Human Kidney Proximal Tubule Research Articles

Human kidney on a chip assessment of polymyxin antibiotic nephrotoxicity.

Drug-induced kidney injury, largely caused by proximal tubular intoxicants, limits development and clinical use of new and approved drugs. Assessing preclinical nephrotoxicity relies on animal models that are frequently insensitive; thus, potentially novel techniques - including human microphysiological systems, or "organs on chips" - are proposed to accelerate drug development and predict safety. Polymyxins are potent antibiotics against multidrug-resistant microorganisms; however, clinical use remains restricted because of high risk of nephrotoxicity and limited understanding of toxicological mechanisms. To mitigate risks, structural analogs of polymyxins (NAB739 and NAB741) are currently in clinical development. Using a microphysiological system to model human kidney proximal tubule, we exposed cells to polymyxin B (PMB) and observed significant increases of injury signals, including kidney injury molecule-1 KIM-1and a panel of injury-associated miRNAs (each P < 0.001). Surprisingly, transcriptional profiling identified cholesterol biosynthesis as the primary cellular pathway induced by PMB (P = 1.22 ×10-16), and effluent cholesterol concentrations were significantly increased after exposure (P < 0.01). Additionally, we observed no upregulation of the nuclear factor (erythroid derived-2)-like 2 pathway, despite this being a common pathway upregulated in response to proximal tubule toxicants. In contrast with PMB exposure, minimal changes in gene expression, injury biomarkers, and cholesterol concentrations were observed in response to NAB739 and NAB741. Our findings demonstrate the preclinical safety of NAB739 and NAB741 and reveal cholesterol biosynthesis as a potentially novel pathway for PMB-induced injury. To our knowledge, this is the first demonstration of a human-on-chip platform used for simultaneous safety testing of new chemical entities and defining unique toxicological pathway responses of an FDA-approved molecule.

Design and in vivo characterization of kidney-targeting multimodal micelles for renal drug delivery

One in three Americans is at risk for developing chronic kidney disease (CKD) and end-stage renal disease (ESRD), leading to the need for dialysis or a kidney transplant. Small-molecule drugs have been proposed as therapies to manage kidney diseases, but high dosages are often required to achieve therapeutic efficacy, generating off-target side effects, some of which are lethal. To address these limitations, we developed a novel, kidney-targeting multimodal micelle (KM) system for drug delivery applications. Specifically, we incorporated the kidney-targeting peptide (Lysine-Lysine-Glutamic acid-Glutamic acid-Glutamic acid)3-Lysine) ((KKEEE)3K) into micelles. This peptide binds to megalin, a multi-ligand cell surface receptor present on renal tubule cells. When incubated with human kidney proximal tubule cells, KMs were found to be biocompatible in vitro. In vivo, KMs showed higher accumulation in the kidneys as compared to a non-targeted (NT) control upon intravenous injection in wild-type C57BL/6J mice. Histological evaluation showed no signs of tissue damage, while blood urea nitrogen (BUN) and creatinine levels were within normal ranges, validating the preservation of kidney health upon micelle administration. To our knowledge, this is the first utilization of (KKEEE)3K in a nanoparticle formulation, and our study offers strong evidence that this novel nanoparticle platform can be used as a candidate drug delivery carrier to direct therapeutics to diseased tissue in CKD.

Differential effect of OCRL1 patient mutations on protein localization, and sensitivity to FDA‐approved drug suppression of cellular phenotypes associated with Lowe syndrome

