Proximal Tubule Epithelial Cells Research Articles

Activation of vitamin D receptor attenuates high glucose-induced cellular injury partially dependent on CYP2J5 in murine renal tubule epithelial cell

AimsVitamin D and its receptor, vitamin D receptor (VDR), have renoprotection effect against diabetic nephropathy (DN). But the exact mechanism has not been fully elucidated. Epoxyeicosatrienoic acids (EETs) are cytochrome P450 (CYP) epoxygenase-derived metabolites of arachidonic acid, protecting against diabetes and DN. Herein, we hypothesized that activation of VDR attenuated high glucose-induced cellular injury in renal tubular epithelial cells partially through up-regulating CYP2J5 expression. Main methodsStreptozotocin (STZ) was injected to induce diabetic in wild type and Vdr−/− mice. The effects of VDR knockout and an activator of VDR, paricalcitol, on the renal injury were detected. In vitro, a murine kidney proximal tubule epithelial cell line BU.MPT induced by high glucose were treated with or without paricalcitol (30 mM) for 12 h or 24 h. Key findingsThe expression of CYP2J5 was significantly decreased both in wild type and Vdr−/− diabetic mice induced by STZ. The STZ-induced kidney architecture damage and apoptosis rate in Vdr−/− mice were more severe. In vitro, high glucose treatment strongly reduced the CYP2J5 expression and the synthesis of 14,15-EET in BU.MPT cells. Supplement of 14,15-EET significantly reduced the lactate dehydrogenase (LDH) release induced by high glucose in BU.MPT cells. Furthermore, treatment with paricalcitol attenuated cellular injury and restored the expression of CYP2J5 reduced by high glucose in BU.MPT cells. SignificanceWe conclude that activation of VDR attenuates high glucose-induced cellular injury partially dependent on CYP2J5 in murine renal tubule epithelial cells and paricalcitol may represent a potential therapy for DN.

Role of the kidneys in the redistribution of heme-derived iron during neonatal hemolysis in mice

Moderate intravascular hemolysis is a common condition in newborns. It is followed by the accumulation of bilirubin, which is a secondary product of the activity of heme oxygenase-1, an enzyme that catalyzes the breakdown of heme released from disrupted erythrocytes and taken up by hepatic macrophages. Although these cells are a major site of enzymatic heme breakdown in adults, we show here that epithelial cells of proximal tubules in the kidneys perform the functions of both heme uptake and catabolism in mouse neonates. A time-course study examining mouse pups during the neonatal period showed a gradual recovery from hemolysis, and concomitant decreases in the expression of heme-related genes and non-heme iron transporters in the proximal tubules. By adjusting the expression of iron-handling proteins in response to the disappearance of hemolysis in mouse neonates, the kidneys may play a role in the detoxification of iron and contribute to its recirculation from the primary urine to the blood.

Src-mediated crosstalk between FXR and YAP protects against renal fibrosis.

Renal fibrosis is the common pathway of chronic kidney disease progression. The nuclear receptor farnesoid X receptor [FXR, NR1H4 (nuclear receptor subfamily 1 group member 4)], a multifunctional transcription factor, plays a pivotal role in protecting against fibrosis. However, the mechanisms underlying these antifibrotic actions of FXR in kidney disease are largely unknown. Here, we show that agonist GW4064-mediated FXR activation inhibits the activity of the nonreceptor tyrosine kinase Src (proto-oncogene tyrosine-protein kinase), which is critical for regulation of yes-associated protein (YAP) phosphorylation and nuclear localization in renal fibrosis. Activation of FXR suppressed renal fibrosis and Tyr416-Src phosphorylation in TGF-β-treated human renal proximal tubule epithelial (HK2) cells. Moreover, GW4064 treatment in HK2 cells increased Ser127 phosphorylation, cytosolic accumulation of YAP, and interaction of the hippo core kinases (Ste20-like kinase 1, large tumor suppressor kinase 1, and salvador homolog 1). Inhibition of Src using PP2 (Src kinase inhibitor) prevented renal fibrosis and increased Ser127 phosphorylation and cytosolic accumulation of YAP. The expression of fibrosis markers, inflammatory genes, and YAP target genes was increased in the kidneys of FXR knockout mice compared with those of wild-type mice. In addition, GW4064 or WAY-362450 (turofexorate isopropyl) treatment protected against unilateral ureteral obstruction-induced renal fibrosis. Collectively, our data support the novel conclusion that Src-mediated crosstalk between FXR and YAP protects against renal fibrosis, making this pathway a possible therapeutic target for chronic kidney disease.-Kim, D.-H., Choi, H.-I., Park, J. S., Kim, C. S., Bae, E. H., Ma, S. K., Kim, S. W. Src-mediated crosstalk between FXR and YAP protects against renal fibrosis.

