Human Kidney Mesangial Cells Research Articles

Abstract 4070: β-arrestin-2 mediated regulation of plasminogen activator inhibitor Type 1.

Abstract Several studies described the elevated levels of plasminogen activated Inhibitor-1 (PAI-1) protein in malignant tissues and indicated the potential use as a therapeutic target. In bladder cancer, higher levels of PAI-1 were found in tumor tissues compared to normal tissues. Urinary levels of PAI-1 protein in combination with other markers resulted in higher sensitivity and specificity in detecting bladder cancer. In human kidney mesangial cells, the thromboxane A2 (TP) receptor agonist stimulation increased PAI-1 mRNA and protein. Our lab indicated that TP receptor is highly expressed in bladder cancer tissues and inversely correlated with patients` survival. We also indicated the TPβ isoform can induce malignant transformation in immortalized non-transformed urothelial cells. Our published data indicated that U46619 dependent stimulation of cell migration is β-arrestin-2 dependent. The purpose of the current study is to investigate the mechanism whereby TP stimulation regulates PAI-1 expression. The protein levels of PAI-1 were found to be increased in bladder cancer cells following stimulation with TP agonist (U46619). Tetracycline inducible shRNA knock-down of β-arrestin-2 resulted in a significant reduction of PAI-1 protein, but not mRNA in bladder cancer cells and also resulted in a significant reduction of trans-well cell migration and invasion. Our data suggest that the agonist mediated increase in PAI-1 is regulated at the post-transcriptional level. The role of β-arrestin-2 KD on agonist dependent stimulation remains to be determined. Citation Format: Philip M. Sobolesky, Tanyau Zhou, Arielle Gorstein, Julie A. Woolworth, Omar Moussa. β-arrestin-2 mediated regulation of plasminogen activator inhibitor Type 1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4070. doi:10.1158/1538-7445.AM2013-4070

Geldanamycin Derivative Ameliorates High Fat Diet-Induced Renal Failure in Diabetes

Diabetic nephropathy is a serious complication of longstanding diabetes and its pathogenesis remains unclear. Oxidative stress may play a critical role in the pathogenesis and progression of diabetic nephropathy. Our previous studies have demonstrated that polyunsaturated fatty acids (PUFA) induce peroxynitrite generation in primary human kidney mesangial cells and heat shock protein 90β1 (hsp90β1) is indispensable for the PUFA action. Here we investigated the effects of high fat diet (HFD) on kidney function and structure of db/db mice, a widely used rodent model of type 2 diabetes. Our results indicated that HFD dramatically increased the 24 h-urine output and worsened albuminuria in db/db mice. Discontinuation of HFD reversed the exacerbated albuminuria but not the increased urine output. Prolonged HFD feeding resulted in early death of db/db mice, which was associated with oliguria and anuria. Treatment with the geldanamycin derivative, 17-(dimethylaminoehtylamino)-17-demethoxygeldanamycin (17-DMAG), an hsp90 inhibitor, preserved kidney function, and ameliorated glomerular and tubular damage by HFD. 17-DMAG also significantly extended survival of the animals and protected them from the high mortality associated with renal failure. The benefit effect of 17-DMAG on renal function and structure was associated with a decreased level of kidney nitrotyrosine and a diminished kidney mitochondrial Ca2+ efflux in HFD-fed db/db mice. These results suggest that hsp90β1 is a potential target for the treatment of nephropathy and renal failure in diabetes.

