Kidney Proximal Tubule Epithelial Cells Research Articles

Modeling cellular responses to serum and vitamin D in microgravity using a human kidney microphysiological system

The microgravity environment aboard the International Space Station (ISS) provides a unique stressor that can help understand underlying cellular and molecular drivers of pathological changes observed in astronauts with the ultimate goals of developing strategies to enable long- term spaceflight and better treatment of diseases on Earth. We used this unique environment to evaluate the effects of microgravity on kidney proximal tubule epithelial cell (PTEC) response to serum exposure and vitamin D biotransformation capacity. To test if microgravity alters the pathologic response of the proximal tubule to serum exposure, we treated PTECs cultured in a microphysiological system (PT-MPS) with human serum and measured biomarkers of toxicity and inflammation (KIM-1 and IL-6) and conducted global transcriptomics via RNAseq on cells undergoing flight (microgravity) and respective controls (ground). Given the profound bone loss observed in microgravity and PTECs produce the active form of vitamin D, we treated 3D cultured PTECs with 25(OH)D3 (vitamin D) and monitored vitamin D metabolite formation, conducted global transcriptomics via RNAseq, and evaluated transcript expression of CYP27B1, CYP24A1, or CYP3A5 in PTECs undergoing flight (microgravity) and respective ground controls. We demonstrated that microgravity neither altered PTEC metabolism of vitamin D nor did it induce a unique response of PTECs to human serum, suggesting that these fundamental biochemical pathways in the kidney proximal tubule are not significantly altered by short-term exposure to microgravity. Given the prospect of extended spaceflight, more study is needed to determine if these responses are consistent with extended (>6 months) exposure to microgravity.

Standalone cell culture microfluidic device-based microphysiological system for automated cell observation and application in nephrotoxicity tests.

Microphysiological systems (MPS) offer an alternative method for culturing cells on microfluidic platforms to model organ functions in pharmaceutical and medical sciences. Although MPS hardware has been proposed to maintain physiological organ function through perfusion culture, no existing MPS can automatically assess cell morphology and conditions online to observe cellular dynamics in detail. Thus, with this study, we aimed to establish a practical strategy for automating cell observation and improving cell evaluation functions with low temporal resolution and throughput in MPS experiments. We developed a versatile standalone cell culture microfluidic device (SCCMD) that integrates microfluidic chips and their peripherals. This device is compliant with the ANSI/SLAS standards and has been seamlessly integrated into an existing automatic cell imaging system. This integration enables automatic cell observation with high temporal resolution in MPS experiments. Perfusion culture of human kidney proximal tubule epithelial cells using the SCCMD improves cell function. By combining the proximal tubule MPS with an existing cell imaging system, nephrotoxicity studies were successfully performed to automate morphological and material permeability evaluation. We believe that the concept of building the ANSI/SLAS-compliant-sized MPS device proposed herein and integrating it into an existing automatic cell imaging system for the online measurement of detailed cell dynamics information and improvement of throughput by automating observation operations is a novel potential research direction for MPS research.

Identification of prognostic biomarkers for antibiotic associated nephrotoxicity in cystic fibrosis

BackgroundOur objective was to discover novel urinary biomarkers of antibiotic-associated nephrotoxicity using an ex-vivo human microphysiological system (MPS) and to translate these findings to a prospectively enrolled cystic fibrosis (CF) population receiving aminoglycosides and/or polymyxin E (colistin) for a pulmonary exacerbation. MethodsWe populated the MPS with primary human kidney proximal tubule epithelial cells (PTECs) from three donors and modeled nephrotoxin injury through exposure to 50 µg/mL polymyxin E for 72 h. We analyzed gene transcriptional responses by RNAseq and tested MPS effluents. We translated candidate biomarkers to a CF cohort via analysis of urine collected prior to, during and two weeks after antibiotics and patients were followed for a median of 3 years after antibiotic use. ResultsPolymyxin E treatment resulted in a statistically significant increase in the pro-apoptotic Fas gene relative to control in RNAseq of MPS: fold-change = 1.63, FDR q-value = 7.29 × 10−5. Effluent analysis demonstrated an acute rise of soluble Fas (sFas) concentrations that correlated with cellular injury. In 16 patients with CF, urinary sFas concentrations were significantly elevated during antibiotic treatment, regardless of development of AKI. Over a median of three years of follow up, we identified seven cases of incident chronic kidney disease (CKD). Urinary sFas concentrations during antibiotic treatment were significantly associated with subsequent development of incident CKD (unadjusted relative risk = 2.02 per doubling of urinary sFas, 95 % CI = 1.40, 2.90, p < 0.001). ConclusionsUsing an ex-vivo MPS, we identified a novel biomarker of proximal tubule epithelial cell injury, sFas, and translated these findings to a clinical cohort of patients with CF.