SUN-141 MOLECULAR PROFILING OF APICAL VERSUS BASOLATERAL EXOSOMES DERVIED FROM HUMAN PROXIMAL TUBULE EPITHELIAL CELLS (PTEC) UNDER NORMAL AND DISEASED CONDITIONS

Proximal tubule epithelial cells (PTEC) are key players in kidney diseases. However, the complex signalling mechanism/s by which polarised PTEC mediate disease progression require investigation. Exosomes are extracellular vesicles with roles in cell-cell communication courtesy of their molecular cargo. Here we examine the microRNA (miRNA) content of exosomes secreted from apical versus basolateral PTEC surfaces under diseased conditions.

SAT-303 A NOVEL DPP4/CD32b/NF-kB CIRCUIT IS ESSENTIAL FOR C-REACTIVE PROTEIN-DRIVEN TYPE-2 DIABETIC NEPHROPATHY

Diabetic nephropathy (DN) is one of the major causes of end-stage renal disease (ESRD), but treatment remains ineffective due to its unclear pathogenesis. The pathogenic role of a risk factor C-reactive protein (CRP) has been increasingly recognized in type-2 DN (T2DN). Recently, a number of studies showed that plasma CRP level can be significantly reduced in diabetic patients by dipeptidyl peptidase-4 (DPP4) inhibitor treatment. A better understanding the underlying mechanism would uncover novel therapeutic targets for ESRD. Expression pattern of renal DPP4 were examined in the human CRP transgenic db/db (CRP-tg db/db), db/db and non-diabetic db/m mice during the development of T2DN. The renoprotective effect of DPP4 inhibition on CPR-driven T2DN was evaluated by treating CRP-tg db/db mice with DPP4 inhibitor Linagliptin. The underlying mechanism was elucidated in the Linagliptin-treated kidneys in vivo and cultured human proximal tubule epithelial HK-2 cells in vitro by IF, Co-IP and ChIP assays. Interestingly, we found that DPP4 expression was increased in the diabetic injured kidney of db/db mice, which further enhanced in the transgenic CRP-tg db/db and associated with a more advanced kidney injury compared with both the db/db and db/m mice. A 12-week-treatment with DPP4 inhibitor Linagliptin significantly ameliorated the renal function loss, inflammation (MCP-1, IL-1beta, TNF-alpha) and fibrosis (Col-I, Col-IV, alpha-SMA) in the CRP-tg db/db mice at 24-week-old. Surprisingly, mechanistic study revealed that CRP not only triggered the dimerization of DPP4 with CD32b in the plasma membrane, but also increased the physical binding of NF-kB on the DPP4 promoter region for transcriptional regulation in human proximal tubule epithelial HK-2 cells in vitro. Unexpectedly, targeting of either DPP4, CD32b or NF-kB with specific inhibitor/blocker can markedly suppressed all these three proteins in the CRP-stimulated HK-2 cells in vitro; suggesting the existence of a novel DPP4/CD32b/NF-kB signaling circuit under CRP-rich condition. More importantly, we demostrated that Linagliptin was capable of shutting down the whole circuit; therefore effectively inhibiting the progression of T2DN in CRP-tg db/db mice in vivo. Here, we discovered a novel DPP4/CD32b/NF-kB signaling circuit and identified its importance in the CRP-driven diabetic kidney injury. Thus, DPP4 may represent as a potential therapeutic target for T2DN-derived ESRD.

Abstract 1901: Collagen remodeling in clear cell renal cell carcinoma: Influence of composition on growth and metabolism