Generation of Induced Pluripotent Stem Cells from Human Kidney Mesangial Cells

Glomerular injury and podocyte loss leads to secondary tubulointerstitial damage and the development of fibrosis. The possibility of genetically reprogramming adult cells, termed induced pluripotent stem cells (iPS), may pave the way for patient-specific stem-cell-based therapies. Here, we reprogrammed normal human mesangial cells to pluripotency by retroviral transduction using defined factors (OCT4, SOX2, KLF4 and c-Myc). The kidney iPS (kiPS) cells resembled human embryonic stem-cell-like colonies in morphology and gene expression: They were alkaline phosphatase-positive; expressed OCT3/4, TRA-1 to 60 and TRA-1 to 81 proteins; and showed downregulation of mesangial cell markers. Quantitative (qPCR) showed that kiPS cells expressed genes analogous to embryonic stem cells and exhibited silencing of the retroviral transgenes by the fourth passage of differentiation. Furthermore, kiPS cells formed embryoid bodies and expressed markers of all three germ layers. The injection of undifferentiated kiPS colonies into immunodeficient mice formed teratomas, thereby demonstrating pluripotency. These results suggest that reprogrammed kidney induced pluripotent stem cells may aid the study of genetic kidney diseases and lead to the development of novel therapies.

TNF-like weak inducer of apoptosis (TWEAK) induces inflammatory and proliferative effects in human kidney cells

Members of the TNF-ligand and receptor superfamilies are important in the pathogenesis of lupus nephritis, a major cause of mortality and morbidity in SLE. TWEAK, a member of the TNF-ligand superfamily, is markedly increased in urine from patients with active lupus nephritis, and urinary TWEAK levels significantly correlate with renal disease activity. To support a possible role of TWEAK in the pathogenesis of lupus nephritis and other inflammatory nephritides, we examined the effects of TWEAK in human kidney mesangial cells, podocytes and tubular cells, following our demonstration of the presence of the TWEAK receptor Fn14 on these cells. We found that TWEAK induces human kidney cells to express multiple inflammatory mediators, including RANTES, MCP-1, IP-10, MIP-1α, ICAM-1, and VCAM-1. Cytokine production is mediated through NF-κB activation, and is inhibited by anti-TWEAK monoclonal antibodies. TWEAK stimulated chemokines induced migration of human PBMC, particularly monocytes/macrophages. Furthermore, we found that TWEAK promotes kidney infiltration of inflammatory cells, and stimulates proliferation of kidney cells in vitro and in vivo. Thus, TWEAK may play an important pathogenic role in the development of glomerulonephritis by promoting a local inflammatory environment and inducing kidney cell proliferation. Blocking TWEAK/Fn14 interactions may be a promising therapeutic target in immune-mediated renal diseases.

MAP-kinase activity necessary for TGFβ1-stimulated mesangial cell type I collagen expression requires adhesion-dependent phosphorylation of FAK tyrosine 397

The signals mediating transforming growth factor beta (TGFbeta)-stimulated kidney fibrogenesis are poorly understood. We previously reported TGFbeta-stimulated, Smad-mediated collagen production by human kidney mesangial cells, and that ERK MAP kinase activity optimizes collagen expression and enhances phosphorylation of the Smad3 linker region. Furthermore, we showed that disrupting cytoskeletal integrity decreases type I collagen production. Focal adhesion kinase (FAK, PTK2) activity could integrate these findings. Adhesion-dependent FAK Y397 phosphorylation was detected basally, whereas FAK Y925 phosphorylation was TGFbeta1-dependent. By immunocytochemistry, TGFbeta1 stimulated the merging of phosphorylated FAK with the ends of thickening stress fibers. Cells cultured on poly-L-lysine (pLL) to promote integrin-independent attachment spread less than those on control substrate and failed to demonstrate focal adhesion (FA) engagement with F-actin. FAK Y397 phosphorylation and ERK activity were also decreased under these conditions. In cells with decreased FAK Y397 phosphorylation from either plating on pLL or overexpressing a FAK Y397F point mutant, serine phosphorylation of the Smad linker region, but not of the C-terminus, was reduced. Y397F and Y925F FAK point mutants inhibited TGFbeta-induced Elk-Gal activity, but only the Y397F mutant inhibited TGFbeta-stimulated collagen-promoter activity. The inhibition by the Y397F mutant or by culture on pLL was prevented by co-transfection of constitutively active ERK MAP kinase kinase (MEK), suggesting that FAK Y397 phosphorylation promotes collagen expression via ERK MAP kinase activity. Finally, Y397 FAK phosphorylation, and both C-terminal and linker-region Smad3 phosphorylation were detected in murine TGFbeta-dependent kidney fibrosis. Together, these data demonstrate adhesion-dependent FAK phosphorylation promoting TGFbeta-induced responses to regulate collagen production.

