Human Renal Tubule Epithelial Cells Research Articles

SFN promotes renal fibrosis via binding with MYH9 in chronic kidney disease

Chronic kidney disease (CKD) is a clinical syndrome characterized by persistent renal function decline. Renal fibrosis is the main pathological process in CKD, but an effective treatment does not exist. Stratifin (SFN) is a highly-conserved, multi-function soluble acidic protein. Therefore, this study explored the effects of SFN on renal fibrosis. First, we found that SFN was highly expressed in patients with CKD, as well as in renal fibrosis animal and cell models. Next, transforming growth factor-beta 1 (TGF-β1) induced injury and fibrosis in human renal tubule epithelial cells, and SFN knockdown reversed these effects. Furthermore, SFN knockdown mitigated unilateral ureteral obstruction (UUO)-induced renal tubular dilatation and renal interstitial fibrosis in mice. Liquid chromatography-tandem mass spectrometry/mass spectrometry (LC-MS/MS), co-immunoprecipitation (Co-IP), and immunofluorescence co-localization assays demonstrated that SFN bound the non-muscle myosin-encoding gene, myosin heavy chain 9 (MYH9), in the cytoplasm of renal tubular epithelial cells. MYH9 knockdown also reduced Col-1 and α-SMA expression, which are fibrosis markers. Finally, silencing SFN decreased MYH9 expression, alleviating renal fibrosis. These results suggest that SFN promotes renal fibrosis in CKD by interacting with MYH9. This study may provide potential strategies for the treatment of CKD.

Modelling BK Polyomavirus dissemination and cytopathology using polarized human renal tubule epithelial cells.

Most humans have a lifelong imperceptible BK Polyomavirus (BKPyV) infection in epithelial cells lining the reno-urinary tract. In kidney transplant recipients, unrestricted high-level replication of donor-derived BKPyV in the allograft underlies polyomavirus-associated nephropathy, a condition with massive epithelial cell loss and inflammation causing premature allograft failure. There is limited understanding on how BKPyV disseminates throughout the reno-urinary tract and sometimes causes kidney damage. Tubule epithelial cells are tightly connected and have unique apical and basolateral membrane domains with highly specialized functions but all in vitro BKPyV studies have been performed in non-polarized cells. We therefore generated a polarized cell model of primary renal proximal tubule epithelial cells (RPTECs) and characterized BKPyV entry and release. After 8 days on permeable inserts, RPTECs demonstrated apico-basal polarity. BKPyV entry was most efficient via the apical membrane, that in vivo faces the tubular lumen, and depended on sialic acids. Progeny release started between 48 and 58 hours post-infection (hpi), and was exclusively detected in the apical compartment. From 72 hpi, cell lysis and detachment gradually increased but cells were mainly shed by extrusion and the barrier function was therefore maintained. The decoy-like cells were BKPyV infected and could transmit BKPyV to uninfected cells. By 120 hpi, the epithelial barrier was disrupted by severe cytopathic effects, and BKPyV entered the basolateral compartment mimicking the interstitial space. Addition of BKPyV-specific neutralizing antibodies to this compartment inhibited new infections. Taken together, we propose that during in vivo low-level BKPyV replication, BKPyV disseminates inside the tubular system, thereby causing minimal damage and delaying immune detection. However, in kidney transplant recipients lacking a well-functioning immune system, replication in the allograft will progress and eventually cause denudation of the basement membrane, leading to an increased number of decoy cells, high-level BKPyV-DNAuria and DNAemia, the latter a marker of allograft damage.

Inhibition of Transforming Growth Factor-β Improves Primary Renal Tubule Cell Differentiation in Long-Term Culture.