Fulvestrant alleviates cisplatin-induced acute kidney injury via repression of BNIP3-mediated apoptosis and autophagy.

Cisplatin-induced acute kidney injury (AKI) is a clinical disease characterized by a sudden loss of renal function within a few hours or days, due to cisplatin uptake. Fulvestrant is an oestrogen receptor alpha (ERα) antagonist used for endocrine therapy. However, the role of fulvestrant in cisplatin-induced AKI remains unclear. In this study, we investigated the effects of fulvestrant on the regulation of apoptotic cell death and autophagic response in cisplatin-induced AKI. The human kidney proximal tubule epithelial cell line (HK-2) was co-treated with fulvestrant and cisplatin. C57BL/6 mice were subcutaneously injected with fulvestrant and cisplatin was administered via intraperitoneal injection. First, cisplatin treatment increased ERα expression, apoptosis, and autophagy in HK-2 cells. Fulvestrant treatment decreased apoptosis and autophagy, which were accompanied by cisplatin treatment in HK-2 cells. Consistent with in vitro results, cisplatin treatment significantly increased ERα expression in vivo. Additionally, cisplatin treatment increased renal injury, apoptosis, and autophagy. Surprisingly, compared to that in the cisplatin-treated mice group, reduced cisplatin-induced renal injury, apoptosis, and autophagy was observed in the cisplatin+fulvestrant-treated mice group. In summary, these results suggest that fulvestrant plays an important role in cisplatin-induced AKI by decreasing apoptosis and autophagy.

Crabtree effect in kidney proximal tubule cells via late-stage glycolytic intermediates

The Crabtree effect is defined as a rapid glucose-induced repression of mitochondrial oxidative metabolism and has been described in yeasts and tumor cells. Using plate-based respirometry, we identified the Crabtree effect in normal (non-tumor) kidney proximal tubule epithelial cells (PTEC) but not in other kidney cells (podocytes or mesangial cells) or mammalian cells (C2C12 myoblasts). Glucose-induced repression of respiration was prevented by reducing glycolysis at the proximal step with 2-deoxyglucose and partially reversed by pyruvate. The late-stage glycolytic intermediates glyceraldehyde 3-phosphate, 3-phosphoglycerate, and phosphoenolpyruvate, but not the early-stage glycolytic intermediates or lactate, inhibited respiration in permeabilized PTEC and kidney cortex mitochondria, mimicking the Crabtree effect. Studies in diabetic mice indicated a pattern of increased late-stage glycolytic intermediates consistent with a similar pattern occurring invivo. Our results show the unique presence of the Crabtree effect in kidney PTEC and identify the major mediators of this effect.

Effects of Albizia julibrissin Durazz through Suppression of Mitochondrial Fission and Apoptosis in Cisplatin-induced Acute Kidney Injury

Albizia julibrissin Durazz. (AJ; family Minosaceae) is widely distributed worldwide, and its stem bark has been used as a traditional herbal medicine. Acute kidney injury (AKI) is a clinical syndrome that results in sudden loss of renal function. This study aimed to investigate the effects of AJ against cisplatin-induced AKI using a human kidney proximal tubule epithelial cell line (HK-2) and cisplatin-treated mice. In vitro, cisplatin treatment increased apoptosis in HK-2 cells. However, AJ treatment decreased apoptosis of cisplatin-treated HK-2 cells. In vivo, cisplatin treatment accelerated renal injury by increasing the levels of renal injury markers, such as blood urea nitrogen, creatinine, kidney injury molecule 1, and neutrophil gelatinase-associated lipocalin, which were reversed by AJ treatment. Histopathologically, AJ treatment resulted in decreased renal damage with less tubular necrosis and brush border desquamation compared with the AKI group. Additionally, cisplatin treatment upregulated mitochondrial fission, a pathological characteristic of AKI, which was downregulated by AJ treatment. Along with increased mitochondrial fission, AJ treatment also reduced cisplatin-induced apoptosis. These results suggest that AJ may be a potential therapeutic agent for cisplatin-induced AKI.

