Human Proximal Tubule Research Articles

Isoliquiritin Ameliorates Cisplatin-Induced Renal Proximal Tubular Cell Injury by Antagonizing Apoptosis, Oxidative Stress and Inflammation.

Acute kidney injury (AKI) is a clinical syndrome characterized by morbidity, mortality, and cost. Cis-diamminedichloroplatinum (cisplatin) is a chemotherapeutic agent used to treat solid tumors and hematological malignancies, but its side effects, especially nephrotoxicity, limit its clinical application. Isoliquiritin (ISL), one of the major flavonoid glycoside compounds in licorice, has been reported to have anti-apoptotic, antioxidant, and anti-inflammatory activities. However, the effect and mechanism of ISL on cisplatin-induced renal proximal tubular cell injury remain unknown. In this study, mouse proximal tubular cells (mPTCs) and human proximal tubule epithelial cells (HK2) were administered increasing concentrations of ISL from 7.8125 to 250 μM. Moreover, mPTC and HK2 cells were pretreated with ISL for 6–8 h, followed by stimulation with cisplatin for 24 h. CCK-8 assay was performed to evaluate the cell viability. Apoptosis and reactive oxygen species (ROS) of cells were measured by using flow cytometer and western blotting. Our results showed that ISL had no obvious effect on cell viability. ISL decreased cisplatin-induced cell injury in a dose-dependent manner. ISL also protected against cisplatin-induced cell apoptosis. Meanwhile, the enhanced protein levels of Bax, cleaved caspase-3/caspase-3 ratio, levels of Pp-65/p-65, levels of IL-6, and the production of ROS induced by cisplatin were significantly attenuated by ISL treatment. Moreover, ISL markedly increased the protein levels of Bcl-2 and SOD2, which were reduced by cisplatin stimulation. These results showed that ISL ameliorated cisplatin-induced renal proximal tubular cell injury by antagonizing apoptosis, oxidative stress and inflammation.

RGS14 regulates PTH- and FGF23-sensitive NPT2A-mediated renal phosphate uptake via binding to the NHERF1 scaffolding protein

Phosphate homeostasis, mediated by dietary intake, renal absorption, and bone deposition, is incompletely understood because of the uncharacterized roles of numerous implicated protein factors. Here, we identified a novel role for one such element, regulator of G protein signaling 14 (RGS14), suggested by genome-wide association studies to associate with dysregulated Pi levels. We show that human RGS14 possesses a carboxy-terminal PDZ ligand required for sodium phosphate cotransporter 2a (NPT2A) and sodium hydrogen exchanger regulatory factor-1 (NHERF1)–mediated renal Pi transport. In addition, we found using isotope uptake measurements combined with bioluminescence resonance energy transfer assays, siRNA knockdown, pull-down and overlay assays, and molecular modeling that secreted proteins parathyroid hormone (PTH) and fibroblast growth factor 23 inhibited Pi uptake by inducing dissociation of the NPT2A–NHERF1 complex. PTH failed to affect Pi transport in cells expressing RGS14, suggesting that it suppresses hormone-sensitive but not basal Pi uptake. Interestingly, RGS14 did not affect PTH-directed G protein activation or cAMP formation, implying a postreceptor site of action. Further pull-down experiments and direct binding assays indicated that NPT2A and RGS14 bind distinct PDZ domains on NHERF1. We showed that RGS14 expression in human renal proximal tubule epithelial cells blocked the effects of PTH and fibroblast growth factor 23 and stabilized the NPT2A–NHERF1 complex. In contrast, RGS14 genetic variants bearing mutations in the PDZ ligand disrupted RGS14 binding to NHERF1 and subsequent PTH-sensitive Pi transport. In conclusion, these findings identify RGS14 as a novel regulator of hormone-sensitive Pi transport. The results suggest that changes in RGS14 function or abundance may contribute to the hormone resistance and hyperphosphatemia observed in kidney diseases.

Dissecting Drug-Induced Cytotoxicity and Metabolic Dysfunction in Conditionally Immortalized Human Proximal Tubule Cells.

