Human Kidney-2 Cells Research Articles

Correlation between Urinary Cyclophilin A and Urinary Albumin Levels on Diabetic Kidney Disease

Introduction: Diabetic kidney disease (DKD) is a complication of diabetes mellitus characterized by albuminuria persisting within 3 to 6 months, the earliest clinical evidence is microalbuminuria (30-299 mg/24hours or 20-199 ug/i). Cyclophilin A (Cyp A) is an 18 k-Da 165-amino acid long cytosolic protein also known as peptidylprolyl isomerase A. In DN, hyperglycemia will cause Cyp A secretion by human kidney-2 (HK-2) cells from PTEC and mesangial-13 cells (MES-13) and causes kidney damage. Material and Methods: This study was an analytic observational, cross sectional study conducted at the clinic and inpatient internal medicine installation at dr. M. Djamil General Hospital Padang for 6 months. Samples were selected by consecutive sampling, as many as 60 people with post prandial blood glucose > 180 mg/dl and urinary albumin > 30 mg/24 hours and met the inclusion and exclusion criteria. The samples were examined for urinary Cyp A and albumin levels. Results: The mean level of urinary Cyp A in patients with DKD is 4.96 (2.03) ng/ml. Median urinary albumin levels in DKD patients is 287.89 (30.79-394.57) mg/24 hours. Correlation analysis between urinary Cyp A and albumin levels showed a significant (p < 0.05) with a positive and strong correlation (r = 0.776) in DKD patients. Conclusion: There was an increase of urinary Cyp A and urinary albumin levels, with a positive and strong correlation between them in DKD patients. Keywords: Urinary Cyclophilin A, urinary albumin, diabetic kidney disease

Triptriolide antagonizes triptolide-induced nephrocyte apoptosis via inhibiting oxidative stress in vitro and in vivo

Triptolide(T9) is a predominant bioactive component extracted from Chinese herb Tripterygium wilfordii Hook F. (TwHF), and has multiple pharmacological activities, such as immunosuppressive and anti-inflammatory activities, et al. However, severe adverse effects and toxicity, particularly nephrotoxicity, limit its clinical application. It has been demonstrated that the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway could alleviate T9-induced nephrocyte damage. The aim of this study was to investigate the potential protective role of triptriolide (T11) against T9-induced nephrocyte apoptosis in vitro and in vivo. Renal injury models were established in human kidney 2 (HK2) cells and BALB/c mice using T9, and the protective effects of T11 were probed in vitro and in vivo, respectively. T9 induced nephrocyte damage in HK2 cells and BALB/c mice by induction of reactive oxygen species (ROS), lactate dehydrogenase (LDH), malondialdehyde (MDA) and glutathione (GSH) and reduction of superoxide dismutase (SOD), which resulted in the apoptosis of nephrocyte and injury of renal function. While, pretreatment of T11 effectively reversed these changes, resulting in the obvious decrease of oxidative stress and renal function parameters, ameliorated nephrocyte apoptosis, improved cell morphology, and higher increase of Nrf2, NAD(P)H: quinine oxidoreductase 1 (NQO1) and heme oxygenase 1 (HO-1) protein levels in vitro and in vivo. Altogether, T11 protected against T9-induced nephrocyte apoptosis possibly via suppressing oxidative stress.

Rac-GTPase promotes fibrotic TGF-β1 signaling and chronic kidney disease via EGFR, p53, and Hippo/YAP/TAZ pathways.

Rac-GTPases are major regulators of cytoskeletal remodeling and their deregulation contributes to numerous pathologies. Whether or how Rac promotes tubulointerstitial fibrosis and chronic kidney disease (CKD) is currently unknown. We showed that the major profibrotic cytokine, TGF-β1 promoted rapid Rac1-GTP loading in human kidney 2 (HK-2) human renal epithelial cells. A Rac-specific chemical inhibitor, EHT 1864, blocked TGF-β1-induced fibrotic reprogramming in kidney epithelial cells and fibroblasts. Stable Rac1 depletion in HK-2 cells, moreover, eliminated TGF-β1-mediated non-SMAD pathway activation [e.g., Src, epidermal growth factor receptor (EGFR), p53] and subsequent plasminogen activator inhibitor-1 (PAI-1), connective tissue growth factor, fibronectin, and p21 induction. Rac1 and p22phox knockdown abrogated free radical generation by TGF-β1 in HK-2 cells, consistent with the role of Rac1 in NAPD(H). TGF-β1-induced renal epithelial cytostasis was also completely bypassed by Rac1, p22phox, p47phox, and PAI-1 silencing. Rac1b isoform expression was robustly induced in the fibrotic kidneys of mice and humans. Intraperitoneal administration of EHT 1864 in mice dramatically attenuated ureteral unilateral obstruction-driven EGFR, p53, Rac1b, yes-associated protein/transcriptional coactivator with PDZ-binding motif activation/expression, dedifferentiation, cell cycle arrest, and renal fibrogenesis evident in vehicle-treated obstructed kidneys. Thus, the Rac1-directed redox response is critical for TGF-β1-driven epithelial dysfunction orchestrated, in part, via PAI-1 up-regulation. Rac pathway inhibition suppressed renal oxidative stress and maladaptive repair, identifying Rac as a novel therapeutic target against progressive CKD.-Patel, S., Tang, J., Overstreet, J. M., Anorga, S., Lian, F., Arnouk, A., Goldschmeding, R., Higgins, P. J., Samarakoon, R. Rac-GTPase promotes fibrotic TGF-β1 signaling and chronic kidney disease via EGFR, p53, and Hippo/YAP/TAZ pathways.