Patient-oriented applications of cell culture include cell therapy of organ failure like chronic renal failure. Clinical deployment of a cell-based device for artificial renal replacement requires qualitative and quantitative fidelity of a cultured cell to its in vivo counterpart. Active specific apicobasal ion transport reabsorbs 90-99% of the filtered load of salt and water in the kidney. In a bioengineered kidney, tubular transport concentrates wastes and eliminates the need for hemodialysis, but renal tubule cells in culture transport little or no salt and water due to dedifferentiation that mammalian cells undergo in vitro thereby losing important cell-type specific functions. We previously identified transforming growth factor-β (TGF-β) as a signaling pathway necessary for in vitro differentiation of renal tubule cells. Inhibition of TGF-β receptor-1 led to active and inhibitable electrolyte and water transport by primary human renal tubule epithelial cells in vitro. Addition of metformin increased transport, in the context of a transient effect on 5'-AMP-activated kinase phosphorylation. These data motivated us to examine whether increased transport was an idiosyncratic effect of SB431542, probe pathways downstream of TGF-β receptors possibly responsible for the improved differentiation, evaluate whether TGF-β inhibition induced a range of differentiated tubule functions, and to explore crosstalk between the effects of SB431542 and metformin. In this study, we use multiple small-molecule inhibitors of canonical and noncanonical pathways to confirm that inhibition of canonical TGF-β signaling caused the increased apicobasal transport. Hallmarks of proximal tubule cell function, including sodium reabsorption, para-amino hippurate excretion, and glucose uptake increased with TGF-β inhibition, and the specificity of the response was shown using inhibitors of each transport protein. We did not find any evidence of crosstalk between metformin and SB431542. These data suggest that the TGF-β signaling pathway governs multiple features of differentiation in renal proximal tubule cells in vitro. Inhibition of TGF-β by pharmacologic or genome engineering approaches may be a viable approach to enhancing differentiated function of tubule cells in vitro. Impact statement Cell therapy of renal failure requires qualitative and quantitative fidelity between in vitro and in vivo phenotypes, which has been elusive. We show that control of transforming growth factor-β signaling can promote differentiation of renal tubule cells grown in artificial environments. This is a key enabling step for cell therapy of renal failure.

Maresin 1 Alleviates Diabetic Kidney Disease via LGR6-Mediated cAMP-SOD2-ROS Pathway.

Background. Chronic hyperglycemia-induced inflammation is recognized as the most important pathophysiological process in diabetic kidney disease (DKD). As maresin 1 (MaR1) is an extensive anti-inflammatory lipid mediator, the present study investigated the protective role of MaR1 in the pathogenesis of DKD and its clinical relevance. Methods. Serum MaR1 concentrations were analyzed in 104 subjects with normal glucose tolerant, type 2 diabetes (T2DM), or DKD. Streptozotocin (STZ) together with high fat diet was used to induce male C57BL/6 J mice into diabetic mice which were treated with MaR1. Human renal tubule epithelial cells (HK-2 cells) were treated by high glucose for glucotoxicity cell model and transfected with LGR6 siRNA for knockdown with MaR1 added，and detected oxidative stress and inflammatory related factors. Results. Serum MaR1 concentrations were significant decreased in T2DM with or without kidney disease compared with normal participant and were lowest in patients with DKD. Serum MaR1 concentrations were negatively correlated with hemoglobin A1c (HbA1c), duration of diabetes, urinary albumin to creatinine ratio (UACR), neutrophil, and neutrophil-lymphocyte ratio and were positively correlated with high-density lipoprotein-cholesterol (HDL-C) and estimated glomerular filtration rate (eGFR). In mouse model, MaR1 injection alleviated hyperglycemia, UACR and the pathological progression of DKD. Interestingly, the renal expression of LGR6 was down-regulated in DKD and high glucose treated HK-2 cells but up-regulated by MaR1 treatment. Mechanistically, MaR1 alleviated inflammation via LGR6-mediated cAMP-SOD2 antioxidant pathway in DKD mice and high glucose treated HK-2 cells. Conclusions. Our study demonstrates that decreased serum MaR1 levels were correlated with the development of DKD. MaR1 could alleviate DKD and glucotoxicity-induced inflammation via LGR6-mediated cAMP-SOD2 antioxidant pathway. Thus, our present findings identify MaR1 as a predictor and a potential therapeutic target for DKD.

