Proximal Tubular Cell Line Research Articles

Sinomenine mitigates cisplatin-induced kidney injury by targeting multiple signaling pathways.

Sinomenine is a pharmacologically active alkaloid with antioxidant and anti-inflammatory properties. We aimed to investigate the mechanism of renoprotective activity of sinomenine in kidney injury induced by cisplatin (CP). Sinomenine (5mg/kg) was administered to mice orally in two doses, the second day after intraperitoneal application of CP (13mg/kg). Sinomenine ameliorated kidney injury and decreased serum levels of urea and creatinine and renal expression of NGAL and KIM-1. The increase in HO-1, 4-HNE, 3-NT and TNF-α renal expression was mitigated by sinomenine. Additionally, sinomenine reduced the expression of p21, Bax, Noxa, caspase-3 and -8 and PARP1 cleavage in mice kidneys as well as the number of TUNEL-positive cells. Sinomenine attenuated CP-induced activation of ERK1/2, Akt, FOXO3a, STAT3 and NF-κB and restored Sirtuin 6 expression. In the human proximal tubular cell line HK2, sinomenine 100μM reduced the toxic effect of CP 30μM. Our current findings suggest that sinomenine suppresses CP-induced oxidative stress, inflammation and apoptosis in mice kidneys by targeting multiple signaling pathways.

LncRNA ENSMUST00000171502 Induced by HIF-1α Ameliorates Ischemic Acute Kidney Injury via Targeting the miR-130b-3p/Mybl-1 Axis.

Background: Numerous studies have suggested that long non-coding RNA (lncRNA) affects the progression of ischemic acute kidney injury (IAKI). However, little information is currently available concerning the mechanisms of lncRNA171502 involved in IAKI. Methods: We applied an RT-qPCR assay for the expression of lncRNA171502 and miRNA-130b-3p, immunoblotting for the detection of Mybl-1-myeloblastosis oncogene-like 1 (Mybl-1) and cleaved caspase-3 (CC3) expression, and flow cytometry (FCM) for the evaluation of apoptosis. Result: Initially, lncRNA171502 was induced by HIF-1α in the mouse proximal tubular (BUMPT) cell line and C57BL/6J mice during ischemic injury. Secondly, ischemic injury-induced BUMPT cell apoptosis was markedly relieved following the overexpression of lncRNA171502. However, this effect was enhanced by the knockdown of lncRNA171502. Mechanistically, lncRNA171502 could sponge miRNA-130b-3p and would subsequently upregulate the expression of Mybl-1 to drive the apoptotic process. Lastly, the overexpression of lncRNA171502 alleviated the development of IAKI by targeting miRNA-130b-3p/Mybl-1 pathways. Conclusions: In summary, the HIF-1α/lncRNA171502/miRNA-130b-3p/Mybl-1 axis prevented the progression of IAKI and might serve as a potential therapeutic target.

Inhibition of sodium-glucose cotransporter 2 suppresses renal stone formation

Background and aimsNephrolithiasis is a common renal disease with no effective medication. Sodium-glucose cotransporter-2 (SGLT2) inhibitors, an anti-diabetic agent, have diuretic and anti-inflammatory properties and could prevent nephrolithiasis. Here, we investigated the potential of SGLT2 inhibition against nephrolithiasis using large-scale epidemiological data, animal models, and cell culture experiments. MethodsThis study included the data of diabetic patients (n = 1,538,198) available in the Japanese administrative database and divided them according to SGLT2 inhibitor prescription status. For animal experiments, renal calcium oxalate stones were induced by ethylene glycol in Sprague-Dawley rats, and phlorizin, an SGLT1/2 inhibitor, was used for the treatment. The effects of SGLT2-specific inhibition for renal stone formation were assessed in SGLT2-deficient mice and a human proximal tubular cell line, HK-2. ResultsNephrolithiasis prevalence in diabetic men was significantly lower in the SGLT2 inhibitor prescription group than in the non-SGLT2 inhibitor prescription group. Phlorizin attenuated renal stone formation and downregulated the kidney injury molecule 1 (Kim1) and osteopontin (Opn) expression in rats, with unchanged water intake and urine volume. It suppressed inflammation and macrophage marker expression, suggesting the role of the SGLT2 inhibitor in reducing inflammation. SGLT2-deficient mice were resistant to glyoxylic acid-induced calcium oxalate stone formation with reduced Opn expression and renal damages. High glucose-induced upregulation of OPN and CD44 and cell surface adhesion of calcium oxalate reduced upon SGLT2-silencing in HK-2 cells. ConclusionOverall, our findings identified that SGLT2 inhibition prevents renal stone formation and may be a promising therapeutic approach against nephrolithiasis.