Blocking connexin 43 hemichannel-mediated ATP release reduces communication within and between tubular epithelial cells and medullary fibroblasts in a model of diabetic nephropathy.

Fibrosis of renal tubules is the final common pathway in diabetic nephropathy and develops in the face of tubular injury and fibroblast activation. Aberrant connexin 43 (Cx43) hemichannel activity has been linked to this damage under euglycaemic conditions, however, its role in glycaemic injury is unknown. This study investigated the effect of a Cx43 blocker (Tonabersat) on hemichannel activity and cell-cell interactions within and between tubular epithelial cells and fibroblasts in an in vitro model of diabetic nephropathy. Human kidney (HK2) proximal tubule epithelial cells and medullary fibroblasts (TK173) were treated in low (5 mM) or high (25 mM) glucose ± transforming growth factor beta-1 (TGFβ1) ± Tonabersat in high glucose. Carboxyfluorescein dye uptake and ATPlite luminescence assessed changes in hemichannel-mediated ATP release, while immunoblotting determined protein expression. Co-incubation with the ATP-diphosphohydrolase apyrase or a P2X7R inhibitor (A438079) assessed ATP-P2X7R signalling. Indirect co-culture with conditioned media from the alternate cell type evaluated paracrine-mediated heterotypic interactions. Tonabersat partially negated glucose/TGFβ1-induced increases in Cx43 hemichannel-mediated ATP release and downstream changes in adherens junction and extracellular matrix (ECM) protein expression in HK2 and TK173 cells. Apyrase and A438079 highlighted the role for ATP-P2X7R in driving changes in protein expression in TK173 fibroblasts. Indirect co-culture studies suggest that epithelial cell secretome increases Tonabersat-sensitive hemichannel-mediated dye uptake in fibroblasts and downstream protein expression. Tonabersat-sensitive hemichannel-mediated ATP release enhances TGFβ1-driven heterotypic cell-cell interaction and favours myofibroblast activation. The data supports the potential benefit of Cx43 inhibition in reducing tubulointerstitial fibrosis in late-stage diabetic nephropathy.

Mitochondria-targeted CoQ10 loaded PLGA-b-PEG-TPP nanoparticles: Their effects on mitochondrial functions of COQ8B-/- HK-2 cells

Coenzyme Q10 (CoQ10) deficiency exhibits signs of multiple organ dysfunctions, particular subtypes present isolated kidney involvement progressing to chronic kidney disease. In these patients, early administration of oral CoQ10 has been shown to decrease proteinuria and to delay development of chronic kidney disease, which suggests that it may have a renoprotective potential in these patients. However, CoQ10 bioavailability in mitochondria is low, therefore its efficacy is limited. We aimed to develop mitochondria-targeted CoQ10 loaded poly(lactic-co-glycolic acid)-poly(ethylene glycol)-triphenylphosphonium nanoparticles (CoQ10-TPP-NPs) that would be more efficient in the treatment of CoQ10 nephropathies. These nanoparticles were found to have a size of approximately 150 nm and a zeta potential of + 20 mV. The entrapment efficiency of the nanoparticles was determined as 40%. Cytotoxicity studies showed no effect on the viability of the human kidney proximal tubule epithelial cells exposed to the nanoparticles. The efficacy of the formulated nanoparticles on in vitro disease model, which was developed in the human kidney proximal tubule epithelial cells by siRNA based silencing of the COQ8B, was evaluated through mitochondrial functions by means of metabolomic analyses. We showed that the treatment of COQ8B-/- cells with mitochondria-targeted nanoparticles was more effective in increasing the tricarboxylic acid cycle rate compared to free-CoQ10. Our formulation would be more effective in treatment of CoQ10-related nephropathies than conventional formulations.