Fumonisin B1 induces nephrotoxicity via autophagy mediated by mTORC1 instead of mTORC2 in human renal tubule epithelial cells

Fumonisin B1 (FB1), a worldwide contaminating mycotoxin, can cause global food issue. It has been reported that FB1 is related to chronic kidney disease of unknown etiology. However, the study of FB1-induced nephrotoxicity in vitro is very limited and the mechanism is unknown. Human renal tubule epithelial (HK-2) cells were used in this study. The results showed that FB1 exposure could decrease cell viability, induce cell apoptosis and up-regulate the expression of Kim-1, collagen I, α-SMA and TGF-β1. In addition, autophagy was activated after FB1 exposure, including the conversion of LC3 and up-regulation of ATGs. Furthermore, autophagy inhibitor 3-MA could block FB1-induced abnormalities. And antioxidant enzymes (Gpx1 and Gpx4) were obviously down-regulated and intracellular ROS levels displayed an ascent trend as FB1 exposure concentrations increased. Employing of antioxidant NAC could suppress FB1-induced nephrotoxicity and autophagy. FB1 inhibited the phosphorylation of p70 S6k, a downstream protein of mTORC1. Also, oxidative stress, autophagy and phosphorylation of p70 S6k induced by FB1 was inhibited by MHY1485, an activator of mTOR. But the phosphorylation of AKT, a downstream protein of mTORC2 showed no change with or without MHY1485. Taken together, FB1 induced nephrotoxicity via autophagy mediated by mTORC1 instead of mTORC2 in HK-2 cells.

Nonesterified free fatty acids enhance the inflammatory response in renal tubules by inducing extracellular ATP release.

In proteinuric renal diseases, excessive plasma nonesterified free fatty acids bound to albumin can leak across damaged glomeruli to be reabsorbed by renal proximal tubular cells and cause inflammatory tubular cells damage by as yet unknown mechanisms. The present study was designed to investigate these mechanisms induced by palmitic acid (PA; one of the nonesterified free fatty acids) overload. Our results show that excess PA stimulates ATP release through the pannexin 1 channel in human renal tubule epithelial cells (HK-2), increasing extracellular ATP concentration approximately threefold compared with control. The ATP release is dependent on caspase-3/7 activation induced by mitochondrial reactive oxygen species. Furthermore, extracellular ATP aggravates PA-induced monocyte chemoattractant protein-1 secretion and monocyte infiltration of tubular cells, enlarging the inflammatory response in both macrophages and HK-2 cells via the purinergic P2X7 receptor-mammalian target of rapamycin-forkhead box O1-thioredoxin-interacting protein/NOD-like receptor protein 3 inflammasome pathway. Hence, PA increases mitochondrial reactive oxygen species-induced ATP release and inflammatory stress, which cause a "first hit," while ATP itself is a "second hit" in amplifying the renal tubular inflammatory response. Thus, inhibition of ATP release or the purinergic P2X7 receptor may be an approach to reduce renal inflammation and improve renal function.

