Cultures Of Renal Cells Research Articles

D-serine, a novel uremic toxin, induces senescence in human renal tubular cells via GCN2 activation

The prevalence of chronic kidney disease (CKD), characterized by progressive renal dysfunction with tubulointerstitial fibrosis, is increasing because of societal aging. Uremic toxins, accumulated during renal dysfunction, cause kidney damage, leading to renal deterioration. A recent metabolomic analysis revealed that plasma D-serine accumulation is associated with faster progression of renal dysfunction in CKD patients. However, the causal relationship and the underlying mechanisms remain unclear. Herein, we demonstrated that D-serine markedly induced cellular senescence and apoptosis in a human proximal tubular cell line, HK-2, and primary culture of human renal tubular cells. The former was accompanied by G2/M cell cycle arrest and senescence-associated secretory phenotype, including pro-fibrotic and pro-inflammatory factors, contributing to tubulointerstitial fibrosis. Integrated stress response mediated by the general control nonderepressible 2 played an important role in D-serine-induced tubular cell toxicity and pro-fibrotic phenotypes, accelerating CKD progression and kidney aging. D-serine upregulated the L-serine synthesis pathway. Furthermore, D-serine-induced suppression of tubular cell proliferation was ameliorated by L-serine administration, indicating that D-serine exposure induced an L-serine-deprived state in tubular cells, compensated by L-serine synthesis. Thus, this study unveils molecular mechanisms underlying D-serine-induced tubular damage and pro-fibrotic phenotypes, suggesting that D-serine is a uremic toxin involved in CKD pathogenesis.

Preconditioning of primary human renal proximal tubular epithelial cells without tryptophan increases survival under hypoxia by inducing autophagy

Hypoxia plays a significant role in the pathogenesis of acute kidney injury (AKI). Autophagy protects from AKI. Amino acid deprivation induces autophagy. The effect of L-tryptophan depletion on survival and autophagy in cultures of renal proximal tubular epithelial cells (RPTECs) under hypoxia was evaluated. RPTECs were preconditioned in a medium containing or not tryptophan, following culture under hypoxia and treatment with or without the autophagy inhibitor chloroquine. Cell survival was assessed by cell imaging, the level of certain proteins by western blotting and cellular ATP fluorometrically. Preconditioning of RPTECs in a medium without tryptophan activated general control nonderepressible 2 kinase and induced changes that favored autophagy and cell survival under hypoxic conditions. Additionally, it increased cellular ATP, while it inhibited apoptosis. Inhibition of autophagy nullified the induced increase in cellular ATP and cell survival by the absence of tryptophan. The absence of tryptophan increased p53, although its effect on p53's transcriptional targets was heterogeneous. In accordance with the decreased apoptosis, expression of p21 increased, while expression of Bax decreased. The expression of BNIP3L, which may be pro-apoptotic or pro-autophagic, increased. Considering the decreased apoptosis, it is likely that tryptophan depletion enhances autophagy through a p53-mediated increase of BNIP3L. Preconditioning of primary human RPTECs in a medium without tryptophan increases their survival under hypoxia by inducing autophagy. Identifying new molecular mechanisms that protect renal tissue from hypoxia could be proved clinically important in the prevention of AKI.

Rapid Renal Regulation of Peroxisome Proliferator-activated Receptor γ Coactivator-1α by Extracellular Signal-Regulated Kinase 1/2 in Physiological and Pathological Conditions

Previous studies have shown that extracellular signal-regulated kinase 1/2 (ERK1/2) directly inhibits mitochondrial function during cellular injury. We evaluated the role of ERK1/2 on the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) gene, a master regulator of mitochondrial function. The potent and specific MEK1/2 inhibitor trametinib rapidly blocked ERK1/2 phosphorylation, decreased cytosolic and nuclear FOXO3a/1 phosphorylation, and increased PGC-1α gene expression and its downstream mitochondrial biogenesis (MB) targets under physiological conditions in the kidney cortex and in primary renal cell cultures. The epidermal growth factor receptor (EGFR) inhibitor erlotinib blocked ERK1/2 phosphorylation and increased PGC-1α gene expression similar to treatment with trametinib, linking EGFR activation and FOXO3a/1 inactivation to the down-regulation of PGC-1α and MB through ERK1/2. Pretreatment with trametinib blocked early ERK1/2 phosphorylation following ischemia/reperfusion kidney injury and attenuated the down-regulation of PGC-1α and downstream target genes. These results demonstrate that ERK1/2 rapidly regulates mitochondrial function through a novel pathway, EGFR/ERK1/2/FOXO3a/1/PGC-1α, under physiological and pathological conditions. As such, ERK1/2 down-regulates mitochondrial function directly by phosphorylation of upstream regulators of PGC-1α and subsequently decreasing MB.

