Proximal Tubular Cell Line HK-2 Research Articles

[HIF-1α activation induces cholesterol homeostasis dysfunction to accelerate progression of diabetic nephropathy in rats].

To investigate the effect of hypoxia inducible factor-1α (HIF-1α) activation on cholesterol homeostasis dysfunction in diabetic nephropathy (DN). Rat models of type 1 diabetes established by intraperitoneal STZ injection were treated with intraperitoneal injection of Lificiguat (YC-1, a HIF-1α inhibitor). Human proximal tubular cell line HK-2 was incubated with cobalt chloride (CoCl2, 100 μmol/L) in the presence or absence of 30 mmol/L glucose for 24 h. Renal injury of the rats was assessed by measuring 24-h urinary total protein level and PAS staining of the renal tubules. Cholesterol deposition in rat kidneys and HK-2 cells were observed using a quantitative assay of total cholesterol and Filipin staining, and HIF-1α protein expression was detected using Western blotting, immunohistochemistry or immunofluorescence assay; the expressions of cholesterol metabolism-related proteins HMGCR, LDLr, CXCL16 and profibrogenic factors including TGF-β1 and CTGF were also analyzed. The diabetic rats showed significantly increased 24-h urinary protein level (P<0.001), obvious renal tubular injury, and increased renal cholesterol content (P<0.05) with significantly increased HIF-1α expression in the renal tubular (P<0.01). YC-1 treatment significantly ameliorated tubulointerstitial injury in the diabetic rats as shown by decreased 24-h urinary total protein (P<0.05) and reduced damage area of the tubules, and effectively decreased renal cholesterol levels and renal expression of HIF-1α (P<0.05). In HK-2 cells, CoCl2 stimulation in the presence of high glucose effectively activated HIF-1α expression (P<0.0001), aggravated cholesterol accumulation (P<0.05), and increased the expressions of HMGCR, LDLr, CXCL16, TGF-β1, and CTGF (P<0.05 or 0.01). Consistent with the in vitro study, YC-1 treatment also significantly decreased the expressions of cholesterol metabolism-related proteins and the profibrogenic factors in the renal tubules of the diabetic rats. HIF-1α activation induces cholesterol homeostasis dysregulation possibly by upregulating the de novo synthesis and uptake of cholesterol, thereby aggravating tubulointerstitial injury in DN.

HK-2 cell response to TGF-β highly depends on cell culture medium formulations

The immortalized human renal proximal tubular epithelial cell line HK-2 is most commonly used to study renal cell physiology and human kidney diseases with tubulointerstitial fibrosis such as diabetic nephropathy, obstructive uropathy or allograft fibrosis. Epithelial-to-mesenchymal transition (EMT) is the main pathological process of tubulointerstitial fibrosis in vitro. Transforming growth factor-beta (TGF-β) is a key inducer of EMT. Several pro-fibrotic gene expression differences have been observed in a TGF-β-induced EMT model of HK-2 cells. However, growth conditions and medium formulations might greatly impact these differences. We investigated gene and protein expression of HK-2 cells cultured in six medium formulations. TGF-β1 increased the expression of ACTA2, TGFB1, COL4A1, EGR2, VIM and CTGF genes while reducing PPARG in all medium formulations. Interestingly, TGF-β1 treatment either increased or decreased EGR1, FN, IL6 and C3 gene expression, depending on medium formulations. The cell morphology was slightly affected, but immunoblots revealed TGFB1 and vimentin protein overexpression in all media. However, fibronectin expression as well as the nuclear translocation of EGR1 was medium dependent. In conclusion, our study demonstrates that, using the HK-2 in vitro model of EMT, the meticulous selection of appropriate cell culture medium formulation is essential to achieve reliable scientific results.

