Kidney Proximal Tubule Epithelial Cells Research Articles

Abstract P1100: Angiotensin-(1-7) Via Angiotensin Type-1 Receptor Synergies Angiotensin Type-2 Receptor Response of Nitrite Formation in Kidney Cells

Angiotensin-(1-7) is evidently considered as an endogenous agonist for Mas receptor. Our recent studies show that MasR and AT 2 R physically interact and in response to their respective agonists Ang-(1-7) and C21 produce interdependent functional response in terms of nitric oxide (NO) generation and natriuresis. Moreover, recent studies suggest that Ang-(1-7) is cardio-protective via AT 1 R stimulation. Considering the complex nature of Ang-(1-7) function, we tested as to how Ang-(1-7) affects AT 2 R function utilizing human kidney proximal tubule epithelial cells (HK-2 cells). Ang-(1-7), C21 or AVE0991 (a MasR selective agonist), alone significantly stimulated NO formation at 1-10 μM (50-100%, n=3), but not at lower concentrations (0.1-100 nM). In the interaction studies, the non-responsive concentrations (0.1-10 nM) of Ang-(1-7) and C21 together produced a synergy in producing NO in HK-2 cells (p<0.05 vs. either ligand alone). The synergy is observed only when the Ang-(1-7) was added to the HK-2 cells 10 min prior to the C21, not if the agonists were added simultaneously. The synergy between Ang-(1-7) and C21 was sensitive to AT 2 R antagonist (PD123319, 10 μM) and AT 1 R antagonist (candesartan, 10 μM), but not MasR antagonist (A779, 10 μM). Interestingly, pre-stimulation with synthetic non-peptide MasR selective agonist AVE0991 at any concentrations (0.1-100 nM) did not show synergism with AT 2 R agonist C21. Collectively, results show Ang-(1-7) at lower concentrations via AT 1 R, not MasR, synergies the AT 2 R function and this phenomenon may have physiological significance.

Activation of vitamin D receptor attenuates high glucose-induced cellular injury partially dependent on CYP2J5 in murine renal tubule epithelial cell

AimsVitamin D and its receptor, vitamin D receptor (VDR), have renoprotection effect against diabetic nephropathy (DN). But the exact mechanism has not been fully elucidated. Epoxyeicosatrienoic acids (EETs) are cytochrome P450 (CYP) epoxygenase-derived metabolites of arachidonic acid, protecting against diabetes and DN. Herein, we hypothesized that activation of VDR attenuated high glucose-induced cellular injury in renal tubular epithelial cells partially through up-regulating CYP2J5 expression. Main methodsStreptozotocin (STZ) was injected to induce diabetic in wild type and Vdr−/− mice. The effects of VDR knockout and an activator of VDR, paricalcitol, on the renal injury were detected. In vitro, a murine kidney proximal tubule epithelial cell line BU.MPT induced by high glucose were treated with or without paricalcitol (30 mM) for 12 h or 24 h. Key findingsThe expression of CYP2J5 was significantly decreased both in wild type and Vdr−/− diabetic mice induced by STZ. The STZ-induced kidney architecture damage and apoptosis rate in Vdr−/− mice were more severe. In vitro, high glucose treatment strongly reduced the CYP2J5 expression and the synthesis of 14,15-EET in BU.MPT cells. Supplement of 14,15-EET significantly reduced the lactate dehydrogenase (LDH) release induced by high glucose in BU.MPT cells. Furthermore, treatment with paricalcitol attenuated cellular injury and restored the expression of CYP2J5 reduced by high glucose in BU.MPT cells. SignificanceWe conclude that activation of VDR attenuates high glucose-induced cellular injury partially dependent on CYP2J5 in murine renal tubule epithelial cells and paricalcitol may represent a potential therapy for DN.

