Human Renal Proximal Tubular Epithelial Cells Research Articles

Ketoconazole-loading strategy to improve antifungal activity and overcome cytotoxicity on human renal proximal tubular epithelial cells

Ketoconazole (KTZ), an antifungal agent used to treat localized or systemic fungal infections by inhibiting ergosterol synthesis, exhibits restricted efficacy within eukaryotic cells owing to its elevated toxicity and limited solubility in water. This study aims to improve the biological activity and overcome cytotoxic effects in the renal system of the hydrophobic KTZ by incorporating it into poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) utilizing biomaterial nano-engineering techniques. KTZ-loaded PLGA NPs (KTZ-NPs) were prepared by single emulsion solvent evaporation method and characterized by using dynamic light scattering (DLS), electrophoretic light scattering (ELS), Fourier transform-infrared (FT-IR) spectroscopy and scanning light microscopy (SEM). Particle size and zeta potential of KTZ-NPs were determined as 182.0 ± 3.27 nm and −27.4 ± 0.56 mV, respectively. Antifungal activity was analyzed with the time-kill and top agar dilution methods on Candida albicans (C. albicans) and Aspergillus flavus (A. flavus). Both KTZ and KTZ-NPs caused a significant decrease in A. flavus cell growth; however, the same effect was only observed in time-killing analysis on C. albicans, indicating a methodological difference in the antifungal analysis. According to the top agar method, the MIC value of KTZ-NPs against A. flavus was 9.1 μg ml−1, while the minimum inhibition concentration (MIC) value of KTZ was 18.2 μg ml−1. The twofold increased antifungal activity indicates that nanoparticular drug delivery systems enhance the water solubility of hydrophobic drugs. In addition, KTZ-NPs were not cytotoxic on human renal proximal tubular epithelial cells (HRPTEpCs) at fungistatic concentration, thus reducing fungal colonization without cytotoxic on renal excretion system cells.

Hederagenin inhibits high glucose-induced fibrosis in human renal cells by suppression of NLRP3 inflammasome activation through reducing cathepsin B expression.

Diabetic nephropathy is a major complication of diabetes mellitus and is related to dysfunction of renal cells. Hederagenin is a triterpenoid saponin from some Chinese herbs with anti-inflammatory and anti-diabetic activities. However, its role in diabetic nephropathy progression is still obscure. This study aimed to explore the effects of hederagenin on renal cell dysfunction in vitro. Human renal mesangial cells (HRMCs) and human renal proximal tubular epithelial cells (HRPTEpiCs) were cultured under high glucose (HG) conditions to mimic diabetic nephropathy-like injury. Cell proliferation was evaluated by CCK-8. mRNA and protein levels were determined by qRT-PCR and western blotting, respectively. The secretion levels of fibrosis-related biomarkers were analyzed by ELISA. Results showed that hederagenin reduced HG-induced proliferation increase in HRMCs and HRPTEpiCs. Hederagenin attenuated HG-induced increase in mRNA and protein expression of NLRP3, ASC, and IL-1β. Hederagenin also suppressed HG-induced increase in mRNA and secretion levels of FN, Col. IV, PAI-1, and TGF-β1. NLRP3 inhibitor MCC950 attenuated HG-induced fibrosis of renal cells, and its activator nigericin reversed the suppressive effect of hederagenin on HG-induced fibrosis. Bioinformatics analysis predicted cathepsin B (CTSB) as a target of hederagenin to modulate NOD-like receptor (NLR) pathway. Hederagenin decreased CTSB level, and CTSB overexpression reversed the suppressive effect of hederagenin on HG-induced NLRP3 inflammasome activation and fibrosis in HRMCs and HRPTEpiCs. In conclusion, hederagenin attenuates HG-induced fibrosis of renal cells by inhibiting NLRP3 inflammasome activation via reducing CTSB expression, indicating a therapeutic potential of hederagenin in diabetic nephropathy.

Hypoxia-inducible Factor-1α Suppresses the Innate Immune Response in Cultured Human Proximal Tubular Cells.