Role of ACSL4 in the chemical-induced cell death in human proximal tubule epithelial HK-2 cells.

Acyl-CoA synthetase long-chain family member 4 (ACSL4) activates polyunsaturated fatty acids (PUFAs) to produce PUFA-derived acyl-CoAs, which are utilised for the synthesis of various biological components, including phospholipids (PLs). Although the roles of ACSL4 in non-apoptotic programmed cell death ferroptosis are well-characterised, its role in the other types of cell death is not fully understood. In the present study, we investigated the effects of ACSL4 knockdown on the levels of acyl-CoA, PL, and ferroptosis in the human normal kidney proximal tubule epithelial (HK-2) cells. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) analyses revealed that the knockdown of ACSL4 markedly reduced the levels of PUFA-derived acyl-CoA, but not those of other acyl-CoAs. In contrast with acyl-CoA levels, the docosahexaenoic acid (DHA)-containing PL levels were preferentially decreased in the ACSL4-knockdown cells compared with the control cells. Cell death induced by the ferroptosis inducers RSL3 and FIN56 was significantly suppressed by treatment with ferrostatin-1 or ACSL4 knockdown, and, unexpectedly, upon treating with a necroptosis inhibitor. In contrast, ACSL4 knockdown failed to suppress the other oxidative stress-induced cell deaths initiated by cadmium chloride and sodium arsenite. In conclusion, ACSL4 is involved in the biosynthesis of DHA-containing PLs in HK-2 cells and is specifically involved in the cell death induced by ferroptosis inducers.

Regulation of Megalin membrane traffic by AMP‐activated protein kinase in kidney proximal tubule epithelial cells

Megalin is a transmembrane receptor protein that belongs to a family of proteins known as low-density lipoprotein receptors (LDLRs) that functions in reabsorption of various molecules from the urine in the proximal tubule epithelial cells (PTECs) of the kidney. Disruption of the regulation of Megalin is associated with various stages of renal disease. Megalin mediates reabsorption by functioning as a scavenger receptor: binding of molecules (cargo) within the kidney proximal tubule lumen to the ectodomain of Megalin followed by internalization of cargo-bound Megalin via clathrin-mediated endocytosis, the first stage in transcytosis of cargo molecules across the kidney epithelium. Kidney proximal tubule cells experience metabolic stress due to low oxygen availability. We have recently shown that metabolic stress resulting in activation of AMP-activated protein kinase (AMPK) regulates the endocytosis of a large number of cell surface proteins, including that of beta1-integrin, that has a similar requirement for dab2 for internalization as does Megalin. How AMPK may regulate Megalin internalization in kidney PTECs is not well understood. Megalin internalization occurs via recruitment of the receptor to clathrin-coated pits (CCPs), which are ~100 nm structures comprised of clathrin and other proteins at the cell surface. Upon assembly and receptor recruitment, CCPs mediate membrane budding and eventually scission, leading to an internalized clathrin-coated vesicle (CCV). Megalin recruitment to CCPs is mediated by the clathrin adaptor protein dab2 that binds to NPXY motif that is present on Megalin and other LDLR family proteins. Here, we examine how metabolic stress and AMPK regulate the formation of CCPs and the dab2-dependent recruitment of Megalin and related receptors for internalization. Using cells engineered to express fluorescently tagged clathrin and other proteins, together with total internal reflection fluorescence microscopy and automated image analysis, we uncovered that AMPK activation triggers a reduction in the size and rate of initiation of CCPs, but an increased efficiency of formation of clathrin-coated vesicles. In addition, AMPK activation resulted in a robust increase in the recruitment of dab2 to CCPs. We have complemented this approach with assays that monitor the cell surface membrane traffic of Megalin and related receptors such as integrins. Collectively, these studies reveal a novel signaling pathway that mediates the regulation of the clathrin internalization of several receptors by AMPK and metabolic stress. A better understanding of signals responsive to cell metabolism and metabolic stress in kidney PTECs and how these lead to remodeling of endocytic membrane traffic may reveal novel ways to treat certain forms of kidney diseases.