Activation of TRPC6 channels contributes to (+)-conocarpan-induced apoptotic cell death in HK-2 cells

(+)-Conocarpan (CNCP), a neolignan frequently found in many medicinal and edible plants displays a broad spectrum of bioactivity. Here, we demonstrated that CNCP induced apoptotic cell death in human kidney-2 (HK-2) cells in a concentration-dependent manner (IC50 = 19.3 μM) and led to the sustained elevation of intracellular Ca2+ ([Ca2+]i). Lower extracellular Ca2+ concentrations from 2.3 mM to 0 mM significantly suppressed the CNCP-induced Ca2+ response by 69.1%. Moreover, the depletion of intracellular Ca2+ stores using thapsigargin normalized CNCP-induced Ca2+ release from intracellular Ca2+ stores, suggesting that the CNCP-induced Ca2+ response involved both extracellular Ca2+ influx and Ca2+ release from intracellular Ca2+ stores. SAR7334, a TRPC3/6/7 channel inhibitor, but neither Pyr3, a selective TRPC3 channel inhibitor, nor Pico145, a TRPC1/4/5 inhibitor, suppressed the CNCP-induced Ca2+ response by 57.2% and decreased CNCP-induced cell death by 53.4%, suggesting a critical role for TRPC6 channels in CNCP-induced Ca2+ influx and apoptotic cell death. Further electrophysiological recording demonstrated that CNCP directly activated TRPC6 channels by increasing channel open probability with an EC50 value of 6.01 μM. Considered together, these data demonstrate that the direct activation of TRPC6 channels contributes to CNCP-induced apoptotic cell death in HK-2 cells. Our data point out the potential risk of renal toxicity from CNCP if used as a therapeutic agent.

Reduced asymmetric dimethylarginine accumulation through inhibition of the type I protein arginine methyltransferases promotes renal fibrosis in obstructed kidneys.

The role of asymmetric dimethylarginine (ADMA) in chronic kidney disease (CKD) is unclear. Through inhibition of type I protein arginine methyltransferases (PRMTs), a novel strategy, we aimed to determine the effect of ADMA on renal fibrosis and explore its underlying working mechanisms. After sham or unilateral ureter ligation (UUO) operation, 20-25 g male c57 mice were treated with vehicle or PT1001B, an inhibitor of type I PRMTs, for 13 d. Moreover, human kidney 2 (HK2) and normal rat kidney 49F (NRK-49F) cells were treated with various concentrations of PT1001B or ADMA in the presence of 2.5 ng/ml TGF-β. We found that treatment with PT1001B increased the deposition of extracellular matrix proteins, the expression of α smooth muscle actin, and connective tissue growth factor in UUO-induced fibrotic kidneys, which is correlated with reduced expression of PRMT1, reduced the production of ADMA, and increased expression of uromodulin. In TGF-β-stimulated HK2 and NRK-49F cells, PT1001B dose-dependently inhibited ADMA production, increased NO concentrations, and enhanced the expression of profibrotic proteins. Exogenous addition of ADMA inhibited the expression of profibrotic proteins dose-dependently and attenuated the profibrotic effect of PT1001B. Moreover, ADMA reduced the NO concentration in PT1001B-treated HK2 cells. Finally, we conclude that ADMA has an antifibrotic effect in obstructed kidneys, and future application of type I PRMT inhibitor should be done cautiously for patients with CKD.-Wu, M., Lin, P., Li, L., Chen, D., Yang, X., Xu, L., Zhou, B., Wang, C., Zhang, Y., Luo, C., Ye, C. Reduced asymmetric dimethylarginine accumulation through inhibition of the type I protein arginine methyltransferases promotes renal fibrosis in obstructed kidneys.