Lowe Syndrome (LS) is a lethal X‐linked genetic disorder caused by mutations in the OCRL1 gene, which encodes the lipid phosphatase Ocrl1. LS children present with congenital cataracts, low molecular weight proteinuria and mental retardation as characteristic clinical symptoms. Progressive renal dysfunction is the main cause of death. Ocrl1 localizes to the trans Golgi network (TGN), early endosomes and the plasma membrane and has been shown to participate in vesicle trafficking. We established that LS patient cells are defective in fluid phase uptake and ciliogenesis, which are critical processes required for kidney function. Over 100 OCRL1 mutations have been identified in LS, but their specific impact on cellular processes is unknown. However, our results indicate that different mutations have diverse effect on Ocrl1 localization and on triggering cellular phenotypes.We expressed GFP‐Ocrl1 (WT and several patient mutants) in human kidney proximal tubule (HK2) cells and LS fibroblasts lacking endogenous Ocrl1. The co‐localization of WT/mutant Ocrl1 with various organelle markers was quantitatively analyzed and different cellular processes/phenotypes were monitored. Our data indicates that in general, N‐terminal PH domain mutations resulted in redistribution of Ocrl1 to the cytoplasm, ASH‐RhoGAP domain mutations prevented localization of Ocrl1 to TGN and mutations in the phosphatase domain resulted in Golgi apparatus (GA) morphological abnormalities.Importantly, using a drug repurposing approach we identified two FDA‐approved compounds able to differentially suppress LS cellular phenotypes; the ability of these drugs to alleviate defects associated with specific Ocrl1 patient mutants was also analyzed. Our data indicate that while some patient OCRL1 mutations require treatment with both drugs, others only necessitate one for LS cellular phenotype suppression.This study determined for the first time the effect of different patient mutation on the Ocrl1 intracellular localization and on various LS‐associated cellular phenotypes. Further, it explored how different OCRL1 mutations determined the sensitivity of cells to the newly identified FDA‐approved drugs with ameliorating effects on LS phenotypes. It also contributes to expand our understanding of the molecular mechanism of LS pathogenesis and provide the basis for the development of a LS‐specific therapeutic.Support or Funding InformationLowe Syndrome TrustNational Institutes of Health (NIH)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Determination of the role of PAR‐associated proteins in ROS‐mediated cell death

2,3,5‐Tris‐(glutathion‐S‐yl)hydroquinone (TGHQ) is a nephrotoxic and nephrocarcinogenic metabolite of hydroquinone. TGHQ can redox cycle, generating ROS which cause DNA strand breaks, hyperactivation of poly(ADP‐ribose) polymerase‐1 (PARP‐1), increases in intracellular calcium concentration (i[Ca2+]), and cell death. PARP‐1 is a nuclear protein involved in DNA repair, transcriptional regulation and intracellular trafficking, and catalyzes the attachment of a posttranscriptional modification, consisting of multi branched ADP‐ribose polymers (PAR) on target proteins. ROS stress promotes PARP‐1 hyperactivation and elevations in i[Ca2+] which are reciprocally coupled, resulting in cell death. The molecular mechanism of this interaction is unclear. The aim of the present study was to identify TGHQ‐induced PAR‐associated proteins and their potential role in cell death. Human kidney proximal tubule cells (HK‐2) were treated with 400 μM TGHQ to induce ROS‐stress leading to PARP‐1 hyperactivation. PAR‐associated proteins were immunoprecipitated, followed by subsequent separation by SDS PAGE and proteomic analysis. A relative protein abundance analysis by spectral counts allowed us to obtain fold‐changes in protein abundance relative to control. 361 PAR‐associated proteins were significantly identified as modified by TGHQ treatment. From this cohort, 276 proteins showed an increased PAR association while 85 were decreased. Additionally, 13 targets had Gene Ontology annotations related to calcium. Of these, neuroblast differentiation‐associated protein (AHNAK), calcium homeostasis endoplasmic reticulum protein (CHERP) and general transcription factor II‐I (TFII‐I) are directly involved in modulation of i[Ca2+]. TFII‐I is a transcription factor that regulates i[Ca2+] by sequestering phospholipase C (PLC) in the cytosol. Under basal conditions free PLC binds and promotes the translocation of the transient receptor potential channel (TRPC3) to the plasma membrane leading to increases in i[Ca2+]. TFII‐I increased PAR‐association might therefore lead to calcium influx via TRPC3. Using co‐immunoprecipitation and immunodetection against TFII‐I, we confirmed increased TFII‐I PAR‐association. The effect of a TRPC3 inhibitor on TGHQ‐dependent modulation of i[Ca2+] was determined by live imaging of HK‐2 cells with the calcium sensitive dye fluo‐8 AM. Inhibition of TRPC3 promoted a delayed but higher increase in i[Ca2+]. Cellular compartmentalization of TFII‐I under TGHQ treatment was determined by cellular fractionation. TGHQ induced TFII‐I translocation from the nucleus to the cytosol as well as decreased tyrosine phosphorylation, which might have a functional effect on the transactivation potential of TFII‐I. In summary, LC‐MS/MS identified proteins with altered PARylation under ROS stress. TFII‐I is a novel PAR interacting protein which participates in signaling pathways with potential importance in TGHQ‐mediated cell death. Currently we are investigating the transcriptional and DNA repair effects of PAR‐associated TFII‐I that might be relevant to the mechanism of ROS‐dependent toxicity.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