Gallic acid ameliorates calcium oxalate crystal-induced renal injury via upregulation of Nrf2/HO-1 in the mouse model of stone formation

BackgroundHigh prevalence and reoccurrence rate of nephrolithiasis bring about serious socioeconomic and healthcare burden, necessitating the need of effective therapeutic agents. Previous study revealed that gallic acid (GAL) alters the nucleation pathway of calcium oxalate (CaOx). On the other hand, it appears protective role against oxidative injury. Whether GAL could protect against crystal-induced lesion in vivo, and its underlying mechanism is yet unsolved. PurposeThis study aims to investigate the protective effects of GAL on the crystal-induced renal injury and its underlying mechanism in the mouse model of stone formation induced by glyoxylic acid. Study design and methodsThe mouse model of stone formation was established via successive intraperitoneal injection of glyoxylate. Proximal tubular epithelial cell line HK-2 treated with calcium oxalate monohydrate (COM) was used as in vitro model. The protective role of GAL on nephrolithiasis was tested by determination of tubular injury, crystal deposition and adhesion, levels of inflammatory cytokines, macrophage infiltration and the redox status of kidney. In vitro, effect of GAL on the ROS level and oxidative tubular injury induced by COM were detected, as well as major antioxidant pathway Nrf2/HO-1. ResultsAdministration of GAL alleviates the renal deposition and adhesion of CaOx stone. Meanwhile, GAL ameliorates the inflammation and renal tubular injury. Level of intracellular ROS, osteopontin and CD44 are reduced, either in the mouse model of stone formation or in the COM-treated HK-2 cells after treatment of GAL. Mechanistically, GAL activates Nrf2/HO-1 pathway in HK-2 cells. Silencing Nrf2 abrogates the protective effect of GAL on the oxidative injury and adhesion of COM in HK-2 cells. ConclusionTaken together, our study demonstrates the protective effect of GAL on the deposition of kidney stone and consequent tubular injury. Induction of the antioxidant pathway Nrf2/HO-1 was found to decrease the level of ROS and oxidative injury, thus implying that GAL could be a potential therapeutic agent for the treatment of nephrolithiasis.

Transcriptomic signatures responding to PKM2 activator TEPP-46 in the hyperglycemic human renal proximal epithelial tubular cells.

Pyruvate kinase M2 (PKM2), as the terminal and last rate-limiting enzyme of the glycolytic pathway, is an ideal enzyme for regulating metabolic phenotype. PKM2 tetramer activation has shown a protective role against diabetic kidney disease (DKD). However, the molecular mechanisms involved in diabetic tubular have not been investigated so far. In this study, we performed transcriptome gene expression profiling in human renal proximal tubular epithelial cell line (HK-2 cells) treated with 25 mM high D-glucose (HG) for 7 days before the addition of 10 μM TEPP-46, an activator of PKM2 tetramerization, for a further 1 day in the presence of HG. Afterwards, we analyzed the differentially expressed (DE) genes and investigated gene relationships based on weighted gene co-expression network analysis. The results showed that 2,902 DE genes were identified (adjusted P-value ≤ 0.05), where 2,509 DE genes (86.46%) were co-expressed in the key module. Four extremely downregulated DE genes (HSPA8, HSPA2, HSPA1B, and ARRB1) and three extremely upregulated DE genes (GADD45A, IGFBP3, and SIAH1) enriched in the downregulated endocytosis (hsa04144) and upregulated p53 signaling pathway (hsa04115), respectively, were validated by qRT-PCR experiments. The qRT-PCR results showed that the relative expression levels of HSPA8 [adjusted P-value = 4.45 × 10-34 and log2(FC) = -1.12], HSPA2 [adjusted P-value = 6.09 × 10-14 and log2(FC) = -1.27], HSPA1B [adjusted P-value = 1.14 × 10-11 and log2(FC) = -1.02], and ARRB1 [adjusted P-value = 2.60 × 10-5 and log2(FC) = -1.13] were significantly different (P-value < 0.05) from the case group to the control group. Furthermore, the interactions and predicted microRNAs of the key genes (HSPA8, HSPA2, HSPA1B, and ARRB1) were visualized in networks. This study identified the key candidate transcriptomic biomarkers and biological pathways in hyperglycemic HK-2 cells responding to the PKM2 activator TEPP-46 that can highlight a possibility of PKM2 tetramerization reshaping the interplay among endocytic trafficking through the versatile networks of Hsp70s and rewiring the crosstalk between EGFR signal transduction circuits and metabolic stress to promote resilience, which will be valuable for further research on PKM2 in DKD.