Abstract The extracellular matrix (ECM) plays an important role in maintaining the structure and function of the kidney. During aggressive fibrotic disease of the kidney, such as chronic kidney disease (CKD), the kidney extracellular matrix is rapidly modified and degraded. Reports have shown that changes in the ECM composition and architecture can have profound influence on kidney function and recovery. The influence of ECM remodeling on disease progression and kidney function in Renal Cell Carcinoma (RCC), is still not well understood. By co-isolating primary cancer cell lines and the extracellular protein fraction from resected ccRCC tumors, we have determined that changes in collagen isoform composition are drastically altered compared to healthy normal kidney via Mass Spectrometry SWATH analysis (NvT COL1 p= 5.2x10-5; COLIII p=0.03; COLVI; COLIV p=0.004). RNA-seq analysis of TCGA data shows that altered collagen composition compared to normal correlates with patient outcomes (5 year survival COL1 HIGH=55%, LOW=72%, p=3.96e-4; COLIII HIGH=60%, LOW=73%, p=6.67e-2; COLIV HIGH=67%, LOW=56%, p=1.06e-2; COLVI HIGH=44%, LOW=71%, p=4.09e-9). Trichrome staining and IHC analysis of COLI, COLIII, and COLIV alongside cryptic-collagen epitopes XL313 and Hu177, suggests that subsets of tumor cell populations degrade surrounding collagen to drastically alter the ECM architecture. H&E scoring of regions of high XL313/Hu177 staining shows increased grade compared to low/no-staining. Isolated primary ccRCC cancer lines were analyzed for differential adherence to collagen isoforms, showing preferential binding preferences. In parallel, isolated normal kidney epithelia and renal proximal tubule epithelial cells (RPTECs) adherence preference was also investigated. Freshly dissociated tumor tissue cultured on a novel ECM cocktail coating in 3D scaffolds show changes in cell viability, growth, and purity compared to standard tissue culture conditions. In this study, we conclude that collagen composition analysis through IHC and Mass Spectrometry can be indicative of patient tumor aggressiveness. Preferential binding to collagen isoforms by tumor cells and the lack of adherence by normal kidney epithelia indicates a novel mechanism for tumor cells to promote local invasion. Additionally, we have generated a novel method by which to isolate human ccRCC cell lines using a collagen cocktail that will be useful for basic research and disease modeling. Note: This abstract was not presented at the meeting. Citation Format: Kyle H. Bond, Anna Deck, Jennifer Caron, Peter Brooks, Calvin Vary, Sunder Sims-Lucas, Leif Oxburgh. Collagen remodeling in clear cell renal cell carcinoma: Influence of composition on growth and metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1901.

MALAT1 accelerates the development and progression of renal cell carcinoma by decreasing the expression of miR-203 and promoting the expression of BIRC5.

ObjectiveWe aimed to investigate the roles of the lncRNA MALAT1 in renal cell carcinoma (RCC) progression.MethodsqRT‐PCR was used for the assessment of BIRC5, miRNA‐203 and MALAT1 expression. Furthermore, the targeted relationships between miR‐203 and BIRC5, as well as MALAT1 and miR‐203, were predicted by the miRanda/starBase database and verified by dual‐luciferase reporter gene assay. The effects of MALAT1, miRNA‐203 and BIRC5 on cell proliferation, cell cycle, cell apoptosis, cell invasion and cell migration were studied by using CCK‐8, flow cytometry, transwell and wound healing assays, respectively. In addition, the effects of MALAT1 on RCC tumorigenesis were evaluated in vivo by nude mouse tumorigenesis.ResultsThe expression levels of BIRC5 and MALAT1 were higher in RCC tissues and cell lines than in adjacent normal tissues and a normal renal cortex proximal tubule epithelial cell line. In contrast, the expression of miRNA‐203 in RCC tissues and cell lines was higher than that in adjacent normal tissues and a normal renal cortex proximal tubule epithelial cell line. BIRC5 and MALAT1 promoted cell proliferation yet decreased the percentage of RCC cells at G0/G1 phase.ConclusionsOur study demonstrated that MALAT1 functions as a miR‐203 decoy to increase BIRC5 expression in RCC.

520-P: Hyperglycemia-Induced Mitochondrial Dysfunction in Diabetic Kidney Disease May Be Mediated by Crabtree Effect in Kidney Proximal Tubule Epithelial Cells