Retinoic acid-inducible gene (RIG)-I like receptors (RLRs) are expressed on renal proximal tubular epithelial cells (RPTECs) in viral nephropathy, indicating the presence of RLR-mediated innate immune responses in RPTECs. Hypoxia is also known to affect innate immunity. This study investigated the effects of hypoxia, and hypoxia-inducible factor (HIF) on innate immunity in RPTECs. Primary human RPTECs were cultured under normoxic or hypoxic conditions and treated with a synthetic analog of double-stranded RNA (polyIC). The expression levels of RIG-I and MDA5, as RLRs, and IFNβ, IL6, and TNFα, as inflammatory mediators were evaluated using quantitative reverse transcription-polymerase chain reaction, western blotting, and lactate dehydrogenase activity (LDH) assays. To further investigate the role of hypoxia, a small interfering RNA was used to knockdown HIF1α. Under normoxic conditions, polyIC increased RIG-I, MDA5, and IFNβ mRNA expression in RPTECs by, 9.4±0.4-, 10.8±0.5-, and 4.0±0.1-fold, respectively, compared to control, and by 5.4±0.1-, 7.4±0.1-, and 2.4±0.3-fold, respectively, under hypoxic conditions, the rate of increase was lower than that under normoxic conditions (p<0.01). Protein expression showed a similar trend. Under hypoxic conditions, polyIC treatment with HIF1α knockdown in RPTECs increased RIG-I, MDA5, and IFNβ mRNA expression by 3.1±0.5-, 2.9±0.4-, and 6.1±0.4-fold, respectively, and cytotoxicity, demonstrated by LDH assay, was increased compared to that without knockdown (all p<0.01). Hypoxia suppresses polyIC-induced RLRs mediated innate immune responses in RPTECs via HIF1α.

Bone marrow mesenchymal stem cell-derived exosomes improve renal fibrosis via regulating Smurf 2/Smad 7.

Exosomes can be secreted from bone marrow mesenchymal stem cells (BMSCs) to extracellular space and exert anti-fibrotic effects, but the underlying mechanisms remain to be elucidated. 5/6 subtotal nephrotomy (SNx) rat models and TGF-β1-induced human renal proximal tubular epithelial cells (HRPTEpiCs) were established to simulate renal fibrosis. Renal function and fibrosis were assessed by Hematoxylin and Eeosin (HE) staining, Masson staining, immunohistochemistry, and western blot. The expression of Smad 7/Smurf 2 was detected in rats and HRPTEpiCs by western blot, and a further potential mechanism was explored using si-Smurf 2. BMSC-Exo improved renal function, reduced the fibrotic region, down-regulated the expression of fibronectin, Collagen-I, α-SMA, and up-regulated E-cadherin in SNx models. In vitro study demonstrated that knocking down the expression of Smurf 2 significantly increased the expression of Smad 7, which could be enhanced by BMSC-Exo. BMSC-Exo could alleviate the fibrosis induced by TGF-β1 in tubular epithelial cells and enhanced the protective effect of si-Smurf 2 on renal fibrosis. BMSC-Exo inhibited renal fibrosis both in vivo and in vitro, partially, by regulating the Smurf 2/Smad 7 axis. BMSC-Exo enhanced the protective effect of si-Smurf 2 on fibrosis induced by transforming growth factor-β1 (TGF-β1).

Role of Aberrantly Activated Lysophosphatidic Acid Receptor 1 Signaling Mediated Inflammation in Renal Aging.

The increasing load of senescent cells is a source of aging, and chronic inflammation plays a pivotal role in cellular senescence. In addition, senescent renal tubular epithelial cells are closely associated with renal aging. Lysophosphatidic acid (LPA) is a bioactive lipid mainly produced by the catalytic action of autotaxin (ATX), and its ligation to LPA receptor-1 (LPAR1) is associated with chronic inflammation and renal fibrosis; however, its role in renal aging is unclear. Male 2-, 12-, and 24-month-old C57BL/6 mice and Human renal proximal tubular epithelial cells (HRPTEpiC) were used in the present study. DNA damage and oxidative stress-induced senescence were simulated using doxorubicin (DOXO) and H2O2, respectively. The aged kidney showed decreased renal function, increased fractional mesangial area, and tubulointerstitial fibrosis. Both aged kidney and senescent cells showed increased levels of LPAR1, Nuclear factor κB (NF-κB), and inflammatory cytokines. In addition, LPAR1-knockdown reduced NF-κB and subsequent inflammatory cytokine induction, and NF-κB-knockdown resulted in decreased LPAR1 expression. Our study revealed a positive feedback loop between LPAR1 and NF-κB, which reinforces the role of inflammatory response, suggesting that blocking of aberrantly activated LPAR1 may reduce excessive inflammation, thereby providing a new possible therapeutic strategy to attenuate renal aging.