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 &lt; 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.

Design of a Mestranol 2-N-Piperazino-Substituted Derivative Showing Potent and Selective in vitro and in vivo Activities in MCF-7 Breast Cancer Models.

Anticancer structure-activity relationship studies on aminosteroid (5α-androstane) derivatives have emerged with a promising lead candidate: RM-133 (2β-[1-(quinoline-2-carbonyl)pyrrolidine-2-carbonyl]-N-piperazine-5α-androstane-3α,17β-diol), which possesses high in vitro and in vivo activities against several cancer cells, and selectivity over normal cells. However, the relatively weak metabolic stability of RM-133 has been a drawback to its progression toward clinical trials. We investigated the replacement of the androstane backbone by a more stable mestranol moiety. The resulting compound, called RM-581 ({4-[17α-ethynyl-17β-hydroxy-3-methoxyestra-1,3,5(10)-trien-2-yl]piperazin-1-yl}[(2S)-1-(quinolin-2-ylcarbonyl)pyrrolidin-2-yl]methanone), was synthesized efficiently in only five steps from commercially available estrone. In comparison with RM-133, RM-581 was found to be twice as metabolically stable, retains potent cytotoxic activity in breast cancer MCF-7 cell culture, and fully blocks tumor growth in a mouse xenograft model of breast cancer. Advantageously, the selectivity over normal cells has been increased with this estrane version of RM-133. In fact, RM-581 showed a better selectivity index (15.3 vs. 3.0) for breast cancer MCF-7 cells over normal breast MCF-10A cells, and was found to be nontoxic toward primary human kidney proximal tubule cells at doses reaching 50 μm.

Characterization of the Mouse and Human Monoacylglycerol O-Acyltransferase 1 (Mogat1) Promoter in Human Kidney Proximal Tubule and Rat Liver Cells.

Monoacylglycerol acyltransferase 1 (Mogat1) catalyzes the conversion of monoacylglycerols (MAG) to diacylglycerols (DAG), the precursor of several physiologically important lipids such as phosphatidylcholine, phosphatidylethanolamine and triacylglycerol (TAG). Expression of Mogat1 is tissue restricted and it is highly expressed in the kidney, stomach and adipose tissue but minimally in the normal adult liver. To understand the transcriptional regulation of Mogat1, we characterized the mouse and human Mogat1 promoters in human kidney proximal tubule-2 (HK-2) cells. In-silico analysis revealed several peroxisome proliferator response element (PPRE) binding sites in the promoters of both human and mouse Mogat1. These sites responded to all three peroxisome proliferator activated receptor (PPAR) isoforms such that their respective agonist or antagonist activated or inhibited the expression of Mogat1. PPRE site mutagenesis revealed that sites located at -592 and -2518 are very effective in decreasing luciferase reporter gene activity. Chromatin immunoprecipitation (ChIP) assay using PPARα antibody further confirmed the occupancy of these sites by PPARα. While these assays revealed the core promoter elements necessary for Mogat1 expression, there are additional elements required to regulate its tissue specific expression. Chromosome conformation capture (3C) assay revealed additional cis-elements located ~10–15 kb upstream which interact with the core promoter. These chromosomal regions are responsive to both PPARα agonist and antagonist.