Background: Recent studies indicate mitochondrial dysfunction is a dominant pathway in diabetic kidney disease (DKD), however underlying mechanisms are unclear. Here we examined if Crabtree effect by glucose is a cause of mitochondrial dysfunction in kidney. Methods: Lactate was measured by by YSI biochemistry analyzer. TCA cycle metabolites were measured by gas chromatography mass spectroscopy in urine samples of type 1 and type 2 DKD mouse models. Changes in oxygen consumption (OCR) and extracellular acidification i(ECAR) were analyzed on a Seahorse extracellular flux analyzer. Immunohistochemical analysis was performed on FFPE-embedded mouse kidney sections. Western blot analysis was performed on kidney cortical sections and HK2 cells. Results: Significantly increased urine and plasma lactate along with a decrease in several TCA cycle metabolites were observed in both type 1 and type 2 diabetic mice. Mitochondrial functional analysis in different kidney cells showed a profound respiratory inhibition and stimulation of lactate by glucose in proximal tubules, but not in mesangial cells and podocytes. Pyruvate at above physiological concentration reversed the glucose-induced inhibition of OCR. Western blotting of kidney cortical tissue sections showed a significant increase in phospho PDH in diabetic kidney tissues and in HK2 cells treated with high glucose. In normal conditions, phospho PDH was predominantly on distal tubules, however in diabetic conditions a significant increase in phospho PDH was noted in proximal tubule cells. Dichloroacetate, an inhibitor of PDK completely reversed the glucose induced respiratory inhibition in proximal tubule epithelial cells. Conclusions: Mitochondrial dysfunction in proximal tubule epithelial cells may potentially be due to high-glucose induced PDH phosphorylation resulting in limited pyruvate oxidation into mitochondria and enhanced lactate production. Disclosure M. Darshi: None. J.J. Kim: None. V. Drel: None. A.N. Murphy: Advisory Panel; Self; Cytokinetics Inc. Consultant; Self; Agilent Technologies. Funding JDRF; U.S. Department of Veterans Affairs

511-P: The Renoprotective Effect of Evogliptin, a Dipeptidyl Peptidase-4 Inhibitor, in a Mouse Model of Renal Fibrosis

Background: Renal tubulointerstitial fibrosis is a major pathologic finding of nearly all cause of end-stage renal disease. Dipeptidyl peptidase-4 (DPP-4) inhibitors are widely used in the treatment of patients with type 2 diabetes and have proven protective effects on diabetic kidney disease. This study aimed to investigate possible beneficial effects of evogliptin on renal fibrosis in mice. Methods: We examined the effects of evogliptin on renal tubulointerstitial fibrosis following unilateral ureteral obstruction (UUO) in mice. We further defined the role of evogliptin on transforming growth factor-β (TGF-β)/Smad signaling pathways in renal tubulointerstitial fibrosis through in vivo and in vitro study. Results: Through hematoxylin/eosin and sirius red staining, we observed that evogliptin effectively ameliorates UUO-induced renal atrophy and fibrosis. Immunohistochemistry in conjunction with western blot analysis revealed that evogliptin treatment inhibited UUO-induced up-regulation of phosphorylated Smad3, α-smooth muscle actin, plasminogen activator inhibitor 1, fibronenctin, and type 1 collagen. In vitro experiments with proximal tubule epithelial cell suggested that the effects of evogliptin on UUO-induced renal fibrosis are mediated by inhibition of the TGF-β/Smad signaling pathway. Conclusion: The present study demonstrates that evogliptin has a protective effect on UUO-induced renal tubulointerstitial fibrosis, suggesting that its clinical applications could extend to the treatment of nondiabetic origin renal disease. Disclosure N. Kim: None. I. Lee: Board Member; Self; NovMetapharm Co, Inc. S. Park: None. M. Kim: None. J. Kim: None. Y. Choi: None. K. Park: None. M. Kim: None. Funding National Research Foundation of Korea

518-P: Involvement of Low-Density Lipoprotein Receptor-Related Protein 5 in Chronic Kidney Diseases via Modulation of TGF-β-Smad2/3 Signaling

Aims: Low-density lipoprotein receptor-related protein 5 (LRP5) is known as a co-receptor of canonical Wnt signaling. This study aimed to investigate LRP5’s role in chronic kidney diseases including diabetic and obstructive nephropathies, and its new role in regulating TGF-β-Smad2/3 signaling in renal fibrogenesis. Methods: The expression of LRP5 was examined in the kidneys of Akita mice, db/db mice and mice with obstructive nephropathy. The regulation of TGF-β signaling by LRP5 was determined in both obstructed kidneys and in human proximal tubule epithelial cells exposed to TGF-β1. Interaction of LRP5 with TGF-β receptors was evaluated by Co-IP assays, immunostainings and cell fractionation. Results: We found that LRP5 levels were substantially up-regulated in the renal tubules of diabetic mice and mice with obstructive nephropathy. In the obstructed kidneys, Lrp5 knockout significantly ameliorated tubulointerstitial fibrosis and attenuated the increases of TGF-β1 and TGF-β receptors (TβRI and TβRII), without affecting canonical Wnt signaling. Utilizing SBE-Luc reporter mice and immunostaining assays, LRP5 depletion was demonstrated to attenuate the activation and nuclear translocation of Smad2/3, whereas LRP5 overexpression enhanced TGF-β-Smad2/3 signaling in tubule epithelial cells. Obstruction-induced LRP5 and TβRI were co-localized in the injured tubules, and furthermore, LRP5 was co-precipitated with TβRI and TβRII. The extracellular domain of LRP5 was essential for its association with TGF-β receptors and profibrotic activity. In addition, LRP5 stabilized TGF-β receptors, increased their membrane presentation in the absence of TGF-β1, and promoted the TGF-β1-induced formation of TβRI/TβRII heterodimers and internalization of these receptors. Conclusion: These findings reveal a novel profibrotic activity of LRP5 by interacting with TGF-β receptors, independent of canonical Wnt signaling. Disclosure X. He: None. R. Cheng: None. C. Huang: None. Y. Chen: None. J. Ma: None.