β-Hydroxybutyrate against Cisplatin-Induced acute kidney injury via inhibiting NLRP3 inflammasome and oxidative stress

Cisplatin, as a commonly used anticancer drug, can easily lead to acute kidney injury (AKI), and has received more and more attention in clinical practice. β-hydroxybutyric acid (BHB) is a metabolite in the body and acts as an inhibitor of oxidative stress and NLRP3 inflammasome, reducing inflammatory responses and apoptosis. However, the role of BHB in cisplatin-induced AKI is currently not fully elucidated. In this study, C57BL/6 male mice were randomly divided into normal control group, cisplatin-induced AKI group and AKI with BHB treatment group. Compared to the control, cisplatin-treated mice exhibited high level of serum creatinine, blood urea nitrogen and severe tubular injury, which accompanied with significantly increased expression level of NLRP3, IL-1β, IL-18, BAX, cleaved-caspase 3, as well as aggravated oxidative stress and renal tubular cell apoptosis. However, these changes were significantly improved in that of BHB treatment. In vitro, our study showed that the expression of cleaved-caspase3, IL-1β and IL-18 were significantly increased in human proximal tubular epithelial cell line (HK-2) treated with cisplatin compared with the control group, while decreased in cells treated with BHB. Furthermore, a significantly increased expression of cGAS and STING in HK-2 cells treated with cisplatin were found, whereas notably decreased in cells treated with BHB. This data indicates that BHB protects against cisplatin-induced AKI and renal tubular damage mediated by NLRP3 inflammasome and cGAS-STING pathway.

Cadmium Toxicity Is Regulated by Peroxisome Proliferator-Activated Receptor δ in Human Proximal Tubular Cells.

Cadmium (Cd) is a toxic heavy metal that is widely present in the environment. Renal proximal tubule disorder is the main symptom of Cd chronic poisoning. Our previous study demonstrated that Cd inhibits the total activities of peroxisome proliferator-activated receptor (PPAR) transcription factors in human and rat proximal tubular cells. In this study, we investigated the involvement of PPAR in Cd renal toxicity using the HK-2 human proximal tubular cell line. Among PPAR isoform genes, only PPARD knockdown significantly showed resistance to Cd toxicity in HK-2 cells. The transcriptional activity of PPARδ was decreased not only by PPARD knockdown but also by Cd treatment. DNA microarray analysis showed that PPARD knockdown changed the expression of apoptosis-related genes in HK-2 cells. PPARD knockdown decreased apoptosis signals and caspase-3 activity induced by Cd treatment. PPARD knockdown did not affect the intracellular Cd level after Cd treatment. These results suggest that PPARδ plays a critical role in the modification of susceptibility to Cd renal toxicity and that the apoptosis pathway may be involved in PPARδ-related Cd toxicity.

Anti-EMT and anti-fibrosis effects of protocatechuic aldehyde in renal proximal tubular cells and the unilateral ureteral obstruction animal model

Context Protocatechuic aldehyde (PCA) is a natural product that has various benefits for fibrosis. Objective This study evaluated the effects of PCA on renal fibrosis. Materials and methods Epithelial–mesenchymal transition (EMT) was induced by 20 ng/mL transforming growth factor-β1 (TGF-β1), followed by treatment with 1 and 5 μM PCA, in the rat renal proximal tubular cell line NRK-52E. Cell viability, protein expression, and scratch wound-healing assays were conducted. Sprague–Dawley (SD) rats underwent unilateral ureteral obstruction (UUO) surgery for renal fibrosis indication and were treated with 50 and 100 mg/kg PCA for 14 days. Results The IC50 of PCA was appropriately 13.75 ± 1.91 μM in NRK-52E cells, and no significant difference at concentrations less than 5 μM. PCA ameliorated TGF-β1-induced EMT, such as enhanced E-cadherin and decreased vimentin. Fibrotic markers collagen IV and α-smooth muscle actin (α-SMA) increased in TGF-β1-induced NRK-52E. Moreover, PCA reduced TGF-β1-induced migration in the wound-healing assay. Analysis of rat kidneys indicated that PCA reduced UUO-induced hydronephrosis (control: 15.11 ± 1.00%; UUO: 39.89 ± 1.91%; UUO + PCA50: 18.37 ± 1.61%; UUO + PCA100: 17.67 ± 1.39%). Protein level demonstrated that PCA not only decreased vimentin expression and enhanced E-cadherin expression, but inhibited UUO-induced collagen IV and α-SMA upregulation, indicating that it could mitigate EMT in a rat model of UUO-induced renal fibrosis. Discussion and conclusions This study suggested that PCA decreases TGF-β1-induced fibrosis and EMT in vitro and in vivo. These findings demonstrate pharmacological effects of PCA and might be a potential strategy for the prevention of organ fibrosis in clinics.

Inactivation of fatty acid amide hydrolase protects against ischemic reperfusion injury-induced renal fibrogenesis