Fourteen to 26 percent of all hospitalized cases of acute kidney injury are explained by drug-induced toxicity, emphasizing the importance of proper strategies to pre-clinically assess renal toxicity. The MTT assay is widely used as a measure of cell viability, but largely depends on cellular metabolic activity. Consequently, MTT as a single assay may not be the best way to assess cytotoxicity of compounds that reduce mitochondrial function and cellular metabolic activity without directly affecting cell viability. Accordingly, we aim to highlight the limitations of MTT alone in assessing renal toxicity of compounds that interfere with metabolic activity. Therefore, we compared toxic effects observed by MTT with a fluorescent assay that determines compromised plasma membrane permeability. Exposure of proximal tubule epithelial cells to nephrotoxic compounds reduced cellular metabolic activity concentration- and time-dependently. We show that compared to our fluorescence-based approach, assessment of cellular metabolic activity by means of MTT provides a composite readout of cell death and metabolic impairment. An approach independent of cellular metabolism is thus preferable when assessing cytotoxicity of compounds that induce metabolic dysfunction. Moreover, combining both assays during drug development enables a first discrimination between compounds having a direct or indirect mitochondrial toxic potential.

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.

The Influence of Betulin and Its Derivatives EB5 and ECH147 on the Antioxidant Status of Human Renal Proximal Tubule Epithelial Cells.

Betulin and its derivatives, 28-propyne derivative EB5 and 29-diethyl phosphonate analog ECH147, are promising compounds in anti-tumor activity studies. However, their effect on kidney cells has not yet been studied. The study aimed to determine whether betulin and its derivatives—EB5 and ECH147—influence the viability and oxidative status of human renal proximal tubule epithelial cells (RPTECs). The total antioxidant capacity of cells (TEAC), lipid peroxidation product malondialdehyde (MDA) level, and activity of antioxidant enzymes (SOD, CAT, and GPX) were evaluated. Additionally, the mRNA level of genes encoding antioxidant enzymes was assessed. Cisplatin and 5-fluorouracil were used as reference substances. Betulin and its derivatives affected the viability and antioxidant systems of RPTECs. Betulin strongly reduced TEAC in a concentration-dependent manner. All tested compounds caused an increase in MDA levels. The activity of SOD, CAT, and GPX, and the mRNA profiles of genes encoding antioxidant enzymes depended on the tested compound and its concentration. Betulin showed an cisplatin-like effect, indicating its nephrotoxic potential. Betulin derivatives EB5 and ECH147 showed different impacts on the antioxidant system, which gives hope that these compounds will not cause severe consequences for the kidneys in vivo.

Inorganic nitrate and nitrite ameliorate kidney fibrosis by restoring lipid metabolism via dual regulation of AMP-activated protein kinase and the AKT-PGC1α pathway

BackgroundRenal fibrosis, associated with oxidative stress and nitric oxide (NO) deficiency, contributes to the development of chronic kidney disease and renal failure. As major energy source in maintaining renal physiological functions, tubular epithelial cells with decreased fatty acid oxidation play a key role in renal fibrosis development. Inorganic nitrate, found in high levels in certain vegetables, can increase the formation and signaling by bioactive nitrogen species, including NO, and dampen oxidative stress. In this study, we evaluated the therapeutic value of inorganic nitrate treatment on development of kidney fibrosis and investigated underlying mechanisms including regulation of lipid metabolism in tubular epithelial cells. MethodsInorganic nitrate was supplemented in a mouse model of complete unilateral ureteral obstruction (UUO)-induced fibrosis. Inorganic nitrite was applied in transforming growth factor β-induced pro-fibrotic cells in vitro. Metformin was administrated as a positive control. Fibrosis, oxidative stress and lipid metabolism were evaluated. ResultsNitrate treatment boosted the nitrate-nitrite-NO pathway, which ameliorated UUO-induced renal dysfunction and fibrosis in mice, represented by improved glomerular filtration and morphological structure and decreased renal collagen deposition, pro-fibrotic marker expression, and inflammation. In human proximal tubule epithelial cells (HK-2), inorganic nitrite treatment prevented transforming growth factor β-induced pro-fibrotic changes. Mechanistically, boosting the nitrate-nitrite-NO pathway promoted AMP-activated protein kinase (AMPK) phosphorylation, improved AKT-mediated peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC1α) activity and restored mitochondrial function. Accordingly, treatment with nitrate (in vivo) or nitrite (in vitro) decreased lipid accumulation, which was associated with dampened NADPH oxidase activity and mitochondria-derived oxidative stress. ConclusionsOur findings indicate that inorganic nitrate and nitrite treatment attenuates the development of kidney fibrosis by targeting oxidative stress and lipid metabolism. Underlying mechanisms include modulation of AMPK and AKT-PGC1α pathways.