Type III interferon networks in systemic lupus erythematosus

Abstract Background Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of nucleic acid autoantibodies and immune complex deposition resulting in organ damage. A type I interferon (IFN) gene signature historically attributed to IFN-α is found in cells from SLE patients and associated with disease activity. Type III IFN (IFN-λ) is relatively understudied, but is expressed in SLE renal tubule epithelial cells (RTEC) and found in patient serum. The objective of this study was to determine if IFN-λ1 allows cross-talk between human RTEC and B cells, which may be relevant for lupus nephritis pathogenesis. Methods SLE peripheral blood B cell phenotyping was completed by flow cytometry (n=26). RNA was isolated for RT-PCR from primary human RTEC and B cells stimulated with Toll-like receptor (TLR) agonists. CD19+ cells were stimulated with or without IFN-λ1. IFN-λ1 protein was measured by ELISA in supernatant and serum. Results SLE IFN-λ1 serum levels positively correlate (p=0.006) with peripheral blood IgD- CD27- CD24- CD21- B cells, a subset which expresses CD11c and T-bet and was expanded in SLE. Healthy B cells treated with IFN-λ1 express IFIT1, ISG15 and IRF7 mRNA. IFN-λ1 promoted plasma cell differentiation in B cells activated with TLR7 agonists. IFN-λ1 mRNA and protein secretion increase in RTEC with TLR3 stimulation. Conclusions RTEC, which constitute the bulk of the kidney, produce IFN-λ1 upon TLR3 stimulation. B lymphocytes express an IFN signature in response to IFN-λ1. IFN-λ1 promotes plasma cells in TLR7-activated B cells. These findings suggest the potential for type III IFN networks between RTEC and B cells in the renal microenvironment which may contribute to SLE and lupus nephritis pathogenesis.

Polyploidy and Mitotic Cell Death Are Two Distinct HIV-1 Vpr-Driven Outcomes in Renal Tubule Epithelial Cells.

Prior studies have found that HIV, through the Vpr protein, promotes genome reduplication (polyploidy) in infection-surviving epithelial cells within renal tissue. However, the temporal progression and molecular regulation through which Vpr promotes polyploidy have remained unclear. Here we define a sequential progression to Vpr-mediated polyploidy in human renal tubule epithelial cells (RTECs). We found that as in many cell types, Vpr first initiates G2 cell cycle arrest in RTECs. We then identified a previously unreported cascade of Vpr-dependent events that lead to renal cell survival and polyploidy. Specifically, we found that a fraction of G2-arrested RTECs reenter the cell cycle. Following this cell cycle reentry, two distinct outcomes occur. Cells that enter complete mitosis undergo mitotic cell death due to extra centrosomes and aberrant division. Conversely, cells that abort mitosis undergo endoreplication to become polyploid. We further show that multiple small-molecule inhibitors of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, including those that target ATR, ATM, and mTOR, indirectly prevent Vpr-mediated polyploidy by preventing G2 arrest. In contrast, an inhibitor that targets DNA-dependent protein kinase (DNA-PK) specifically blocks the Vpr-mediated transition from G2 arrest to polyploidy. These findings outline a temporal, molecularly regulated path to polyploidy in HIV-positive renal cells.IMPORTANCE Current cure-focused efforts in HIV research aim to elucidate the mechanisms of long-term persistence of HIV in compartments. The kidney is recognized as one such compartment, since viral DNA and mRNA persist in the renal tissues of HIV-positive patients. Further, renal disease is a long-term comorbidity in the setting of HIV. Thus, understanding the regulation and impact of HIV infection on renal cell biology will provide important insights into this unique HIV compartment. Our work identifies mechanisms that distinguish between HIV-positive cell survival and death in a known HIV compartment, as well as pharmacological agents that alter these outcomes.

Effect of propofol on human renal tubule epithelial cell fibrosis induced by ATP depletion/recovery: the role of TAK1