Although cannabinoid receptors (CB) are recognized as targets for renal fibrosis, the roles of endogenous cannabinoid anandamide (AEA) and its primary hydrolytic enzyme, fatty acid amide hydrolase (FAAH), in renal fibrogenesis remain unclear. The present study used a mouse model of post-ischemia-reperfusion renal injury (PIR) to test the hypothesis that FAAH participates in the renal fibrogenesis. Our results demonstrated that PIR showed upregulated expression of FAAH in renal proximal tubules, accompanied with decreased AEA levels in kidneys. Faah knockout mice recovered the reduced AEA levels and ameliorated PIR-triggered increases in blood urea nitrogen, plasma creatinine as well as renal profibrogenic markers and injuries. Correspondingly, a selective FAAH inhibitor, PF-04457845, inhibited the transforming growth factor-beta 1 (TGF-β1)–induced profibrogenic markers in human proximal tubular cell line (HK-2 cells) and mouse primary cultured tubular cells. Knockdown of FAAH by siRNA in HK-2 cells had similar effects as PF-04457845. Tubular cells isolated from Faah−/− mice further validated the protection against TGF-β1–induced damages. The CB 1 or CB2 receptor antagonist and exogenous FAAH metabolite arachidonic acid failed to reverse the protective effects of FAAH inactivation in HK-2 cells. However, a substrate-selective inhibitor of AEA-cyclooxygenase-2 (COX-2) pathway significantly suppressed the anti-profibrogenic actions of FAAH inhibition. Further, the AEA-COX-2 metabolite, prostamide E2 exerted anti-fibrogenesis effect. These findings suggest that FAAH activation and the consequent reduction of AEA contribute to the renal fibrogenesis, and that FAAH inhibition protects against fibrogenesis in renal cells independently of CB receptors via the AEA-COX-2 pathway by the recovery of reduced AEA.

ZIP14 is involved in iron deposition and triggers ferroptosis in diabetic nephropathy.

Ferroptosis is caused by lipid peroxidation and iron accumulation and can cause cell death. Abnormally expressed iron transporters are involved in ferroptosis in a variety of diseases. ZRT/IRT-like protein 14 (ZIP14) is a transport protein that can mediate cellular uptake of iron, zinc, and manganese. Herein, we have tested the hypothesis that the divalent metal transporter ZIP14 is involved in the initiation of ferroptosis in diabetic nephropathy (DN). DN was induced in 8-week-old male rats by streptozotocin before analysis of the degree of renal tubular injury. In addition, an in vitro model of DN in human kidney proximal tubular cell line was used. We showed that ZIP14 was up-regulated and ferrous iron (Fe2+) levels increased both in vivo and in vitro. Expression of glutathioneperoxidase4 and the level of glutathione were reduced, whereas that of malondialdehyde (MDA) increased. Ferrostatin-1 (Fer-1) treatment reduced the expression of ZIP14 and the levels of Fe2+ and MDA, which is consistent with ferroptosis. Fer-1 improved kidney function in DN rats. This was characterized by urine levels of protein-to-creatinine ratio, α1-microglobulin, and N-acetyl-β-D-glucosaminidase. Our study demonstrates a novel role for ZIP14 in diabetic kidney injury mediated by ferroptosis, and suggests a potential new therapeutic approach for the treatment of diabetic nephropathy.

Berberine protects diabetic nephropathy by suppressing epithelial-to-mesenchymal transition involving the inactivation of the NLRP3 inflammasome

Accumulating evidence has implicated that berberine (BBR) has a beneficial effect on diabetic kidney disease (DKD), but its mechanism is not clear. The aim of this study was to assess whether berberine could alleviate tubulointerstitial fibrosis and attenuate epithelial-to-mesenchymal transition (EMT) and its possible molecular mechanism. High-fat diet (HFD) followed by injection of STZ was used to induce diabetic rats in vivo. After the onset of diabetes, rats were treated with either BBR or saline for 12 weeks. In vitro, the human renal proximal tubular epithelial cell line (HK-2) was exposed to high glucose, with or without BBR. The influence of berberine on renal tubulointerstitial histological changes, markers of epithelial-to-mesenchymal transition (EMT) and (NOD-like receptor pyrin domain-containing protein 3) NLRP3 inflammasome expression were examined. Results showed that in vivo, BBR could significantly ameliorate microalbumin and renal pathologic changes in diabetic rats. Immunofluorescence showed that BBR could inhibit EMT. Furthermore, BBR could down-regulate the level of the NLRP3 inflammasome in diabetic rats. Consistently, in vitro, BBR suppressed high glucose-induced EMT and activation of NLRP3 inflammasome in HK-2. Our study demonstrated that BBR could inhibit high glucose-induced EMT and renal interstitial fibrosis by suppressing the NLRP3 inflammasome. BBR might be used as a novel drug to ameliorate tubulointerstitial fibrosis in DKD.