Etoposide induced cytotoxicity mediated by ROS and ERK in human kidney proximal tubule cells.

Etoposide (ETO) is a commonly used chemotherapeutic drug that inhibits topoisomerase II activity, thereby leading to genotoxicity and cytotoxicity. However, ETO has limited application due to its side effects on normal organs, especially the kidney. Here, we report the mechanism of ETO-induced cytotoxicity progression in human kidney proximal tubule (HK-2) cells. Our results show that ETO perpetuates DNA damage, activates mitogen-activated protein kinase (MAPK), and triggers morphological changes, such as cell and nuclear swelling. When NAC, a well-known reactive oxygen species (ROS) scavenger, is co-treated with ETO, it inhibits an ETO-induced increase in mitochondrial mass, mitochondrial DNA (ND1 and ND4) copy number, intracellular ATP level, and mitochondrial biogenesis activators (TFAM, PGC-1α and PGC-1β). Moreover, co-treatment with ETO and NAC inhibits ETO-induced necrosis and cell swelling, but not apoptosis. Studies using MAPK inhibitors reveal that inhibition of extracellular signal regulated kinase (ERK) protects ETO-induced cytotoxicity by inhibiting DNA damage and caspase 3/7 activity. Eventually, ERK inhibitor treated cells are protected from ETO-induced nuclear envelope (NE) rupture and DNA leakage through inhibition of caspase activity. Taken together, these data suggest that ETO mediates cytotoxicity in HK-2 cells through ROS and ERK pathways, which highlight the preventive avenues in ETO-induced cytotoxicity in kidney.

Development of a microphysiological model of human kidney proximal tubule function

The kidney proximal tubule is the primary site in the nephron for excretion of waste products through a combination of active uptake and secretory processes and isalso a primary target of drug-induced nephrotoxicity. Here, we describe the development and functional characterization of a 3-dimensional flow-directed human kidney proximal tubule microphysiological system. The system replicates the polarity of the proximal tubule, expresses appropriate marker proteins, exhibits biochemical and synthetic activities, as well as secretory and reabsorptive processes associated with proximal tubule function invivo. This microphysiological system can serve as an ideal platform for exvivo modeling of renal drug clearance and drug-induced nephrotoxicity. Additionally, this novel system can be used for preclinical screening of new chemical compounds prior to initiating human clinical trials.

Low dose ouabain stimulates Na[sbnd]K ATPase α1 subunit association with angiotensin II type 1 receptor in renal proximal tubule cells

Our laboratory has recently demonstrated that low concentrations of ouabain increase blood pressure in rats associated with stimulation of NaK ATPase activity and activation of the Src signaling cascade in NHE1-dependent manner. Proteomic analysis of human kidney proximal tubule cells (HKC11) suggested that the Angiotensin II type 1 receptor (AT1R) as an ouabain-associating protein. We hypothesize that ouabain-induced stimulation of NaK ATPase activity is mediated through AT1R. To test this hypothesis, we examined the effect of ouabain on renal cell angiotensin II production, the effect of AT1R inhibition on ouabain-stimulated NKA activity, and the effect of ouabain on NKA-AT1R association. Ouabain increased plasma angiotensin II levels in rats treated with ouabain (1μg/kg body wt./day) for 9days and increased angiotensin II levels in cell culture media after 24h treatment with ouabain in human (HKC11), mouse (MRPT), and human adrenal cells. Ouabain 10pM stimulated NKA-mediated 86Rb uptake and phosphorylation of EGFR, Src, and ERK1/2. These effects were prevented by the AT1R receptor blocker candesartan. FRET and TIRF microscopy using Bodipy-labeled ouabain and mCherry-NKA or mCherry-AT1R demonstrated association of ouabain with AT1R and NKA. Further our FRET and TIRF studies demonstrated increased association between AT1R and NKA upon treatment with low dose ouabain. We conclude that ouabain stimulates NKA in renal proximal tubule cells through an angiotensin/AT1R-dependent mechanism and that this pathway contributes to cardiac glycoside associated hypertension.

