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Transforming growth factor (TGF)-β1 contributes to podocyte injury in various glomerular diseases, including diabetic kidney disease, probably at least in part by attenuating the expression of Wilms' tumor 1 (WT1). However, the precise mechanisms remain to be defined. We performed miRNA microarray analysis in a human podocyte cell line cultured with TGF-β1 to examine the roles of miRNAs in podocyte damage. The microarray analysis identified miR-143-3p as the miRNA with the greatest increase following exposure to TGF-β1. Quantitative RT-PCR confirmed a significant increase in the miR-143-3p/145-5p cluster in TGF-β1-supplemented cultured podocytes and demonstrated upregulation of miR-143-3p in the glomeruli of mice with type 2 diabetes. Ectopic expression of miR-143-3p and miR-145-5p suppressed WT1 expression in cultured podocytes. Furthermore, inhibition of Smad or mammalian target of rapamycin signaling each partially reversed the TGF-β1-induced increase in miR-143-3p/145-5p and decrease in WT1. In conclusion, TGF-β1 induces expression of miR-143-3p/145-5p in part through Smad and mammalian target of rapamycin pathways, and miR-143-3p/145-5p reduces expression of WT1 in cultured human podocytes. miR-143-3p/145-5p may contribute to TGF-β1-induced podocyte injury. [ABSTRACT FROM AUTHOR]

Apolipoprotein L1 (ApoL1) genetic variants are strongly associated with kidney diseases. We investigated the role of ApoL1 variants in monocyte differentiation and eicosanoid production in macrophages, as activated tissue macrophages in kidney might contribute to kidney injury. In human monocyte THP-1 cells, transient overexpression of ApoL1 (G0, G1, G2) by transfection resulted in a 5- to 11-fold increase in CD14 and CD68 gene expression, similar to that seen with phorbol-12-myristate acetate treatment. All ApoL1 variants caused monocytes to differentiate into atypical M1 macrophages with marked increase in M1 markers CD80, TNF, IL1B, and IL6 and modest increase in the M2 marker CD163 compared with control cells. ApoL1-G1 transfection induced additional CD206 and TGFB1 expression, and ApoL1-G2 transfection induced additional CD204 and TGFB1 expression. Gene expression of prostaglandin E2 (PGE2) synthase and thromboxane synthase and both gene and protein expression of cyclooxygenase-2 (COX-2) were increased by ApoL1-G1 and -G2 variants compared with -G0 transfection. Higher levels of PGE2 and thromboxane B2, a stable metabolite of thromboxane A2, and transforming growth factor (TGF)-β1 were released into the supernatant of cultured THP-1 cells transfected with ApoL1-G1 and -G2, but not -G0. The increase in PGE2, thromboxane B2, and TGF-β1 was inhibited by COX-2-specific inhibitor CAY10404 but not by COX-1-specific inhibitor SC-560. These results demonstrate a novel role of ApoL1 variants in the regulation of monocyte differentiation and eicosanoid metabolism, which could modify the immune response and promote inflammatory signaling within the local targeted organs and tissues including the kidney. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are noncoding RNAs that regulate posttranscriptional gene expression. In this study we characterized the circulating and urinary miRNA pattern associated with reduced glomerular filtration rate, using Affymetrix GeneChip miR 4.0 in 28 patients with chronic kidney disease (CKD). Top miRNA discoveries from the human studies were validated in an Alb/TGF β mouse model of CKD, and in rat renal proximal tubular cells (NRK52E) exposed to TGFβ1. Plasma and urinary levels of procollagen III N-terminal propeptide and collagen IV were elevated in patients with decreased estimated glomerular filtration rate (eGFR). Expression of 384 urinary and 266 circulatory miRNAs were significantly different between CKD patients with eGFR ≥30 vs. <30 ml⋅min-1⋅1.73 m-2. Pathway analysis mapped multiple miRNAs to TGFβ signaling-related mRNA targets. Specifically, Let-7a was significantly downregulated, and miR-130a was significantly upregulated, in urine of patients with eGFR <30; miR-1825 and miR-1281 were upregulated in both urine and plasma of patients with decreased eGFR; and miR-423 was significantly downregulated in plasma of patients with decreased eGFR. miRNA expression in urine and plasma of Alb/TGFβ mice generally resembled and confirmed most, although not all, of the observations from the human studies. In response to TGFβ1 exposure, rat renal proximal tubular cells overexpressed miR-1825 and downregulated miR-423. Thus, miRNA are associated with kidney fibrosis, and specific urinary and plasma miRNA profile may have diagnostic and prognostic utility in CKD. [ABSTRACT FROM AUTHOR]