#5453 LIPID NEPHROTOXICITY INDUCED BY HIF-1α ACTIVATION ACCELERATES INTERSTITIAL INJURY IN DIABETIC NEPHROPATHY

Abstract Background and Aims Our previous study demonstrated lipid accumulation contributes to tubulointerstitial injury in diabetic nephropathy. However, the exact mechanism of which accelerate lipid accumulation in tubulointerstitium has not been completely elucidated. Increasing studies suggest that chronic hypoxia plays an important pathophysiologic role in diabetic nephropathy. This study aimed to investigate the effect of hypoxia inducible factor-1α (HIF-1α) activation induced by chronic hypoxia on lipid accumulation in diabetic kidney and to explore its potential mechanism. Method Human proximal tubular cell line HK-2 was incubated with or without cobalt chloride (CoCl2, 100 μM) which stimulates hypoxic condition in the presence or absence of glucose (30 mM) for 24 h. Specific siRNA was transfected in cells to knock down HIF-1α expression. Type 1 diabetic rats model were induced by STZ injection, and Lificiguat (YC-1) was used as HIF-1α inhibitor by intraperitoneal injection. Immunofluorescence staining and Western blot were used to determine the cellular localization and expression of HIF-1α. Periodic Acid-Schiff (PAS) staining was used to observe basic structure and pathological changes of kidney. The expression of carnitine palmitoyl transferase 1A (CPT1A), tissue growth factor-β1 (TGF-β1), connective tissue growth factor (CTGF) were measured by immunohistochemical staining and Western blot. Lipid accumulation was detected by oil red O staining and cellular triglyceride quantification assay. Results In in vitro study, CoCl2 effectively increased the expression of HIF-1α and its translocation from cytoplasm to nucleus. Meanwhile, HIF-1α activation induced by CoCl2 significantly increased lipid accumulation in HK-2 cells. Moreover, CoCl2 treatment increased TGF- β and CTGF expression in HK-2 cells. The presence of glucose (30mM) made no significant difference to HK-2 cells incubated with CoCl2. HIF-1α siRNA treatment significantly reduced lipid deposition in HK-2 cells incubated with CoCl2. In vivo study showed YC-1 treatment decreased the expression of HIF-1α in renal tubules of diabetic rats, which was accompanied by less lipid accumulation compared with the diabetes mellitus (DM) group. PAS staining showed the desquamation and necrosis of tubular epithelial cells in the YC-1 group were alleviated compared with the DM group. YC-1 treatment did not affect the ratio of kidney weight to body weight, whereas decreased the levels of blood glucose, urine albumin creatinine ratio and NAG creatinine ratio in diabetic rats. The expressions of fibrosis factor TGF- β and CTGF were decreased in the YC-1 group compared with the DM group. Furthermore, the protein expression of CPT1A, the key enzyme of fatty acid oxidation, which was decreased in the DM group compared with the controls, was regained by YC-1 treatment. Conclusion Our findings demonstrated that HIF-1α activation contributed to interstitial injury in diabetic nephropathy by inducing lipid accumulation. HIF-1α activation might contribute to lipid nephrotoxicity by downregulating fatty acid oxidation.

LTBP4 Protects Against Renal Fibrosis via Mitochondrial and Vascular Impacts.

As a part of natural disease progression, acute kidney injury (AKI) can develop into chronic kidney disease via renal fibrosis and inflammation. LTBP4 (latent transforming growth factor beta binding protein 4) regulates transforming growth factor beta, which plays a role in renal fibrosis pathogenesis. We previously investigated the role of LTBP4 in chronic kidney disease. Here, we examined the role of LTBP4 in AKI. LTBP4 expression was evaluated in human renal tissues, obtained from healthy individuals and patients with AKI, using immunohistochemistry. LTBP4 was knocked down in both C57BL/6 mice and human renal proximal tubular cell line HK-2. AKI was induced in mice and HK-2 cells using ischemia-reperfusion injury and hypoxia, respectively. Mitochondrial division inhibitor 1, an inhibitor of DRP1 (dynamin-related protein 1), was used to reduce mitochondrial fragmentation. Gene and protein expression were then examined to assess inflammation and fibrosis. The results of bioenergetic studies for mitochondrial function, oxidative stress, and angiogenesis were assessed. LTBP4 expression was upregulated in the renal tissues of patients with AKI. Ltbp4-knockdown mice showed increased renal tissue injury and mitochondrial fragmentation after ischemia-reperfusion injury, as well as increased inflammation, oxidative stress, and fibrosis, and decreased angiogenesis. in vitro studies using HK-2 cells revealed similar results. The energy profiles of Ltbp4-deficient mice and LTBP4-deficient HK-2 cells indicated decreased ATP production. LTBP4-deficient HK-2 cells exhibited decreased mitochondrial respiration and glycolysis. Human aortic endothelial cells and human umbilical vein endothelial cells exhibited decreased angiogenesis when treated with LTBP4-knockdown conditioned media. Mitochondrial division inhibitor 1 treatment ameliorated inflammation, oxidative stress, and fibrosis in mice and decreased inflammation and oxidative stress in HK-2 cells. Our study is the first to demonstrate that LTBP4 deficiency increases AKI severity, consequently leading to chronic kidney disease. Potential therapies focusing on LTBP4-associated angiogenesis and LTBP4-regulated DRP1-dependent mitochondrial division are relevant to renal injury.