Inhibition of lymphatic proliferation by the selective VEGFR-3 inhibitor SAR131675 ameliorates diabetic nephropathy in db/db mice

Recent studies have demonstrated that chronic inflammation-induced lymphangiogenesis plays a crucial role in the progression of various renal diseases, including diabetic nephropathy. SAR131675 is a selective vascular endothelial cell growth factor receptor-3 (VEGFR-3)-tyrosine kinase inhibitor that acts as a ligand for VEGF-C and VEGF-D to inhibit lymphangiogenesis. In this study, we evaluated the effect of SAR131675 on renal lymphangiogenesis in a mouse model of type 2 diabetes. Male C57BLKS/J db/m and db/db mice were fed either a regular chow diet or a diet containing SAR131675 for 12 weeks from 8 weeks of age. In addition, we studied palmitate-induced lymphangiogenesis in human kidney-2 (HK2) cells and RAW264.7 monocytes/macrophages, which play a major role in lymphangiogenesis in the kidneys. SAR131475 ameliorated dyslipidemia, albuminuria, and lipid accumulation in the kidneys of db/db mice, with no significant changes in glucose and creatinine levels and body weight. Diabetes-induced systemic inflammation as evidenced by increased systemic monocyte chemoattractant protein-1 and tumor necrosis factor-α level was decreased by SAR131475. SAR131475 ameliorated the accumulation of triglycerides and free fatty acids and reduced inflammation in relation to decreased chemokine expression and pro-inflammatory M1 macrophage infiltration in the kidneys. Downregulation of VEGF-C and VEGFR-3 by SAR131475 inhibited lymphatic growth as demonstrated by decreased expression of LYVE-1 and podoplanin that was further accompanied by reduced tubulointerstitial fibrosis, and inflammation in relation to improvement in oxidative stress and apoptosis. Treatment with SAR131475 improved palmitate-induced increase in the expression of VEGF-C, VEGFR-3, and LYVE-1, along with improvement in cytosolic and mitochondrial oxidative stress in RAW264.7 and HK2 cells. Moreover, the enhanced expression of M1 phenotypes in RAW264.7 cells under palmitate stress was reduced by SAR131475 treatment. The results suggest that modulation of lymphatic proliferation in the kidneys is a new treatment approach for type 2 diabetic nephropathy and that SAR131675 is a promising therapy to ameliorate renal damage by reducing lipotoxicity-induced lymphangiogenesis.

Protective effect of salvianolic acid B against oxidative injury associated with cystine stone formation

The aim of this study was to investigate the role of oxidative stress in cystine crystal formation and whether salvianolic acid B, a natural antioxidant, could prevent cystine-mediated oxidative injury in vivo and in vitro. The levels of oxidative stress and antioxidase activity in cystine stone patients were assessed. Then, the oxidative stress exerted by cystine on human kidney-2 (HK-2) cell viability and biochemical parameters including antioxidase activity and antioxidant protein expression were evaluated, and the protective action of salvianolic acid B was also examined. Finally, salvianolic acid B was tested to determine whether it could prevent or reduce renal crystal formation in Slc7a9 knockout mice. The activity levels of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were decreased, and the amount of malondialdehyde (MDA) was increased in patients with cystine stones compared with people without cystine stones (p < 0.05). Significant reductions in cell viability, antioxidase activity and antioxidant protein expression levels were found in the cystine group compared with controls. However, such oxidative injuries were prevented by salvianolic acid B. In the animal study, loose crystals with white spots were seen in the renal parenchyma, bilateral renal pelvis and bladders in the Slc7a9 knockout group. In contrast, no renal crystals were seen in the control group, and markedly fewer crystals with significantly higher antioxidase activity and diminished oxidative stress were detected in the salvianolic acid B group. Cystine cytotoxicity in vitro and cystine stone formation in vivo were associated with oxidative stress, and salvianolic acid B could protect against cystine stone-induced injury.

Hypoxia preconditioned renal tubular epithelial cell-derived extracellular vesicles alleviate renal ischaemia-reperfusion injury mediated by the HIF-1α/Rab22 pathway and potentially affected by microRNAs.