Influence of vitamin D treatment on functional expression of drug disposition pathways in human kidney proximal tubule cells during simulated uremia

Effects of cholecalciferol (VitD3) and calcitriol (1,25-VitD3), on the expression and function of major vitamin D metabolizing enzymes (cytochrome P450 [CYP]2R1, CYP24A1) and select drug transport pathways (ABCB1/P-gp, SLCO4C1/OATP4C1) were evaluated in human kidney proximal tubule epithelial cells (hPTECs) under normal and uraemic serum conditions. hPTECs were incubated with 10% normal or uraemic serum for 24 h followed by treatment with 2% ethanol vehicle, or 100 and 240 nM doses of VitD3, or 1,25-VitD3 for 6 days. The effects of treatment on mRNA and protein expression and functional activity of select CYP enzymes and transporters were assessed Under uraemic serum, treatment with 1,25-VitD3 resulted in increased mRNA but decreased protein expression of CYP2R1. Activity of CYP2R1 was not influenced by serum or VitD analogues. CYP24A1 expression was increased with 1,25-VitD3 under normal as well as uraemic serum, although to a lesser extent. ABCB1/P-gp mRNA expression increased under normal and uraemic serum, with exposure to 1,25-VitD3. SLCO4C1/OATP4C1 exhibited increased mRNA but decreased protein expression, under uraemic serum + 1,25-VitD3. Functional assessments of transport showed no changes regardless of exposure to serum or 1,25-VitD3. Key findings indicate that uraemic serum and VitD treatment led to differential effects on the functional expression of CYPs and transporters in hPTECs.

Basic science: Beta cells, islets and complications

Introduction: Inflammation has been linked to hemichannel-mediated release of adenosine triphosphate (ATP) and downstream fibrosis in various tissue type-specific complications of diabetes. In the current study we examined how inflammatory cytokines alter markers of renal tubular injury ahead of evaluating the efficacy of the connexin-43 (Cx43) hemichannel blocker Tonabersat in negating these effects. Methods: Human Kidney (HK-2) proximal tubule epithelial cells were cultured in low (5mM) or high (25mM) glucose ± interleukin (IL)-1β (10ng) and tumour necrosis factor alpha (TNFα; 10ng) for 48 hours. Immunoblotting determined expression of key proteins, whilst carboxyfluorescein (200µM) dye uptake examined hemichannel activity in treated cells +/- Tonabersat (10µM). Results: Under high glucose conditions, IL-1β and TNFα significantly increased hemichannel activity to 161±16%, (P<0.05, n=5) an effect which was negated when co-incubated with hemichannel blocker Tonabersat (123±8.5%, n=5). Moreover, stimulation with IL-1β and TNFα increased expression of the inflammatory protein IL-6 (206±42%, P<0.05, n=5) and the extracellular matrix proteins, fibronectin (365±81%, P<0.05, n=4), collagen-I (516±36%, P<0.05, n=4) and collagen-IV (830±217%, P<0.01, n=5) as compared to low glucose control. The effect was augmented at high (25mM) glucose for IL-6 (491±164%, P<0.05, n=5), fibronectin (455±112%, n=6), collagen-I (725±85%, P<0.05, n=4) and colIagen-IV (868±77%, P<0.001, n=6). Conclusion: We demonstrate that IL1β and TNFα increase expression of inflammatory and profibrotic proteins in glucose treated HK-2 cells. Paralleled by a Tonabersat-sensitive increase in Cx43 hemichannel activity, we believe that blocking hemichannels has therapeutic potential and protects against changes associated with tubular injury in the diabetic kidney.

The Crucial Role of Xanthine Oxidase in CKD Progression Associated with Hypercholesterolemia.