520-P: Hyperglycemia-Induced Mitochondrial Dysfunction in Diabetic Kidney Disease May Be Mediated by Crabtree Effect in Kidney Proximal Tubule Epithelial Cells

Background: Recent studies indicate mitochondrial dysfunction is a dominant pathway in diabetic kidney disease (DKD), however underlying mechanisms are unclear. Here we examined if Crabtree effect by glucose is a cause of mitochondrial dysfunction in kidney. Methods: Lactate was measured by by YSI biochemistry analyzer. TCA cycle metabolites were measured by gas chromatography mass spectroscopy in urine samples of type 1 and type 2 DKD mouse models. Changes in oxygen consumption (OCR) and extracellular acidification i(ECAR) were analyzed on a Seahorse extracellular flux analyzer. Immunohistochemical analysis was performed on FFPE-embedded mouse kidney sections. Western blot analysis was performed on kidney cortical sections and HK2 cells. Results: Significantly increased urine and plasma lactate along with a decrease in several TCA cycle metabolites were observed in both type 1 and type 2 diabetic mice. Mitochondrial functional analysis in different kidney cells showed a profound respiratory inhibition and stimulation of lactate by glucose in proximal tubules, but not in mesangial cells and podocytes. Pyruvate at above physiological concentration reversed the glucose-induced inhibition of OCR. Western blotting of kidney cortical tissue sections showed a significant increase in phospho PDH in diabetic kidney tissues and in HK2 cells treated with high glucose. In normal conditions, phospho PDH was predominantly on distal tubules, however in diabetic conditions a significant increase in phospho PDH was noted in proximal tubule cells. Dichloroacetate, an inhibitor of PDK completely reversed the glucose induced respiratory inhibition in proximal tubule epithelial cells. Conclusions: Mitochondrial dysfunction in proximal tubule epithelial cells may potentially be due to high-glucose induced PDH phosphorylation resulting in limited pyruvate oxidation into mitochondria and enhanced lactate production. Disclosure M. Darshi: None. J.J. Kim: None. V. Drel: None. A.N. Murphy: Advisory Panel; Self; Cytokinetics Inc. Consultant; Self; Agilent Technologies. Funding JDRF; U.S. Department of Veterans Affairs

The Effect of Overexpression of the Enhancer of Zeste Homolog 1 (EZH1) Gene on Aristolochic Acid-Induced Injury in HK-2 Human Kidney Proximal Tubule Cells In Vitro.

BackgroundAcute kidney injury (AKI) involves the renal tubular epithelium. The enhancer of zeste homolog 1 (EZH1) gene has a role in cell development and differentiation. This study aimed to investigate the effect of overexpression of the EZH1 gene on aristolochic acid-induced injury in HK-2 human kidney proximal tubule epithelial cells in vitro.Material/MethodsThe HK-2 cells were cultured and treated with aristolochic acid and the effects of aristolochic acid-injury were evaluated using a cell counting kit-8 (CCK-8) assay. Overexpression of EZH1 used gene plasmid transfection into HK-2 cells. The cell apoptosis rate and levels of intracellular reactive oxygen species (ROS) were measured using flow cytometry. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were performed to determine the expressions of inflammatory cytokines including interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α), apoptosis-related genes, and the downstream target genes of NF-κB signaling pathway, including NFKBIA, CXCL8, and cyclin D1.ResultsAristolochic acid inhibited HK-2 cell viability, induced cell apoptosis, increased the levels of ROS and inflammatory cytokines, including IL-1β, IL-6, TNF-α, and activated the NF-κB pathway. Overexpression the EZH1 gene inhibited HK-2 cell apoptosis, reduced ROS levels, and down-regulated the expressions of IL-1β, IL-6, TNF-α, Bax and Cyt C mRNA and protein, and increased the expressions of Bcl-2 and NFKBIA, CXCL8 and cyclin D1, indicating that overexpression of EZH1 suppressed NF-κB signaling in aristolochic acid-injured HK-2 cells.ConclusionsOverexpression of EZH1 reduced HK-2 cell injury induced by aristolochic acid in vitro by inhibition of NF-κB signaling.

Cyclosporin A aggravates hydrogen peroxide-induced cell death in kidney proximal tubule epithelial cells.