We previously found that hypoxia induced renal tubular epithelial cells (RTECs) release functional extracellular vesicles (EVs), which mediate the protection of remote ischaemic preconditioning (RIPC) for kidney ischaemia-reperfusion (I/R) injury. We intend to investigate whether the EVs were regulated by hypoxia-inducible factor 1α (HIF-1α) and Rab22 during RIPC. We also attempted to determine the potentially protective cargo of the EVs and reveal their underlying mechanism. Hypoxia preconditioning (HPC) of human kidney 2 (HK2) cells was conducted at 1% oxygen (O2) for different amounts of time to simulate IPC in vitro. EVs were isolated and then quantified. HIF-1α- and Rab22-inhibited HK2 cells were used to investigate the role of the HIF-1α/Rab22 pathway in HPC-induced EV production. Both normoxic and HPC EVs were treated in vivo to assess the protective effect of I/R injury. Moreover, microRNA (miRNA) sequencing analysis and bioinformatics analysis was performed. We revealed that the optimal conditions for simulating IPC in vitro was no more than 12 h under the 1% O2 culture circumstance. HPC enhanced the production of EVs, and the production of EVs was regulated by the HIF-1α/Rab22 pathway during HPC. Moreover, HPC EVs were found to be more effective at attenuating mice renal I/R injury. Furthermore, 16 miRNAs were upregulated in HPC EVs. Functional and pathway analysis indicated that the miRNAs may participate in multiple processes and pathways by binding their targets to influence the biochemical results during RIPC. We demonstrated that HIF-1α/Rab22 pathway mediated RTEC-derived EVs during RIPC. The HPC EVs protected renal I/R injury potentially through differentially expressed miRNAs. Further study is needed to verify the effective EV-miRNAs and their underlying mechanism.

Atg7 mediates renal tubular cell apoptosis in vancomycin nephrotoxicity through activation of PKC-δ.

Recent studies have shown that autophagy exhibits a renoprotective role in various models of acute kidney injury (AKI). However, its role in vancomycin (Van)-induced AKI remains largely unclarified. This study was the first to indicate that autophagy was rapidly activated in both human kidney-2 cells and renal tissues, and mammalian target of rapamycin (mTOR) was inactivated via the suppression of ERK1/2 and mTOR during Van treatment. Interestingly, for both in vitro and in vivo experiments, the suppression of autophagy via chloroquine and PT-Atg7-KO significantly ameliorated Van-induced kidney injury and renal tubular cell apoptosis. Global gene expression analysis indicated that the expression levels of 6159 genes were induced by Van treatment in the kidney cortical tissues of PT-Atg7 wild-type mice, and 18 of them were notably suppressed in PT-Atg7-KO mice. These 18 genes were further classified as programmed cell death, protein binding, signal transduction, E3 ubiquitin ligase, nucleoside diphosphate kinase activity, and E1-like activating enzyme. Unexpectedly, following Van treatment, PKC-δ expression was found to be highest among the 4 genes related to cell death, which was remarkably suppressed in vitro and in PT-Atg7-KO mice. In addition, Atg7 could induce renal cell apoptosis during Van treatment via binding to PKC-δ. Likewise, the inhibition of PKCδ ameliorated Van-induced apoptosis in human kidney-2 cells and kidney tissues. Furthermore, the data showed that PT-Atg7-KO exerted a renoprotective effect against Van-induced nephrotoxicity, but this effect was lost after injection with myc-tagged PKCδ. Taken altogether, these results indicate that Van induces autophagy by suppressing the activation of the ERK1/2 and mTOR signaling pathway. In addition, Atg7 mediates Van-induced AKI through the activation of PKCδ. In sum, autophagy inhibition may serve as a novel therapeutic target for treating nephrotoxic AKI induced by Van.-Xu, X., Pan, J., Li, H., Li, X., Fang, F., Wu, D., Zhou, Y., Zheng, P., Xiong, L., Zhang, D. Atg7 mediates renal tubular cell apoptosis in vancomycin nephrotoxicity through activation of PKC-δ.

Downregulation of TIMP2 attenuates sepsis-induced AKI through the NF-κb pathway

Acute kidney injury (AKI) is a frequent complication of sepsis and contributes to increased morbidity and mortality. Urinary tissue inhibitor of metalloproteinases-2 (TIMP2) has been recently recognized as an early biomarker to predict AKI in critically ill patients. However, the biological functions of TIMP2 remain largely unknown. In this study, we investigated the role of TIMP2 in mediating inflammation and tubular cell apoptosis in AKI. In kidney tissue taken from mice exposed to cecal ligation and puncture (CLP) and in human kidney 2 (HK-2) cells exposed to lipopolysaccharide (LPS) in culture, TIMP2 expression was significantly upregulated. The expression of TIMP2 in the kidney tissue correlated with the severity of AKI in vivo. In cultured HK-2 cells, LPS challenge markedly induced cytokine release, and recombinant cytokines promoted TIMP2 expression and apoptosis. However, TIMP2 silencing ameliorated LPS-induced cytokine release, apoptosis, and cell injury. We further found that the effects of downregulation of TIMP2 on a suppression of release of inflammatory cytokines were mediated by p-P65. Stable, kidney-specific TIMP2 knockdown mice were transduced by injecting the TIMP2 knockdown lentiviral vector into kidney parenchyma. TIMP2 silencing ameliorated CLP-induced proinflammatory cytokines, kidney dysfunction as measured by serum creatinine level, and histopathological changes. Downregulation of TIMP2 showed renoprotective effects on endotoxin-induced AKI, which was associated with the anti-inflammatory activity through inhibition of the nuclear factor (NF)-κB pathway. Collectively, our results indicate that TIMP2 plays an important role in mediating sepsis-induced AKI through regulation of NF-κB. These findings reveal the pathogenic role of TIMP2 in AKI and suggest a novel target for the treatment of AKI.