FC022: IPI3K/AKT/MTOR Pathway Regulates Renal Expression of Klotho

Abstract BACKGROUND AND AIMS Chronic kidney disease (CKD) represents an important and growing burden on health worldwide, mainly driven by the increase in the incidence of associated risk factors like diabetes. As kidney function declines, changes in mineral metabolism occur, which contribute to the increased risk of cardiovascular disease in CKD patients. In this respect, Klotho was described as an anti-ageing protein, mainly expressed in the kidney, where it acts as an obligatory co-receptor of the Fibroblast Growth Factor (FGF23), which is involved in urinary phosphate excretion. The earliest mineral metabolism change in CKD is the fall in renal and plasma Klotho levels, followed by an increase in FGF23. Klotho loss is related to inflammatory and fibrotic processes contributing not only to the progression of CKD but also to the increase in cardiovascular risk. Clinically, there is a growing interest in maintaining renal Klotho expression in patients, but the mechanisms in Klotho decline remain unknown. PTEN is the main negative regulator of PI3K-AKT-mTOR pathway. PTEN degrades PIP3 to PIP2 decreasing the activation of the canonical insulin pathway. The involvement of PTEN in cancer has been broadly investigated. Furthermore, the PI3K/PTEN signalling pathways are involved in a wide variety of diseases including cardiac hypertrophy, heart failure, hypertension and acute kidney injury. However, its potential role in CKD has not been properly assessed. The objective of this study was to investigate the role of PTEN and the PI3K/AKT/mTOR pathways in kidney Klotho levels and the modulation of phosphorus metabolism. METHOD We generated a proximal tubule conditional PTEN knockout mouse model (PT-PTEN-cKO), in which a high phosphorus diet (HPD) and rapamycin treatment were administered in order to analyse how PI3K/AKT/mTOR pathway participates in phosphorus metabolism and kidney Klotho levels. We used human proximal tubular cell (PTC) line HK-2 to knock down PTEN by means of transfection with shPTEN lentiviral vector, to study the direct effect of PTEN elimination in Klotho levels in vitro. RESULTS PT-PTEN-cKO mice present a specific activation of the PI3K/AKT/mTOR pathway in the proximal tubule. PTEN mRNA decrease correlates with a drop in kidney Klotho levels, accompanied by an increment of inflammatory and fibrotic markers. In addition, our model showed an increase in both, plasma phosphate and FGF23, with a decrease in the fractional excretion of phosphate (% FEPi). This fact can be explained by the inability of kidney cells to respond to FGF23, because of Klotho loss. Moreover, parathyroid hormone (PTH) and bone resorption were increased. We observe that PT-PTEN-KO mice in an HPD needed higher FGF23 levels in order to excrete phosphate than control mice. Rapamycin administration restored Klotho levels. In vitro, PTEN silencing in PTC, leads to an increase in PI3K/AKT/mTOR activity and also to a reduction in Klotho expression. Finally, the inhibition of mTOR phosphorylation by rapamycin reestablished Klotho expression in vitro. CONCLUSION The overactivation of PI3K/AKT/mTOR pathway, in PTC, decreases Klotho kidney levels. Our findings constitute an important breakthrough in the research of new therapeutic targets in order to maintain renal Klotho levels and it may be useful in the treatment of kidney disease patients.

MO455: Fatty Acid-Associated Albumin Induces Proliferation and Senescence of Proximal Tubular Cells

Abstract BACKGROUND AND AIMS Proteinuria is a major risk factor for the progression of chronic kidney disease. Protein overload in the proximal tubular epithelial cells causes oxidative stress, lysosomal dysfunction, inflammation and apoptosis, resulting in proximal tubule dysfunction. Recent studies have focussed on the association between proximal tubule injury and cellular senescence and the development of drugs targeting and removing senescent proximal tubular cells. Senescent cells show resistance to apoptosis and persistently secrete inflammatory cytokines, resulting in chronic inflammation. It has been suggested that excess proliferation induced by fatty acids causes senescence of proximal tubular cells. However, the association between protein overload, proliferation and cellular senescence in proximal tubular cells remains unclear. The present study aimed to clarify the effect of protein overload on cell proliferation and senescence in a proteinuric mouse model and in an immortalized proximal tubular epithelial cell line. Moreover, it was evaluated whether the endocytic receptors for protein uptake, megalin and cubilin, affect protein overload-induced proliferation and senescence using knockout (KO) of megalin and cubilin in the proteinuric mouse model. METHOD Experiments were performed using podocin-KO (proteinuric mouse model) and megalin-cubilin-podocin triple-KO mice. Kidneys from these mice were analysed using immunohistochemical and immunofluorescent staining. Immortalized proximal tubular cells (RPTEC/TERT1, ATCC) were used for in vitro experiments, wherein RPTEC/TERT1 cells were incubated with 0.1–10 mg/mL of human serum albumin (HSA; Sigma), fatty acid-free HSA (Sigma), and transferrin (Sigma). Western blotting, quantitative rt-PCR, senescence-associated beta-galactosidase (SA-β-gal) staining and immunofluorescence were performed to analyse cellular senescence. The effect of a PKC activator (phorbol 12-myristate 13-acetate) and an inhibitor (Go6983) on proliferation and senescence was also evaluated. RESULTS The proliferation markers EdU incorporation, PCNA (Figure 1) and Ki-67 were detected in proximal tubular cells of podocin-KO mice, but not in wild-type mice. In triple-KO mice, a decrease in PCNA-positive tubules was observed. This suggests that protein reabsorption via megalin and cubilin provoked cell proliferation. Light microscopy analysis and a proliferation assay with BrdU incorporation revealed that HSA overload-induced the proliferation of RPTEC/TERT1 cells, whereas fatty acid-free HSA or transferrin had no effect on the proliferation of RPTEC/TERT1 cells. Western blot analysis showed that HSA treatment, but not fatty acid-free HSA treatment, induced alterations in proliferation (PCNA and Ki-67), cell cycle (cyclin A, D, and Rb), cellular senescence (p21 and p16), and DNA injury (γ-H2AX). Using immunofluorescence, an increase in p21 and p16 expression was also observed in HSA-treated cells. Moreover, the HSA-treated cells showed positive staining for SA-β-gal. These results suggest that fatty acids bound to HSA induce cell proliferation and senescence. Furthermore, results showed that a PKC inhibitor suppressed HSA-induced proliferation and senescence, while a PKC activator accelerated these alterations without HSA treatment. CONCLUSION The present study showed that fatty acid-associated albumin induced proliferation and senescence of the proximal tubule cells, which were dependent on megalin/cubilin endocytosis of filtered protein. PKC activation is at least in part related to cell proliferation and senescence. It is unknown which molecular switch determine the cell cycle fate.