Mechanisms of Inflammatory Injury of Renal Tubular Cells in a Cellular Model of Pyelonephritis.

Previously, we have assembled a cellular model of pyelonephritis which contains a primary culture of renal tubular epithelial cells, mononuclear leukocytes, and bacterial lysate or lipopolysaccharide. After cocultivation of renal cells with leukocytes and bacterial lysate, proinflammatory changes were observed in the renal cells, followed by nitrosative and oxidative stress and cell death. The interaction of bacterial antigens not only with leukocytes, but also with epithelial cells of the renal tubules, was partially mediated by signaling pathways involving Toll-like receptors (TLR2 and TLR4). Activation of these receptors led to increased levels of oxidative stress and synthesis of proinflammatory cytokines (TNF, IL-6, IL-1α) in the renal epithelium, while TLR4 blockade decreased the severity of these processes. Apart from the fact that activation of inflammatory signaling in response to bacterial antigens is observed directly in the renal cells, the presence of leukocytes significantly amplifies the inflammatory response as measured by the level of cytokines generated in the ensemble. In the presence of activated leukocytes, higher expression of TLR2 on the surface of renal cells was observed in response to exposure to bacterial components, which might explain the increased inflammatory response in the presence of leukocytes. The synthesis of IL-1α in the epithelial cells of the renal tubules in this inflammatory model leads to its accumulation in the nuclei, which has been reduced by the TLR4 antagonist polymyxin. TLR2 agonists also led to increased levels of IL-1α. The elevation in the content of IL-1α in nuclei was accompanied by increased acetylation of nuclear proteins, which has been reduced to control values after exposure to protective agents (Trolox, mitochondria-targeted antioxidant SkQR1 or LiCl). The high level of acetylation of histones is probably regulated by proinflammatory cytokines, and to some extent it is a marker of inflammation, which can indirectly be reduced by protective agents.

Effects of shiga toxin 2 on cellular regeneration mechanisms in primary and three-dimensional cultures of human renal tubular epithelial cells

Shiga toxin (Stx)-producing Escherichia coli (STEC) causes post-diarrheal Hemolytic Uremic Syndrome (HUS), which is one of the most common causes of acute renal failure in children in Argentine. The aim of the present work was to study the effects of Shiga toxin type 2 (Stx2) on regenerative mechanisms of primary cultures of human cortical renal tubular epithelial cells (HRTEC) and three-dimensional (3D) cultures of HRTEC. Primary cultures of HRTEC were able to develop tubular structures when grown in matrigel, which showed epithelial cells surrounding a central lumen resembling the original renal tubules. Exposure to Stx2 inhibited tubulogenesis in 3D-HRTEC cultures. Moreover, a significant increase in apoptosis, and decrease in cell proliferation was observed in tubular structures of 3D-HRTEC exposed to Stx2. A significant reduction in cell migration and vimentin expression levels was observed in HRTEC primary cultures exposed to Stx2, demonstrating that the holotoxin affected HRTEC dedifferentiation. Furthermore, a decreased number of cells expressing CD133 progenitor marker was found in HRTEC cultures treated with Stx2. The CD133 positive cells also expressed the Stx receptor globotriaosylceramide, which may explain their sensitivity to Stx2. In conclusion, Stx2 affects the regenerative processes of human renal tubular epithelial cells in vitro, by inhibiting cell dedifferentiation mechanisms, as well as tubules restoration. The development of 3D-HRTEC cultures that resemble original human renal proximal tubules is a novel in vitro model to study renal epithelial repair mechanisms after injury.