The renal glomerulus forms a selective filtration barrier that allows the passage of water, ions, and small solutes into the urinary space while restricting the passage of cells and macromolecules. The three layers of the glomerular filtration barrier include the vascular endothelium, glomerular basement membrane (GBM), and podocyte epithelium. Podocytes are capable of internalizing albumin and are hypothesized to clear proteins that traverse the GBM. The present study followed the fate of FITC-labeled albumin to establish the mechanisms of albumin endocytosis and processing by podocytes. Confocal imaging and total internal reflection fluorescence microscopy of immortalized human podocytes showed FITC-albumin endocytosis occurred preferentially across the basal membrane. Inhibition of clathrin-mediated endocytosis and caveolae-mediated endocytosis demonstrated that the majority of FITC-albumin entered podocytes through caveolae. Once internalized, FITC-albumin colocalized with EEA1 and LAMP1, endocytic markers, and with the neonatal Fc receptor, a marker for transcytosis. After preloading podocytes with FITC-albumin, the majority of loaded FITC-albumin was lost over the subsequent 60 min of incubation. A portion of the loss of albumin occurred via lysosomal degradation as pretreatment with leupeptin, a lysosomal protease inhibitor, partially inhibited the loss of FITC-albumin. Consistent with transcytosis of albumin, preloaded podocytes also progressively released FITC-albumin into the extracellular media. These studies confirm the ability of podocytes to endocytose albumin and provide mechanistic insight into cellular mechanisms and fates of albumin handling in podocytes. [ABSTRACT FROM AUTHOR]

Transforming growth factor (TGF)-β has been associated with podocyte injury; we have examined its effect on podocyte bioenergetics. We studied transformed mouse podocytes, exposed to TGF-β1, using a label-free assay system, Seahorse XF24, which measures oxygen consumption rates (OCR) and extracellular acidification rates (ECAR). Both basal OCR and ATP generation-coupled OCR were significantly higher in podocytes exposed to 0.3-10 ng/ml of TGF-β1 for 24, 48, and 72 h. TGF-β1 (3 ng/ml) increased oxidative capacity 75%, and 96% relative to control after 48 and 72 h, respectively. ATP content was increased 19% and 30% relative to control after a 48- and 72-h exposure, respectively. Under conditions of maximal mitochondrial function, TGF-β1 increased palmitate-driven OCR by 49%. Thus, TGF-β1 increases mitochondrial oxygen consumption and ATP generation in the presence of diverse energy substrates. TGF-β1 did not increase cell number or mitochondrial DNA copy number but did increase mitochondrial membrane potential (MMP), which could explain the OCR increase. Reactive oxygen species (ROS) increased by 32% after TGF-β1 exposure for 48 h. TGF-β activated the mammalian target of rapamycin (mTOR) pathway, and rapamycin reduced the TGF-β1-stimulated increases in OCR, ECAR, ATP generation, cellular metabolic activity, and protein generation. Our data suggest that TGF-β1, acting, in part, via mTOR, increases mitochondrial MMP and OCR, resulting in increased ROS generation and that this may contribute to podocyte injury. [ABSTRACT FROM AUTHOR]

Gasser DL, Winkler CA, Peng M, An P, McKenzie LM, Kirk GD, Shi Y, Xie LX, Marbois BN, Clarke CF, Kopp JB. Focal segmental glomerulosclerosis is associated with a PDSS2 haplotype and, independently, with a decreased content of coenzyme Q10. Am J Physiol Renal Physiol 305: F1228-F1238, 2013. First published August 7, 2013; doi:10.1152/ajprenal.00143.2013.--Focal segmental glomerulosclerosis (FSGS) and collapsing glomerulopathy are common causes of nephrotic syndrome. Variants in >20 genes, including genes critical for mitochondrial function, have been associated with these podocyte diseases. One such gene, PDSS2, is required for synthesis of the decaprenyl tail of coenzyme Q10 (Q10) in humans. The mouse gene Pdss2 is mutated in the kd/kd mouse model of collapsing glomerulopathy. We examined the hypothesis that human PDSS2 polymorphisms are associated with podocyte diseases. We genotyped 377 patients with primary FSGS or collapsing glomerulopathy, together with 900 controls, for 9 single-nucleotide polymorphisms in the PDSS2 gene in a case-control study. Subjects included 247 African American (AA) and 130 European American (EA) patients and 641 AA and 259 EA controls. Among EAs, a pair of proxy SNPs was significantly associated with podocyte disease, and patients homozygous for one PDSS2 haplotype had a strongly increased risk for podocyte disease. By contrast, the distribution of PDSS2 genotypes and haplotypes was similar in AA patients and controls. Thus a PDSS2 haplotype, which has a frequency of 13% in the EA control population and a homozygote frequency of 1.2%, is associated with a significantly increased risk for FSGS and collapsing glomerulopathy in EAs. Lymphoblastoid cell lines from FSGS patients had significantly less Q10 than cell lines from controls; contrary to expectation, this finding was independent of PDSS2 haplotype. These results suggest that FSGS patients have Q10 deficiency and that this deficiency is manifested in patient-derived lymphoblastoid cell lines. [ABSTRACT FROM AUTHOR]