Periostin induces kidney fibrosis after acute kidney injury via the p38 MAPK pathway.

Periostin plays a crucial role in fibrosis, and acute kidney injury results in a high risk of progression to chronic kidney disease. Therefore, we hypothesized that periostin was involved in the progression of acute kidney injury to kidney fibrosis. Unilateral ischemia-reperfusion injury (UIRI) was induced in 7- to 8-wk-old male wild-type and periostin-null mice, and the animals were observed for 6 wk. In vitro, human kidney-2 cells and primary-cultured human tubular epithelial cells were incubated under hypoxic conditions (5% O2, 5% CO2, and 90% N2) for 5 days. The cells were also cultured with recombinant periostin (rPeriostin) and a p38 mitogen-activated protein kinase (MAPK) inhibitor in a hypoxic incubator. At 6 wk after UIRI, interstitial fibrosis/tubular atrophy was significantly alleviated in periostin-null mice compared with wild-type controls. In addition, periostin-null mice had attenuated expression of fibrosis/apoptosis markers and phosphorylated-p38 MAPK compared with wild-type controls. In vitro, hypoxic injury increased the expression of fibrosis markers, periostin, and phosphorylated-p38 MAPK, which was comparable to or substantially greater than their expression levels following treatment with recombinant transforming growth factor-β1 under normoxic conditions. Furthermore, rPeriostin treatment under hypoxic conditions enhanced fibrosis/apoptosis markers and phosphorylated-p38 MAPK. In contrast, p38 MAPK inhibition ameliorated hypoxia-induced fibrosis, and the addition of the p38 MAPK inhibitor to rPeriostin significantly ameliorated the changes induced by rPeriostin. In conclusion, periostin promotes kidney fibrosis via the p38 MAPK pathway following acute kidney injury triggered by a hypoxic or ischemic insult. Periostin ablation may protect against chronic kidney disease progression.

Artemisinin attenuates tubulointerstitial inflammation and fibrosis via the NF-κB/NLRP3 pathway in rats with 5/6 subtotal nephrectomy.

Artemisinin (Art) is isolated from Artemisia annua L. and known as the most effective antimalaria drugs. Previous studies demonstrated that it could exert an immune-regulatory effect on autoimmune diseases. In this study, we first investigated its potential role in tubulointerstitial inflammation and fibrosis in rats with 5/6 nephrectomy. Subtotal nephrectomized (SNx) rats were orally administered Art (100 mg·kg -1 ·d - 1) for 16 weeks. Blood and urine samples were collected for biochemical examination. Kidney tissues were collected for immunohistochemistry and Western blot analyses. Ang II-induced injury of the human kidney 2 (HK-2) cells was used for in vitro study. It was shown that Art could significantly attenuate the renal function decline in SNx rats compared with control. More importantly, Art treatment significantly reduced the tubulointerstitial inflammation and fibrosis, as demonstrated by the evaluation of renal pathology. Furthermore, Art inhibited the activation of NLRP3 inflammasome and NF-κB in the kidneys. In in vitro study, Art pretreatment could significantly prevent the activation of NLRP3 inflammasome and NF-κB in Ang II-treated HK-2 cells, while BAY11-7082 (an inhibitor of NF-κB) significantly inhibited Ang II-induced NLRP3 inflammasome activation. This study suggested that Art could provide renoprotective role by attenuating the tubulointerstitial inflammation and fibrosis in SNx rats by downregulating the NF-κB/NLRP3 signaling pathway.

Arsenic induces fibrogenic changes in human kidney epithelial cells potentially through epigenetic alterations in DNA methylation.