Objective To investigate the effect of propofol on human renal tubule epithelial cell(HK-2 cells)fibrosis induced by ATP depletion/recovery and the role of transforming growth factor β activated kinase 1(TAK1)in it. Methods HK-2 cells were seeded in 96-well plates, and randomly divided into 4 groups(n=36 each)using a random number table: control group(group C), ATP depletion/recovery group(group D/R), propofol group(group P), and TAK1 over-expression group(group T). HK-2 cells were exposed to antimycin A for l h and then returned to normal culture medium to establish the model of ATP depletion/recovery-induced injury.At 1 h before ATP depletion, the cells were incubated for 1 h in the DMEM liquid culture medium containing propofol with the final concentration of 20 μmol/L in group P, and the cells were incubated for 1 h in the DMEM liquid culture medium containing propofol with the final concentration of 20 μmol/L and TAK1 with the titer of 2×107 TU/ml in group T, and the other treatments were similar to those previously described in group D/R.At 12 h after ATP recovery, the cell viability was evaluated by methyl thiazolyl tetrazolium assay, and cell apoptosis was detected using TUNEL and scored.The expression of TAK1 was detected using Western blot at 12, 24 and 48 h after ATP recovery.The expression of α-smooth muscle actin(αSMA), fibronectin(FN), and collagen protein 1(COL1)was measured at 48 h after ATP recovery. Results Compared with group C, the cell viability was significantly decreased, the apoptosis score was increased, and the expression of TAK1, COL1, αSMA and FN was up-regulated after ATP recovery in D/R, P and T groups(P<0.05). Compared with group D/R, the cell viability was significantly increased, the apoptosis score was decreased, and the expression of TAK1, COL1, αSMA and FN was down-regulated after ATP recovery in P and T groups(P<0.05). Compared with group P, the cell viability was significantly decreased, the apoptosis score was increased, and the expression of TAK1, COL1, αSMA and FN was up-regulated after ATP recovery in group T(P<0.05). Conclusion Propofol can reduce HK-2 cell fibrosis induced by ATP depletion/recovery, and the mechanism may be related to down-regulation of TAK1 expression. Key words: Propofol; Reperfusion injury; Kidney; Fibrosis; MAP kinase kinase kinases

A bio-artificial renal epithelial cell system conveys survival advantage in a porcine model of septic shock.

Renal cell therapy using the hollow fiber based renal assist device (RAD) improved survival time in an animal model of septic shock (SS) through the amelioration of cardiac and vascular dysfunction. Safety and ability of the RAD to improve clinical outcomes was demonstrated in a Phase II clinical trial, in which patients had high prevalence of sepsis. Even with these promising results, clinical delivery of cell therapy is hampered by manufacturing hurdles, including cell sourcing, large-scale device manufacture, storage and delivery. To address these limitations, the bioartificial renal epithelial cell system (BRECS) was developed. The BRECS contains human renal tubule epithelial cells derived from adult progenitor cells using enhanced propagation techniques. Cells were seeded onto trabeculated disks of niobium-coated carbon, held within cryopreservable, perfusable, injection-moulded polycarbonate housing. The study objective was to evaluate the BRECS in a porcine model of SS to establish conservation of efficacy after necessary cell sourcing and design modifications; a pre-clinical requirement to move back into clinical trials. SS was incited by peritoneal injection of E. coli simultaneous to insertion of BRECS (n=10) or control (n=15), into the ultrafiltrate biofeedback component of an extracorporeal circuit. Comparable to RAD, prolonged survival of the BRECS cohort was conveyed through stabilization of cardiac output and vascular leak. In conclusion, the demonstration of conserved efficacy with BRECS therapy in a porcine SS model represents a crucial step toward returning renal cell therapy to the clinical setting, initially targeting ICU patients with acute kidney injury requiring continuous renal replacement therapy. Copyright © 2014 John Wiley & Sons, Ltd.

TGF-Beta Auto-Induction and Connective Tissue Growth Factor Expression in Human Renal Tubule Epithelial Cells Requires N-Ras