Tuberin regulates reactive oxygen species in renal proximal cells, kidney from rodents, and kidney from patients with tuberous sclerosis complex

Reactive oxygen species (ROS) are an important endogenous source of DNA damage and oxidative stress in all cell types. Deficiency in tuberin resulted in increased oxidative DNA damage in renal cells. In this study, the role of tuberin in the regulating of ROS and NADPH oxidases was investigated. Formation of ROS and activity of NADPH oxidases were significantly higher in mouse embryonic fibroblasts and in primary culture of rat renal proximal tubular epithelial tuberin‐deficient cells compared to wild‐type cells. In addition, expression of NADPH oxidase (Nox)1, Nox2, and Nox4 (Nox isoforms) was higher in mouse embryonic fibroblasts and renal proximal tubular epithelial tuberin‐deficient cells compared to wild‐type cells. Furthermore, activity levels of NADPH oxidases and protein expression of all Nox isoforms were higher in the renal cortex of rat deficient in tuberin. However, treatment of tuberin‐deficient cells with rapamycin showed significant decrease in protein expression of all Nox. Significant increase in protein kinase C βII expression was detected in tuberin‐deficient cells, whereas inhibition of protein kinase C βII by bisindolylmaleimide I resulted in decreased protein expression of all Nox isoforms. In addition, treatment of mice deficient in tuberin with rapamycin resulted in significant decrease in all Nox protein expression. Moreover, protein and mRNA expression of all Nox were highly expressed in tumor kidney tissue of patients with tuberous sclerosis complex compared to control kidney tissue of normal subjects. These data provide the first evidence that tuberin plays a novel role in regulating ROS generation, NADPH oxidase activity, and Nox expression that may potentially be involved in development of kidney tumor in patients with tuberous sclerosis complex.

Abstract 4583: Potential role of neuropilin-2 in the regulation of c-MET-induced PD-L1 expression in renal cancer cells

Abstract Neuropilin-1 and -2 (NRP1 and NRP2) are the transmembrane glycoproteins that act as co-receptors for class III semaphorins and several members of the vascular endothelial growth factor (VEGF) family. In conjunction with VEGF receptors and plexins, NRPs and semaphorins modulate various biological responses. Recent studies suggest that NRPs are up-regulated in many types of cancer cells, and may play a major role in tumor growth. In addition to semaphorins and VEGF, NRP1 and NRP2 can also bind to hepatocyte growth factor (HGF), which is the ligand for the tyrosine kinase receptor c-MET. c-MET is highly over-expressed in renal cancer cells; however, the impact of NRPs on c-MET-mediated tumorigenic pathways have not been studied. Recently, we have reported that HGF-/c-MET-induced signaling increases the expression of immunosuppressive and negative-co-stimulatory molecule programmed death-1 ligand (PD-L1) on renal cell carcinoma (RCC) cells, and it protects tumor cells from immune-mediated killing. In this study, we investigated whether NRPs and its ligands can modulate the expression of HGF-/c-MET-induced PD-L1. We have observed that NRP2, and to a lesser extent NRP1, is over-expressed in human RCC cell lines (786-O, ACHN and Caki-1) compared with normal renal epithelial cells at both mRNA and protein level. siRNA-mediated silencing of NRP1 and NRP2 promoted apoptosis of renal cancer cells, suggesting possible role(s) of NRPs in tumor growth. The knock-down of NRP2 down-regulated the cell surface expression of PD-L1 possibly through the reduced interaction of growth-promoting ligands (like, HGF) with NRP2. Interestingly, SEMA3F, a class III semaphorin ligand for both NRP1 and NRP2, has been reported to have anti-tumorigenic effects. We observed that SEMA3F was significantly down-regulated in RCC cells compared with normal renal epithelial cells; and when we added SEMA3F to the cultures of renal cancer cells, it down-regulated both basal and HGF-/c-MET-induced PD-L1 expression. Together, our findings suggest that NRPs (primarily NRP2) can play a critical role in regulating HGF-/c-MET-induced PD-L1 expression in renal cancer cells to mediate immune escape of tumor cells. Therefore targeting NRP2 and/or its activation through SEMA3F/synthetic ligands may restrict c-MET-PD-L1-induced tumor promoting pathways in renal cancer cells. Citation Format: Murugabaskar Balan, Soumitro Pal. Potential role of neuropilin-2 in the regulation of c-MET-induced PD-L1 expression in renal cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4583.