Cyclosporin A (CsA) does not only exert a toxic effect on kidney parenchymal cells, but also protects them against necrotic cell death by inhibiting opening of mitochondrial permeability transition pore. However, whether CsA plays a role in hydrogen peroxide-induced kidney proximal tubular cell death is currently unclear. In the present study, treatment with CsA further increased apoptosis and necrosis in HK-2 human kidney proximal tubule epithelial cells during exposure to hydrogen peroxide. In addition, hydrogen peroxide-induced p53 activation and BH3 interacting-domain death agonist (BID) expression were higher in CsA-treated cells than those in non-treated cells, whereas hydrogen peroxide-induced activation of mitogen-activated protein kinases including p38, c-Jun N-terminal kinase, and extracellular signal-regulated kinase and activation of protein kinase B were not significantly altered by treatment with CsA. In oxidant-antioxidant system, reactive oxygen species (ROS) production induced by hydrogen peroxide was further enhanced by treatment with CsA. However, expression levels of antioxidant enzymes including manganese superoxide dismutase, copper/zinc superoxide dismutase, and catalase were not altered by treatment with hydrogen peroxide or CsA. Treatment with CsA further enhanced mitochondrial membrane potential induced by exposure to hydrogen peroxide, although it did not alter endoplasmic reticulum stress based on expression of glucose-regulated protein 78 and 94. Taken together, these data suggest that CsA can aggravate hydrogen peroxide-induced cell death through p53 activation, BID expression, and ROS production.

Extraneural CGRP Induces Oxidative Stress in Kidney Proximal Tubule Epithelial Cells

Extraneural CGRP Induces Oxidative Stress in Kidney Proximal Tubule Epithelial Cells

Fluorescent Ca2+ indicators directly inhibit the Na,K-ATPase and disrupt cellular functions.

Fluorescent Ca2+ indicators have been essential for the analysis of Ca2+ signaling events in various cell types. We showed that chemical Ca2+ indicators, but not a genetically encoded Ca2+ indicator, potently suppressed the activity of Na+- and K+-dependent adenosine triphosphatase (Na,K-ATPase), independently of their Ca2+ chelating activity. Loading of commonly used Ca2+ indicators, including Fluo-4 acetoxymethyl (AM), Rhod-2 AM, and Fura-2 AM, and of the Ca2+ chelator BAPTA AM into cultured mouse or human neurons, astrocytes, cardiomyocytes, or kidney proximal tubule epithelial cells suppressed Na,K-ATPase activity by 30 to 80%. Ca2+ indicators also suppressed the agonist-induced activation of the Na,K-ATPase, altered metabolic status, and caused a dose-dependent loss of cell viability. Loading of Ca2+ indicators into mice, which is carried out for two-photon imaging, markedly altered brain extracellular concentrations of K+ and ATP. These results suggest that a critical review of data obtained with chemical Ca2+ indicators may be necessary.

Toxicity of sub-micron and micron calcium oxalate monohydrate on renal epithelial cells

[Objective] This study aims to synthesize calcium oxalate monohydrate (COM) crystals with a size of 300 nm and 5 μm, respectively, and to compare their damage and adhesion ability toward human kidney proximal tubule epithelial cells (HKC). [Method] The injury degree of HKC caused by COM crystals was measured by detecting the cell viability, lactate dehydrogenase (LDH) release amounts, reactive oxygen species (ROS), cell death rate, hyaluronic acid (HA) expression and the crystal adhesion amount on cell surface. [Result] HKC cell viability, LDH release quantity, cell death rate and HA expression were closely associated with the size of COM crystals. The damage ability of 300 nm COM crystals on normal cells was higher than that of COM-5 μm crystals. The adhesive amount of small size COM crystals (62.4 μg/cm2) was higher than micron COM crystals (26.7 μg/cm2). The increased adhesion amount of small size COM crystal are related to its larger specific surface area, crystal face changes and larger cell contact probability. [Conclusions] The cytotoxicity of COM crystals toward HKC cells is size dependent. This study is helpful to further clarify the formation mechanism of COM calculi, and provide inspiration for its prevention and treatment.

Kaempferol increases levels of coenzyme Q in kidney cells and serves as a biosynthetic ring precursor

Coenzyme Q (Q) is a lipid-soluble antioxidant essential in cellular physiology. Patients with Q deficiencies, with few exceptions, seldom respond to treatment. Current therapies rely on dietary supplementation with Q10, but due to its highly lipophilic nature, Q10 is difficult to absorb by tissues and cells. Plant polyphenols, present in the human diet, are redox active and modulate numerous cellular pathways. In the present study, we tested whether treatment with polyphenols affected the content or biosynthesis of Q. Mouse kidney proximal tubule epithelial (Tkpts) cells and human embryonic kidney cells 293 (HEK 293) were treated with several types of polyphenols, and kaempferol produced the largest increase in Q levels. Experiments with stable isotope 13C-labeled kaempferol demonstrated a previously unrecognized role of kaempferol as an aromatic ring precursor in Q biosynthesis. Investigations of the structure-function relationship of related flavonols showed the importance of two hydroxyl groups, located at C3 of the C ring and C4′ of the B ring, both present in kaempferol, as important determinants of kaempferol as a Q biosynthetic precursor. Concurrently, through a mechanism not related to the enhancement of Q biosynthesis, kaempferol also augmented mitochondrial localization of Sirt3. The role of kaempferol as a precursor that increases Q levels, combined with its ability to upregulate Sirt3, identify kaempferol as a potential candidate in the design of interventions aimed on increasing endogenous Q biosynthesis, particularly in kidney.