Exosomal microRNA-342-5p secreted from adipose-derived mesenchymal stem cells mitigates acute kidney injury in sepsis mice by inhibiting TLR9

BackgroundSepsis-related acute kidney injury (AKI) is an inflammatory disease associated with extremely high mortality and health burden. This study explored the possibility of exosomes secreted by adipose-derived mesenchymal stem cells (AMSCs) serving as a carrier for microRNA (miR)-342-5p to alleviate sepsis-related AKI and investigated the possible mechanism.MethodsSerum was obtained from 30 patients with sepsis-associated AKI and 30 healthy volunteers for the measurement of miR-342-5p, blood urea nitrogen (BUN), and serum creatinine (SCr) levels. For in vitro experiments, AMSCs were transfected with LV-miR-342-5p or LV-miR-67 to acquire miR-342-5p-modified AMSCs and miR-67-modified AMSCs, from which the exosomes (AMSC-Exo-342 and AMSC-Exo-67) were isolated. The human renal proximal tubular epithelial cell line HK-2 was induced by lipopolysaccharide (LPS) to construct a cellular model of sepsis. The expression of Toll-like receptor 9 (TLR9) was also detected in AKI cells and mouse models. The interaction between miR-342-5p and TLR9 was predicted by dual luciferase reporter gene assay.ResultsDetection on clinical serum samples showed that BUN, SCr, and TLR9 were elevated and miR-342-5p level was suppressed in the serum of patients with sepsis-associated AKI. Transfection with LV-miR-342-5p reinforced miR-342-5p expression in AMSCs and AMSC-secreted exosomes. miR-342-5p negatively targeted TLR9. LPS treatment enhanced TLR9 expression, reduced miR-342-5p levels, suppressed autophagy, and increased inflammation in HK-2 cells, while the opposite trends were observed in LPS-induced HK-2 cells exposed to AMSC-Exo-342, Rapa, miR-342-5p mimic, or si-TLR9. Additionally, the effects of AMSC-Exo-342 on autophagy and inflammation in LPS-induced cells could be weakened by 3-MA or pcDNA3.1-TLR9 treatment. Injection of AMSC-Exo-342 enhanced autophagy, mitigated kidney injury, suppressed inflammation, and reduced BUN and SCr levels in sepsis-related AKI mouse models.ConclusionmiR-342-5p transferred by exosomes from miR-342-5p-modified AMSCs ameliorated AKI by inhibiting TLR9 to accelerate autophagy.Graphical

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.

Amelioration of Cyclosporine A-Induced Acute Nephrotoxicity by Cordyceps cicadae Mycelia via Mg+2 Reabsorption and the Inhibition of GRP78-IRE1-CHOP Pathway: In Vivo and In Vitro.

Fruiting bodies of Cordyceps cicadae (CC) have been reported to have a therapeutic effect in chronic kidney disease. Due to the rare and expensive resources from natural habitats, artificially cultivated mycelia using submerged liquid cultivation of CC (CCM) have been recently developed as an alternative to scarce sources of CC. However, little is known regarding potential protective effects of CCM against cyclosporine A (CsA)-induced acute nephrotoxicity in vivo and in vitro. In this study, male Sprague-Dawley rats were divided into six groups: control, CCM (40 mg and 400 mg/kg, orally), CsA (10 mg/kg, oral gavage), and CsA + CCM (40 mg and 400 mg/kg, orally). At the end of the study on day 8, all rats were sacrificed, and the blood and kidneys retrieved. CsA-induced acute nephrotoxicity was evident by increased levels of blood urea nitrogen (BUN). Levels of the endoplasmic reticulum (ER) resident chaperone glucose regulated protein 78 (GRP 78) were increased significantly in rats with acute nephrotoxicity. BUN and GRP 78 were significantly ameliorated in synchronous oral groups of CCM (40 or 400 mg/kg) plus CsA. Examination of hematoxylin and eosin stained kidney tissues revealed that the combined treatment of CCM slightly improved vacuolization in renal tubules upon CsA-induced damage. CsA-induced down-regulation of protein expression of magnesium ion channel proteins and transient receptor potential melastatin 6 and 7 were abolished by the combined treatment of CCM. CCM has the potential to protect the kidney against CsA-induced nephrotoxicity by reducing magnesium ion wasting, tubular cell damage, and ER stress demonstrated further by human renal proximal tubular epithelial cell line HK-2. Our results contribute to the in-depth understanding of the role of polysaccharides and nucleobases as the main secondary metabolites of CCM in the defense system of renal functions in CsA-induced acute nephrotoxicity.