Kidney-on-a-chip technology for renal proximal tubule tissue reconstruction

The renal proximal tubule epithelium is responsible for active secretion of endogenous and exogenous waste products from the body and simultaneous reabsorption of vital compounds from the glomerular filtrate. The complexity of this transport machinery makes investigation of processes such as tubular drug secretion a continuous challenge for researchers. Currently available renal cell culture models often lack sufficient physiological relevance and reliability. Introducing complex biological culture systems in a 3D microfluidic design improves the physiological relevance of in vitro renal proximal tubule epithelium models. Organ-on-a-chip technology provides a promising alternative, as it allows the reconstruction of a renal tubule structure. These microfluidic systems mimic the in vivo microenvironment including multi-compartmentalization and exposure to fluid shear stress. Increasing data supports that fluid shear stress impacts the phenotype and functionality of proximal tubule cultures, for which we provide an extensive background. In this review, we discuss recent developments of kidney-on-a-chip platforms with current and future applications. The improved proximal tubule functionality using 3D microfluidic systems is placed in perspective of investigating cellular signalling that can elucidate mechanistic aberrations involved in drug-induced kidney toxicity.

Albuminuria confers renal resistance to loop diuretics via the stimulation of NLRP3 inflammasome/prostaglandin signaling in thick ascending limb.

Renal resistance to loop diuretics is a frequent complication in a number of kidney disease patients with elusive mechanism. Employing human renal biopsy specimens, albumin overload mouse model, and primary cultures of mouse renal tubular cells, albuminuria effect on NKCC2 expression and function and the underlying mechanisms were investigated. In the renal biopsy specimens of albuminuric patients, we found that NKCC2 was significantly downregulated with a negative correlation with albuminuria severity as examined by immunohistochemistry. Meanwhile, NLRP3 and mPGES-1 were stimulated in NKCC2 positive tubules (thick ascending limb, TAL) paralleled with increased urinary PGE2 excretion. To examine the role of albuminuria in the downregulation of NKCC2 and the potential role of NLRP3/prostaglandin signaling in NKCC2 downregulation, an albumin overload mouse model was employed. Interestingly, we discovered that albuminuria downregulated NKCC2 protein expression in murine kidney and impaired the renal response to loop diuretic furosemide. Specifically, albuminuria suppressed NKCC2 expression and function through NLRP3/prostaglandin dependent signaling in TAL. In primary cultures of renal tubular cells, albumin directly reduced NKCC2 but enhanced NLRP3, COX-2, and mPGES-1 expression. These novel findings demonstrated that albuminuria is of importance in mediating the renal resistance to loop diuretics via NLRP3/prostaglandin signaling-dependent NKCC2 downregulation in TAL. This may also offer novel, effective targets for dealing with the resistance of loop diuretics in proteinuric renal diseases.

Albuminuria enhances NHE3 and NCC via stimulation of mitochondrial oxidative stress/angiotensin II axis.

Imbalance of salt and water is a frequent and challenging complication of kidney disease, whose pathogenic mechanisms remain elusive. Employing an albumin overload mouse model, we discovered that albuminuria enhanced the expression of NHE3 and NCC but not other transporters in murine kidney in line with the stimulation of angiotensinogen (AGT)/angiotensin converting enzyme (ACE)/angiotensin (Ang) II cascade. In primary cultures of renal tubular cells, albumin directly stimulated AGT/ACE/Ang II and upregulated NHE3 and NCC expression. Blocking Ang II production with an ACE inhibitor normalized the upregulation of NHE3 and NCC in cells. Interestingly, albumin overload significantly reduced mitochondrial superoxide dismutase (SOD2), and administration of a SOD2 mimic (MnTBAP) normalized the expression of NHE3, NCC, and the components of AGT/ACE pathway affected by albuminuria, indicating a key role of mitochondria-derived oxidative stress in modulating renin-angiotensin system (RAS) and renal sodium transporters. In addition, the functional data showing the reduced urinary excretion of Na and Cl and enhanced response to specific NCC inhibitor further supported the regulatory results of sodium transporters following albumin overload. More importantly, the upregulation of NHE3 and NCC and activation of ACE/Ang II signaling pathway were also observed in albuminuric patient kidneys, suggesting that our animal model accurately replicates the human condition. Taken together, these novel findings demonstrated that albuminuria is of importance in resetting renal salt handling via mitochondrial oxidative stress-initiated stimulation of ACE/Ang II cascade. This may also offer novel, effective therapeutic targets for dealing with salt and water imbalance in proteinuric renal diseases.

