these pathologic changes remain largely unknown. Here, in a rat kidney proximal tubular cell line (RPTC), albumin induced apoptosis in a time- and dose-dependent manner. Caspase activation accompanied albumin-induced apoptosis, and general caspase inhibitors could suppress this activation. In addition, Bcl-2 transfection inhibited apoptosis and attenuated albumin-induced Bax translocation to mitochondria and cytochrome c release from the organelles, further confirming a role for the intrinsic pathway of apoptosis in albuminuria-associated tubular apoptosis. We observed phosphorylation and activation of PKC-delta early during treatment of RPTC cells with albumin. Rottlerin, a pharmacologic inhibitor of PKC-delta, suppressed albumin-induced Bax translocation, cytochrome c release, and apoptosis. Moreover, a dominant-negative mutant of PKC-delta blocked albumin-induced apoptosis in RPTC cells. In vivo, we observed activated PKC-delta in proteinuric kidneys of streptozotocin-induced diabetic mice and in kidneys after direct albumin overload. Notably, albumin overload induced apoptosis in renal tubules, which was less severe in PKC-delta-knockout mice. Taken together, these results suggest that activation of PKC-delta promotes tubular cell injury and death during albuminuria, broadening our understanding of the pathogenesis of progressive proteinuric kidney diseases.

Topics: Acetophenones; Albumins; Animals; Apoptosis; bcl-2-Associated X Protein; Benzopyrans; Caspases; Cell Line; Cytochromes c; Diabetes Mellitus, Experimental; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Kidney Tubules, Proximal; Male; Mice; Mice, Knockout; Protein Kinase C-delta; Proteinuria; Proto-Oncogene Proteins c-bcl-2; Rats; Streptozocin