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.

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.

Human kidney proximal tubule cells are vulnerable to the effects of Rauwolfia serpentina.

Rauwolfia serpentina (or Snake root plant) is a botanical dietary supplement marketed in the USA for maintaining blood pressure. Very few studies have addressed the safety of this herb, despite its wide availability to consumers. Its reported pleiotropic effects underscore the necessity for evaluating its safety. We used a human kidney cell line to investigate the possible negative effects of R. serpentina on the renal system in vitro, with a specific focus on the renal proximal tubules. We evaluated cellular and mitochondrial toxicity, along with a variety of other kidney-specific toxicology biomarkers. We found that R. serpentina was capable of producing highly detrimental effects in our in vitro renal cell system. These results suggest more studies are needed to investigate the safety of this dietary supplement in both kidney and other target organ systems.

Etoposide Induces Necrosis Through p53-Mediated Antiapoptosis in Human Kidney Proximal Tubule Cells.

The p53 protein is an important transcription factor that modulates signaling pathways for both cell death and survival. Its antiapoptotic mechanisms that correlate with necrotic and apoptotic cell death are not well understood. Here, we report that etoposide promotes progression of the DNA damage response as well as necrotic morphological changes including plasma membrane rupture using carbon nanotube-tipped/atomic force microscopy (CNT/AFM) probes in human kidney proximal tubule (HK-2) cells. Inhibition of p53 abrogated cell cycle arrest and led to a decrease in the expression levels of repair proteins that were induced by DNA damage. Mitochondrial biogenesis and cytosolic production of reactive oxygen species were also reduced after p53 inhibition; the latter change induced mitochondrial superoxide accumulation and mitochondrial damage, which triggered the activation of caspase 3. Inhibition of p53 also led to a loss of cell adhesion and converted necrotic cell death to apoptotic cell death, with appreciable cell shrinkage and appearance of apoptotic bodies that were observed using CNT/AFM probes. Thus, our study demonstrated that p53 protects against apoptosis, and leads to etoposide-induced necrosis. These results are expected to aid in the understanding of mechanism of antiapoptosis and its relationship to cell death.

Design and Characterization of a Multicellular, Three‐Dimensional (3D) Tissue Model of the Human Kidney Proximal Tubule

Renal toxicity is a major cause of drug attrition at the clinical trial stage, and the primary site of this toxicity is within the proximal tubule. Conventional renal cell culture models lack the complexity of native tissue and thus have a limited capacity for predicting tissue‐level responses. In addition, the predictive potential of pre‐clinical animal trials is limited due to species‐specific differences between human and animal renal functions, including differential sensitivity to insults. We designed and fabricated a human three‐dimensional (3D) tissue model of the tubulointerstitial interface in which human renal interstitial tissue is supporting proximal tubule epithelial cells to facilitate their optimal morphology and function. Histological characterization demonstrated that the interstitial layer is viable and well organized, containing well‐developed CD31+ endothelial cell networks. Method optimization resulted in the formation of a polarized layer of renal epithelium on top of the interstitial layer, and formation of a basement membrane between the layers. Gene expression analysis showed that the renal tissues expressed key enzymes involved in metabolism and protein processing (CYPs, renin‐angiotensin system), suggesting that physiologic function is retained. These bioprinted human tissues may provide an opportunity to accurately study how compounds affect the renal proximal tubule as well as modeling pathogenic processes that involve tubular transport, cell‐cell interactions, and the development of tubulointerstitial fibrosis.This work was supported with internal funds.