Protein interactions with metallothionein-3 promote vectorial active transport in human proximal tubular cells.

Metallothionein 3 (MT-3) is a small, cysteine-rich protein that binds to essential metals required for homeostasis, as well as to heavy metals that have the potential to exert toxic effects on cells. MT-3 is expressed by epithelial cells of the human kidney, including the cells of the proximal tubule. Our laboratory has previously shown that mortal cultures of human proximal tubular (HPT) cells express MT-3 and form domes in the cell monolayer, a morphological feature indicative of vectorial active transport, an essential function of the proximal tubule. However, an immortalized proximal tubular cell line HK-2 lacks the expression of MT-3 and fails to form domes in the monolayer. Transfection of HK-2 cells with the MT-3 gene restores dome formation in these cells suggesting that MT-3 is required for vectorial active transport. In order to determine how MT-3 imparts this essential feature to the proximal tubule, we sought to identify proteins that interact either directly or indirectly with MT-3. Using a combination of pulldowns, co-immunoprecipitations, and mass spectrometry analysis, putative protein interactants were identified and subsequently confirmed by Western analysis and confocal microscopy, following which proteins with direct physical interactions were investigated through molecular docking. Our data shows that MT-3 interacts with myosin-9, aldolase A, enolase 1, β-actin, and tropomyosin 3 and that these interactions are maximized at the periphery of the apical membrane of doming proximal tubule cells. Together these observations reveal that MT-3 interacts with proteins involved in cytoskeletal organization and energy metabolism, and these interactions at the apical membrane support vectorial active transport and cell differentiation in proximal tubule cultures.

Activation of aryl hydrocarbon receptor stimulates GLUT‐mediated glucose transport in human renal proximal tubular cells

Aryl hydrocarbon receptor (AhR), a ligand‐activated transcription factor, plays a major role in detoxification of xenobiotics. AhR deficiency has been shown to attenuate renal injury induced by diabetes. It is interesting whether the beneficial effect of AhR deficiency on renal injury in diabetes involve in alteration of intracellular glucose in renal tubular cells. The aim of this study was to investigate the effect and mechanism of AhR on glucose uptake into human renal proximal tubular cells. Exposure HK‐2 cells, human renal proximal tubular cell line, with omeprazole, an AhR agonist, increased glucose transporter (GLUT)‐mediated [3H]‐2‐Deoxy‐D glucose uptake into the cells in concentration‐dependent manner. The stimulatory effect of omeprazole was reduced by co‐incubation with CH223191, an AhR antagonist. These results indicate that the stimulatory effect of omeprazole on GLUT transport function regards activation of AhR. The kinetic study showed that omeprazole significantly decreased half‐maximal transport (Kt) but not maximal rate of transport (Jmax) of GLUT, suggesting that AhR might increase affinity of GLUT‐mediated glucose uptake into the cells. In addition, activation of AhR significantly induced cellular level of ROS. Inhibition of ROS using N‐acetyl‐L‐cysteine (NAC) inhibited the stimulatory effect of AhR on GLUT‐mediated glucose uptake. These results support that activation of AhR increases GLUT‐mediated glucose accumulation via increase in ROS production in human renal proximal tubular cells. Inhibition of AhR might be a target for treatment of high glucose‐induced renal injury from diabetes.