Inflammatory cytokines disrupt LDL-receptor feedback regulation and cause statin resistance: a comparative study in human hepatic cells and mesangial cells

LDL receptor (LDLr) is widely expressed in both liver and peripheral tissue. We aimed to clarify tissue-specific regulation of LDLr in hepatic cell line (HepG2) cells and human kidney mesangial cells (HMCs) under physiological and inflammatory conditions. We have demonstrated that the concentration of LDL required for 50% inhibition of LDLr mRNA expression (IC50) in HepG2 was 75 microg/ml, but only 30 microg/ml in HMCs. The concentration of mevastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, which achieved 200% upregulation of LDLr (UC200) in HepG2 cells, was 0.7 microM, which is much lower than 2.8 microM in HMCs. Inflammatory stress increased IC50 to 80 and 75 microg/ml of LDL, UC(200) to 2.8 microM, and 4.2 microM of mevastatin in HepG2 and HMCs. There was obvious sterol-regulatory element binding protein cleavage-activating protein accumulation in the Golgi in HepG2 cells, but not in HMCs in the presence of high concentration of LDL. IL-1beta further increased sterol-regulatory element binding protein cleavage-activating protein accumulation in HepG2 and HMCs in the presence of high concentration of LDL. These results indicate that LDLr in HepG2 cells have a relative resistant phenotype for downregulation, while LDLr in HMCs is very sensitive for downregulation. Inflammatory cytokine disrupts LDLr negative feedback regulation induced by intracellular cholesterol in both cell types, to a greater degree in HMCs, which could be one reason why HMCs are more prone to become foam cells under inflammatory stress. Inflammation also causes statin resistance; therefore, a high concentration of statin may be required to achieve the same biological effect.

Beilong virus, a novel paramyxovirus with the largest genome of non-segmented negative-stranded RNA viruses

During a subtraction study on gene expression in human kidney mesangial cells (HMCs), cDNA clones with sequence homology to paramyxovirus P, M and F genes were isolated. Subsequent investigation revealed that this particular HMC line was infected with a previously unknown paramyxovirus. Here, we report the isolation and genome characterization of this new virus, now named Beilong virus (BeV). The genome of BeV is 19,212 nucleotides (nt) in length and is the largest among all known members of the order Mononegavirales. The BeV genome contains eight genes in the order 3′-N-P/V/C-M-F-SH-TM-G-L-5′. The SH and TM genes code for a small hydrophobic protein of 76 aa and a transmembrane protein of 254 aa, respectively. The BeV G gene, at 4527 nt, codes for an attachment protein of 734 aa and contains two additional open reading frames (ORFs) in the 3′ half of the gene, coding for putative proteins of 299 and 394 aa in length. Although the exact origin of BeV is presently unknown, we provide evidence indicating that BeV was present in a rat mesangial cell line used in the same laboratory prior to the acquisition of the HMC line, suggesting a potential rodent origin for BeV.

Activation of the Nrf2/antioxidant response pathway increases IL-8 expression

Oxidant stress can initiate or enhance inflammatory responses during tissue injury, possibly through activation of redox-sensitive chemokines. Because the transcription factor Nrf2 (NF-E2-related factor 2) is responsive to oxidative stress, and induces expression of cytoprotective and antioxidant genes that attenuate tissue injury, we postulated that Nrf2 may also regulate chemokine expression. To test this hypothesis, Nrf2 expression was directly increased in primary human kidney mesangial cells and aortic endothelial cells, or cell lines with an adenoviral construct, and the effects on the pro-inflammatory chemokine interleukin-8 (IL-8) were assessed. Nrf2 expression significantly increased IL-8 mRNA levels and protein secretion. Nrf2 caused only a weak induction of IL-8 transcription, but significantly increased the half-life of IL-8 mRNA. These data demonstrate that activation of the Nrf2/antioxidant response pathway induces expression of IL-8. The dominant mechanism of Nrf2-mediated IL-8 induction is through mRNA stabilization. Considering the evidence that Nrf2 activation is mainly cytoprotective, these observations raise the possibility that under certain circumstances IL-8 may serve an anti-inflammatory role and thereby contribute to the resolution of tissue injury.