Critical Conditions for Studying Interleukin-11 Signaling In Vitro and Avoiding Experimental Artefacts.

Interleukin (IL) 11 is a member of the IL6 family of cytokines which require the ubiquitous gp130 receptor to activate canonical (JAK/STAT) and non-canonical (e.g., ERK) signaling pathways. The IL11 cytokine is upregulated in a number of fibro-inflammatory diseases and cancer, where it binds the cognate IL11 receptor alpha subunit (IL11RA) to form a hexameric IL11:IL11RA:gp130 signaling complex. The specific IL11RA receptor is highly expressed on cells of the stromal and parenchymal niche but expressed at low levels on immune cells, highly passaged cells, or transformed cell lines. Consequently, primary cells such as hepatic stellate cells, fibroblasts, and hepatocytes are ideal experimental systems to study IL11 signaling in vitro. In contrast to immortalized cell lines, primary cells better display relevant cellular physiology and pathobiology. This collection of protocols details experimental and culturing conditions for primary cells that preserve meaningful cellular states and physiological responses ex vivo in conventional 2D cell culture systems. Readouts of cellular activity are chosen carefully to capture the non-canonical, post-transcriptional activity of IL11 signaling. Our data suggest that cell type, cell culture conditions, passage number, concentrations of stimuli, timing, and other factors have major implications for studies of IL11 signaling. In vitro experiments with primary cell material need to be planned and executed with great caution. Otherwise, physiologically relevant mechanisms may become dysfunctional and reproducible experimental artefacts can obscure our view of true cytokine biology. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expansion of primary human hepatic stellate cells (HSCs) and human renal proximal tubular epithelial cells (HRPTEpiCs) Basic Protocol 2: Expansion of primary human lung fibroblasts (HLFs) Alternate Protocol 1: Isolation and expansion of primary mouse lung fibroblasts Support Protocol 1: Freezing and thawing of primary cells Support Protocol 2: Operetta high-content imaging-based phenotyping Support Protocol 3: Colorimetric assay of solubilized collagen Support Protocol 4: Quantification of fibrosis marker secretion Support Protocol 5: Western blotting studies of IL11 signaling in HSCs, HLFs, and HRPTEpiCs Basic Protocol 3: IL11 stimulation of primary human hepatocytes Alternate Protocol 2: IL11 stimulation of primary mouse hepatocytes Support Protocol 6: Alanine transaminase (ALT) secretion by human and mouse hepatocytes.

LincRNA-p21 Inhibits Cisplatin-Induced Apoptosis of Human Renal Proximal Tubular Epithelial Cells by Sponging miR-449a

Introduction: LincRNA-p21 is predicted to interact with miR-449a, which plays a protective role in cisplatin-induced acute kidney injury (CIA). Objective: This study aimed to analyze the involvement of lincRNA-p21 in breast cancer patients with CIA. Methods: Levels of lincRNA-p21 in plasma from CIA, triple negative breast cancer, and control groups were measured by performing RT-qPCR. The potential interaction between lincRNA-p21 and miR-449a was first predicted by RT-qPCR. The relationship between lincRNA-p21 and miR-449a was analyzed by overexpression experiment. Results: We found that lincRNA-p21 is downregulated in CIA. Dual luciferase activity assay showed that lincRNA-p21 and miR-449a can interact with each other, while overexpression of lincRNA-p21 and miR-449a failed to affect the expression of each other. In human renal proximal tubular epithelial cells (HRPTEpCs), cisplatin led to the upregulated miR-449a but downregulated lincRNA-p21. Interestingly, lincRNA-p21 overexpression led to reduced enhancing effects of miR-449a on the cisplatin-induced apoptosis of HRPTEpCs. Conclusion: Therefore, lincRNA-p21 is downregulated in CIA and may sponge miR-449a to inhibit cisplatin-induced apoptosis of HRPTEpCs.

Angiotensin‑converting enzyme‑2 improves diabetic nephropathy by targeting Smad7 for ubiquitin degradation.