Blocking pannexin-1 alleviates cisplatin-induced acute kidney injury in mice by reducing renal inflammatory cell infiltration

To investigate the effect of blocking pannexin-1 against acute kidney injury induced by cisplatin. Twenty-six male C57BL/6 mice aged 6-8 weeks were randomly divided into control group, cisplatin model (Cis) group and cisplatin + carbenoxolone treatment group (Cis + CBX). In Cis group and Cis + CBX group, the mice were injected intraperitoneally with 20 mg/kg of cisplatin and with CBX (20 mg/kg) at 30 min before and 24 and 48 h after cisplatin inhjection, respectively. All the mice were sacrificed at 72 h after cisplatin injection, and plasma and kidney samples were collected for testing mRNA and protein expression levels of pannexin-1 in the renal tissue using RT-qPCR and Western blotting and for detecting plasma creatinine and BUN levels; the pathological changes in the renal tissues were observed using Periodic Acid-Schiff staining. The expression of kidney injury molecule 1 (KIM-1) was examined using immunohistochemistry and the mRNA expressions of KIM-1 and neutrophil gelatinase- related lipid transport protein (NGAL) were detected by RT-qPCR to evaluate the injuries of the renal tubules. The infiltration of F4/80-positive macrophages and CD4-positive T cells were observed by immunofluorescence. In the in vitro experiment, human proximal tubule epithelial cell line HK-2 was stimulated with 50 μmol/L cisplatin to establish a cell model of acute kidney injury, and the mRNA and protein expressions of pannexin-1 were detected by RT-qPCR and Western blotting at 4, 6, 12, 18 and 24 h after the stimulation. Compared with the control mice, the cisplatin-treated mice showed significantly up-regulated protein levels (P < 0.05) and mRNA levels (P < 0.005) of pannexin-1 in the kidney tissue. Cisplatin stimulation also caused significant increases in the protein levels (P < 0.005) and mRNA levels (P < 0.005) of pannexin-1 in cultured HK-2 cells. Compared with cisplatin-treated mice, the mice treated with both cisplatin and the pannexin-1 inhibitor CBX showed obviously lessened kidney pathologies and milder renal tubular injuries with significantly reduced plasma BUN and Scr levels (P < 0.01), expressions of KIM-1 and NGAL in the kidney (P < 0.05), and infiltration of F4/80-positive macrophages (P < 0.01) and CD4- positive T cells (P < 0.05) in the kidney tissues. In cisplatin induced acute kidney injury mice model, Pannexin-1 expression is up-regulated in the kidneys tissue, and blocking pannexin-1 alleviates the acute kidney injury via reducing renal inflammatory cell infiltration.

Silica coated iron oxide nanoparticles-induced cytotoxicity, genotoxicity and its underlying mechanism in human HK-2 renal proximal tubule epithelial cells

Iron oxide nanoparticles (IONPs) have a great potential with regard to cell labelling, cell tracking, cell separation, magnetic resonance imaging, magnetic hyperthermia, targeted drug and gene delivery. However, a growing body of research has raised concerns about the possible unwanted adverse cytotoxic effects of IONPs. In the present study, the in vitro cellular uptake, antiproliferative activity, cytotoxicity, genotoxicity, prooxidant, microtubule-disrupting and apoptosis-inducing effect of Fe3O4@SiO2 and passivated Fe3O4@SiO2-NH2 nanoparticles on human renal proximal tubule epithelial cells (HK-2) have been studied. Both investigated silica coated IONPs were found to have cell growth-inhibitory activity in a time- and dose-dependent manner. Determination of cell cycle phase distribution by flow cytometry demonstrated a G1 and G2/M phase accumulation of HK-2 cells. A tetrazolium salt cytotoxicity assay at 24 h following treatment demonstrated that cell viability was reduced in a dose-dependent manner. Microscopy observations showed that both Fe3O4@SiO2 and Fe3O4@SiO2-NH2 nanoparticles accumulated in cells and appeared to have microtubule-disrupting activity. Our study also revealed that short term 1 h exposure to 25 and 100 μg/mL of silica coated IONPs causes genotoxicity. Compared with vehicle control cells, a significantly higher amount of γH2AX foci correlating with an increase in DNA double-strand breaks was observed in Fe3O4@SiO2 and Fe3O4@SiO2-NH2-treated and immunestained HK-2 cells. The investigated nanoparticles did not trigger significant ROS generation and apoptosis-mediated cell death. In conclusion, these findings provide new insights into the cytotoxicity of silica coated IONPs that may support their further safer use.