Renal Wilms' tumor-1 (WT-1) staining is used to detect podocyte loss in kidney biopsies. We aimed to determine if urinary exosomal WT-1 could serve as a noninvasive biomarker of podocyte injury. We examined WT-1 by Western blot in a human podocyte-like cell line, a mouse model of podocyte injury, and human subjects with podocyte disorders. WT-1 was detected in exosomal fraction of the conditioned media from podocytes and increased 48 h after hTGF-1 stimulation. Cellular WT-1 decreased in podocytes following hTGF-1 incubation. In mice with induced podocyte injury, urinary exosomal WT-1 was detected 1 wk earlier than albuminuria and also tracked the effects of angiotensin receptor blocker (ARB) treatment. In addition, urinary exosomal WT-1 levels at 1 wk post-injury correlated with the severity of glomerular injury at 3 wk later. In human subjects, urinary exosomal WT-1 was significantly increased in focal segmental glomerulosclerosis (FSGS) patients compared with healthy volunteers or steroid-sensitive nephrotic syndrome (SSNS) patients. Urinary exosomal WT-1 was also significantly decreased in patients in remission for either FSGS or SSNS or following steroid treatment in six SSNS subjects. We conclude that urinary exosomal WT-1 is a promising noninvasive biomarker with apparent podocyte specificity that can detect early progression and treatment-induced regression of podocyte injury in FSGS or SSNS. These results warrant longitudinal, prospective studies in a large cohort with a range of podocyte diseases. [ABSTRACT FROM AUTHOR]

We transformed mous podocytes by ectopic expression of cyclin-dependent kinase 4 (CDK4). Compared with podocytes transformed with a thermo-sensitive SV40 large T antigen mutant tsA58U19 (tsT podocytes), podocytes transformed with CDK4 (CDK4 podocytes) exhibited significantly higher expression of nephrin mRNA. Synaptopodin mRNA expression was significantly lower in CDK4 podocytes and in tsT podocytes under growth-permissive conditions (33°C) compared with tsT podocytes under growth-restricted conditions (37°C), which suggests a role for cell cycle arrest in synaptopodin mRNA expression. Confluent CDK4 podocytes showed significantly higher mRNA expression levels for nephrin, synaptopodin, Wilms tumor I, podocalyxin, and P-cadherin compared with subconfluent cultures. We carried out experiments to clarify roles of various factors in the confluent podocyte cultures; our findings indicate that cell-cell contact promotes expression of five podocyte marker genes studied, that cellular quiescence increases synaptopodin and podocalyxin mRNA expression, and that soluble factors play a role in nephrin mRNA expression. Our findings suggest that CDK4 podocytes are useful tools to study podocyte biology. Furthermore, the role of cell-cell contact in podocyte gene expression may have relevance for podocyte function in vivo. [ABSTRACT FROM AUTHOR]

The podocyte secretory proteome may influence the phenotype of adjacent podocytes, endothelial cells, parietal epithelial cells, and tubular epithelial cells but has not been systematically characterized. We have initiated studies to characterize this proteome, with the goal of further understanding the podocyte cell biology. We cultured differentiated conditionally immortalized human podocytes and subjected the proteins in conditioned medium to mass spectrometry. At a false discovery rate of <3%, we identified 111 candidates from conditioned medium, including 44 proteins that have signal peptides or are described as secreted proteins in the UniProt database. As validation, we confirmed that one of these proteins, insulin-like growth factor-binding protein-related protein-I (IGFBP-rPI), was expressed in mRNA and protein of cultured podocytes. In addition, transforming growth factor-J31 stimulation increased IGFBP-rPI in conditioned medium. We analyzed IGFBP-rP1 glomerular expressio in a mouse model of human immunodeficiency virus-associated nephropathy. IGFBP-rP 1 was absent from podocytes of normal mice and was expressed in podocytes and pseudocrescents of transgenic mice, where it was coexpressed with desmin, a podocyte injury marker. We conclude that IGFBP-rP1 may be a product of injured podocytes. Further analysis of the podocyte secretory proteomc may identify biomarkers of podocyte injury. [ABSTRACT FROM AUTHOR]

