Human Glomerular Podocytes Research Articles

#2474 TPPP3 maintains the stability of foot cytoskeleton architecture

Abstract Background and Aims Podocytes have a unique cytoskeleton architecture, and the disturbance of the skeleton architecture can lead to foot processes fusion and then proteinuria. Actin microfilaments dominate the cytoskeleton of the foot process, and microtubules and intermediate filaments dominate the synapses of the foot cells. The structural destruction of foot process actin is closely related to the injury of podocytes. The microtubule and actin often work together in cellular processes through stable connections. As the core structure of the skeleton network, microtubules play a key role in supporting and maintaining the skeleton network. However, the mechanism of microtubule structure destruction and podocyte injury remains unclear. Method Immunofluorescence and western blot were used to confirm the correlation between actin injury and decreased acetylation of tubulin in podocytes in vitro and in vivo. Single-cell RNA sequencing comprehensively characterized the expression of TPPP3 in human glomeruli. Immunofluorescence, immunohistochemical, western blot were used to determine the expression of TPPP3, α-tubulin, acetylated tubulin, actin microfilaments. Results In the case of podocyte injury, the acetylation level of tubulin decreased (P < 0.05). Reducing tubulin acetylation can lead to microfilaments injury (P < 0.05) and weakened podocyte motility (P < 0.05). TPPP3 was specifically expressed in human glomerular podocytes, and its expression level was significantly decreased after podocyte injury (P < 0.05). Knockdown of TPPP3 leaded to the level of microtubule acetylation decreased (P < 0.05) and microfilaments were damaged (P < 0.05) in podocytes. High acetylation levels of tubulin can partially restore actin microfilament damage caused by TPPP3 reduction in vivo and in vitro (P < 0.05). Conclusion TPPP3 is involved in maintaining the normal morphology and function of podocytes by stabilizing the actin microfilament skeleton structure by regulating the acetylation level of microtubules.

Down-Regulation Assession of Methyl CpG Binding Protein 2 in Diabetic Nephropathy

Diabetes nephropathy (DN) is the most significant microvascular complication of diabetes worldwide due to hyperglycemiainduced podocyte injury and apoptosis. The role of methyl CpG binding protein 2 (MECP2) has been observed, but its specific involvement in DN remains unclear. In this study, an in vitro DN model was established using human glomerular podocytes exposed to high glucose (HG, 30 mM). MECP2 expression was assessed using qRT-PCR and Western Blot. Proliferation and migration were evaluated through CCK-8 and transwell assays in both the HG group and the HG + MECP2 knockdown group. Apoptosis was assessed by flow cytometry and Western Blot. RNA-Sequencing identified differentially expressed genes (DEGs) between HG and HG+MECP2 knockdown groups, with subsequent enrichment analysis using KEGG and GSEA databases. Our results revealed elevated MECP2 expression in HG-treated podocytes compared to the control group. Podocytes with MECP2 knockdown displayed increased proliferation and migration compared to the HG group. MECP2 knockdown significantly inhibited HG-induced apoptosis in podocytes, with lower expression of pro-apoptotic protein (cleaved-caspase3, Bax, BAD, Desmin) and higher expression of anti-apoptotic protein Bcl-2 in the HG+MECP2 shRNA group. RNA sequencing identified 123 upregulated and 129 downregulated DEGs. Enrichment analysis highlighted apoptosis-related pathways like PPAR, TNF, p53, RELAXIN, WNT, and RAP1 signaling. Podocytes with MECP2 knockdown showed reduced apoptosis upon HG treatment. In summary, downregulation of MECP2 in podocytes effectively mitigated apoptosis caused by high glucose, suggesting a potential strategy to improve diabetes nephropathy outcomes.