The Role of Internalization in Transforming Growth Factor β1-induced Smad2 Association with Smad Anchor for Receptor Activation (SARA) and Smad2-dependent Signaling in Human Mesangial Cells

Recent data investigating the role of the Smad anchor for receptor activation (SARA) in TGF-beta signaling have suggested that it has a crucial function in both aiding the recruitment of Smad to the TGF-beta receptor, and ensuring appropriate subcellular localization of the activated receptor-bound complex. The FYVE domain in SARA directs its localization to early endosomal compartments where it can interact with both the TGF-beta receptors and Smads. However, the necessity of endocytosis in the TGF-beta response remains controversial. We sought to examine the role of internalization in TGF-beta/Smad signaling in human kidney mesangial cells. Using co-immunoprecipitation studies, we show that endogenous Smad2 interacts with SARA after TGF-beta1 stimulation. Inhibition of clathrin-mediated internalization only slightly affects TGF-beta1-stimulated association between SARA and Smad2, Smad2 phosphorylation, or Smad2 interaction with Smad4. However, endocytosis inhibition decreases TGF-beta1-induced Smad2 nuclear translocation and thus abrogates Smad2-dependent transcriptional responses. The TGF-beta1-stimulated association between SARA and Smad2 peaks at 30 min followed by separation of the complex components. However, under conditions of inhibited endocytosis, Smad2 remains bound to SARA for at least 6 h without a significant decline in associated levels. This lack of complex dissociation correlates with a lack of Smad2 nuclear accumulation and reduction of Smad2-dependent ARE-Luc reporter activity. Our data therefore suggest that endocytosis plays a critical role in TGF-beta signaling in mesangial cells, and that internalization enhances the dissociation of Smad2 from the TGF-beta receptor-SARA complex, allowing Smad2 to accumulate in the nucleus and modulate target gene transcription.

Regulation of lipoprotein trafficking in the kidney: role of inflammatory mediators and transcription factors.

Inflammation and dyslipidaemia both play important roles in the development of glomerular atherosclerosis in renal diseases. We have demonstrated that inflammatory mediators induced Scr (scavenger receptor) expression and the formation of foam cells, and that AP-1 (activator protein 1)/ets were necessary transcriptional factors for Scr induction in HMCs (human kidney mesangial cells). Most cells are protected from excessive native LDL (low-density lipoprotein) accumulation by tight feedback regulation of the LDLr (LDL receptor). However, we observed that HMCs formed foam cells via the LDLr pathway when incubated with IL-1beta (interleukin-1beta; 5 ng/ml) and unmodified LDL (200 microg/ml), suggesting that inflammatory mediators may disrupt the cholesterol-mediated feedback regulation. This feedback involves cholesterol-mediated down-regulation of LDLr controlled by SCAP [SREBP (sterol responsive element-binding protein) cleavage-activating protein]. We have also demonstrated that both tumour necrosis factor alpha and IL-1beta increased nuclear SREBP-1 levels by increasing SCAP mRNA expression, even in the presence of a high concentration of LDL. Since intracellular lipid content is governed by both influx and efflux mechanisms, we set out to examine the impact of inflammatory cytokines on cholesterol efflux, a process mediated by the protein ABCA1 (ATP binding cassette A1). IL-1beta inhibited [(3)H]cholesterol efflux from HMCs by inhibition of the peroxisome-proliferator-activated receptor/LXR (liver X receptor)/ABCA1 pathway. Taken together, our results suggest that inflammatory mediators increase lipid accumulation in HMCs not only by promoting increased lipoprotein uptake by Scr and LDLr, but also by inhibiting ABCA1-mediated cholesterol efflux to high-density lipoprotein.