Impact of Membrane Voltage on Formation and Stability of Human Renal Proximal Tubules in Vitro.

More than 15% of adults in the United States suffer from some form of chronic kidney disease (CKD). Current strategies for CKD consist of dialysis or kidney transplant, which, however, can take several years. In this light, tissue engineering and regenerative medicine approaches are the key to improving people's living conditions by advancing previous tissue engineering approaches and seeking new targets as intervention methods for kidney repair or replacement. The membrane voltage (Vm) dynamics of a cell have been associated with cell migration, cell cycle progression, differentiation, and pattern formation. Furthermore, bioelectrical stimuli have been used as a means in the treatment of diseases and wound healing. Here, we investigated the role of Vm as a novel target to guide and manipulate in vitro renal tissue models. Human-immortalized renal proximal tubule epithelial cells (RPTECs-TERT1) were cultured on Matrigel to support the formation of 3D proximal tubular-like structures with the incorporation of a voltage-sensitive dye indicator─bis-(1,3-dibutylbarbituric acid)timethine oxonol (DiBAC). The results demonstrated a correlation between the depolarization and the reorganization of human renal proximal tubule cells, indicating Vm as a candidate variable to control these events. Accordingly, Vm was pharmacologically manipulated using glibenclamide and pinacidil, KATP channel modulators, and proximal tubule formation and tubule stability over 21 days were assessed. Chronic manipulation of KATP channels induced changes in the tubular network topology without affecting lumen formation. Thus, a relationship was found between the preluminal tubulogenesis phase and KATP channels. This relationship may provide future options as a control point during kidney tissue development, treatment, and regeneration goals.

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.

Platelets Derived Transthyretin Participate in The Development of Sepsis Associated Acute Kidney Injury by Inducing Oxidative Stress and Apoptosis of Renal Tubular Epithelial Cells.

The pathophysiology of sepsis-associated acute kidney injury (S-AKI) is not well elucidated. Platelets have been reported to play a critical role in the pathogenesis of AKI, but the true mechanism remains unknown. Herein, we established a mouse model of S-AKI by cecal ligation and puncture (CLP). Ticagrelor was given 24 h before and after CLP by gastric gavage. Platelets were isolated and analyzed by the label-free proteome approach to identify platelet-derived damage-associated molecular patterns (DAMPs). Our results demonstrated that, among all differentially expressed proteins (DEPs), platelet-derived transthyretin (TTR) exerted effects in S-AKI. To examine the direct effects of platelet TTR on human renal proximal tubule epithelial (HK2) cells damage, platelets were co-cultured with HK2 cells. The results indicated that platelet TTR can cause reactive oxygen species production and apoptosis in HK2 cells. Further research found that platelet TTR can also result in increased levels of mRNA and protein for protein kinase B (AKT), phosphatidylinositol 3-kinase (PI3K), and extracellular regulated protein kinase (ERK), as analyzed by real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting. In conclusion, platelet-derived TTR may be one kind of DAMPs that plays an important role in the development of S-AKI.

Stx2 Induces Differential Gene Expression and Disturbs Circadian Rhythm Genes in the Proximal Tubule.

Shiga toxin-producing Escherichia coli (STEC) causes proximal tubular defects in the kidney. However, factors altered by Shiga toxin (Stx) within the proximal tubules are yet to be shown. We determined Stx receptor Gb3 in murine and human kidneys and confirmed the receptor expression in the proximal tubules. Stx2-injected mouse kidney tissues and Stx2-treated human primary renal proximal tubular epithelial cell (RPTEC) were collected and microarray analysis was performed. We compared murine kidney and RPTEC arrays and selected common 58 genes that are differentially expressed vs. control (0 h, no toxin-treated). We found that the most highly expressed gene was GDF15, which may be involved in Stx2-induced weight loss. Genes associated with previously reported Stx2 activities such as src kinase Yes phosphorylation pathway activation, unfolded protein response (UPR) and ribotoxic stress response (RSR) showed differential expressions. Moreover, circadian clock genes were differentially expressed, suggesting Stx2-induced renal circadian rhythm disturbance. Circadian rhythm-regulated proximal tubular Na+-glucose transporter SGLT1 (SLC5A1) was down-regulated, indicating proximal tubular functional deterioration, and mice developed glucosuria confirming proximal tubular dysfunction. Stx2 alters gene expression in murine and human proximal tubules through known activities and newly investigated circadian rhythm disturbance, which may result in proximal tubular dysfunctions.