#4996 TPPP3 REGULATES THE MICROTUBULE ACETYLATION AND PROMOTING YAP NUCLEAR RECRUITMENT IN HUMAN PODOCYTES

Abstract Background and Aims Minimal change disease (MCD) is a podocytopathy more commonly seen in children, but it also accounts for 10%–25% of adult NS. Patients with MCD manifest with abundant albuminuria, which is related to the damage of the glomerular filtration barrier. Podocytes are major components of the glomerular filtration barrier and play a crucial role in maintaining the integrity of the glomerular filtration barrier. Alterations in podocyte actin critically affect podocyte function. Tubulin polymerization-promoting protein family member 3 (TPPP3) is regulator of microtubule dynamic that has microtubule bundling activity. However, the mechanism of TPPP3 involved in podocyte apoptosis is remains unknown. Method Single-cell RNA sequencing comprehensively characterized the expression of TPPP3 in human glomeruli. We cultured human immortalized podocyte cells (HPC) in vitro, and constructed the HPC injury model. Immunofluorescence, western blot were used to determine the expression of TPPP3, α-tubulin, acetylated tubulin, phosopho-YAP1, Podocyte apoptosis was determined by Annexin V APC Apoptosis Detection Kit. Results TPPP3 was specifically expressed in human glomerular podocytes, and its expression level was significantly decreased in the cases of MCD and ADR induced podocyte injury(P<0.05). Knockdown of TPPP3 leaded to decreasing expression of acetylated tubulin in podocytes(P<0.05). Phosopho-YAP1 was also observed in the TPPP3 knockdown HPCs(P<0.05). Apoptosis was increased in the TPPP3 knockdown HPCs, suggesting TPPP3 may be related to the podocytes injury and apoptosis. Conclusion TPPP3 is involved with the apoptosis of podocytes by regulates nuclear recruitment of YAP which was promoted by microtubule acetylation.

Serum miRNA-30a-5p in Steroid Sensitive Idiopathic Nephrotic Syndrome in Indian Children: A Case-control Study

Introduction: Childhood Nephrotic Syndrome (NS) is a podocytopathy. Micro Ribonucleic Acid (miRNA), composed of 21-25 non coding nucleotides, regulates gene expression by inhibiting protein transcription by binding to complementary messenger RNA. The microRNA-30a is expressed in the human glomerular podocytes and collecting ducts. This microRNA protects the podocytes by targeting the Calcineurin-nuclear Factor of Activated T cells (NFATc) pathways. Serum microRNA30a-5p is a validated biomarker which is upregulated in NS. Aim: To determine the serum miRNA-30a-5p in steroid sensitive idiopathic NS in Indian children. Materials and Methods: This case-control study was conducted at the Department of Paediatrics, KS Hegde Medical Academy, Mangalore, Karnataka, India, from from January 2018 to June 2019. Thirty children with NS and age and gender matched controls were recruited. Relative expression of microRNA-30a5p was analysed by Real-Time quantitative Polymerase Chain Reaction (RT-qPCR). Estimations were done both in cases and controls at enrollment and also at four weeks when in remission in cases. The fold change was calculated as a power of cycle threshold. Statistical tests Kolmogorov-Smirnov test was used to establish the normality using Statistical Package for Social Sciences (SPSS) version 22.0. Results: There was upregulation of microRNA-30a-5p expression among children with NS with a significant fold change (~184) at enrollment. The levels declined, but remained above baseline (~6) after four weeks of treatment when compared to controls. The mean differences in delta threshold cycle and threshold cycle between the three groups were significant (p<0.001). There was no correlation with the biochemical parameters. Conclusion: The present study concludes that serum microRNA-30a-5p expression is upregulated in children with steroid sensitive NS in Indian children.

Apolipoprotein L1 (APOL1) cation current in HEK-293 cells and in human podocytes.

Two heterozygous missense variants (G1 and G2) of Apolipoprotein L1 (APOL1) found in individuals of recent African ancestry can attenuate the severity of infection by some forms of Trypanosoma brucei. However, these two variants within a broader African haplotype also increase the risk of kidney disease in Americans of African descent. Although overexpression of either variant G1 or G2 causes multiple pathogenic changes in cultured cells and transgenic mouse models, the mechanism(s) promoting kidney disease remain unclear.Human serum APOL1 kills trypanosomes through its cation channel activity, and cation channel activity of recombinant APOL1 has been reconstituted in lipid bilayers and proteoliposomes. Although APOL1 overexpression increases whole cell cation currents in HEK-293 cells, the ion channel activity of APOL1 has not been assessed in glomerular podocytes, the major site of APOL1-associated kidney diseases.We characterize APOL1-associated whole cell and on-cell cation currents in HEK-293T-Rex cells and demonstrate partial inhibition of currents by anti-APOL antibodies. We detect in primary human podocytes a similar cation current inducible by interferon-γ (IFNγ) and sensitive to inhibition by anti-APOL antibody as well as by a fragment of T. brucei Serum Resistance-Associated protein (SRA). CRISPR knockout of APOL1 in human primary podocytes abrogates the IFNγ-induced, antibody-sensitive current.Our novel characterization in HEK-293 cells of heterologous APOL1-associated cation conductance inhibited by anti-APOL antibody and our documentation in primary human glomerular podocytes of endogenous IFNγ-stimulated, APOL1-mediated, SRA and anti-APOL-sensitive ion channel activity together support APOL1-mediated channel activity as a therapeutic target for treatment of APOL1-associated kidney diseases.