Adhesion of cultured human kidney mesangial cells to native entactin: role of integrin receptors.

Entactin is an extracellular matrix glycoprotein which binds to laminin and is found in most renal basement membranes and in the glomerular mesangial matrix. In the present study, we have characterized specific integrin receptors on cultured human mesangial cells (CHMC) responsible for adhesion to native entactin. The integrin receptors alpha 2 beta 1, alpha 3 beta 1, alpha 5 beta 1, alpha v beta 3, alpha v beta 5, and alpha 6 complexed with either beta 1 or beta 4 could be immune precipitated from detergent extracts of metabolically labeled CHMC. Adhesion assays with inhibitory anti integrin monoclonal antibodies (mab) demonstrated that CHMC use both alpha v beta 3 and a beta 1-containing integrin to bind surfaces coated with native entactin. Optimal binding of CHMC to native entactin required the participation of cations. Using wild type and mutant recombinant entactin fragments, the binding site for the alpha v beta 3 receptor was localized to the RGD sequence on the rod or E domain of entactin. CHMC adhesion to mutant full length recombinant entactin ligands lacking the E domain RGD sequence confirmed the presence of ligand binding site(s) for beta 1 integrin receptor(s). Differences in CHMC binding characteristics to recombinant and full length entactin compared to native bovine basement membrane entactin were observed. This suggests that tertiary molecular structure may contribute to entactin ligand binding properties. Primary amino acid residue sequences and tertiary structure of entactin may play roles in forming functional cell attachment sites in native basement membrane entactin.

Expression of components of the superoxide generating NADPH oxidase by human leucocytes and other cells

NADPH oxidase of phagocytic leucocytes contains a membrane cytochromeb with two subunits, gp91phox and p22phox, together with three cytosolic proteins, p47phox, p67phox and p2rac. The presence of some of these components has been sought in non-phagocytes, using Western blot analysis for protein expression and PCR to amplify and detect mRNA. All components were detected in EBV-transformed B lymphocytes and peripheral blood B lymphocytes. Fibroblasts and human kidney mesangial cells contained mRNA for p67phox, p47phox, and p22phox but not gp91phox. Levels of expression varied with growth conditions, but it appears possible than an isozyme of cytochromeb which lacks gp9phox is present in these cells. Proteins of p47phox and p67phox were expressed, in low concentrations, in these two cell types. Expression of mRNA for p47phox and p67phox was found to be widespread in many cell types.

Effects of matrix glycation on mesangial cell adhesion, spreading and proliferation

We examined the effects of in vitro nonenzymatic glycosylation (NEG) of the extracellular matrix (ECM) on various aspects of the adhesive interaction between the ECM and human kidney mesangial (HKM) cells, and also on the ability of HKM cells to spread and proliferate. Isolated type IV collagen (tIV) or intact complexes of glomerular basement membrane and mesangial matrix (GBM/MM) were used as substrates following control incubation or modest glycation. We observed that HKM cells adhered less effectively to glycated tIV at early time intervals and were delayed in attaining maximal levels of adhesion compared to cells interacting with control tIV. The nature of the adhesive interaction was also different since antibodies which blocked the function of beta 1-containing integrins more effectively inhibited adhesion between HKM cells and glycated tIV than cells and control tIV. HKM cells interacting with glycated tIV also demonstrated less cell surface microspike and ruffle formation at five minutes after plating, less extensive spreading throughout the examined time intervals (> or = 90 min after plating), and slightly increased cell numbers 5 to 10 days after plating when compared to cells interacting with control tIV. However, increased cell numbers were not observed when HKM cells were grown on glycated GBM/MM. Similar changes in response to glycated substrate were observed when HKM cells were grown in either 5 or 25 mM glucose. In conclusion, relatively modest glycation of the ECM alone was sufficient to result in specific changes in HKM cell behavior in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell-specific regulation of plasminogen activator inhibitor 1 and tissue type plasminogen activator release by human kidney mesangial cells