Anti‐Diabetic Agent Sodium Tungstate Induces the Secretion of Pro‐ and Anti‐Inflammatory Cytokines by Human Kidney Cells

Diabetic kidney disease (DKD) is the major cause of end stage renal disease. Sodium tungstate (NaW) exerts anti-diabetic and immunomodulatory activities in diabetic animal models. Here, we used primary cultures of renal proximal tubule epithelial cells derived from type-2-diabetic (D-RPTEC) and non-diabetic (N-RPTEC) subjects as in vitro models to study the effects of NaW on cytokine secretion, as these factors participate in intercellular regulation of inflammation, cell growth and death, differentiation, angiogenesis, development, and repair, all processes that are dysregulated during DKD. In basal conditions, D-RPTEC cells secreted higher levels of prototypical pro-inflammatory IL-6, IL-8, and MCP-1 than N-RPTEC cells, in agreement with their diabetic phenotype. Unexpectedly, NaW further induced IL-6, IL-8, and MCP-1 secretion in both N- and D-RPTEC, together with lower levels of IL-1 RA, IL-4, IL-10, and GM-CSF, suggesting that it may contribute to the extent of renal damage/repair during DKD. Besides, NaW induced the accumulation of IκBα, the main inhibitor protein of one major pathway involved in cytokine production, suggesting further anti-inflammatory effect in the long-term. A better understanding of the mechanisms involved in the interplay between the anti-diabetic and immunomodulatory properties of NaW will facilitate future studies about its clinical relevance. J. Cell. Physiol. 232: 355-362, 2017. © 2016 Wiley Periodicals, Inc.

Rab35 GTPase couples cell division with initiation of epithelial apico-basal polarity and lumen opening.

Establishment and maintenance of apico-basal polarity in epithelial organs must be tightly coupled with cell division, but the underlying molecular mechanisms are largely unknown. Using 3D cultures of renal MDCK cells (cysts), we found that the Rab35 GTPase plays a crucial role in polarity initiation and apical lumen positioning during the first cell division of cyst development. At the molecular level, Rab35 physically couples cytokinesis with the initiation of apico-basal polarity by tethering intracellular vesicles containing key apical determinants at the cleavage site. These vesicles transport aPKC, Cdc42, Crumbs3 and the lumen-promoting factor Podocalyxin, and are tethered through a direct interaction between Rab35 and the cytoplasmic tail of Podocalyxin. Consequently, Rab35 inactivation leads to complete inversion of apico-basal polarity in 3D cysts. This novel and unconventional mode of Rab-dependent vesicle targeting provides a simple mechanism for triggering both initiation of apico-basal polarity and lumen opening at the centre of cysts.

Nephrotoxicity of Combining 2-Phenethylamine and N, N-Dimethyl-β-Phenethylamine

Abstract Phenethylamines are ingredients in many dietary supplements aimed at supporting weight loss or maintaining heightened energy levels for productive workouts. This class of compounds affects the central nervous and cardiac systems, but there is evidence that 2-phenethylamine itself or its derivatives can accumulate in the kidneys and induce nephrotoxicity. As such, we investigated the possible nephrotoxic effects of two naturally occurring compounds: 2-phenethylamine and N, N-dimethyl-β-phenethylamine, alone and in combination, because they are currently used in dietary supplements presently on the market. Our approach entailed testing these compounds using an established in vitro renal cell culture method. Human proximal tubule cells were directly exposed to increasing concentrations of these compounds for 24 hours and evaluated for evidence of cytotoxicity and mitochondrial dysfunction. We also specifically tested for nephrotoxic effects using biomarkers of proximal tubule cell injury. We found t...