Angiotensin‑converting enzyme 2(ACE2), an important component of the renin‑angiotensin system, protects against renal tubulointerstitial fibrosis, but its level of involvement in the mechanism of diabetic nephropathy(DN) currently remains unclear. Herein, the effects of ACE2 in DN and the associated mechanisms were investigated using serum and renal biopsy specimens from patients with DN and control participants, and human renal proximal tubular epithelial cells (HRPTEpiCs). The present study determined that the circulating concentration of ACE2 was high, but renal ACE2 expression was markedly lower, and there was abundant expression of Arkadia, an E3 ubiquitin ligase, in patients with DN. Invitro, ACE2 attenuated high‑glucose‑induced tubular epithelial to mesenchymal cell transition(EMT), which was demonstrated by increased expression of α‑SMA and loss of E‑cadherin expression, as demonstrated by western blot analysis and reverse transcription‑quantitative PCR. Adenovirus‑mediated ACE2 overexpression was also revealed to significantly inhibit Arkadia expression and alleviated high‑glucose‑induced EMT, while ACE2 inhibition had the opposite effects. Furthermore, western blot analysis demonstrated that ACE2‑alleviated EMT was associated with downregulated Arkadia and increased SMAD family member 7(Smad7) protein, followed by TGF‑β/Smad pathway inhibition in HRPTEpiCs. In conclusion, ACE2 is protective in DN, which may be due to the inhibition of Arkadia‑mediated Smad7 degradation, whereby TGF‑β/Smad‑mediated EMT is ameliorated in high‑glucose‑stimulated HRPTEpiCs.

LncRNA RPSAP52 regulates miR-423-5p/GSTM1 axis to suppress hypoxia-induced renal proximal tubular epithelial cell apoptosis

This study aimed to investigate the roles of RPSAP52 in renal failure. Our results showed that RPSAP52 was upregulated in plasma of renal failure patients in comparison to healthy controls. Dual luciferase reporter assay showed that RPSAP52 could interact with miR-423-5p, while overexpression of RPSAP52 and miR-423-5p did not alter the expression of each other in human renal proximal tubular epithelial cells (HRPTEpCs). In addition, overexpression of RPSAP52 increased the expression levels of GSTM1 in HRPTEpCs. Cell apoptosis assay showed that overexpression of RPSAP52 and GSTM1 decreased the apoptotic rate of HRPTEpCs under hypoxia conditions. MiR-423-5p played an opposite role and attenuated the effects of overexpressing RPSAP52 and GSTM1. Therefore, RPSAP52 may regulate miR-423-5p/GSTM1 axis to suppress hypoxia-induced HRPTEpC apoptosis.

Metformin rescues Parkin protein expression and mitophagy in high glucose-challenged human renal epithelial cells by inhibiting NF-κB via PP2A activation

Our preliminary research revealed that metformin, a classic anti-diabetic drug, could rescue Parkin protein expression and mitophagy in high glucose-challenged human renal epithelial cells in vitro, but the molecular mechanism remains to be explored. In the study, Human Renal Cortical Epithelial Cells (HRCEpiC) and Human Renal Proximal Tubular Epithelial Cells (HRPTEpic) were challenged with high glucose with or without metformin pre-treatment to monitor Parkin mRNA and protein expression level change. PRKN gene knockdown was performed by lentiviral-based shRNA delivery. Cell viability, apoptosis and mitophagy were monitored after treatment. Mitochondrial damage was evaluated by analyzing mitochondrial permeability transition pore opening, membrane potential change, mitochondrial superoxide accumulation and cytochrome C release. Protein levels of activating transcription factor 4 (ATF4), p53 phospho-Ser15, IκBα phosphor-Ser32, IKKα phosphor-Ser176/180 in whole cell lysate and nuclear entry of p50/p65 were assessed by western blot. Okadaic acid was used to inhibit protein phosphatase 2A (PP2A). The data suggested high glucose challenge significantly reduced PRKN gene expression, mitophagy, mitochondria integrity and cell viability in vitro, which was rescued by metformin co-treatment. The effects of metformin were crippled by PRKN gene knockdown. Metformin increased PRKN gene transcription while reducednuclear factor kappa B (NF-κB) activation but not that of p53 or ATF4. Inhibiting PP2A weakened NF-κB inhibition and PRKN induction by metformin in high glucose-challenged cells, reducing its mitochondrial protective and cytoprotective effect. So, we concluded thatmetformin protects human renal epithelial cells from high glucose-induced apoptosis by restoring Parkin protein expression and mitophagy via PP2A activation and NF-κB inhibition.