Transcriptomic profiling of tight junctions dysfunction in endothelial cells and epithelial cells in renal failure

BackgroundChronic kidney disease (CKD) has been recently identified as a significant risk factor for vascular diseases such as cerebral small‐vessel diseases (CSVD), and up to 50% of deaths in patients with end‐stage renal failure are due to cardiovascular disease (CVD). There is a 10 to 30‐fold higher cardiovascular mortality in CKD patients than in the age‐matched general population, despite adjustment for classical cardiovascular risk factors. Tight junctions (TJ) are specialized membrane domains that play multiple functions in kidney epithelial cells, including the maintenance of cellular polarity and work as a primary regulatory barrier particularly in cerebral microvasculatures and kideny. Studies indicate emerging links between TJ dysfunction and the development of kidney disease and following CVD and CSVD. The goal of this study was to elucidate the transcription profiling related to TJ dysfunction and its role in preventing renal failure through the regulation of TJs.MethodsTo profile the exhaustive signatures in TJ dysfunction human endothelium and epithelium, we performed whole transcriptome analysis to explore novel genes in primary human aortic endothelial cells (HAECs), human brain microvascular endothelial cells (HBMECs) and immortalized human kidney proximal tubule epithelial cells (HK‐2) using our CKD models, in vitro. In addition, we analyzed human arteries from CVD and CKD patients. Total RNA was extracted using the Nucleospin RNA isolation kit (Clontech). cDNA was synthesized and fragmented using SMART‐seq2 and Nextera XT (Illumina), respectively to generate libraries for sequencing on Hi‐seq 2000 (Illumina). The abundancies of each gene, (FPKM, fragment per kilo‐base exon per million mapped reads), were estimated using RNA‐seq by Expectation Maximization (RSEM). Log 2 transformed FPKM were normalized by quantile normalization, which were then subjected to differential expression analysis to calculate significant fold change (Gene‐E, Broad Institute). Gene selections were performed by the combinations of fold‐changes on log 2 ratio, and p value < 0.05. Data were analyzed using Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg/) and The Database for Annotation, Visualization and Integrated Discovery v6.7 (DAVID, http://david.abcc.ncifcrf.gov/).ResultsPathway analysis among the differentially expressed genes in comparing normal samples to CKD groups revealed that these genes are mainly involved in cytoskeleton formation, cell metabolic, proliferation, and apoptosis processes. Among these TJ dysfunction related genes, heat shock protein 70 families (HSPA1, HSPB7), SYT, SNAP25, and aryl hydrocarbon receptor nuclear translocator (ARNT) in HIF‐1signaling pathways were identified. In addition, MT‐RNR1 and cannabinoid receptor 1 were also detected that are involved in ATP production in the mitochondria and as co‐chaperone for HSP70.ConclusionWe have identified a group of genes differentially expressed in healthy and CKD endothelial cells and epithelial cells. Our data contribute to our understanding of the direct effects in the presence of TJ dysfunction at endothelial cells and epithelial cells. This gene set may have pathophysiological significance and may serve to inform rational development of therapeutic strategies to prevent CVD adverse effects in CKD patients.