Arsenic contamination is a significant public health issue, and kidney is one of the target organ for arsenic-induced adverse effects. Renal fibrosis is a well-known pathological stage frequently observed in progressive chronic kidney disease (CKD). Epidemiological studies implicate arsenic exposure to CKD, but the role of arsenic in kidney fibrosis and the underlying mechanism is still unclear. It is in this context that the current study evaluated the effects of long-term arsenic exposure on the cellular response in morphology, and marker genes expression with respect to fibrosis using human kidney 2 (HK-2) epithelial cells. Results of this study revealed that in addition to increased growth, HK-2 cells underwent phenotypic, biochemical and molecular changes indicative of epithelial-mesenchymal transition (EMT) in response to the exposure to arsenic. Most importantly, the arsenic-exposed cells acquired the pathogenic features of fibrosis as supported by increased expression of markers for fibrosis, such as Collagen I, Fibronectin, transforming growth factor β, and α-smooth muscle actin. Upregulation of fibrosis associated signaling molecules such as tissue inhibitor of metalloproteinases-3 and matrix metalloproteinase-2 as well as activation of AKT was also observed. Additionally, the expression of epigenetic genes (DNA methyltransferases 3a and 3b; methyl-CpG binding domain 4) was increased in arsenic-exposed cells. Treatment with DNA methylation inhibitor 5-Aza-2'-dC reversed the EMT properties and restored the level of phospho-AKT. Together, these data for the first time suggest that long-term exposure to arsenic can increase the risk of kidney fibrosis. Additionally, our data suggest that the arsenic-induced fibrotic changes are, at least in part, mediated by DNA methylation and therefore potentially can be reversed by epigenetic therapeutics.

MiR-373 exacerbates renal injury and fibrosis via NF-κB/MatrixMetalloproteinase-9 signaling by targeting Sirtuin1

BackgroundRenal fibrosis is a final common pathway of chronic kidney disease. SIRT1, a NAD+-dependent protein deacetylase, deacetylates the p65 of NF-κB and shows protective effects in kidney disorders. miR-373 directly targets the 3′UTR of SIRT1. However, roles of miR-373 in renal fibrosis are unclear. MethodsTGF-β1, a critical regulator of fibrosis, was used to stimulate human kidney-2 cells to establish cell model for renal fibrosis. Unilateral ureteral obstruction (UUO) was performed as an in vivo model. ResultsTGF-β1 induced the level of miR-373, reduced level of SIRT1, and promoted p65 acetylation and MMP-9 expression. These effects were reversed by the miR-373 inhibitor. In the animal model, UUO caused a consistent pattern as demonstrated in vitro. ConclusionThese results indicated an undesired effect of miR-373 in the regulation of renal injury and fibrosis by targeting SIRT1-mediated NF-κB/MMP-9 signaling, which might provide a potential therapeutic strategy for renal fibrosis.

Increased transforming growth factor beta (TGF-\u03b2) and pSMAD3 signaling in a Murine Model for Contrast Induced Kidney Injury

We tested the hypothesis that post-contrast acute kidney injury (PC-AKI) occurs due to increase in transforming growth factor beta (Tgf-β) and pSMAD3 signaling in a murine model of PC-AKI. Mice had nephrectomy performed and twenty-eight days later, 100-μL of radio-contrast (Vispaque 320) or saline was administered via the jugular vein. Animals were sacrificed at 2, 7, and 28 days later and the serum BUN, creatinine, urine protein levels, and kidney weights were assessed. In human kidney-2 (HK-2) cells, gene and protein expression with cellular function was assessed following inhibition of TGFβR-1 plus contrast exposure. After contrast administration, the average serum creatinine is significantly elevated at all time points. The average gene expression of connective tissue growth factor (Ctgf), Tgfβ-1, matrix metalloproteinase-9 (Mmp-9), and collagen IVa (Col IVa) are significantly increased at 2 days after contrast administration (P < 0.05). Cellular proliferation is decreased and there is increased apoptosis with tubulointerstitial fibrosis. Contrast administered to HK-2 cells results in increased pSMAD3 levels and gene expression of Ctgf, Tgfβ-1, Tgfβ-2, Col IVa, Mmp-9, and caspase/7 activity with a decrease in proliferation (all, P < 0.05). TGFβR-1 inhibition decreased the expression of contrast mediated pro-fibrotic genes in HK-2 cells with no change in the proliferation and apoptosis.

Caspase-independent programmed cell death triggers Ca2PO4 deposition in an in vitro model of nephrocalcinosis.

Nephrocalcinosis involves the deposition of microscopic crystals in the tubular lumen or interstitium. While the clinical, biochemical, and genetic aspects of the diseases causing nephrocalcinosis have been elucidated, little is known about the cellular events in this calcification process. We previously reported a phenomenon involving the spontaneous formation of Ca2PO4 nodules in primary papillary renal cells from a patient with medullary nephrocalcinosis harboring a rare glial cell-derived neurotrophic factor (GDNF) gene variant. We also demonstrated that cultivating GDNF-silenced human kidney-2 (HK-2) cells in osteogenic conditions for 15 days triggered Ca2PO4 deposits. Given the reportedly close relationship between cell death and pathological calcification, aim of the present study was to investigate whether apoptosis is involved in the calcification of GDNF-silenced HK-2 cells under osteogenic conditions. Silenced and control cells were cultured in standard and osteogenic medium for 1, 5, and 15 days, and any Ca2PO4 deposition was identified by means of von Kossa staining and environmental SEM (ESEM) analyses. Based on the results of annexin V and propidium iodide (PI) analysis, and terminal deoxynucleotidyl transferase dUTP-biotin nick end labeling (TUNEL) assay, the silenced cells in the osteogenic medium showed a significant increase in the percentage of cells in the late phase of apoptosis and an increased Ca2PO4 deposition at 15 days. The results of quantitative real-time PCR (qRT-PCR) of BAX and BCL2, and in-cell Western analysis of caspases indicated that the cell death process was independent of caspase-3, -6, -7, and -9 activation, however. Using this model, we provide evidence of caspase-independent cell death triggering the calcification process in GDNF-silenced HK-2 cells.