Y+LAT1 and y+LAT2 contribution to arginine uptake in different human cell models: Implications in the pathophysiology of Lysinuric Protein Intolerance.

y+LAT1 (encoded by SLC7A7), together with y+LAT2 (encoded by SLC7A6), is the alternative light subunits composing the heterodimeric transport system y+L for cationic and neutral amino acids. SLC7A7 mutations cause lysinuric protein intolerance (LPI), an inherited multisystem disease characterized by low plasma levels of arginine and lysine, protein‐rich food intolerance, failure to thrive, hepatosplenomegaly, osteoporosis, lung involvement, kidney failure, haematologic and immunological disorders. The reason for the heterogeneity of LPI symptoms is thus far only poorly understood. Here, we aimed to quantitatively compare the expression of SLC7A7 and SLC7A6 among different human cell types and evaluate y+LAT1 and y+LAT2 contribution to arginine transport. We demonstrate that system y+L‐mediated arginine transport is mainly accounted for by y+LAT1 in monocyte‐derived macrophages (MDM) and y+LAT2 in fibroblasts. The kinetic analysis of arginine transport indicates that y+LAT1 and y+LAT2 share a comparable affinity for the substrate. Differences have been highlighted in the expression of SLC7A6 and SLC7A7 mRNA among different cell models: while SLC7A6 is almost equally expressed, SLC7A7 is particularly abundant in MDM, intestinal Caco‐2 cells and human renal proximal tubular epithelial cells (HRPTEpC). The characterization of arginine uptake demonstrates that system y+L is operative in renal cells and in Caco‐2 where, at the basolateral side, it mediates arginine efflux in exchange with leucine plus sodium. These findings explain the defective absorption/reabsorption of arginine in LPI. Moreover, y+LAT1 is the prevailing transporter in MDM sustaining a pivotal role in the pathogenesis of immunological complications associated with the disease.

Hypoxia-inducible factor-1\u03b1 is the therapeutic target of the SGLT2 inhibitor for diabetic nephropathy

Previous studies have demonstrated intrarenal hypoxia in patients with diabetes. Hypoxia-inducible factor (HIF)-1 plays an important role in hypoxia-induced tubulointerstitial fibrosis. Recent clinical trials have confirmed the renoprotective action of SGLT2 inhibitors in diabetic nephropathy. We explored the effects of an SGLT2 inhibitor, luseogliflozin on HIF-1α expression in human renal proximal tubular epithelial cells (HRPTECs). Luseogliflozin significantly inhibited hypoxia-induced HIF-1α protein expression in HRPTECs. In addition, luseogliflozin inhibited hypoxia-induced the expression of the HIF-1α target genes PAI-1, VEGF, GLUT1, HK2 and PKM. Although luseogliflozin increased phosphorylated-AMP-activated protein kinase α (p-AMPKα) levels, the AMPK activator AICAR did not changed hypoxia-induced HIF-1α expression. Luseogliflozin suppressed the oxygen consumption rate in HRPTECs, and subsequently decreased hypoxia-sensitive dye, pimonidazole staining under hypoxia, suggesting that luseogliflozin promoted the degradation of HIF-1α protein by redistribution of intracellular oxygen. To confirm the inhibitory effect of luseogliflozin on hypoxia-induced HIF-1α protein in vivo, we treated male diabetic db/db mice with luseogliflozin for 8 to 16 weeks. Luseogliflozin attenuated cortical tubular HIF-1α expression, tubular injury and interstitial fibronectin in db/db mice. Together, luseogliflozin inhibits hypoxia-induced HIF-1α accumulation by suppressing mitochondrial oxygen consumption. The SGLT2 inhibitors may protect diabetic kidneys by therapeutically targeting HIF-1α protein.

492-P: A Novel Renoprotective Target of SGLT2 Inhibitor: HIF-1α Inhibition and AMPK Activation in Renal Proximal Tubular Epithelial Cells