Phosphorodiamidate morpholino targeting the 5′ untranslated region of the ZIKV RNA inhibits virus replication

BackgroundZika virus (ZIKV) infection has been associated with microcephaly in infants. Currently there is no treatment or vaccine. Here we explore the use of a morpholino oligonucleotide targeted to the 5′ untranslated region (5′-UTR) of the ZIKV RNA to prevent ZIKV replication. MethodsMorpholino DWK-1 inhibition of ZIKV replication in human glomerular podocytes was examined by qRT-PCR, reduction in ZIKV genome copy number, western blot analysis, immunofluorescence and proinflammatory cytokine gene expression. ResultsPodocytes pretreated with DWK-1 showed reduced levels of both viral mRNA and ZIKV E protein expression compared to controls. We observed suppression in proinflammatory gene expression for IFN-β (interferon β) RANTES (regulated on activation, normal T cell expressed and secreted), MIP-1α (macrophage inflammatory protein-1α), TNF-α (tumor necrosis factor-α) and IL1-α (interleukin 1-α) in ZIKV-infected podocytes pretreated with DWK-1. ConclusionsMorpholino DWK-1 targeting the ZIKV 5′-UTR effectively inhibits ZIKV replication and suppresses ZIKV-induced proinflammatory gene expression.

Diet-Induced Podocyte Dysfunction in Drosophila and Mammals

Diabetic nephropathy is a major cause of end-stage kidney disease. Characterized by progressive microvascular disease, most efforts have focused on injury to the glomerular endothelium. Recent work has suggested a role for the podocyte, a highly specialized component of the glomerular filtration barrier. Here, we demonstrate that the Drosophila nephrocyte, a cell analogous to the mammalian podocyte, displays defects that phenocopy aspects of diabetic nephropathy in animals fed chronic high dietary sucrose. Through functional studies, we identify an OGT-Polycomb-Knot-Sns pathway that links dietary sucrose to loss of the Nephrin ortholog Sns. Reducing OGT through genetic or drug means is sufficient to rescue loss of Sns, leading to overall extension of lifespan. We demonstrate upregulation of the Knot ortholog EBF2 in glomeruli of human diabetic nephropathy patients and a mouse ob/ob diabetes model. Furthermore, we demonstrate rescue of Nephrin expression and cell viability in ebf2(-/-) primary podocytes cultured in high glucose.

Effects of adiponectin on endoplasmic reticulum stress-mediated apoptosis and cell cytoskeleton and its mechanism of podocytes cultured in high glucose

To explore the effects of adiponectin (APN) on high glucose-induced endoplasmic reticulum stress (ERS)-mediated apoptosis and cytoskeleton. The conditionally immortal human glomerular podocytes were divided into normal glucose, mannitol, high glucose and high glucose with adiponectin groups. Flow cytometry was employed to assess cell apoptosis.Real-time polymerase chain reaction (PCR) and Western blot were used to detect the expressions of such ERS molecules as GRP78, CHOP and caspase 12 and desmin and TRPC6. Immunofluorescent staining was used to detect the changes in the skeleton of podocyte. (1) The apoptosis rate in high glucose group is significantly higher than the other groups (26.15% ± 1.38% vs 2.39% ± 0.58%, 4.84% ± 0.87%, 16.71% ± 1.15%, all P < 0.05). Compared with control group, the mRNA and protein expressions of GRP78, CHOP and caspase 12 were all up-regulated significantly in high glucose group (P < 0.05). The high glucose with adiponectin group could reduce the podocyte apoptosis by 10% and down-regulated the mRNA and protein expressions of GRP78, CHOP and caspase 12 versus high glucose group (all P < 0.05). (2) High glucose-induced podocyte caused the up-regulated expressions of TRPC6 and desmin (all P < 0.05) and it was inhibited by adiponectin (all P < 0.05). Additionally, immunofluorescent assay of high glucose-induced podocyte cytoskeleton showed disorderly F-actin and absent tensile fiber. Adiponectin prevented the F-actin cytoskeleton disruption under high glucose. Adiponectin plays a protective role in high glucose-induced podocyte through reducing endoplasmic reticulum stress-induced apoptosis and blunting the injury of cytoskeleton.