In the present study, we investigated the effects of xanthine oxidase (XO) inhibition on cholesterol-induced renal dysfunction in chronic kidney disease (CKD) mice, and in low-density lipoprotein (LDL)-treated human kidney proximal tubule epithelial (HK-2) cells. ApoE knockout (KO) mice underwent uninephrectomy to induce CKD, and were fed a normal diet or high-cholesterol (HC) diet along with the XO inhibitor topiroxostat (1 mg/kg/day). HK-2 cells were treated with LDL (200 µg/mL) and topiroxostat (5 µM) or small interfering RNA against xanthine dehydrogenase (siXDH; 20 nM). In uninephrectomized ApoE KO mice, the HC diet increased cholesterol accumulation, oxidative stress, XO activity, and kidney damage, while topiroxostat attenuated the hypercholesterolemia-associated renal dysfunction. The HC diet induced cholesterol accumulation by regulating the expressions of genes involved in cholesterol efflux (Nr1h3 and Abca1) and synthesis (Srebf2 and Hmgcr), which was reversed by topiroxostat. Topiroxostat suppressed the expressions of genes related to hypercholesterolemia-associated inflammation and fibrosis in the unilateral kidney. LDL stimulation evoked changes in the cholesterol metabolism, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and NF-κB pathways in HK-2 cells, which were mitigated by XO inhibition with topiroxostat or siXDH. These findings suggest that XO inhibition exerts renoprotective effects against hypercholesterolemia-associated kidney injury. XO could be a novel therapeutic target for hypercholesterolemia-associated kidney injury in uninephrectomized patients.

Integrative Omics Analyses Reveal Epigenetic Memory in Diabetic Renal Cells Regulating Genes Associated With Kidney Dysfunction.

Diabetic kidney disease (DKD) is a major complication of diabetes and the leading cause of end-stage renal failure. Epigenetics has been associated with metabolic memory in which prior periods of hyperglycemia enhance the future risk of developing DKD despite subsequent glycemic control. To understand the mechanistic role of such epigenetic memory in human DKD and to identify new therapeutic targets, we profiled gene expression, DNA methylation, and chromatin accessibility in kidney proximal tubule epithelial cells (PTECs) derived from subjects with and without type 2 diabetes (T2D). T2D-PTECs displayed persistent gene expression and epigenetic changes with and without transforming growth factor-β1 treatment, even after culturing in vitro under similar conditions as nondiabetic PTECs, signified by deregulation of fibrotic and transport-associated genes (TAGs). Motif analysis of differential DNA methylation and chromatin accessibility regions associated with genes differentially regulated in T2D revealed enrichment for SMAD3, HNF4A, and CTCF transcription factor binding sites. Furthermore, the downregulation of several TAGs in T2D (including CLDN10, CLDN14, CLDN16, SLC16A2, and SLC16A5) was associated with promoter hypermethylation, decreased chromatin accessibility, and reduced enrichment of HNF4A, histone H3-lysine-27-acetylation, and CTCF. Together, these integrative analyses reveal epigenetic memory underlying the deregulation of key target genes in T2D-PTECs that may contribute to sustained renal dysfunction in DKD.

Long Term Culture of Human Kidney Proximal Tubule Epithelial Cells Maintains Lineage Functions and Serves as an Ex vivo Model for Coronavirus Associated Kidney Injury.

The mechanism of how SARS-CoV-2 causes severe multi-organ failure is largely unknown. Acute kidney injury (AKI) is one of the frequent organ damage in severe COVID-19 patients. Previous studies have shown that human renal tubule cells could be the potential host cells targeted by SARS-CoV-2. Traditional cancer cell lines or immortalized cell lines are genetically and phenotypically different from host cells. Animal models are widely used, but often fail to reflect a physiological and pathogenic status because of species tropisms. There is an unmet need for normal human epithelial cells for disease modeling. In this study, we successfully established long term cultures of normal human kidney proximal tubule epithelial cells (KPTECs) in 2D and 3D culture systems using conditional reprogramming (CR) and organoids techniques. These cells had the ability to differentiate and repair DNA damage, and showed no transforming property. Importantly, the CR KPTECs maintained lineage function with expression of specific transporters (SLC34A3 and cubilin). They also expressed angiotensin-converting enzyme 2 (ACE2), a receptor for SARS-CoV and SARS-CoV-2. In contrast, cancer cell line did not express endogenous SLC34A3, cubilin and ACE2. Very interestingly, ACE2 expression was around twofold higher in 3D organoids culture compared to that in 2D CR culture condition. Pseudovirion assays demonstrated that SARS-CoV spike (S) protein was able to enter CR cells with luciferase reporter. This integrated 2D CR and 3D organoid cultures provide a physiological ex vivo model to study kidney functions, innate immune response of kidney cells to viruses, and a novel platform for drug discovery and safety evaluation.Electronic supplementary materialThe online version of this article (10.1007/s12250-020-00253-y) contains supplementary material, which is available to authorized users.