The renoprotective mechanism of SGLT2 inhibitors has not been fully elucidated. Tubular hypoxia is major driving force for proximal tubulopathy in diabetic kidney. In addition, diabetes causes dysregulation of AMP-activated protein kinase (AMPK) in kidney cortex. In this study, we assessed the effects of luseogliflozin, an SGLT2 inhibitor, on hypoxia inducible factor-1α (HIF-1α) expression and AMPK phosphorylation in cultured human renal proximal tubular epithelial cells (HRPTECs) and on tubulointerstitial pathological changes in diabetic mice. Luseogliflozin inhibited hypoxia (1%O2, 24h)-induced HIF-1α protein expression in HRPTECs in a dose-dependent manner (1-100µM). In addition, luseogliflozin increased AMPKα protein phosphorylation (Th172) in normoxic and hypoxic condition. Intriguingly, luseogliflozin suppressed oxygen consumption rate (OCR) measured by oxygen quenching phosphorescent probe by 69% in HRPTECs from normoxic condition. Hypoxia decreased OCR by 52% and luseogliflozin further decreased OCR by 33% from hypoxic condition. Moreover, luseogliflozin rescued hypoxic state in HRPTECs even under hypoxic conditions assessed by a hypoxia-sensitive dye, pimonidazole. These data indicate luseogliflozin inhibits HIF-1α expression through suppression of mitochondrial OCR, which leads to restore intracellular hypoxia. We next treated db/db mice with 15 mg/kg/day luseogliflozin for 8 weeks. Luseogliflozin ameliorated tubular injury compared to non-treated db/db mice. Furthermore, immunostaining of HIF-1α and fibronectin in cortical tubules revealed that luseogliflozin decreased HIF-1α and fibronectin expression in db/db mice. In conclusion, luseogliflozin decreases mitochondrial OCR and ameliorates tubular fibrosis at least partly by suppressing HIF-1α expression and activating AMPK in renal proximal tubules of diabetic mice. These results may provide a novel renoprotective target of SGLT2 inhibitor. Disclosure R. Bessho: None. Y. Takiyama: None. T. Takiyama: None. T. Ota: None.

C1q/tumor necrosis factor-related protein-3 improves renal fibrosis via inhibiting notch signaling pathways.

C1q/tumor necrosis factor-related protein-3 (CTRP3) has been extensively reported as an important role involved in antifibrosis, antiapoptosis, and anti-inflammation. However, the role of CTRP3 involved in renal fibrosis remains unclear. Our current study explored the role of CTRP3 in renal fibrosis and its underlying mechanisms by using serums and renal biopsy specimens from renal fibrosis patients and control subjects, rats models with the surgery of unilateral ureteral obstruction (UUO) and human renal proximal tubular epithelial cells (HRPTEpiCs). We found that circulating levels of CTRP3 had no significant difference between renal fibrosis patients and healthy subjects; however, renal CTRP3 expression was markedly downregulated in the fibrotic region with an abundant expression of collagen-I. In UUO rat models, circulating levels of CTRP3 have not changed with the prolonged obstruction of the kidney; renal CTRP3 expression was decreased with the severity of renal fibrosis; adenovirus-mediated CTRP3 treatment inhibited renal interstitial fibrosis. In vitro experiments revealed that CTRP3 attenuates TGF-β1 induced tubular epithelial cells fibrotic changes; CTRP3 knockdown facilitates the expression of fibrotic markers in TGF-β1-induced HRPTEpiCs; recombinant CTRP3 or adenovirus-mediated CTRP3 overexpression significantly inhibited the Notch signaling pathway-associated factors, and knockdown of CTRP3 increased TGF-β1-mediated activation of the Notch signaling pathways. Collectively, our current study found that CTRP3 could improve renal fibrosis, to some extent, through inhibiting the Notch pathway.

Effects of resveratrol on Th17 cell-related immune responses under tacrolimus-based immunosuppression

BackgroundWe previously reported that tacrolimus (Tac) does not decrease T helper 17 cells (Th17) response in kidney transplantation. In this study, we evaluated whether Resveratrol (Resv) has immunosuppressive effects by decreasing Th17 responses in Tac-based immunosuppression.MethodsWe investigated the effects of Resv under Tac-treatment conditions, on CD4+ T cell differentiation to Th17 cells in peripheral blood mononuclear cells (PBMCs), and proliferation of CD4+ T cells co-cultured with human renal proximal tubular epithelial cells (HRPTEpiCs). The effects of Resv on Th17 cells were tested in the murine skin transplant model.ResultsIn PBMCs, Tac did not but combination of Tac and Resv further suppressed Th17 immune response. In the co-culture study, combination of Resv to Tac significantly decreased HRPTEpiC-induced T cell proliferation compared to Tac alone. Resv treatment in the Jurkat cell induced the expression of AMP-activated protein kinase and suppressed the expression of mammalian target of rapamycin (mTOR), suggesting blocking Th17 pathway by Resv. In the murine skin transplant model, combination of Resv to Tac significantly prolonged skin graft survival accompanied by the suppression of Th17 cells, compared to either the Tac-alone or control groups.ConclusionThe results of our study suggest that Resv provides additional immunosuppressive effects to Tac by suppressing effector CD4+ T cells, especially Th17 cells, in the transplantation setting.