&amp;lt;i&amp;gt;In Vitro&amp;lt;/i&amp;gt; Study of the Nephrotoxicity of Tripterygium Tablet Extract and Triptolide in Monolayer HK-2 Cells Cultured in a Transwell Chamber

We established a monolayer polarized cell model using human kidney 2 (HK-2) cells cultured in a transwell chamber to examine the changes in the morphology and physiological functions of human-derived renal proximal tubular epithelial cells caused by tripterygium tablet extract (TTE) and triptolide. HK-2 cells were cultured on PCF membranes to form a complete monolayer of cells. A MTT assay was used to select 10, 40, 160, 640 μg·ml-1 TTE or 4, 16, 64, 256 ng·ml-1 triptolide to treat HK-2 monolayer cells. After 24 hours, a FITC permeability assay was performed; GGT, LDH and NAG secretion on the apical (AP) and basolateral (BL) sides of the cells by HK-2 cells were examined. The morphology and the monolayer structure of HK-2 cells was observed via optical microscope and scanning electron microscope, respectively. The effect on the cytoskeleton of HK-2 cells was observed under a fluorescence microscope. The IC50 of TTE was 277.122 μg·ml-1, and the IC50 of triptolide was 148.035 ng·ml-1. Compared with the DMSO group, the FITC leakage rate with TTE 160, 640 μg·ml-1 treated group and 4 - 256 ng·ml-1 triptolide dose group exhibited statistically significant increase. TTE significantly increased secretion of GGT and LDH at 160, 640 μg·ml-1, meanwhile, dramatically increased the AP/BL ratio of LDH at 160 μg·ml-1; triptolide significantly increased secretion and AP/BL ratio of GGT and LDH at 256 ng·ml-1. The morphological observations via optical and electron microscope indicated various degrees of damage to HK-2 cells by TTE and triptolide, and the degree of damage correlated positively with the dosage of the tested articles. Compared with DMSO group, the cellular damage degrees at TTE dosages of 40 - 640 μg·ml-1 and triptolide dose group at 16, 256 ng·ml-1 exhibited statistically significant differences via observation under optical microscope. Both TTE and triptolide caused various degrees of shortening and thickening of intracellular F-actin bundles of HK-2 cells; aggravation of these changes was observed with increasing drug dosage. Thus, we conclude both TTE and triptolide caused damage to human renal proximal tubular epithelial cells at certain dosages; TTE dosages of 40 μg·ml-1 and above and triptolide dose group at 16 ng·ml-1 and above exhibited the changes in the morphology, meanwhile, TTE dosages of 160 μg·ml-1 and above and triptolide dose group at 256 ng·ml-1 exhibited the changes in the physiological functions such as secretion of HK-2 cell.

Massive Proteinuria-Induced Injury of Tubular Epithelial Cells in Nephrotic Syndrome is Not Exacerbated by Furosemide

Background/Aims: Massive proteinuria, a significant sign of nephrotic syndrome (NS), has the potential to injure tubular epithelial cells (TECs). Furosemide is widely used for the treatment of edema, a common manifestation of NS. However, whether furosemide treatment affects massive proteinuria-induced TEC injury in patients with NS is unknown. Methods: The effect of furosemide on TEC damage was investigated in vitro. In addition, a clinical study was conducted to study whether the short-term treatment of nephrotic edema with furosemide could exacerbate TEC injury. Results: The proliferation of in vitro human kidney-2 (HK-2) cells exposed to massive urinary protein (8 mg/mL) significantly decreased (P<0.05), while the levels of kidney injury molecule-1 (Kim-1) and neutrophil gelatinase associated lipocalin (NGAL) in the supernatants significantly increased (P<0.05). Importantly, furosemide treatment did not further increase the expression of Kim-1 and NGAL in HK-2 cells upregulated by massive proteinuria. For the clinical study, 26 patients with NS, all prescribed the recommended dosage of prednisone (1 mg/kg/day), were randomly assigned to two groups. One group (n=13) received furosemide (60-120 mg/day, intravenously) for 1 week; the remaining participants (control group) did not receive furosemide or any other diuretics. The results showed that the 24-h urine volume in the furosemide-treated group was slightly, but not significantly, higher than that in the control group (P>0.05). In addition, serum levels of BUN, Scr, Cys C, and urinary Kim-1 and NGAL were not significantly different between the two groups (all P>0.05). Twenty-three patients underwent a renal biopsy. Of these, 22 patients exhibited vacuolar degeneration of the TECs; 8 patients showed brush border membrane shedding of the TECs; and 12 patients showed protein casts. However, there were no significant differences between the two groups (all P>0.05). Conclusion: In summary, massive proteinuria induced the injury of TECs in patients with NS, and furosemide treatment did not aggravate this injury.