Icariin improves renal interstitial fibrosis in a rat model of chronic renal failure by regulating mitochondrial dynamics

This study aims to explore the effect of icariin(ICA) on mitochondrial dynamics in a rat model of chronic renal failure(CRF) and to investigate the molecular mechanism of ICA against renal interstitial fibrosis(RIF). CRF was induced in male Sprague-Dawley(SD) rats with 5/6(ablation and infarction, A/I) surgery(right kidney ablation and 2/3 infarction of the left kidney). Four weeks after surgery, the model rats were randomized into the following groups: 5/6(A/I) group, 5/6(A/I)+low-dose ICA group, and 5/6(A/I)+high-dose ICA group. Another 12 rats that received sham operation were randomly classified into 2 groups: sham group and sham+ICAH group. Eight weeks after treatment, the expression of collagen-Ⅰ(Col-Ⅰ), collagen-Ⅲ(Col-Ⅲ), mitochondrial dynamics-related proteins(p-Drp1 S616, p-Drp1 S637, Mfn1, Mfn2), and mitochondrial function-related proteins(TFAM, ATP6) in the remnant kidney tissues was detected by Western blot. The expression of α-smooth muscle actin(α-SMA) was examined by immunohistochemical(IHC) staining. The NRK-52 E cells, a rat proximal renal tubular epithelial cell line, were cultured in vitro and treated with ICA of different concentration. Cell viability was detected by CCK-8 assay. In NRK-52 E cells stimulated with 20 ng·mL~(-1) TGF-β1 for 24 h, the effect of ICA on fibronectin(Fn), connective tissue growth factor(CTGF), p-Drp1 S616, p-Drp1 S637, Mfn1, Mfn2, TFAM, and ATP6 was detected by Western blot, and the ATP content and the mitochondrial morphology were determined. The 20 ng·mL~(-1) TGF-β1-stimulated NRK-52 E cells were treated with or without 5 μmol·L~(-1) ICA+10 μmol·L~(-1) mitochondrial fusion promoter M1(MFP-M1) for 24 h and the expression of fibrosis markers Fn and CTGF was detected by Western blot. Western blot result showed that the levels of Col-Ⅰ, Col-Ⅲ, and p-Drp1 S616 were increased and the levels of p-Drp1 S637, Mfn1, Mfn2, TFAM, and ATP6 were decreased in 5/6(A/I) group compared with those in the sham group. The levels of Col-Ⅰ, Col-Ⅲ, and p-Drp1 S616 were significantly lower and the levels of p-Drp1 S637, Mfn1, Mfn2, TFAM, and ATP6 were significantly higher in ICA groups than that in 5/6(A/I) group. IHC staining demonstrated that for the expression of α-SMA in the renal interstitium was higher in the 5/6(A/I) group than in the sham group and that the expression in the ICA groups was significantly lower than that in the 5/6(A/I) group. Furthermore, the improvement in the fibrosis, mitochondrial dynamics, and mitochondrial function were particularly prominent in rats receiving the high dose of ICA. The in vitro experiment revealed that ICA dose-dependently inhibited the increase of Fn, CTGF, and p-Drp1 S616, increased p-Drp1 S637, Mfn1, Mfn2, TFAM, and ATP6, elevated ATP content, and improved mitochondrial morphology of NRK-52 E cells stimulated by TGF-β1. ICA combined with MFP-M1 further down-regulated the expression of Fn and CTGF in NRK-52 E cells stimulated by TGF-β1 compared with ICA alone. In conclusion, ICA attenuated RIF of CRF by improving mitochondrial dynamics.

Cigarette Smoke Exposure Increases Glucose-6-phosphate Dehydrogenase, Autophagy, Fibrosis, and Senescence in Kidney Cells In Vitro and In Vivo.

Cigarette smoke (CS) is a risk factor for chronic obstructive pulmonary disease. We attempted to investigate fully the possible effects of CS on kidney cells. We found that the viability of a human kidney proximal tubular epithelial cell line (HK-2 cells) was decreased after treatment with CS extract (CSE). In particular, the effects of CSE at low concentrations did not change the expression of apoptosis and necrosis. Furthermore, CSE increased autophagy- and fibrosis-related proteins in HK-2 cells. Senescence-related proteins and the senescence-associated secretory phenotype (SASP) increased after HK-2 cells were treated with CSE. In addition, both RNA sequencing and gene set enrichment analysis data revealed that glucose-6-phosphate dehydrogenase (G6PD) in the reactive oxygen species (ROS) pathway is responsible for the changes in CSE-treated HK-2 cells. CSE increased G6PD expression and its activity. Moreover, the inhibition of G6PD activity increased senescence in HK-2 cells. The inhibition of autophagy reinforced senescence in the CSE-treated cells. In a mouse model of CS exposure, CS caused kidney damage, including tubular injury and glomerulosclerosis. CS increased fibrosis, autophagy, and G6PD expression in kidney tissue sections. In conclusion, CS induced G6PD expression, autophagy, fibrosis, and senescence in kidney cells. G6PD has a protective role in CS-induced nephrotoxicity.

Transcriptome Analysis Reveal Candidate Genes and Pathways Responses to Lactate Dehydrogenase Inhibition (Oxamate) in Hyperglycemic Human Renal Proximal Epithelial Tubular Cells.