Functional transepithelial transport measurements to detect nephrotoxicity in vitro using the RPTEC/TERT1 cell line.

The kidney is a frequent target for organ-specific toxicity as a result of its primary function in controlling body fluids, for example, via resorption of amino acids, peptides, nutrients, ions, xenobiotics and water from the primary urine as well as excretion of metabolic waste products and hydrophilic and amphiphilic xenobiotics. Compounds exhibiting dose-limiting nephrotoxicity include drugs from highly diverse classes and chemical structures, e.g., antibiotics (gentamicin), chemotherapeutics (cisplatin), immunosuppressants (cyclosporine A and tacrolimus) or bisphosphonates (zoledronate). All of these compounds elicit nephrotoxicity primarily by injuring renal proximal tubule epithelial cells (RPTECs). However, prediction of a compound's nephrotoxic potential in humans to support early unmasking of risk-bearing drug candidates remains an unmet challenge, mainly due to the complex kidney anatomy as well as pronounced inter- and intraspecies differences and lack of relevant and validated human in vitro models. Accordingly, we used the recently established human RPTEC/TERT1 cell line to carry out toxicity studies with a focus on impairment of functional characteristics, i.e., transepithelial electrical resistance (TEER), vectorial transport of water, cations, and anions. Results were compared to real-time cytotoxicity assessments using cellular impedance (xCELLigence assay) and the routine cell viability readout (MTT). As expected, most toxins caused exposure time- and concentration-dependent cytotoxicity. However, for some compounds (cyclosporine A and tacrolimus), transport processes were strongly impaired in absence of a concomitant decrease in cell viability. In conclusion, these data demonstrate that functional parameters are important, highly sensitive and meaningful additional readouts for nephrotoxicity assessment in human renal proximal tubule epithelial cells.

FKN Facilitates HK-2 Cell EMT and Tubulointerstitial Lesions via the Wnt/β-Catenin Pathway in a Murine Model of Lupus Nephritis.

Fractalkine (FKN), also known as chemokine (C-X3-C motif) ligand 1, constitutes an intriguing chemokine with a documented role in the development of numerous inflammatory diseases including autoimmune disease. Specifically, it has been reported that FKN is involved in the disease progression of lupus nephritis (LN). The epithelial-mesenchymal transition (EMT) plays a significant role in the formation of tubulointerstitial lesions (TIL), which are increasingly recognized as a hallmark of tissue fibrogenesis after injury. However, the correlation between FKN and EMT or TIL in LN has not been determined. To investigate the potential role of FKN in EMT and TIL, MRL lymphoproliferation (MRL/lpr) strain mice were treated with an anti-FKN antibody, recombinant-FKN chemokine domain, or isotype antibody. Our results revealed that treatment with the anti-FKN antibody improved EMT, TIL, and renal function in MRL/lpr mice, along with inhibiting activation of the Wnt/β-catenin signaling pathway. In contrast, administration of the recombinant-FKN chemokine domain had the opposite effect. Furthermore, to further explore the roles of FKN in EMT, we assessed the levels of EMT markers in FKN-depleted or overexpressing human proximal tubule epithelial HK-2 cells. Our results provide the first evidence that the E-cadherin level was upregulated, whereas α-SMA and vimentin expression was downregulated in FKN-depleted HK-2 cells. In contrast, overexpression of FKN in HK-2 cells enhanced EMT. In addition, inhibition of the Wnt/β-catenin pathway by XAV939 negated the effect of FKN overexpression, whereas activation of the Wnt/β-catenin pathway by Ang II impaired the effect of the FKN knockout on EMT in HK-2 cells. Together, our data indicate that FKN plays essential roles in the EMT progression and development of TIL in MRL/lpr mice, most likely through activation of the Wnt/β-catenin signaling pathway.