Bardoxolone methyl modulates efflux transporter and detoxifying enzyme expression in cisplatin-induced kidney cell injury

Cisplatin is prescribed for the treatment of solid tumors and elicits toxicity to kidney tubules, which limits its clinical use. Nuclear factor erythroid 2-related factor 2 (Nrf2, NFE2L2) is a critical transcription factor that has been shown to protect against kidney injury through activation of antioxidant mechanisms. We aimed to evaluate the ability of short-term treatment with the Nrf2 activator bardoxolone methyl (CDDO-Me) to protect against cisplatin-induced kidney cell toxicity. Cell viability was assessed in human kidney proximal tubule epithelial cells (hPTCs) exposed to low, intermediate, and high cisplatin concentrations in the presence and absence of CDDO-Me, administered either prior to or after cisplatin. Treatment with cisplatin alone resulted in reductions in hPTC viability, while CDDO-Me administered prior to or after cisplatin exposure yielded significantly higher cell viability (17%–71%). Gene regulation (mRNA expression) studies revealed the ability of CDDO-Me to modify protective pathways including Nrf2 induced detoxifying genes [GCLC (increased 1.9-fold), NQO1 (increased 9.3-fold)], and an efflux transporter [SLC47A1 (increased 4.5-fold)] at 12h. Protein assessments were in agreement with gene expression. Immunofluorescence revealed localization of GCLC and NQO1 to the nucleus and cytosol, respectively, with CDDO-Me administered prior to or after cisplatin exposure. The findings of enhanced cell viability and increased expression of detoxifying enzymes (GCLC and NQO1) and the multidrug and toxin extrusion protein 1 (MATE1) efflux transporter (SLC47A1) in hPTCs exposed to CDDO-Me, suggest that intermittent treatment with CDDO-Me prior to or after cisplatin exposure may be a promising approach to mitigate acute kidney injury.

SIRT1 activator ameliorates the renal tubular injury induced by hyperglycemia in vivo and in vitro via inhibiting apoptosis.

We aimed to explore the role of SIRT1 in apoptosis in human kidney proximal tubule epithelial (HK-2) cells, and to determine whether resveratrol (RSV, a SIRT1 activator) could ameliorate apoptosis in rats with streptozotocin-induced diabetes mellitus (DM) and/or in high glucose (HG, 30mM) - stimulated HK-2 cells. Rats were distributed randomly into three groups: 1) control group, 2) DM group, and 3) DM with RSV group (DM+RSV; rats treated with 30mg/kg/d of RSV for 16 weeks). The physical, biochemical, and morphological parameters were then examined. Additionally, the deacetylase activity of SIRT1, and the expression levels of SIRT1 and of representative apoptosis markers, such as p53, acetylated p53, cleaved caspase-3, caspase-9, and cleaved PARP, were measured. HK-2 cells were stimulated by HG for different lengths of time to study the effect of HG on apoptosis. HK-2 cells were treated with or without RSV (25μM) to investigate if RSV has a protective effect on HG-induced apoptosis. A gene-specific small interfering RNA against SIRT1 was used to study the role of SIRT1 in apoptosis. More apoptosis was found in the DM rats than in the control rats. Similarly, the expression levels of cleaved caspase-3, cleaved PARP, and acetylated p53 were significantly higher, and the level of SIRT1 was significantly lower, in the HK-2 cells that were cultured under HG conditions than those in the HK-2 cells that were cultured under low glucose (5.5mM) conditions. Notably, treatment with RSV lessened the HG-induced changes in the levels of apoptosis indicators, and this inhibition of HG-induced apoptosis in HK-2 cells by RSV treatment was abolished by SIRT1 silencing. Our study showed that hyperglycemia contributes to apoptosis in rat kidney and HK-2 cells. SIRT1 activation by RSV can reduce urinary albumin excretion and proximal tubule epithelial apoptosis both in vitro and in vivo. Based on our study, SIRT1/p53 axis played an important role in the hyperglycemia induced apoptosis. These findings indicated that the increased expression of SIRT1, mediated by RSV, is a possible mechanism by which RSV prevents renal tubular injury in diabetic nephropathy (DN). So RSV has great clinical significance and could provide the basis for the new way to effective treatment to contain the morbidity and mortality associated with DN.

Poly(ADP-ribose) polymerase regulates glycolytic activity in kidney proximal tubule epithelial cells.