Immunosuppressive calcineurin inhibitor cyclosporine A induces proapoptotic endoplasmic reticulum stress in renal tubular cells

Current immunosuppressive strategies in organ transplantation rely on calcineurin inhibitors cyclosporine A (CsA) or tacrolimus (Tac). Both drugs are nephrotoxic, but CsA has been associated with greater renal damage than Tac. CsA inhibits calcineurin by forming complexes with cyclophilins, whose chaperone function is essential for proteostasis. We hypothesized that stronger toxicity of CsA may be related to suppression of cyclophilins with ensuing endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in kidney epithelia. Effects of CsA and Tac (10 µM for 6 h each) were compared in cultured human embryonic kidney 293 (HEK 293) cells, primary human renal proximal tubule (PT) cells, freshly isolated rat PTs, and knockout HEK 293 cell lines lacking the critical ER stress sensors, protein kinase RNA–like ER kinase or activating transcription factor 6 (ATF6). UPR was evaluated by detection of its key components. Compared with Tac treatment, CsA induced significantly stronger UPR in native cultured cells and isolated PTs. Evaluation of proapoptotic and antiapoptotic markers suggested an enhanced apoptotic rate in CsA-treated cells compared with Tac-treated cells as well. Similar to CsA treatment, knockdown of cyclophilin A or B by siRNA caused proapoptotic UPR, whereas application of the chemical chaperones tauroursodeoxycholic acid or 4-phenylbutyric acid alleviated CsA-induced UPR. Deletion of protein kinase RNA–like ER kinase or ATF6 blunted CsA-induced UPR as well. In summary, inhibition of cyclophilin chaperone function with ensuing ER stress and proapoptotic UPR aggravates CsA toxicity, whereas pharmacological modulation of UPR bears potential to alleviate renal side effects of CsA.

Amplifying the impact of kidney microphysiological systems: predicting renal drug clearance using mechanistic modelling based on reconstructed drug secretion.

Accurate prediction of pharmacokinetic parameters, such as renal clearance, is fundamental to the development of effective and safe new treatments for patients. However, conventional renal models have a limited ability to predict renal drug secretion, a process that is dependent on transporters in the proximal tubule. Improvements in microphysiological systems (MPS) have extended our in vitro capabilities to predict pharmacokinetic parameters. In this study a kidney-MPS model was developed that successfully recreated renal drug secretion. Human proximal tubule cells grown in the kidney-MPS, resem­bling an in vivo phenotype, actively secreted the organic cation drug metformin and organic anion drug cidofovir, in contrast to cells cultured in conventional culture formats. Metformin and cidofovir renal secretory clearance were predicted from kid­ney-MPS data within 3.3- and 1.3-fold, respectively, of clinically reported values by employing a semi-mechanistic drug distribution model using kidney-MPS drug transport parameters together with in vitro to in vivo extrapolation. This approach introduces an effective application of a kidney-MPS model coupled with pharmacokinetic modelling tools to evaluate and predict renal drug clearance in humans. Kidney-MPS renal clearance predictions can potentially complement pharma-cokinetic animal studies and contribute to the reduction of pre-clinical species use during drug development.

Transcription Factor ChREBP Mediates High Glucose-Evoked Increase in HIF-1α Content in Epithelial Cells of Renal Proximal Tubules.

Hyperglycemia/diabetes appears to be accompanied by the state of hypoxia, which especially affects kidneys. The aim of the study was to elucidate the mechanism of high glucose action on HIF-1α expression in renal proximal tubule epithelial cells. The research hypotheses included: (1) the participation of transcription factor ChREBP; and (2) the involvement of the effects resulting from pseudohypoxia, i.e., lowered intracellular NAD+/NADH ratio. The experiments were performed on HK-2 cells and primary cells: D-RPTEC (Diseased Human Renal Proximal Tubule Epithelial Cells—Diabetes Type II) and RPTEC (Renal Proximal Tubule Epithelial Cells). Protein and mRNA contents were determined by Western blot and RT-qPCR, respectively. ChREBP binding to DNA was detected applying chromatin immunoprecipitation, followed by RT-qPCR. Gene knockdown was performed using siRNA. Sirtuin activity and NAD+/NADH ratio were measured with commercially available kits. It was found that high glucose in HK-2 cells incubated under normoxic conditions: (1) activated transcription of HIF-1 target genes, elevated HIF-1α and ChREBP content, and increased the efficacy of ChREBP binding to promoter region of HIF1A gene; and (2), although it lowered NAD+/NADH ratio, it affected neither sirtuin activity nor HIF-1α acetylation level. The stimulatory effect of high glucose on HIF-1α expression was not observed upon the knockdown of ChREBP encoding gene. Experiments on RPTEC and D-RPTEC cells demonstrated that HIF-1α content in diabetic proximal tubular cells was lower than that in normal ones but remained high glucose-sensitive, and the latter phenomenon was mediated by ChREBP. Thus, it is concluded that the mechanism of high glucose-evoked increase in HIF-1α content in renal proximal tubule endothelial cells involves activation of ChREBP, indirectly capable of HIF1A gene up-regulation.