P27—Uptake of α-Galactosidase a in Human Glomerular Podocytes is Mediated by Various Receptors in Fabry Disease

P27—Uptake of α-Galactosidase a in Human Glomerular Podocytes is Mediated by Various Receptors in Fabry Disease

Cubilin is expressed in rat and human glomerular podocytes

The bulk of proteins filtered in the glomeruli are reabsorbed in the proximal tubule by endocytosis mediated by two multiligand receptors operating in concert, megalin and cubilin. Podocytes can also internalize protein and megalin; this was initially reported in rat proximal tubular and glomerular epithelial cells and has recently also been demonstrated in human podocytes. Cubilin, crucial for albumin reabsorption in the proximal tubule, has not been identified in glomerular epithelial cells. In the present study, we used immunocytochemistry and reverse transcription-polymerase chain reaction on laser-captured glomeruli to demonstrate synthesis and expression of cubilin in rat and human glomeruli. In parallel experiments, the expression of cubilin was studied in cultured podocytes. This study identifies cubilin in rat and human glomeruli according to a pattern similar to that reported for megalin. Cubilin revealed a surface expression but also intracellular expression in the podocytes. Our findings show that the podocytes display the two endocytic receptors which are responsible for the only documented process for protein reabsorption in proximal tubule cells.

Nephrin Is Critical for the Action of Insulin on Human Glomerular Podocytes

The leading causes of albuminuria and end-stage renal failure are secondary to abnormalities in the production or cellular action of insulin, including diabetes and hyperinsulinemic metabolic syndrome. The human glomerular podocyte is a critical cell for maintaining the filtration barrier of the kidney and preventing albuminuria. We have recently shown this cell to be insulin sensitive with respect to glucose uptake, with kinetics similar to muscle cells. We now show that the podocyte protein nephrin is essential for this process. Conditionally immortalized podocytes from two different patients with nephrin mutations (natural human nephrin mutant models) were unresponsive to insulin. Knocking nephrin down with siRNA in wild-type podocytes abrogated the insulin response, and stable nephrin transfection of nephrin-deficient podocytes rescued their insulin response. Mechanistically, we show that nephrin allows the GLUT1- and GLUT4-rich vesicles to fuse with the membrane of this cell. Furthermore, we show that the COOH of nephrin interacts with the vesicular SNARE protein VAMP2 in vitro and ex vivo (using yeast-2 hybrid and coimmunoprecipitation studies). This work demonstrates a previously unsuspected role of nephrin in vesicular docking and insulin responsiveness of podocytes.

Upregulation of Id-1 via BMP-2 receptors induces reactive oxygen species in podocytes

Bone morphogenetic proteins (BMPs) are secreted signaling molecules, which play a major role in kidney development and disease. Here, we show the existence of mRNA for BMP-2 and for the BMP receptors BMPR1A, BMPR1B, BMPRII, ACVR1A, ACVR2, and ACVR2B in differentiated mouse podocytes and the protein expression of BMPR1A in human glomerular podocytes. BMP-2 dose dependently increases the free cytosolic Ca(2+) concentration in podocytes proving the existence of a functional receptor in these cells. Recent data indicate that in a myoblastic cell line and in a breast cancer cell line, BMP-2 increases the expression of Id-1, a negative regulator of basic helix-loop-helix transcription factors, but the role of BMP-2 stimulated Id-1 expression in the kidney has not been further characterized. Here, we show that BMP-2 increases the expression of Id-1 in differentiated podocytes. To investigate a role of Id-1 for podocyte function, overexpression of Id-1 was induced in differentiated mouse podocytes. Id-1-overexpressing podocytes show an increased NADPH-dependent production of reactive oxygen species (ROS). This effect can be evoked by BMP-2 and can be antagonized by anti-Id-1 antisense oligonucleotides. The data indicate that BMP-2 may, via an increased expression of Id-1 and an increased generation of ROS, contribute to important cellular functions in podocytes. ROS supposedly play a major role in cell adhesion, cell injury, ion transport, fibrogenesis, angiogenesis and are involved in the pathogenesis of membranous nephropathy.