Ibuprofen transport in renal cell cultures: characterization of an ibuprofen transporter upregulated by hyperosmolarity

An ibuprofen transporter localizes to the apical and basolateral membrane of MDCK I cells is upregulated by hyperosmotic exposure. Ibuprofen uptake is inhibited by other NSAIDs and ibuprofen metabolites.

Protection against Gluten-mediated Tight Junction Injury with a Novel Lignite Extract Supplement

Background: Tight junctions are found in epithelial cells and function as selective gatekeepers to regulate absorption. PT-gliadin is the gluten protein segment that is known to impair the functioning of tight junctions. This study aimed to examine the effects of a lignite extract dietary supplement (RESTORE) on tight junction function in small intestine (IEC-6) and colon (Caco-2) epithelial cells. The study also evaluated the biologic safety of the same supplement as established by the rates of apoptosis in the intestinal and proximal renal tubule cell cultures treated with the supplement. Methods: IEC-6 and Caco-2 cells were incubated until a stable trans-epithelial electrical resistance (TEER) was measured. The dietary supplement at 20% concentration or a control were placed on the cells and left overnight. These cells were then treated with and without PT-gliadin. Tight junction expression was determined by immunofluorescent microscopy. The rate of apoptosis was established in cell culture with the lignite extract at 20% concentration in order to assess a toxic concentration in normal cell lines: IEC-6, Caco-2, and human renal proximal tubule cell (RPTC) lines. Results: The lignite extract supplement increased the TEER in IEC-6 (58%) and Caco-2 (15%) compared to control. PT-gliadin dramatically decreased the TEER in both control IEC-6 (49%) and control Caco-2 (27%) membranes. The lignite supplement prevented PT-gliadin-mediated decrease in TEER. The supplement reduced apoptosis in RPTC (44%), IEC-6 (13%), and Caco-2 (24%) cell cultures. Conclusion: The lignite supplement blocked a PT-gliadin dependent decrease in TEER in small intestine and colon cell line membranes. The lignite extract was not toxic on intestinal or renal cells at high concentration, and demonstrated a statistically significant reduction in apoptosis in RPTCs. Human clinical trials are needed to evaluate the use of RESTORE to support health in gluten-sensitive individuals.

Data on Na,K-ATPase in primary cultures of renal proximal tubule cells treated with catecholamines.

This data article is concerned with chronic regulation of Na,K-ATPase by catecholamines. After a chronic treatment, inhibition of Na,K-ATPase activity was observed in cultures with dopamine, while a stimulation was observed in cultures treated with norepinephrine. Following a chronic incubation with guanabenz, an α adrenergic agonist, an increase in Na,K-ATPase α and β subunit mRNAs was observed. This data supports the research article entitled, “Renal proximal tubule Na, K-ATPase is controlled by CREB regulated transcriptional coactivators as well as salt inducible kinase 1” (Taub et al. 2015) [1].

Methionine restriction fundamentally supports health by tightening epithelial barriers

Dietary methionine restriction (MR) has been found to affect one of the most primary tissue-level functions of an organism: the efficiency with which the epithelial linings of major organs separate the fluid compartments that they border. This process, epithelial barrier function, is basic for proper function of all organs, including the lung, liver, gastrointestinal tract, reproductive tract, blood-brain barrier, and kidney. Specifically, MR has been found to modify the protein composition of tight junctional complexes surrounding individual epithelial cells in a manner that renders the complexes less leaky. This has been observed in both a renal epithelial cell culture model and in gastrointestinal tissue. In both cases, MR increased the transepithelial electrical resistance across the epithelium, while decreasing passive leak of small nonelectrolytes. However, the specific target protein modifications involved were unique to each case. Overall, this provides an example of the primary level on which MR functions to modify, and improve, an organism.

Requirement of clusterin expression for prosurvival autophagy in hypoxic kidney tubular epithelial cells.