MicroRNA 1228 Mediates the Viability of High Glucose-Cultured Renal Tubule Cells through Targeting Thrombospondin 2 and PI3K/AKT Signaling Pathway

Aim: The present study aimed to elucidate the potential function of microRNA 1228 (miR-1228) on the high glucose (HG)-damaged human renal proximal tubule cells (HK-2) and the underlying mechanism. Methods: The datasets GSE47185 and GSE51674 were downloaded from the Gene Expression Omnibus database for mining differently expressed mRNAs and miRNAs, respectively. Bioinformatics online tools were applied to predict the binding sites between miR-1228 and thrombospondin 2 (THBS2), which was confirmed by dual-luciferase assay. Real-time quantitative polymerase chain reaction was used to detect the mRNA level of miR-1228/THBS2. Western blot was used to detect the protein level of THBS2 and the PI3K/AKT signaling pathway-associated markers. HK-2 cells were cultured in HG (30 mM) to mimic hyperglycemia. Cell counting kit 8 and flow cytometry assays were utilized to determine the cell proliferation and apoptosis. Results: The expression of THBS2 was significantly upregulated in diabetic nephropathy (DN) based on bioinformatics tools and identified as a direct target of miR-1228. miR-1228 was downregulated in DN and HG-damaged HK-2 cells. HG notably reduced HK-2 cell proliferation. This negative effect was attenuated by transfecting with an miR-1228 mimic and aggravated by transfecting with an miR-1228 inhibitor. However, under basal condition, there was no significant effect on the HK-2 cell proliferation among blank control, mimic, and inhibitor groups. Overexpression of THBS2 abolished the elevating effect of the miR-1228 mimic on the HG-damaged HK-2 cell proliferation, while restored the inhibitory effects of the miR-1228 mimic on the cell apoptosis. On the contrary, the suppressive effects on the proliferation and the enhancive effects on the apoptosis by silencing miR-1228 in HK-2 cells stimulated with HG can be weakened by recommendation of THBS2 small interference RNAs. Furthermore, we also found that HG significantly enhanced the phosphorylation levels of PI3K and AKT. In terms of overexpression and knockdown experiments, Western blot analysis further revealed that miR-1228 inhibited the activation of the PI3K/AKT signaling pathway in HG-damaged HK-2 cells by regulating THBS2. Conclusion: The findings illustrated that miR-1228 improved survivability and inhibited apoptosis in HK-2 cells stimulated with HG partly by restraining the activation of the PI3K/AKT signaling pathway.

Human Proximal Tubule Epithelial Cells (HK-2) as a Sensitive In Vitro System for Ochratoxin A Induced Oxidative Stress.

Ochratoxin A (OTA) is a mycotoxin that is potentially carcinogenic to humans. Although its mechanism remains unclear, oxidative stress has been recognized as a plausible cause for the potent renal carcinogenicity observed in experimental animals. The effect of OTA on oxidative stress parameters in two cell lines of LLC-PK1 and HK-2 derived from the kidneys of pig and human, respectively, were investigated and compared. We found that the cytotoxicity of OTA on LLC-PK1 and HK-2 cells was dose- and time-dependent in both cell lines. Furthermore, increased intracellular reactive oxygen species (ROS) induced by OTA in both cell lines were observed in a time-dependent manner. Glutathione (GSH) was depleted by OTA at >48 h in HK-2 but not in LLC-PK1 cells. While the mRNA levels of glucose-6-phosphate dehydrogenase (G6PD) and glutathione peroxidase 1 (GPX1) in LLC-PK1 were down-regulated by 0.67- and 0.66-fold, respectively, those of catalase (CAT), glutathione reductase (GSR), and superoxide dismutase 1 (SOD) in HK-2 were up-regulated by 2.20-, 2.24-, and 2.75-fold, respectively, after 72 h exposure to OTA. Based on these results, we conclude that HK-2 cells are more sensitive to OTA-mediated toxicity than LLC-PK1, and OTA can cause a significant oxidative stress in HK-2 as indicated by changes in the parameter evaluated.