Studies have shown that certain cells of the glomerular tuft begin to express proteins considered unique to other cell types upon injury. Little is known about the response of parietal epithelial cells (PEC) to injury. To determine whether PECs change their phenotype upon injury to also express proteins traditionally considered podocyte specific, the following four models of glomerular disease were studied: the transforming growth factor (TGF)-β1 transgenic mouse model of global glomerulosclerosis, the adriamycin model of focal segmental glomerulosclerosis (FSGS), the anti-glomerular basement membrane (GBM) model of crescentic glomerulonephritis, and the passive Heymann nephritis model of membranous nephropathy. Double immunostaining was performed with antibodies to podocyte-specific proteins (synaptopodin and Wilms' tumor 1) and antibodies to PEC specific proteins (paired box gene 8 and claudin-1). No double staining was detected in normal mice. In contrast, the results showed a statistical increase in the number of cells attached to Bowman basement membrane that were double-positive for both podocyte/PEC proteins in TGF-β;1 transgenic, anti-GBM, and membranous animals. Double-positive cells for both podocyte and PEC proteins were also statistically increased in the glomerular tuft in TGF-β1 transgenic, anti-GBM, and FSGS mice. These results are consistent with glomerular cells coexpressing podocyte and PEC proteins in experimental glomerular disease, but not under normal circumstances. [ABSTRACT FROM AUTHOR]

Evidence suggests that loss of podocytes into urine contributes to development of glomerular diseases; shed podocytes are frequently viable and proliferate in culture conditions. To determine the phenotypic characteristics of viable urinary cells derived from human subjects, we established long-term urinary cell culture from two patients with focal segmental glomerulosclerosis and two healthy volunteers, via transformation with the thermosensitive SV40 large T antigen (U19tsA58) together with human telomerase (hTERT). Characterization of arbitrarily selected two clonal cell lines from each human subject was carried out. mRNA expression for the podocyte markers synaptopodin, nestin, and CD2AP were detected in all eight clones. Podocin mRNA was absent from all eight clones. The expression of nephrin, Wilms' tumor 1 (WT1), and podocalyxin mRNA varied among the clones, which may be due to transformation and/or cloning. These results suggest that podocyte cell lines can be established consistently from human urine. The generation of podocyte cell lines from urine of patients and healthy volunteers is novel and will help to advance studies of podocyte cell biology. Further improvements in the approaches to cell transformation and/or cell culture techniques are needed to allow cultured podocytes to fully reproduce in vivo characteristics. [ABSTRACT FROM AUTHOR]

Conditional transgenic mice that express one of the human immunodeficiency virus (HIV)- I accessory genes, vpr, selectively in podocytes using a podocin promoter and a tetracycline-inducible system develop renal injuries similar to those of patients with HIV-associated nephropathy (HIVAN). We have shown that a heminephrectomy accelerates podocyte injury, which is alleviated by angiotensin II (ANG II) type 1 receptor blocker (ARB). The current study further explores the role of ANG II in the genesis of HI VAN in this murine model. With ANG II infusion, heavy proteinuria was observed at I wk after the initiation of doxycycline administration to induce vpr expression in podocytes. Severe morphological and phenotypical changes in the podocytes were observed at 2 wk, together with extensive glomerulosclerosis. Norepinephrine infusion, instead of ANG II, increased the systemic blood pressure to the same level as that achieved using ANG II. However, albuminuria and glomerular injury were modest in norepinephrine-infused mice. Treatment with an ARB, olmesartan, almost completely inhibited glomerular injury. In contrast, lowering the blood pressure with a vasodilator, hydralazine, partially decreased albuminuria but did not produce any histological changes. ANG IT infusion alone without doxycycline resulted in a lower level of albuminuria and minimal histological changes. These data demonstrate that excessive ANG II accelerates vpr-induced podocyte injury in a mouse model of HTVAN. [ABSTRACT FROM AUTHOR]