Cellular autophagy is a prosurvival mechanism in the kidney against ischemia-reperfusion injury (IRI), but the molecular pathways that activate the autophagy in ischemic kidneys are not fully understood. Clusterin (CLU) is a chaperone-like protein, and its expression is associated with kidney resistance to IRI. The present study investigated the role of CLU in prosurvival autophagy in the kidney. Renal IRI was induced in mice by clamping renal pedicles at 32°C for 45 min. Hypoxia in renal tubular epithelial cell (TEC) cultures was induced by exposure to a 1% O2 atmosphere. Autophagy was determined by either light chain 3-BII expression with Western blot analysis or light chain 3-green fluorescent protein aggregation with confocal microscopy. Cell apoptosis was determined by flow cytometric analysis. The unfolded protein response was determined by PCR array. Here, we showed that autophagy was significantly activated by IRI in wild-type (WT) but not CLU-deficient kidneys. Similarly, autophagy was activated by hypoxia in human proximal TECs (HKC-8) and WT mouse primary TECs but was impaired in CLU-null TECs. Hypoxia-activated autophagy was CLU dependent and positively correlated with cell survival, and inhibition of autophagy significantly promoted cell death in both HKC-8 and mouse WT/CLU-expressing TECs but not in CLU-null TECs. Further experiments showed that CLU-dependent prosurvival autophagy was associated with activation of the unfolded protein response in hypoxic kidney cells. In conclusion, these data suggest that activation of prosurvival autophagy by hypoxia in kidney cells requires CLU expression and may be a key cytoprotective mechanism of CLU in the protection of the kidney from hypoxia/ischemia-mediated injury.

Quantitative Proteomic Analysis of Enriched Nuclear Fractions from BK Polyomavirus-Infected Primary Renal Proximal Tubule Epithelial Cells.

Polyomaviruses are a family of small DNA viruses that are associated with a number of severe human diseases, particularly in immunocompromised individuals. The detailed virus-host interactions during lytic polyomavirus infection are not fully understood. Here, we report the first nuclear proteomic study with BK polyomavirus (BKPyV) in a primary renal proximal tubule epithelial cell culture system using stable isotope labeling by amino acids in cell culture (SILAC) proteomic profiling coupled with liquid chromatography-tandem mass spectrometry. We demonstrated the feasibility of SILAC labeling in these primary cells and subsequently performed reciprocal labeling-infection experiments to identify proteins that are altered by BKPyV infection. Our analyses revealed specific proteins that are significantly up- or down-regulated in the infected nuclear proteome. The genes encoding many of these proteins were not identified in a previous microarray study, suggesting that differential regulation of these proteins may be independent of transcriptional control. Western blotting experiments verified the SILAC proteomic findings. Finally, pathway and network analyses indicated that the host cell DNA damage response signaling and DNA repair pathways are among the cellular processes most affected at the protein level during polyomavirus infection. Our study provides a comprehensive view of the host nuclear proteomic changes during polyomavirus lytic infection and suggests potential novel host factors required for a productive polyomavirus infection.

A bioartificial environment for kidney epithelial cells based on a supramolecular polymer basement membrane mimic and an organotypical culture system.

Renal applications in healthcare, such as renal replacement therapies and nephrotoxicity tests, could potentially benefit from bioartificial kidney membranes with fully differentiated and functional human tubular epithelial cells. A replacement of the natural environment of these cells is required to maintain and study cell functionality cell differentiation in vitro. Our approach was based on synthetic supramolecular biomaterials to mimic the natural basement membrane (BM) on which these cells grow and a bioreactor to provide the desired organotypical culture parameters. The BM mimics were constructed from ureidopyrimidinone (UPy)-functionalized polymer and bioactive peptides by electrospinning. The resultant membranes were shown to have a hierarchical fibrous BM-like structure consisting of self-assembled nanofibres within the electrospun microfibres. Human kidney-2 (HK-2) epithelial cells were cultured on the BM mimics under organotypical conditions in a custom-built bioreactor. The bioreactor facilitated in situ monitoring and functionality testing of the cultures. Cell viability and the integrity of the epithelial cell barrier were demonstrated inside the bioreactor by microscopy and transmembrane leakage of fluorescently labelled inulin, respectively. Furthermore, HK-2 cells maintained a polarized cell layer and showed modulation of both gene expression of membrane transporter proteins and metabolic activity of brush border enzymes when subjected to a continuous flow of culture medium inside the new bioreactor for 21 days. These results demonstrated that both the culture and study of renal epithelial cells was facilitated by the bioartificial in vitro environment that is formed by synthetic supramolecular BM mimics in our custom-built bioreactor. Copyright © 2015 John Wiley & Sons, Ltd.