No Inhibition of MATE1/2K-Mediated Renal Creatinine Secretion Predicted With Ritonavir or Cobicistat

Cobicistat has been reported to increase serum creatinine clinically without affecting glomerular filtration. This was ascribed to transient inhibition of MATE1-mediated renal creatinine secretion. Interestingly, a structurally similar drug, ritonavir, has not been associated with serum creatinine increases at the pharmacoenhancer dose. The present study was aimed to investigate the translation of in vitro MATE1/2K inhibition to clinical creatinine increase (cobicistat) and lack of it (ritonavir) considering their intracellular concentrations in renal proximal tubules. Uptake studies showed ritonavir and cobicistat are unlikely substrates for OCT2. The steady-state unbound concentration in the cytosol of human renal proximal tubule epithelial cells was comparable with the extracellular unbound concentration, suggesting that the entry of these compounds is predominantly mediated by passive diffusion. Ritonavir and cobicistat are MATE1 and MATE2K inhibitors with IC50 values of 3.1 and 90 μM (ritonavir), and 4.4 and 3.2 μM (cobicistat), respectively. However, the unbound cytosolic concentrations (Cu,cytosol) of ritonavir and cobicistat in human renal proximal tubule epithelial cells, 0.065 and 0.10 μM, respectively, after incubation with the clinical maximum total plasma concentrations at pharmacoenhancer doses does not support inhibition in vivo; Cu,cytosol >30 fold lower than IC50s. These results demonstrate that MATE1/2K inhibition is unlikely the mechanism of the clinical creatinine elevations with cobicistat.

γ‐secretase inhibitor DAPT mitigates cisplatin‐induced acute kidney injury by suppressing Notch1 signaling

Organ toxicity, including kidney injury, limits the use of cisplatin for the treatment of multiple human cancers. Hence, interventions to alleviate cisplatin‐induced nephropathy are of benefit to cancer patients. Recent studies have demonstrated that pharmacological inhibition of the Notch signaling pathway enhances cisplatin efficacy against several cancer cells. However, whether augmentation of the anti‐cancer effect of cisplatin by Notch inhibition comes at the cost of increased kidney injury is unclear. We show here that treatment of mice with cisplatin resulted in a significant increase in Notch ligand Delta‐like 1 (Dll1) and Notch1 intracellular domain (N1ICD) protein expression levels in the kidneys. N‐ [N‐ (3,5‐difluorophenacetyl) ‐L‐alanyl]‐S‐phenylglycine t‐butyl ester (DAPT), a γ‐secretase inhibitor reversed cisplatin‐induced increase in renal N1ICD expression and plasma or urinary levels of predictive biomarkers of acute kidney injury (AKI). DAPT also mitigated cisplatin‐induced tubular injury and reduction in glomerular filtration rate. Real‐time multiphoton microscopy revealed marked necrosis and peritubular vascular dysfunction in the kidneys of cisplatin‐treated mice which were abrogated by DAPT. Cisplatin‐induced Dll1/Notch1 signaling was recapitulated in a human proximal tubule epithelial cell line (HK‐2). siRNA‐mediated Dll1 knockdown and DAPT attenuated cisplatin‐induced Notch1 cleavage and cytotoxicity in HK‐2 cells. These data suggest that Dll1‐mediated Notch1 signaling contributes to cisplatin‐induced AKI. Hence, the Notch signaling pathway could be a potential therapeutic target to alleviate renal complications associated with cisplatin chemotherapy.Support or Funding InformationWe acknowledge a fellowship (to A. Adebiyi) from the O'Brien Center for Advanced Renal Microscopic Analysis (NIH‐NIDDKP30DK079312).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hydrogen Gas Alleviates Chronic Intermittent Hypoxia-Induced Renal Injury through Reducing Iron Overload.

Iron-induced oxidative stress has been found to be a central player in the pathogenesis of kidney injury. Recent studies have indicated H2 can be used as a novel antioxidant to protect cells. The present study was designed to investigate the protective effects of H2 against chronic intermittent hypoxia (CIH)-induced renal injury and its correlation mechanism involved in iron metabolism. We found that CIH-induced renal iron overloaded along with increased apoptosis and oxidative stress. Iron accumulates mainly occurred in the proximal tubule epithelial cells of rats as showed by Perl’s stain. Moreover, we found that CIH could promote renal transferrin receptor and divalent metal transporter-1 expression, inhibit ceruloplasmin expression. Renal injury, apoptosis and oxidative stress induced by CIH were strikingly attenuated in H2 treated rats. In conclusion, hydrogen may attenuate CIH-induced renal injury at least partially via inhibiting renal iron overload.