Progressive fibrosis is a cause of progressive organ dysfunction. Lack of quantitative in vitro models of fibrosis accounts, at least partially, for the slow progress in developing effective antifibrotic drugs. Here, we report two complementary in vitro models of fibrosis suitable for high-throughput screening. We found that, in mesangial cells and renal fibroblasts grown in eight-well chamber slides, transforming growth factor-β1 (TGF-β1) disrupted the cell monolayer and induced cell migration into nodules in a dose-, time- and Smad3-dependent manner. The nodules contained increased interstitial collagens and showed an increased collagen I:IV ratio. Nodules are likely a biological consequence of TGF-β1-induced matrix overexpression since they were mimicked by addition of collagen 1 to the cell culture medium. TGF-β1-induced nodule formation was inhibited by vacuum ionized gas treatment of the plate surface. This blockage was further enhanced by precoating plates with matrix proteins but was prevented, at least in part, by poly-L-lysine (PLL). We have established two cell-based models of TGF-β-induced fibrogenesis, using mesangial cells or fibroblasts cultured in matrix protein or PLL-coated 96-well plates, on which TGF-β1-induced two-dimensional matrix accumulation, three-dimensional nodule formation, and monolayer disruption can be quantitated either spectrophotometrically or by using a colony counter, respectively. As a proof of principle, chemical inhibitors of Alk5 and the antifibrotic compound tranilast were shown to have inhibitory activities in both assays. [ABSTRACT FROM AUTHOR]

Urinary exosomes are excreted from all nephron segments and may serve as biomarkers for classifying renal diseases. Isolation of urinary exosomes by the established ultracentrifugation method has some limitations for use in a clinical laboratory. We sought a rapid and simple way to obtain urinary exosomes. We used a commercially available nanomembrane concentrator to enrich exosomes from urine by centrifugation at 3,000 g for 10-30 min. Urinary exosomal markers tumor susceptibility gene 101, aquaporin-2, neuron-specific enolase, annexin V, angiotensin-converting enzyme, and podocalyxin (PODXL) were recovered from the nanomembrane concentrator and detected by Western blotting, and typical features of urinary vesicles were found by electron microscopy. Exosomal markers were detected in as little as 0.5 ml of urine. By the nanomembrane method, exosomal proteins could be recovered from urine samples frozen at -80°C or refrigerated over- night at 4°C then stored at -80°C. By enriching exosomes we could detect PODXL, a podocyte marker, which decreased by 71% in five male patients with focal segmental glomerulosclerosis and abundant proteinuria. We conclude that 1) use of a nanomembrane concentrator simplifies and accelerates the enrichment of urinary exosomes; and 2) the nanomembrane concentrator can concentrate exosomal proteins from clinical urine samples. This enhanced method may accelerate the translation of urinary exosomal biomarkers from bench to bedside for the diagnosis, classification, and prognostication of renal diseases. [ABSTRACT FROM AUTHOR]

Gasser DL, Winkler CA, Peng M, An P, McKenzie LM, Kirk GD, Shi Y, Xie LX, Marbois BN, Clarke CF, Kopp JB. Focal segmental glomerulosclerosis is associated with a PDSS2 haplotype and, independently, with a decreased content of coenzyme Q10. Am J Physiol Renal Physiol 305: F1228-F1238, 2013. First published August 7, 2013; doi:10.1152/ajprenal.00143.2013.--Focal segmental glomerulosclerosis (FSGS) and collapsing glomerulopathy are common causes of nephrotic syndrome. Variants in >20 genes, including genes critical for mitochondrial function, have been associated with these podocyte diseases. One such gene, PDSS2, is required for synthesis of the decaprenyl tail of coenzyme Q10 (Q10) in humans. The mouse gene Pdss2 is mutated in the kd/kd mouse model of collapsing glomerulopathy. We examined the hypothesis that human PDSS2 polymorphisms are associated with podocyte diseases. We genotyped 377 patients with primary FSGS or collapsing glomerulopathy, together with 900 controls, for 9 single-nucleotide polymorphisms in the PDSS2 gene in a case-control study. Subjects included 247 African American (AA) and 130 European American (EA) patients and 641 AA and 259 EA controls. Among EAs, a pair of proxy SNPs was significantly associated with podocyte disease, and patients homozygous for one PDSS2 haplotype had a strongly increased risk for podocyte disease. By contrast, the distribution of PDSS2 genotypes and haplotypes was similar in AA patients and controls. Thus a PDSS2 haplotype, which has a frequency of 13% in the EA control population and a homozygote frequency of 1.2%, is associated with a significantly increased risk for FSGS and collapsing glomerulopathy in EAs. Lymphoblastoid cell lines from FSGS patients had significantly less Q10 than cell lines from controls; contrary to expectation, this finding was independent of PDSS2 haplotype. These results suggest that FSGS patients have Q10 deficiency and that this deficiency is manifested in patient-derived lymphoblastoid cell lines. [ABSTRACT FROM AUTHOR]