MiR-152 Attenuates the Severity of Lupus Nephritis Through the Downregulation of Macrophage Migration Inhibitory Factor (MIF)-Induced Expression of COL1A1.

Background: The role of miR-152 in lupus nephritis has not been elucidated. The aim of this study was to investigate the role of miR-152 in the pathogenesis of lupus nephritis (LN).Methods: miR-152 expression was detected using RT-PCR in LN tissue and normal controls. The miR-152 expression was compared with clinical parameters such as 24 h urine protein excretion level, serum creatinine, and serum complement level and SLEDAI score. The function of miR-152 was examined using human renal proximal tubular epithelial cells (HRPTE). miR-152 mimics and inhibitors were transfected to HRPTEs to ascertain the effects of miR-152.Results: miR-152 expression was downregulated in LN tissue. There was an inverse correlation between miR-152 expression in LN tissue and clinical parameters like 24 h urine protein excretion levels and serum creatinine, but not serum complement levels or SLEDAI. Further analysis showed that macrophage migration inhibitory factor (MIF) was a direct target of miR-152. Downregulation of MIF through complementary binding of miR-152 inhibited the renal expression of COL1A1.Conclusion: miR-152 expression was tapered in LN tissue and miR-152 expression was inversely correlated with chronicity index (CI), serum creatinine and severity of proteinuria. miR-152 may attenuate the severity of LN through the downregulation of MIF-induced expression of COL1A1. These findings suggest that miR-152 may be a potential target for the treatment of LN.

Luseogliflozin Inhibits HIF-1α Expression in Renal Proximal Tubular Epithelial Cells

Recent clinical trials have demonstrated the renoprotective effects of SGLT2 inhibitors in diabetic nephropathy (DN). However, the mechanisms of SGLT2 inhibitors on prevention of DN have not been fully elucidated. Hypoxia-induced tubulointerstitial fibrosis is considered to be a common pathway for various progressive kidney diseases including DN. Hypoxia inducible factor (HIF)-1α plays an important role in these pathological processes. In this study, we assessed the effects of luseogliflozin, an SGLT2 inhibitor, on HIF-1α expression in both cultured human renal proximal tubular epithelial cells (HRPTECs) and kidney of diabetic mice. Hypoxia (1%O2 for 24h) markedly increased HIF-1α protein in HRPTEC, whereas luseogliflozin inhibited expression of hypoxia-induced HIF-1α protein. In addition, luseogliflozin inhibited mRNA expression for HIF-1α-targeted genes, PAI-1, VEGF and GLUT-1 under hypoxia. The inhibitors of mitochondrial respiratory complex I, rotenone or complex III, antimycin A equally suppressed hypoxia-induced HIF-1α expression. We next treated db/db mice with 15 mg/kg/day luseogliflozin for 8 weeks. Luseogliflozin administration lowered plasma glucose levels and decreased glomerular mesangial matrix expansion as well as glomerular and interstitial fibronectin accumulation in db/db mice. Electron microscopic study revealed that luseogliflozin attenuated the thickening of glomerular basement membrane in db/db mice. Intriguingly, luseogliflozin decreased the length of mitochondria in the renal proximal tubules of the S1 segment. Furthermore, luseogliflozin attenuated diabetes-induced HIF-1α accumulation in the kidney of db/db mice. These results suggest that luseogliflozin inhibits hypoxia-induced HIF-1α accumulation at least partly by suppressing mitochondrial respiration. SGLT2 inhibitors may protect diabetic kidney from hypoxia-induced renal fibrosis by attenuating the expression of HIF-1α and profibrotic molecules. Disclosure R. Bessho: None. Y. Takiyama: None. T. Ota: None.