Diabetic kidney disease (DKD) is the leading cause of both chronic kidney disease (CKD) and end-stage renal disease (ESRD). Previous studies showed that oxamate could regulate glycemic homeostasis and impacted mitochondria respiration in a hyperglycemia-dependent manner in the rat proximal tubular cells. To explore the transcriptome gene expression profiling of kidney tissues in human renal proximal epithelial tubular cell line (HK-2), we treated HK-2 cells with high D-glucose (HG) for 7 days before the addition of 40 mM oxamate for a further 24 hours in the presence of HG in this study. Afterwards, we identified 3,884 differentially expressed (DE) genes based on adjusted P-value ≤ 0.05 and investigated gene relationships based on weighted gene co-expression network analysis (WGCNA). After qRT-PCR validations, MAP1LC3A, MAP1LC3B (P-value < 0.01) and BECN1 were found to show relatively higher expression levels in the treated groups than the control groups, while PGC1α (P-value < 0.05) showed the lower expressions. Accordingly, enrichment analyses of GO terms and KEGG pathways showed that several pathways [e.g., lysosome pathway (hsa04142) and p53 signaling pathway (hsa04115)] may be involved in the response of HK-2 cells to oxamate. Moreover, via WGCNA, we identified two modules: both the turquoise and blue modules were enriched in pathways associated with lysosome. However, the p53 signaling pathway was only found using all 3,884 DE genes. Furthermore, the key hub genes IGFBP3 (adjusted P-value = 1.34×10-75 and log2(FC) = 2.64) interacted with 6 up-regulated and 12 down-regulated DE genes in the network that were enriched in the p53 signaling pathway. This is the first study reporting co-expression patterns of a gene network after lactate dehydrogenase inhibition in HK-2 cells. Our results may contribute to our understanding of the underlying molecular mechanism of in vitro reprogramming under hyperglycemic stress that orchestrates the survival and functions of HK-2 cells.

Fenofibrate reduces cisplatin-induced apoptosis by inhibiting the p53/Puma/Caspase-9 pathway and the MAPK/Caspase-8 pathway rather than by promoting autophagy in murine renal proximal tubular cells

The main lesion of cisplatin nephrotoxicity is damage to proximal tubular cells due to increased apoptosis via the mitochondrial and death receptor pathways, which may be alleviated by appropriate promotion of autophagy. Fenofibrate, a peroxisome proliferator-activated receptor-alpha (PPAR-α) activator, is recently reported to promote autophagy as well as protect against cisplatin nephrotoxicity, although the mechanisms were only partially analyzed. Here, the detailed mechanisms of these putative protective effects were investigated in a murine renal proximal tubular (mProx) cell line. Fenofibrate attenuated cisplatin-induced apoptosis of mProx cells based on flow cytometry. As for the mitochondrial apoptotic pathway, the reagent reduced cisplatin-stimulated caspase-3 activation by decreasing the phosphorylation of p53, JNK, and 14-3-3, cytosolic and mitochondrial Puma accumulation, cytochrome C release to the cytosol, and resulting cytosolic caspase-9 activation. Fenofibrate also decreased cisplatin-stimulated activation of caspases-8 by suppressing MAPK and NFkB pathways and reducing the gene expression of TNF-α, TL1A, and Fas, main mediators of the death receptor apoptotic pathway. Autophagy defined by p62 reduction and an increase in LC3 II/I was promoted by fenofibrate in mProx cells under starvation. Autophagy inhibition using 3-MA further increased basal and cisplatin-induced caspase-3 and -8 activation, but had no influence on the inhibitory effects of fenofibrate on caspase activation. In conclusion, our study suggests fenofibrate to be a candidate agent to mitigate cisplatin nephrotoxicity by inhibiting the mitochondrial and death apoptotic pathways rather than by promoting autophagy.

A single-domain i-body, AD-114, attenuates renal fibrosis through blockade of CXCR4.

The G protein–coupled CXC chemokine receptor 4 (CXCR4) is a candidate therapeutic target for tissue fibrosis. A fully human single-domain antibody-like scaffold i-body AD-114-PA600 (AD-114) with specific high binding affinity to CXCR4 has been developed. To define its renoprotective role, AD-114 was administrated in a mouse model of renal fibrosis induced by folic acid (FA). Increased extracellular matrix (ECM) accumulation, macrophage infiltration, inflammatory response, TGF-β1 expression, and fibroblast activation were observed in kidneys of mice with FA-induced nephropathy. These markers were normalized or partially reversed by AD-114 treatment. In vitro studies demonstrated AD-114 blocked TGF-β1–induced upregulated expression of ECM, matrix metalloproteinase-2, and downstream p38 mitogen-activated protein kinase (p38 MAPK) and PI3K/AKT/mTOR signaling pathways in a renal proximal tubular cell line. Additionally, these renoprotective effects were validated in a second model of unilateral ureteral obstruction using a second generation of AD-114 (Fc-fused AD-114, also named AD-214). Collectively, these results suggest a renoprotective role of AD-114 as it inhibited the chemotactic function of CXCR4 as well as blocked CXCR4 downstream p38 MAPK and PI3K/AKT/mTOR signaling, which establish a therapeutic strategy for AD-114 targeting CXCR4 to limit renal fibrosis.