Bridging the gap between in silico and in vivo by modeling opioid disposition in a kidney proximal tubule microphysiological system

Opioid overdose, dependence, and addiction are a major public health crisis. Patients with chronic kidney disease (CKD) are at high risk of opioid overdose, therefore novel methods that provide accurate prediction of renal clearance (CLr) and systemic disposition of opioids in CKD patients can facilitate the optimization of therapeutic regimens. The present study aimed to predict renal clearance and systemic disposition of morphine and its active metabolite morphine-6-glucuronide (M6G) in CKD patients using a vascularized human proximal tubule microphysiological system (VPT-MPS) coupled with a parent-metabolite full body physiologically-based pharmacokinetic (PBPK) model. The VPT-MPS, populated with a human umbilical vein endothelial cell (HUVEC) channel and an adjacent human primary proximal tubular epithelial cells (PTEC) channel, successfully demonstrated secretory transport of morphine and M6G from the HUVEC channel into the PTEC channel. The in vitro data generated by VPT-MPS were incorporated into a mechanistic kidney model and parent-metabolite full body PBPK model to predict CLr and systemic disposition of morphine and M6G, resulting in successful prediction of CLr and the plasma concentration–time profiles in both healthy subjects and CKD patients. A microphysiological system together with mathematical modeling successfully predicted renal clearance and systemic disposition of opioids in CKD patients and healthy subjects.

ERK and p38 MAPK inhibition controls NF-E2 degradation and profibrotic signaling in renal proximal tubule cells

AimsTransforming growth factor-β (TGF-β) mediates fibrotic manifestations of diabetic nephropathy. We demonstrated proteasomal degradation of anti-fibrotic protein, nuclear factor-erythroid derived 2 (NF-E2), in TGF-β treated human renal proximal tubule (HK-11) cells and in diabetic mouse kidneys. The current study examined the role of mitogen-activated protein kinase (MAPK) pathways in mediating NF-E2 proteasomal degradation and stimulating profibrotic signaling in HK-11 cells. Main methodsHK-11 cells were pretreated with vehicle or appropriate proteasome and MAPK inhibitors, MG132 (0.5 μM), SB203580 (1 μM), PD98059 (25 μM) and SP600125 (10 μM), respectively, followed by treatment with/without TGF-β (10 ng/ml, 24 h). Cell lysates and kidney homogenates from FVB and OVE26 mice treated with/without MG132 were immunoblotted with appropriate antibodies. pUse vector and pUse-NF-E2 cDNA were transfected in HK-11 cells and effects of TGF-β on JNK MAPK phosphorylation (pJNK) was examined. Key findingsWe demonstrated activation of p38, ERK, and JNK MAPK pathways in TGF-β treated HK-11 cells. Dual p38 and ERK MAPK blockade prevented TGF-β-induced pSer82Hsp27, fibronectin and connective tissue growth factor (CTGF) expression while preserving NF-E2 expression. Blockade of JNK MAPK inhibited TGF-β-induced CTGF expression without preserving NF-E2 expression. MG132 treatment prevented TGF-β-induced pJNK in HK-11 cells and in type 1 diabetic OVE26 mouse kidneys, demonstrating that TGF-β- and diabetes-induced pJNK occurs downstream of proteasome activation. A direct role for NF-E2 in modulating pJNK activation was demonstrated by NF-E2 over-expression. SignificanceERK and p38 MAPK promotes NF-E2 proteasomal degradation while proteasome activation promotes pJNK and profibrotic signaling in renal proximal tubule cells.