Clinical significance of CCR7+CD8+ T cells in kidney transplant recipients with allograft rejection

The regulatory function of CCR7+CD8+ T cells against effector T-cells involved in T-cell mediated rejection (TCMR) in kidney transplant recipients was investigated. In vitro experiments explored the ability of CCR7+CD8+ T cells to suppress T-cell proliferation under T-cell activation conditions or during coculture with human renal proximal tubular epithelial cells (HRPTEpiC). In an ex vivo experiment, the proportion of CCR7+/CD8+, FOXP3+/CCR7+CD8+ T and effector T-cell subsets were compared between the normal biopsy control (NC, n = 17) and TCMR group (n = 17). The CCR7+CD8+ T cells significantly suppressed the proliferation of CD4+ T cells and significantly decreased the proportion of IFN-γ+ and IL-17+/CD4+ T cells and inflammatory cytokine levels (all p < 0.05). After coculturing with HRPTEpiC, CCR7+CD8+ T cells also suppressed T-cell differentiation into IL-2+, IFN-γ+, and IL-17+/CD4+ T cells (all p < 0.05). The TCMR group had significantly fewer CCR7+/CD8+ and FOXP3+/CCR7+CD8+ T in comparison with the NC group, but the proportions of all three effector T-cell subsets were increased in the TCMR group (all p < 0.05). The proportion of CCR7+/CD8+ T was inversely correlated with those of effector T-cell subsets. The results indicate that CCR7+CD8+ T cells may regulate effector T-cells involved in TCMR in an in vitro and in an ex vivo transplant model.

Engineered extracellular matrices with controlled mechanics modulate renal proximal tubular cell epithelialization.

Acute kidney injury (AKI) is common and associated with significant morbidity and mortality. Recovery from many forms of AKI involves the proliferation of renal proximal tubular epithelial cells (RPTECs), but the influence of the microenvironment in which this recovery occurs remains poorly understood. Here we report the development of a poly(ethylene glycol) (PEG) hydrogel platform to study the influence of substrate mechanical properties on the proliferation of human RPTECs as a model for recovery from AKI. PEG diacrylate based hydrogels were generated with orthogonal control of mechanics and cell-substrate interactions. Using this platform, we found that increased substrate stiffness promotes RPTEC spreading and proliferation. RPTECs showed similar degrees of apoptosis and Yes-associated protein (YAP) nuclear localization regardless of stiffness, suggesting these were not key mediators of the effect. However, focal adhesion formation, cytoskeletal organization, focal adhesion kinase (FAK) activation, and extracellular signal-regulated kinase (ERK) activation were all enhanced with increasing substrate stiffness. Inhibition of ERK activation substantially attenuated the effect of stiffness on proliferation. In long-term culture, hydrogel stiffness promoted the formation of more complete epithelial monolayers with tight junctions, cell polarity, and an organized basement membrane. These data suggest that increased stiffness potentially may have beneficial consequences for the renal tubular epithelium during recovery from AKI.

Preconditioning of primary human renal proximal tubular epithelial cells without tryptophan increases survival under hypoxia by inducing autophagy

Hypoxia plays a significant role in the pathogenesis of acute kidney injury (AKI). Autophagy protects from AKI. Amino acid deprivation induces autophagy. The effect of L-tryptophan depletion on survival and autophagy in cultures of renal proximal tubular epithelial cells (RPTECs) under hypoxia was evaluated. RPTECs were preconditioned in a medium containing or not tryptophan, following culture under hypoxia and treatment with or without the autophagy inhibitor chloroquine. Cell survival was assessed by cell imaging, the level of certain proteins by western blotting and cellular ATP fluorometrically. Preconditioning of RPTECs in a medium without tryptophan activated general control nonderepressible 2 kinase and induced changes that favored autophagy and cell survival under hypoxic conditions. Additionally, it increased cellular ATP, while it inhibited apoptosis. Inhibition of autophagy nullified the induced increase in cellular ATP and cell survival by the absence of tryptophan. The absence of tryptophan increased p53, although its effect on p53's transcriptional targets was heterogeneous. In accordance with the decreased apoptosis, expression of p21 increased, while expression of Bax decreased. The expression of BNIP3L, which may be pro-apoptotic or pro-autophagic, increased. Considering the decreased apoptosis, it is likely that tryptophan depletion enhances autophagy through a p53-mediated increase of BNIP3L. Preconditioning of primary human RPTECs in a medium without tryptophan increases their survival under hypoxia by inducing autophagy. Identifying new molecular mechanisms that protect renal tissue from hypoxia could be proved clinically important in the prevention of AKI.