After renal injury, selective damage occurs in the proximal tubules as a result of inhibition of glycolysis. The molecular mechanism of damage is not known. Poly(ADP-ribose) polymerase (PARP) activation plays a critical role of proximal tubular cell death in several renal disorders. Here, we studied the role of PARP on glycolytic flux in pig kidney proximal tubule epithelial LLC-PK1 cells using XFp extracellular flux analysis. Poly(ADP-ribosyl)ation by PARP activation was increased approximately 2-fold by incubation of the cells in 10 mM glucose for 30 minutes, but treatment with the PARP inhibitor 3-aminobenzamide (3-AB) does-dependently prevented the glucose-induced PARP activation (approximately 14.4% decrease in 0.1 mM 3-AB–treated group and 36.7% decrease in 1 mM 3-AB–treated group). Treatment with 1 mM 3-AB significantly enhanced the glucose-mediated increase in the extracellular acidification rate (61.1±4.3 mpH/min vs. 126.8±6.2 mpH/min or approximately 2-fold) compared with treatment with vehicle, indicating that PARP inhibition increases only glycolytic activity during glycolytic flux including basal glycolysis, glycolytic activity, and glycolytic capacity in kidney proximal tubule epithelial cells. Glucose increased the activities of glycolytic enzymes including hexokinase, phosphoglucose isomerase, phosphofructokinase-1, glyceraldehyde-3-phosphate dehydrogenase, enolase, and pyruvate kinase in LLC-PK1 cells. Furthermore, PARP inhibition selectively augmented the activities of hexokinase (approximately 1.4-fold over vehicle group), phosphofructokinase-1 (approximately 1.6-fold over vehicle group), and glyceraldehyde-3-phosphate dehydrogenase (approximately 2.2-fold over vehicle group). In conclusion, these data suggest that PARP activation may regulate glycolytic activity via poly(ADP-ribosyl)ation of hexokinase, phosphofructokinase-1, and glyceraldehyde-3-phosphate dehydrogenase in kidney proximal tubule epithelial cells.

HuangQi Decoction Ameliorates Renal Fibrosis via TGF-β/Smad Signaling Pathway In Vivo and In Vitro

Objective: Traditional Chinese Medicine compound HuangQi decoction is widely used in clinical treatment of chronic kidney disease, but its role on renal interstitial fibrosis and the underlying mechanism remains unclear. The aim of this study is to investigate the effect of HuangQi decoction on renal interstitial fibrosis and its association with the TGF-β/Smad signaling pathway Methods: A total of 120 C57/BL mice were randomly divided into six groups: sham group, sham plus high-dose HuangQi decoction (1.08g/kg) group, unilateral ureteral obstruction (UUO) model group, and UUO model plus low to high doses of HuangQi decoction (0.12g/kg, 0.36g/kg and 1.08g/kg respectively) groups. Animals were sacrificed 14 days after the administration and ipsilateral kidney tissue was sampled for pathologic examinations. Immunohistochemistry, PCR and western blot were used to detect the expressions of related molecules in the TGF-β/Smad signaling pathway. TGF-β1 was used in in vitro experiments to induce human kidney proximal tubule epithelial cells (HK2). Results: HuangQi decoction improved ipsilateral kidney fibrosis in UUO mice and downregulated the expressions of TGF-β1, TβRI, TβRII, Smad4, Smad2/3, P-Smad2/3, α-SMA, collagen type I, III and IV in a dose-dependent manner while upregulated the expression of Smad7 in the same fashion. Similar results were found in in vitro studies. Conclusion: The protective effect of HuangQi decoction for unilateral ureteral obstruction kidney damage in mice was mediated by downregulating the TGF-β/Smad signaling pathway.

The potential role of regucalcin in kidney cell regulation: Involvement in renal failure (Review).

The kidneys play a physiologic role in the regulation of urine formation and nutrient reabsorption in the proximal tubule epithelial cells. Kidney development has been shown to be regulated through calcium(Ca2+) signaling processes that are present through numerous steps of tubulogenesis and nephron induction during embryonic development of the kidneys. Ca2+-binding proteins, such as calbindin-D28k and regucalcin are important proteins that are commonly used as biomarkers in pronephric tubules, and the ureteric bud and metanephric mesenchyme. Previous research on regucalcin focused on Ca2+ sensors that are involved in renal organogenesis and the link between Ca2+-dependent signals and polycystins. Moreover, regucalcin has been highlighted to play a multifunctional role in kidney cell regulation. The regucalcin gene, which is localized on the Xchromosome, is regulated through various transcription factors. Regucalcin has been found to regulate intracellular Ca2+ homeostasis in kidney proximal tubule epithelial cells. Regucalcin has been demonstrated to regulate the activity of various enzymes that are involved in intracellular signaling pathways. It has been noted that regucalcin suppresses DNA synthesis and regulates the gene expression of various proteins related to mineral transport, transcription factors, cell proliferation and apoptosis. The overexpression of regucalcin has been shown to exert suppressive effects on cell proliferation and apoptotic cell death, which are stimulated by various stimulatory factors. Moreover, regucalcin gene expression was found to to be involved in various pathophysiological states, including renal failure. This review discusses recent findings concerning the potential role of regucalcin as a regulatory protein in the kidney proximal tubule epithelial cells.