Differential dopamine receptor subtype regulation of adenylyl cyclases in lipid rafts in human embryonic kidney and renal proximal tubule cells

Dopamine D1-like receptors (D1R and D5R) stimulate adenylyl cyclase (AC) activity, whereas the D2-like receptors (D2, D3 and D4) inhibit AC activity. D1R, but not the D5R, has been reported to regulate AC activity in lipid rafts (LRs). We tested the hypothesis that D1R and D5R differentially regulate AC activity in LRs using human embryonic kidney (HEK) 293 cells heterologously expressing human D1 or D5 receptor (HEK-hD1R or HEK-hD5R) and human renal proximal tubule (hRPT) cells that endogenously express D1R and D5R. Of the AC isoforms expressed in HEK and hRPT cells (AC3, AC5, AC6, AC7, and AC9), AC5/6 was distributed to a greater extent in LRs than non-LRs in HEK-hD1R (84.5±2.3% of total), HEK-hD5R (68.9±3.1% of total), and hRPT cells (66.6±2.2% of total) (P<0.05, n=4/group). In HEK-hD1R cells, the D1-like receptor agonist fenoldopam (1μM/15min) increased AC5/6 protein (+17.2±3.9% of control) in LRs but decreased it in non-LRs (−47.3±5.3% of control) (P<0.05, vs. control, n=4/group). By contrast, in HEK-hD5R cells, fenoldopam increased AC5/6 protein in non-LRs (+67.1±5.3% of control, P<0.006, vs. control, n=4) but had no effect in LRs. In hRPT cells, fenoldopam increased AC5/6 in LRs but had little effect in non-LRs. Disruption of LRs with methyl-β-cyclodextrin decreased basal AC activity in HEK-D1R (−94.5±2.0% of control) and HEK-D5R cells (−87.1±4.6% of control) but increased it in hRPT cells (6.8±0.5-fold). AC6 activity was stimulated to a greater extent by D1R than D5R, in agreement with the greater colocalization of AC5/6 with D1R than D5R in LRs. We conclude that LRs are essential not only for the proper membrane distribution and maintenance of AC5/6 activity but also for the regulation of D1R- and D5R-mediated AC signaling.

Critical Role of Interleukin-11 in Isoflurane-mediated Protection against Ischemic Acute Kidney Injury in Mice

Isoflurane releases renal tubular transforming growth factor-β1 (TGF-β1) and protects against ischemic acute kidney injury. Recent studies suggest that TGF-β1 can induce a cytoprotective cytokine interleukin (IL)-11. In this study, the authors tested the hypothesis that isoflurane protects against ischemic acute kidney injury by direct induction of renal tubular IL-11 synthesis. Human kidney proximal tubule cells were treated with 1.25-2.5% isoflurane or carrier gas (room air + 5% carbon dioxide) for 0-16 h. The authors also anesthetized C57BL/6 mice with 1.2% isoflurane or with equianesthetic dose of pentobarbital for 4 h. In addition, the authors subjected IL-11 receptor (IL-11R) wild-type, IL-11R-deficient, or IL-11 neutralized mice to 30-min renal ischemia followed by reperfusion under 4 h of anesthesia with pentobarbital or isoflurane (1.2%). Isoflurane increased IL-11 synthesis in human (approximately 300-500% increase, N = 6) and mouse (23 ± 4 [mean ± SD] fold over carrier gas group, N = 4) proximal tubule cells that were attenuated by a TGF-β1-neutralizing antibody. Mice anesthetized with isoflurane showed significantly increased kidney IL-11 messenger RNA (13.8 ± 2 fold over carrier gas group, N = 4) and protein (31 ± 9 vs. 18 ± 2 pg/mg protein or approximately 80% increase, N = 4) expression compared with pentobarbital-anesthetized mice, and this increase was also attenuated by a TGF-β1-neutralizing antibody. Furthermore, isoflurane-mediated renal protection in IL-11R wild-type mice was absent in IL-11R-deficient mice or in IL-11R wild-type mice treated with IL-11-neutralizing antibody (N = 4-6). In this study, the authors suggest that isoflurane induces renal tubular IL-11 via TGF-β1 signaling to protect against ischemic acute kidney injury.