Human mesangial cells in culture synthesize and secrete plasminogen activator inhibitor 1 (PAI-1) and tissue-type plasminogen activator (t-PA). Phorbol myristate acetate (PMA), a known activator of protein kinase C, induces a three to four-fold increase in t-PA and PAI-1 release over a period of 24 h, whereas cell-associated t-PA and PAI-1 levels remain relatively stable. A similar effect is obtained with oleylacetyl glycerol, a more physiologic protein kinase C activator. The effect of PMA is suppressed in the presence of H7, an inhibitor of cellular protein kinases, and by cycloheximide and actinomycin D, indicating a requirement for de novo protein and RNA synthesis, respectively. Northern blot analysis of PMA-treated cells reveals a rapid and transient increase in PAI-1 mRNA reaching a maximum after 4–8 h, whereas increase in t-PA mRNA levels requires 24 h. Activation of protein kinase A by addition of 8-bromocyclic AMP (8-bromo cAMP) has no significant effect on PAI-1 release but inhibits the PMA-mediated increases in PAI-1 antigen and mRNA. Addition of 8-bromo cAMP alone does not affect t-PA release. When added to PMA-stimulated cells, 8-bromo cAMP inhibits t-PA release in a dose-dependent manner, but causes a superinduction of t-PA mRNA. 8-bromo cAMP also induces a decrese in PMA-stimulated intracellular t-PA release. Similar inhibition is observed after stimulation of endogenous adenylate cyclase with prostaglandin E1 or isoproterenol. This indicates that protein kinase A activation may inhibit PMA-stimulated t-PA release via a post-transcriptional effect, e.g. inhibition of protein synthesis or activation of protein degradation. In conclusion, hormones or mediators which activate protein kinase C can stimulate t-PA and PAI-1 synthesis in human mesangial cells. Protein kinase A activation has no effect on the basal release of PAI-1 and t-PA by human mesangial cells, and, in contrast to endothelial cells, it inhibits both PMA-stimulated PAI-1 and t-PA releases. This cell-specific regulation of t-PA and PAI-1 seems to be mediated by differenial transcriptional and post transcriptional mechanisms.

Human glomerular mesangial cells inactivate leukotriene B 4 by reduction into dihydro-leukotriene B 4 metabolites

Due to its potent chemotactic properties leukotriene B 4 is an important mediator of inflammatory reactions. Cultured human kidney mesangial cells converted exogenously added leukotriene B 4 efficiently into three different more lipophilic metabolites, two of them probably representing dihydro-leukotriene B 4 isomers. This represents an alternative metabolic pathway, in contrast to leukotriene B 4 omega-oxidation found in human polymorphonuclear leukocytes. Both dihydro-leukotriene B 4 isomers had nearly completely lost their ability to induce leukocyte chemotaxis as compared to leukotriene B 4.

Cytomegalovirus replicates efficiently in human kidney mesangial cells.

Human cytomegalovirus (HCMV) is a major renal pathogen in congenitally infected infants and renal allograft recipients. We postulated that a specific renal cell type was involved in HCMV infection and reactivation. Human fetal kidney cortex cell cultures were assayed for their ability to support HCMV infection. Infectious center assays indicated that the low level of viral replication observed by virus yield assay occurred from a fraction of the cells in the mixed cultures. Virus-specific immunofluorescence and in situ hybridization documented the presence of HCMV-specific protein and nucleic acid, respectively, in a morphologically distinct cell type. These cells were purified, were identified as kidney mesangial cells, and were observed to support efficient HCMV replication. Our research identifies mesangial cells as a renal cell type that supports HCMV replication and provides evidence to implicate these cells in the pathogenesis of HCMV-induced renal disease.