TRPM2 mediates ischemic kidney injury and oxidant stress through RAC1.

Ischemia is a leading cause of acute kidney injury. Kidney ischemia is associated with loss of cellular ion homeostasis; however, the pathways that underlie ion homeostasis dysfunction are poorly understood. Here, we evaluated the nonselective cation channel transient receptor potential melastatin 2 (TRPM2) in a murine model of kidney ischemia/reperfusion (I/R) injury. TRPM2-deficient mice were resistant to ischemic injury, as reflected by improved kidney function, reduced histologic damage, suppression of proapoptotic pathways, and reduced inflammation. Moreover, pharmacologic TRPM2 inhibition was also protective against I/R injury. TRPM2 was localized mainly in kidney proximal tubule epithelial cells, and studies in chimeric mice indicated that the effects of TRPM2 are due to expression in parenchymal cells rather than hematopoietic cells. TRPM2-deficient mice had less oxidative stress and lower levels of NADPH oxidase activity after ischemia. While RAC1 is a component of the NADPH oxidase complex, its relation to TRPM2 and kidney ischemic injury is unknown. Following kidney ischemia, TRPM2 promoted RAC1 activation, with active RAC1 physically interacting with TRPM2 and increasing TRPM2 expression at the cell membrane. Finally, inhibition of RAC1 reduced oxidant stress and ischemic injury in vivo. These results demonstrate that TRPM2-dependent RAC1 activation increases oxidant stress and suggest that therapeutic approaches targeting TRPM2 and/or RAC1 may be effective in reducing ischemic kidney injury.

Autophagy protects kidney proximal tubule epithelial cells from mitochondrial metabolic stress

Chronic metabolic stress is related to diseases, whereas autophagy supplies nutrients by recycling the degradative products. Cyclosporin A (CsA), a frequently used immunosuppressant, induces metabolic stress via effects on mitochondrial respiration, and thereby, its chronic usage is often limited. Here we show that autophagy plays a protective role against CsA-induced metabolic stress in kidney proximal tubule epithelial cells. Autophagy deficiency leads to decreased mitochondrial membrane potential, which coincides with metabolic abnormalities as characterized by decreased levels of amino acids, increased tricarboxylic acid (TCA) ratio (the levels of intermediates of the latter part of the TCA cycle, over levels of intermediates in the earlier part), and decreased products of oxidative phosphorylation (ATP). In addition to the altered profile of amino acids, CsA decreased the hyperpolarization of mitochondria with the disturbance of mitochondrial energy metabolism in autophagy-competent cells, i.e., increased TCA ratio and worsening of the NAD+/NADH ratio, coupled with decreased energy status, which suggests that adaptation to CsA employs autophagy to supply electron donors from amino acids via intermediates of the latter part of the TCA cycle. The TCA ratio of autophagy-deficient cells was further worsened with decreased levels of amino acids in response to CsA, and, as a result, the deficiency of autophagy failed to adapt to the CsA-induced metabolic stress. Deterioration of the TCA ratio further worsened energy status. The CsA-induced metabolic stress also activated regulatory genes of metabolism and apoptotic signals, whose expressions were accelerated in autophagy-deficient cells. These data provide new perspectives on autophagy in conditions of chronic metabolic stress in disease.

Insulin receptor substrate‐2 is expressed in kidney epithelium and up‐regulated in diabetic nephropathy