Assessing the recovery from prerenal and renal acute kidney injury after treatment with single herbal medicine via activity of the biomarkers HMGB1, NGAL and KIM-1 in kidney proximal tubular cells treated by cisplatin with different doses and exposure times

BackgroundAcute kidney injury (AKI) is an initial factor in many kidney disorders. Pre- and intra-renal AKI biomarkers have recently been reported. Recovery from AKI by herbal medicine has rarely been reported. Thus, this study aimed to investigate the dose- and time-dependent effects of herbal medicines to protect against AKI in cisplatin-induced human kidney 2 (HK-2) cells by assessing the activities of high-mobility group box protein 1 (HMGB1), neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1).MethodsProximal tubular HK-2 cell lines were treated with either 400 μM of cisplatin for 6 h or 10 μM of cisplatin for 24 h and then exposed to ten types of single herbal medicines, including Nelumbo nymphaea (NY) at a dose of 100 μg/mL. The AKI biomarkers HMGB1, NGAL and KIM-1 were repeatedly measured by an ELISA assay at 2, 4, and 6 h in the group treated with 400 μM of cisplatin to confirm necrotic cell death and at 6, 24, and 48 h in the group treated with 10 μM of cisplatin to examine apoptotic cell death. Recovery confirm was conducted through in vivo study using ICR mice for 3 day NY or Paeonia suffruticosa intake.ResultsCisplatin treatment at a concentration of 10 μM decreased cell viability. Treatment with 400 μM of cisplatin reduced HMBG1 activity and resulted in lactate dehydrogenase release. In longer exposure durations (up to 48 h), NGAL and KIM-1 exhibited activity from 24 h onward. Additionally, NY treatment resulted in an approximately 50% change in all three biomarkers. The time-dependent profiles of HMGB1, NGAL and KIM-1 activities up to 48 h were notably different; HMGB1 exhibited a 7-fold change at 6 h, and NGAL and KIM-1 exhibited 1.7-fold changes at 24 h, respectively. Consistently, serum and urine NGAL and KIM-1 activities were all reduced in ICR mice.ConclusionsSeveral single herbal medicines, including NY, have a potential as effectors of AKI due to their ability to inhibit the activation of HMGB1, NGAL and KIM-1 in an in vitro AKI-mimicked condition and simple in vivo confirm. Furthermore, an in vivo proof-of-concept study is needed.

Metabolic detoxification of bakuchiol is mediated by oxidation of CYP 450s in liver microsomes

Bakuchiol, one of bioactive compounds isolated from the dried ripe fruits of Psoralea corylifolia L., possesses a variety of pharmacological activities. In this study, the metabolites of bakuchiol in rat liver microsomes as well as their cytotoxicities were studied. A total of eight metabolites were isolated and identified as 14-carboxylbakuchiol (M1), 14,15-dihydroxybakuchiol (M2), 12,13-dihydroxybakuchiol (M3), 15-hydroxybakuchiol (M4), 14-hydroxybakuchiol (M5), bakuchiol hydrate (M6), 15-hydroxybakuchiol acetate (M7), and 14-hydroxybakuchiol acetate (M8). All the metabolites are new compounds except for M3. The main type of biotransformation is oxidation reaction, including hydroxylation, epoxidation and carboxylation. Cytotoxicities of bakuchiol and its metabolites against human kidney-2 (HK-2) cell line were evaluated. The median inhibition concentration (IC50) values of bakuchiol, M4, M6 and M8 were (29.48 ± 0.22) μM, (67.51 ± 6.80) μM, (90.23 ± 3.89) μM, and (86.62 ± 6.08) μM, respectively, and the IC50 values of M1, M2, M3, M5, and M7 were all in excess of 100 μM. To further verify the metabolic reliability, the metabolits of bakuchiol in vivo and the metabolic species variations in human and rat liver microsomes were studied using UPLC-MS/MS method. This study provides valuable information for further investigation of metabolism and toxicity of bakuchiol in vivo.