Open microfluidic coculture reveals paracrine signaling from human kidney epithelial cells promotes kidney specificity of endothelial cells.

Endothelial cells (ECs) from different human organs possess organ-specific characteristics that support specific tissue regeneration and organ development. EC specificity is identified by both intrinsic and extrinsic cues, among which the parenchyma and organ-specific microenvironment are critical contributors. These extrinsic cues are, however, largely lost during ex vivo cultures. Outstanding challenges remain to understand and reestablish EC organ specificity for in vitro studies to recapitulate human organ-specific physiology. Here, we designed an open microfluidic platform to study the role of human kidney tubular epithelial cells in supporting EC specificity. The platform consists of two independent cell culture regions segregated with a half wall; culture media are added to connect the two culture regions at a desired time point, and signaling molecules can travel across the half wall (paracrine signaling). Specifically, we report that in the microscale coculture device, primary human kidney proximal tubule epithelial cells (HPTECs) rescued primary human kidney peritubular microvascular EC (HKMEC) monolayer integrity and fenestra formation and that HPTECs upregulated key HKMEC kidney-specific genes (hepatocyte nuclear factor 1 homeobox B, adherens junctions-associated protein 1, and potassium voltage-gated channel subfamily J member 16) and endothelial activation genes (vascular cell adhesion molecule-1, matrix metalloproteinase-7, and matrix metalloproteinase-10) in coculture. Coculturing with HPTECs also promoted kidney-specific genotype expression in human umbilical vein ECs and human pluripotent stem cell-derived ECs. Compared with culture in HPTEC conditioned media, coculture of ECs with HPTECs showed increased upregulation of kidney-specific genes, suggesting potential bidirectional paracrine signaling. Importantly, our device is compatible with standard pipettes, incubators, and imaging readouts and could also be easily adapted to study cell signaling between other rare or sensitive cells.

Predictive Nephrotoxicity Profiling of a Novel Antifungal Small Molecule in Comparison to Amphotericin B and Voriconazole.

Background and Purpose Candida albicans is the major fungal species associated with superficial mucosal infections such as oral candidiasis as well as systemic mycoses with high morbidity and mortality. On top of the rising drug resistance, currently available antifungal agents have significant adverse effects. Nephrotoxicity is the major treatment complication associated with antifungal agents.Recently, we discovered a novel antifungal small molecule SM21 with promising antifungal activity. The present study aimed to comparatively evaluate the in vivo and in vitro nephrotoxicity of SM21 comparing with Amphotericin B and voriconazole.Experimental ApproachNephrotoxicity of SM21 and its analogue were comparatively evaluated with Amphotericin B (AmB) and voriconazole. Immortalized human kidney proximal tubule epithelial cells (HK-2) were used for in vitro analysis of nephrotoxicity using cytotoxicity assays and qPCR gene expression analysis (Kim-1/HAVcr-1, CASP3). Sprague Dawley (SD) rat model was used to evaluate the nephrotoxicity in vivo using classical (SCr and BUN) and next-generation kidney injury urinary biomarkers (Kim-1, CLU, ALB, NGAL, β2M, and Cys C) alongside histopathological and immunohistochemical standards.Key ResultsAmB treatment showed a stronger cytotoxic impact on HK-2 viability and gene expression of cell death markers (Kim-1/HAVcr-1, CASP3) compared with SM21 and SM21 analogue in vitro (P < 0.01). In vivo data further demonstrated that SM21 did not significantly increase classical as well as novel nephrotoxic biomarkers, and minimal renal tubular necrosis and abnormalities were observed (15 mg kg−1 BW/day).Conclusions and ImplicationsSM21 had a significantly better safety profile in terms of nephrotoxicity with no major tubular epithelial abnormalities observed in kidney cells and no augmentation of kidney injury biomarkers compared to AmB. Kim-1 and CLU were the most sensitive biomarkers for detection of AmB-induced kidney damage. Future clinical trials should consider inclusion of these novel biomarkers as early indicators of acute kidney injury in antifungal-induced nephrotoxicity.