Diabetic nephropathy (DN) is a progressive fibrotic condition that may lead to end-stage renal disease and kidney failure. Transforming growth factor-β1 and bone morphogenetic protein-7 (BMP7) have been shown to induce DN-like changes in the kidney and protect the kidney from such changes, respectively. Recent data identified insulin action at the level of the nephron as a crucial factor in the development and progression of DN. Insulin requires a family of insulin receptor substrate (IRS) proteins for its physiological effects, and many reports have highlighted the role of insulin and IRS proteins in kidney physiology and disease. Here, we observed IRS2 expression predominantly in the developing and adult kidney epithelium in mouse and human. BMP7 treatment of human kidney proximal tubule epithelial cells (HK-2 cells) increases IRS2 transcription. In addition, BMP7 treatment of HK-2 cells induces an electrophoretic shift in IRS2 migration on SDS/PAGE, and increased association with phosphatidylinositol-3-kinase, probably due to increased tyrosine/serine phosphorylation. In a cohort of DN patients with a range of chronic kidney disease severity, IRS2 mRNA levels were elevated approximately ninefold, with the majority of IRS2 staining evident in the kidney tubules in DN patients. These data show that IRS2 is expressed in the kidney epithelium and may play a role in the downstream protective events triggered by BMP7 in the kidney. The specific up-regulation of IRS2 in the kidney tubules of DN patients also indicates a novel role for IRS2 as a marker and/or mediator of human DN progression.

Topographically-patterned porous membranes in a microfluidic device as an in vitro model of renal reabsorptive barriers.

Models of reabsorptive barriers require both a means to provide realistic physiologic cues to and quantify transport across a layer of cells forming the barrier. Here we have topographically-patterned porous membranes with several user-defined pattern types. To demonstrate the utility of the patterned membranes, we selected one type of pattern and applied it to a membrane to serve as a cell culture support in a microfluidic model of a renal reabsorptive barrier. The topographic cues in the model resemble physiological cues found in vivo while the porous structure allows quantification of transport across the cell layer. Sub-micron surface topography generated via hot-embossing onto a track-etched polycarbonate membrane, fully replicated topographical features and preserved porous architecture. Pore size and shape were analyzed with SEM and image analysis to determine the effect of hot embossing on pore morphology. The membrane was assembled into a bilayer microfluidic device and a human kidney proximal tubule epithelial cell line (HK-2) and primary renal proximal tubule epithelial cells (RPTEC) were cultured to confluency on the membrane. Immunofluorescent staining of both cell types revealed protein expression indicative of the formation of a reabsorptive barrier responsive to mechanical stimulation: ZO-1 (tight junction), paxillin (focal adhesions) and acetylated α-tubulin (primary cilia). HK-2 and RPTEC aligned in the direction of ridge/groove topography of the membrane in the device, evidence that the device has mechanical control over cell response. This topographically-patterned porous membrane provides an in vitro platform on which to model reabsorptive barriers with meaningful applications for understanding biological transport phenomenon, underlying disease mechanisms, and drug toxicity.

In vitro evaluation of biomarkers for cisplatin-induced nephrotoxicity using HK-2 human kidney epithelial cells

The non-animal in vitro test methods, especially for assessment of kidney toxicity, have become invaluable tools due to the target organ-selective nature of many nephrotoxic xenobiotics. In vitro evaluation of biomarkers for nephrotoxicity assessment using human cell lines, which can provide more reliable information for toxicological risk evaluation in humans than animal cells, has not been well established to date. The present study investigated the potential use of biomarkers for cisplatin-induced nephrotoxicity assessment in vitro using HK-2 cells derived from human kidney proximal tubule epithelial cells. Cisplatin induced apoptosis of HK-2 cells in which down-regulation of Bcl-2 and activation of caspase-3 were possibly involved. We investigated the effect of cisplatin on the protein levels of kidney injury molecule (KIM)-1, clusterin, calbindin, tissue inhibitor of metalloproteinase (TIMP)-1, cystatin C (CysC), β2-microglobulin (β2-M) and neutrophil gelatinase associated lipocalin (NGAL), which have been recently identified as in vivo biomarkers of nephrotoxicity. The protein levels of KIM-1, calbindin and TIMP-1 were significantly increased in the conditioned media of HK-2 cells treated with cisplatin, while β2-M, CysC, NGAL and clusterin were not affected by cisplatin treatment. The mRNA levels of KIM-1, calbindin and TIMP-1 were increased by cisplatin, indicating that cisplatin-induced up-regulation involves transcriptional activation. The levels of KIM-1, calbindin and TIMP-1 were significantly increased in urine of cisplatin-treated rats, providing in vivo validation of the in vitro results. Taken together, our results clearly demonstrate that among the known in vivo nephrotoxic biomarkers, KIM-1, calbindin and TIMP-1 can be effectively used as in vitro biomarkers for cisplatin-induced nephrotoxicity using a HK-2 human kidney cell system.