Combination of zinc and selenium alleviates ochratoxin A-induced fibrosis via blocking ROS-dependent autophagy in HK-2 cells

BackgroundOchratoxin A (OTA) is a mycotoxin produced by Aspergillus and Penicillium. The key target organ of OTA toxicity is the kidney, which has a significant impact on human health. Recently, nutrition regulation is suggested to be an effective protection against mycotoxins contamination. The current study investigated the combined protective effects of zinc and selenomethionine (SeMet) (a major component of organic selenium) on OTA-induced renal fibrosis and their potential mechanisms in human renal proximal tubule epithelial cells (HK-2 cells). MethodsCytotoxicity of different concentrations of OTA, zinc and SeMet on HK-2 cells was detected by cell viability, lactate dehydrogenase (LDH) and apoptotic nuclei assays. The expression of fibrosis biomarkers was detected by Real-Time PCR, western blotting and indirect immunofluorescence assays. The production of reactive oxygen species (ROS) was detected by ROS assay kit. The protein expression of autophagy biomarkers was detected by western blotting assay. ResultsCytotoxicity was induced by OTA treatment in a dose-dependent manner, and it was attenuated by zinc or SeMet application in HK-2 cells. Zinc or SeMet application also down-regulated the expression of fibrosis biomarkers, and the combination of them displayed better effects. In addition, OTA increased intracellular ROS level and activated autophagy in a dose-dependent manner, and it was reversed by zinc and SeMet combined application. With the treatment of hydrogen peroxide (H2O2) or rapamycin (the specific activator of autophagy), the combined protective effects of zinc and SeMet were abolished. ConclusionsZinc and SeMet application alleviated OTA-induced cytotoxicity and fibrosis in HK-2 cells. Combination of them was more effective than its individual application. The present study manifest novel insight about the alleviation of OTA-induced nephrotoxicity by nutrition regulation, and had a guiding effect on the clinical supplementation of nutritional elements.

Role of HRTPT in kidney proximal epithelial cell regeneration: Integrative differential expression and pathway analyses using microarray and scRNA-seq.

Damage to proximal tubules due to exposure to toxicants can lead to conditions such as acute kidney injury (AKI), chronic kidney disease (CKD) and ultimately end‐stage renal failure (ESRF). Studies have shown that kidney proximal epithelial cells can regenerate particularly after acute injury. In the previous study, we utilized an immortalized in vitro model of human renal proximal tubule epithelial cells, RPTEC/TERT1, to isolate HRTPT cell line that co‐expresses stem cell markers CD133 and CD24, and HREC24T cell line that expresses only CD24. HRTPT cells showed most of the key characteristics of stem/progenitor cells; however, HREC24T cells did not show any of these characteristics. The goal of this study was to further characterize and understand the global gene expression differences, upregulated pathways and gene interaction using scRNA‐seq in HRTPT cells. Affymetrix microarray analysis identified common gene sets and pathways specific to HRTPT and HREC24T cells analysed using DAVID, Reactome and Ingenuity software. Gene sets of HRTPT cells, in comparison with publicly available data set for CD133+ infant kidney, urine‐derived renal progenitor cells and human kidney‐derived epithelial proximal tubule cells showed substantial similarity in organization and interactions of the apical membrane. Single‐cell analysis of HRTPT cells identified unique gene clusters associated with CD133 and the 92 common gene sets from three data sets. In conclusion, the gene expression analysis identified a unique gene set for HRTPT cells and narrowed the co‐expressed gene set compared with other human renal–derived cell lines expressing CD133, which may provide deeper understanding in their role as progenitor/stem cells that participate in renal repair.

Experimental Cell Line Models for Nephrotoxicity Screening

The aim of the study was to review literature data on cell models for experimental assessment of drug nephrotoxicity in vitro. Because of nephrotoxicity, 2% of new investigational medicinal products are discarded at the stage of preclinical in vivo studies in laboratory animals, and 19%—after phase 3 clinical trials. Prediction of toxicity in cell models could make drug development more cost-effective and help to reduce/avoid animal testing. At present, there are no official international guidelines for assessment of nephrotoxicity in vitro, but there is a lot of research underway. The main toxicity target in kidneys is renal proximal tubule epithelial cells, therefore the main research is focused on the development of renal proximal tubule epithelial cell lines with stable functional characteristics. Another important aspect in nephrotoxicity modeling is the choice of relevant test methods and end points which would reflect potential toxicity mechanisms. The paper reviews existing human renal proximal tubule epithelial cell lines and current test methods for assessing cytotoxicity. Promising areas for future development of cell models for nephrotoxicity assessment— are optimisation and standardisation of in vitro systems that would help to make preclinical predictions of drug nephrotoxicity in vivo.