Synergism between Angiotensin AT1 and AT2 Receptors function: Role of Angiotensin‐(1‐7) via AT1R in Modulating AT2R Agonist Response on Nitric Oxide in Kidney Cells

Angiotensin‐(1‐7) is evidently considered as an endogenous agonist for Mas receptor. Our recent studies show that MasR and AT2R 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 AT1R stimulation. Considering the complex nature of Ang‐(1‐7) function, we tested as to how Ang‐(1‐7) affects AT2R function utilizing human kidney proximal tubule epithelial cells (HK‐2 cells). The agonists of MasR [(Ang‐(1‐7), AVE0991] or AT2R (C21), alone significantly stimulated NO formation at 10−6–10−5M (50–300%), but not at lower concentrations (10−12‐10−7M). Contrarily, Ang‐II alone reduced NO formation. In the interaction studies, the non‐responsive concentrations of Ang‐(1‐7) (10−12‐10−7M) synergized NO formation of C21 (10−12‐10−9M) or ang‐II (10−12‐10−9M) in HK‐2 cells (p&lt;0.05 vs. either agonist alone). The synergy is observed only when the Ang‐(1‐7) was added to the HK‐2 cells 10 min prior to the C21/ang‐II, not when the agonists were added simultaneously. The synergy between Ang‐(1‐7) and C21/ang‐II was sensitive to AT2R antagonist (PD123319, 10−5M) and AT1R antagonist (candesartan, 10−5M), but not MasR antagonist (A779, 10−5M). Interestingly, pre‐stimulation with synthetic non‐peptide MasR selective agonist AVE0991 at any concentrations (10−10‐10−7M) did not show synergism with the AT2R agonist C21. Collectively, results show Ang‐(1‐7) at lower concentrations via AT1R, not MasR, synergies the AT2R function of NO formation and this phenomenon may have a physiological significance.Support or Funding InformationNIH R01 DK61578 and R01 DK117495

Extraneural CGRP Upregulates TGF-β1 through RAMP1 Signaling during Mechanical Stretch in Kidney Proximal Tubule Epithelial Cells

Extraneural CGRP Upregulates TGF-β1 through RAMP1 Signaling during Mechanical Stretch in Kidney Proximal Tubule Epithelial Cells

Abstract P144: Integrin β1 in Tubular Epithelium Protects From Acute Injury Induced Fibrosis

Acute kidney injury (AKI) results in the sudden loss of renal function primarily due to renal tubular injury. Repeated injury or maladaptive recovery lead to chronic kidney diseases. All forms of chronic kidney disease are characterized by excessive extracellular matrix (ECM) accumulation. The proximal tubule cells that are injured but not detached from the basement membrane are critical for the restoration of renal functions after the injury incidence. Renal tubular epithelial cell adhesion, migration, and proliferation are required for the kidney to recover from sub-lethal injury. These processes require the ability of cells to interact with the ECM. Integrin, which exists as a complex of α and β subunits, are the principal receptors for ECM proteins. Integrin β1 is the major integrin subunit present in kidney and critically regulates renal epithelial cell functions. We investigated the importance of Integrin β1 in kidney proximal tubule epithelial cells. Integrin β1 was deleted from these cells using the γ-gt cre. These KO mice had no morphological abnormalities but have a higher urine osmolality (2369±881 mΩ) compared to WT (2098±945 mΩ). By using a nephrotoxic injury model, we investigated the role of integrin β1 in AKI. Samples were collected on 3, 5, 7 and 14 days after injury to assess the nature of injury and recovery. Integrin β1 KO mice were more susceptible to injury as evident by protein casts and tubule dilation of tubules. The blood urea nitrogen (BUN) from the plasma showed increased BUN level in KO animals compared to WT controls (26.78±8.23 and 34.05±7.11). After the injury the level was increased to 52.26±18.37 in WT and 60.34±11.58 KO suggesting both were injured to a similar extent. After 14 days the WT recovered better than KO animals (BUN 37.33±10.12 and 48.73±15.92). After 14 KO mice had more collagen deposition compared to WT animals. This data suggests the importance of integrin β1 in the tubular epithelium in promoting the recovery from injury and preventing its progression to chronic kidney disease.