Characterization of a B2-bradykinin receptor in human glomerular podocytes.

We characterized bradykinin (BK) receptors in a human line of glomerular visceral epithelial cells (hGVEC) transfected by the SV40 virus. [3H]BK bound specifically in a manner consistent with a single high-affinity site. Scatchard analysis yielded dissociation constant and maximum binding values of 0.28 +/- 0.04 nM and 76.6 +/- 4.9 fmol/mg, respectively. Competition binding studies with selective BK type 2 (Hoe-140) receptor antagonist and type 1 ([des-Arg9]BK) receptor agonist showed that hGVEC only expressed type 2 receptors, and this was confirmed by reverse transcriptase-polymerase chain reaction and ribonuclease protection assay. BK stimulated intracellular calcium ion concentration ([Ca2+]i) release in a dose-dependent manner with a threshold at 1 nM. Hoe-140, in contrast with [des-Arg9]BK, abolished this effect. [Ca2+]i stimulation was also inhibited by thapsigargin, an inhibitor of Ca(2+)-adenosinetriphosphatase. Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid attenuated but did not suppress the [Ca2+]i peak. These results associated with the stimulatory effect of BK on inositol phosphate production indicated that [Ca2+]i stimulation was produced both by [Ca2+] mobilization from its intracellular stores and by [Ca2+] entry into the cells. In conclusion, hGVEC express specific type 2 BK receptors that enable specific BK-induced responses.

Localization of parathyroid hormone/parathyroid hormone-related peptide receptor mRNA in kidney.

In kidney, parathyroid hormone (PTH) exerts differential distal effects along the nephron. To define cells expressing receptors for PTH in kidney, we localized PTH/ PTH-related peptide (PTH/PTHrP) receptor mRNA in rat kidney by in situ hybridization. PTH/PTHrP receptor mRNA is localized to glomerular podocytes, convoluted and straight proximal tubules, the cortical portion of thick ascending limbs, and distal convoluted tubules, but was not detected in the thin limb of Henle's loop or in collecting ducts. Northern blot analysis showed that cultured human glomerular podocytes express a unique 4.0-kb PTH/PTHrP receptor transcript but do not express detectable levels of the common approximately 2.4-kb transcript found in whole kidney and in many other tissues. Whereas the tubular localization of PTH/PTHrP receptor mRNA coincides well with previously known sites of PTH action, the intense expression and the unique size of the PTH/PTHrP receptor transcript in glomerular podocytes suggest that PTH and/or PTHrP may play a role(s) in glomerular function.

Functional endothelin 1 receptors on human glomerular podocytes and mesangial cells.

To determine if endothelin 1 (Et1) receptors are present in human glomeruli, and which glomerular cells possess these receptors, 125I Et1 binding to isolated glomeruli and cultured glomerular mesangial and epithelial cells was studied. The latter were identified as podocytes. We demonstrated that Et1 binds specifically and reversibly to isolated human glomeruli and to cultured glomerular mesangial and epithelial cells. Scatchard analysis of competitive inhibition of 125I Et1 binding gave the following results (m +/- SEM, n = 3): isolated glomeruli, Kd = 4.2 +/- 2.1 x 10(-10) M, Bmax = 8.1 +/- 1.2 x 10(10) sites/mg protein; mesangial cells, Kd = 5.2 +/- 1.5 x 10(-10) M, Bmax = 1.87 +/- 0.49 x 10(4) sites/cell; epithelial cells, Kd = 7.2 +/- 1.5 x 10(-10) M, Bmax = 2.46 +/- 0.15 x 10(4) sites/cell. These receptors seem to be functional, since in both mesangial and epithelial cells Et1 induces a rapid and transient increase in intracellular [Ca2+]i. All these results indicate that Et1 may regulate glomerular filtration rate through an autocrine-paracrine pathway on mesangial cells and on podocytes.