Background/Aims: Transforming growth factor (TGF) β is strongly implicated in the progression of renal fibrosis. TGFβ1 is reported to cause epithelial-mesenchymal transition, inhibition of epithelial cell proliferation, increased apoptosis, auto-induction of TGFβ production and induction of secondary mediators of tissue fibrosis such as connective tissue growth factor (CTGF, CCN2). The aims of this study were to investigate the role of the Ras/MAP kinase pathway in TGFβ1 inhibition of proliferation, TGFβ auto-induction and TGFβ1-induced CTGF expression in HKC human renal tubule epithelial cells. Methods and Results: TGFβ1 (0–25 ng/ml) inhibited proliferation of HKC cells and at 25 ng/ml also induced apoptosis. After 5–10 min of incubation, TGFβ1 increased cellular levels of phospho-ERK1/2 and phospho-AKT with a bell-shaped dose-response curve with a maximally effective concentration of 2.5 ng/ml. TGFβ3 caused an increase in extracellular TGFβ1, which was significantly reduced in the presence of PD 98059. TGFβ1 increased cellular and secreted CTGF protein in HKC cells in a MEK-dependent manner. To identify the Ras isoform involved, specific antisense oligonucleotides targeted to Ha-Ras, Ki-Ras and N-Ras were employed. Only inhibition of N-Ras resulted in a significant reduction of auto-induced TGFβ1 secretion and TGFβ1-induced cellular and secreted CTGF. Conclusion: These results establish that the Ras/MAP kinase pathway, specifically through N-Ras, mediates TGFβ1 auto-induction and TGFβ1-induced CTGF expression in human renal tubule epithelial cells.

Cell lines with extended in vitro growth potential from human renal proximal tubule: Characterization, response to inducers, and comparison with established cell lines

Few model systems exist for the study of injury to human renal proximal tubule epithelium. Optimized differentiated human renal epithelial cell lines with extended in vitro growth potential would provide an alternative model system to primary culture or other available non-human mammalian kidney cell lines. For this purpose, human renal tubule epithelial cells were isolated from normal kidney cortex and exposed in culture to a hybrid immortalizing virus, adenovirus 12-SV40. Cell lines were developed by limiting dilution, and three selected cell lines were screened for growth pattern, production of immortalizing virus, tumorigenicity, and ploidy. Cell lines were also monitored for response to inducer agents and matrix factors and were screened for expression of biochemical properties and differentiation markers of renal epithelium. All three are nonproducers of the immortalizing virus and are nontumorigenic. They grow in monolayer, have intermediate growth kinetics, and express markers of renal proximal tubular epithelium by immunohistochemistry. They also express biochemical properties comparable to other widely used proximal tubular cell lines including LLC-RK1, OK, and HK-2 and comparable to human tubular cells in stable culture. Growth medium containing low levels of fetal calf serum, or epidermal growth factor combined with parathyroid hormone, produced optimal growth characteristics, brush border enzyme expression, biochemical properties, and glucose transport in a selected cell line. The addition of dimethyl sulfoxide allows maintenance in morphologically intact monolayers for prolonged periods. These cell lines should be useful model systems for the study of human renal proximal tubular injury or disease.

In vitro and in vivo interleukin-8 production in human renal cortical epithelia.

The signals resulting in leukocytes infiltrating the tubulointerstitial compartment during renal inflammatory disease are not well understood. A recently described cytokine, interleukin-8 (IL-8), has been demonstrated to be chemotactic for lymphocytes and neutrophils at picomolar and nanomolar concentrations, respectively. Cytokeratin positive, renal cortical epithelial cells (RCEC) with tubular attributes were cultured from kidney tissue from six human subjects. We report that these human renal cortical epithelial cells in primary cell culture respond to either IL-1 beta, TNF or LPS in both a time- and dose-dependent manner by expressing IL-8 mRNA and secreting antigenic IL-8 peptide. In addition, RCEC were found to be strongly positive for cell-associated antigenic IL-8 peptide by immunostaining after 24 hour incubation with IL-1 beta, TNF and LPS. To ascertain whether IL-8 was present in renal disease associated with infiltrating leukocytes, we performed immunohistochemistry on renal biopsy specimens from patients with acute allograft rejection. Both proximal and distal tubular epithelial cells were found to be strongly positive for cell-associated antigenic IL-8. These findings suggest that the human renal tubule epithelial cell may actively participate in acute inflammatory processes in the kidney, including allograft rejection, by effecting and directing leukocyte chemotaxis via the production of IL-8.