Renal control of disease tolerance to malaria

Malaria, the disease caused by Plasmodium spp. infection, remains a major global cause of morbidity and mortality. Host protection from malaria relies on immune-driven resistance mechanisms that kill Plasmodium However, these mechanisms are not sufficient per se to avoid the development of severe forms of disease. This is accomplished instead via the establishment of disease tolerance to malaria, a defense strategy that does not target Plasmodium directly. Here we demonstrate that the establishment of disease tolerance to malaria relies on a tissue damage-control mechanism that operates specifically in renal proximal tubule epithelial cells (RPTEC). This protective response relies on the induction of heme oxygenase-1 (HMOX1; HO-1) and ferritin H chain (FTH) via a mechanism that involves the transcription-factor nuclear-factor E2-related factor-2 (NRF2). As it accumulates in plasma and urine during the blood stage of Plasmodium infection, labile heme is detoxified in RPTEC by HO-1 and FTH, preventing the development of acute kidney injury, a clinical hallmark of severe malaria.

Toxicity and photosensitizing assessment of gelatin methacryloyl-based hydrogels photoinitiated with lithium phenyl-2,4,6-trimethylbenzoylphosphinate in human primary renal proximal tubule epithelial cells

Gelatin methacryloyl (GelMA) and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) photoinitiator are commonly used in combination to produce a photosensitive polymer but there are concerns that must be addressed: the presence of unreacted monomer is well known to be cytotoxic, and lithium salts are known to cause acute kidney injury. In this study, acellular 10% GelMA hydrogels cross-linked with different LAP concentrations and cross-linking illumination times were evaluated for their cytotoxicity, photosensitizing potential, and elastic moduli. Alamar Blue and CyQuant Direct Cell viability assays were performed on human primary renal proximal tubule epithelial cells (hRPTECs) exposed to extracts of each formulation. UV exposure during cross-linking was not found to affect extract cytotoxicity in either assay. LAP concentration did not affect extract cytotoxicity as determined by the Alamar Blue assay but reduced hRPTEC viability in the CyQuant Direct cell assay. Photocatalytic activity of formulation extracts toward NADH oxidation was used as a screening method for photosensitizing potential; longer UV exposure durations yielded extracts with less photocatalytic activity. Finally, elastic moduli determined using nanoindentation was found to plateau to approximately 20-25 kPa after exposure to 342 mJ/cm2 at 2.87 mW of UV-A exposure regardless of LAP concentration. LAP at concentrations commonly used in bioprinting (<0.5% w/w) was not found to be cytotoxic although the differences in cytotoxicity evaluation determined from the two viability assays imply cell membrane damage and should be investigated further. Complete cross-linking of all formulations decreased photocatalytic activity while maintaining predictable final elastic moduli.

The Effect of Overexpression of the Enhancer of Zeste Homolog 1 (EZH1) Gene on Aristolochic Acid-Induced Injury in HK-2 Human Kidney Proximal Tubule Cells In Vitro.

BackgroundAcute kidney injury (AKI) involves the renal tubular epithelium. The enhancer of zeste homolog 1 (EZH1) gene has a role in cell development and differentiation. This study aimed to investigate the effect of overexpression of the EZH1 gene on aristolochic acid-induced injury in HK-2 human kidney proximal tubule epithelial cells in vitro.Material/MethodsThe HK-2 cells were cultured and treated with aristolochic acid and the effects of aristolochic acid-injury were evaluated using a cell counting kit-8 (CCK-8) assay. Overexpression of EZH1 used gene plasmid transfection into HK-2 cells. The cell apoptosis rate and levels of intracellular reactive oxygen species (ROS) were measured using flow cytometry. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were performed to determine the expressions of inflammatory cytokines including interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α), apoptosis-related genes, and the downstream target genes of NF-κB signaling pathway, including NFKBIA, CXCL8, and cyclin D1.ResultsAristolochic acid inhibited HK-2 cell viability, induced cell apoptosis, increased the levels of ROS and inflammatory cytokines, including IL-1β, IL-6, TNF-α, and activated the NF-κB pathway. Overexpression the EZH1 gene inhibited HK-2 cell apoptosis, reduced ROS levels, and down-regulated the expressions of IL-1β, IL-6, TNF-α, Bax and Cyt C mRNA and protein, and increased the expressions of Bcl-2 and NFKBIA, CXCL8 and cyclin D1, indicating that overexpression of EZH1 suppressed NF-κB signaling in aristolochic acid-injured HK-2 cells.ConclusionsOverexpression of EZH1 reduced HK-2 cell injury induced by aristolochic acid in vitro by inhibition of NF-κB signaling.