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.

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.

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.

In vitro study on effect of bardoxolone methyl on cisplatin-induced cellular senescence in human proximal tubular cells

Bardoxolone methyl [methyl-2-cyano-3, 12-dioxooleana-1, 9(11)dien-28-oate (CDDO-Me)], an activator of the nuclear factor erythroid-derived 2-related factor2 pathway, is a potential therapeutic candidate for the treatment of kidney diseases. However, its effect against cellular senescence remains unclear. This study aimed to investigate whether CDDO-Me protects cells against cisplatin-induced cellular senescence using an in vitro model. The human renal proximal tubular epithelial cell line HK-2 was treated with cisplatin for 6 h, followed by treatment with or without CDDO-Me (0.1 or 0.2 μmol/L). Senescence markers were analyzed using western blotting and real-time PCR. Apoptosis was evaluated through TUNEL staining. Cisplatin induced changes in the levels of markers specific for proliferation, cell cycle, and senescence in a time- and dose-dependent manner. Furthermore, IL-6 and IL-8 levels in the culture medium increased markedly. These data suggested that cellular senescence-like alterations occurred in HK-2 cells exposed to cisplatin. CDDO-Me treatment reversed the cisplatin-mediated alterations in the levels of cellular senescence markers. The antioxidant enzymes, HO1, NQO1, GPX1, and CAT were upregulated by CDDO-Me treatment. Furthermore, CDDO-Me treatment induced apoptosis in cisplatin-exposed HK-2 cells. Pretreatment with Ac-DEVD-CHO, the caspase inhibitor, suppressed the reversal effect of CDDO-Me against cisplatin-induced cellular senescence-like alterations. This study showed that CDDO-Me attenuated cisplatin-induced premature senescence of HK-2 cells. This beneficial effect may be related to Nrf2 activation. Our findings also showed that CDDO-Me induced apoptosis in cisplatin-treated HK-2 cells, potentially protecting the kidneys from cellular senescence. CDDO-Me appears to be a candidate treatment for acute kidney injury.

Glaucocalyxin A Ameliorates Hypoxia/Reoxygenation-Induced Injury in Human Renal Proximal Tubular Epithelial Cell Line HK-2 Cells

Ischemia-reperfusion injury is one of the major causes of acute kidney injury (AKI), which is increasingly prevalent in clinical settings. Glaucocalxin A (GLA), a biologically ent-kauranoid diterpenoid, has various pharmacological effects like antioxidation, immune regulation, and antiatherosclerosis. In this study, the effect of GLA on AKI and its mechanism were studied in vitro. HK-2 human renal tubular epithelial cells were exposed to hypoxia/reoxygenation (H/R), which were established as an in vitro AKI model. Subsequently, the mRNA expressions of inflammatory and antioxidant factors were determined by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Reactive oxygen species (ROS) production and cell death were detected by fluorescence-activated cell sorting. GLA pre-treatment improved the cell viability of HK-2 cells exposed to H/R. GLA suppressed the H/R-induced ROS production in HK-2 cells. GLA also elevated the activities of superoxide dismutase of HK-2 cells exposed to H/R. Moreover, GLA prevented H/R-induced cell death in HK-2 cells. Furthermore, GLA ameliorated the activation of the protein kinase B (Akt)/nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway in HK-2 cells exposed to H/R. Our findings suggested that GLA protected HK-2 cells from H/R-induced oxidative damage, which was mediated by the Akt/Nrf2/HO-1 signaling pathway. These results indicate that GLA may serve as a promising therapeutic drug for AKI.

Alamandine, a derivative of angiotensin-(1-7), alleviates sepsis-associated renal inflammation and apoptosis by inhibiting the PI3K/Ak and MAPK pathways

Sepsis is a frequent cause of kidney injury. The present study investigated whether Alamandine (Ala) could alleviate sepsis-associated renal injury by reducing inflammation and apoptosis. In addition, we investigated downstream signaling pathways modulated by Ala. Studies were performed in mice treated with lipopolysaccharide (LPS) and in the human proximal tubular epithelial cell line HK-2. The increase in serum creatinine, blood urea nitrogen, cystatin C and Fg, and neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 in the kidneys of mice treated with LPS were reduced after administration of Ala. Exposure to LPS increased interleukin-1 beta (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α) in mice and HK-2 cells, but were reduced after Ala treatment. Furthermore, increased levels of cleaved caspase 3, cleaved caspase 7, cleaved caspase 9, cleaved poly (ADP-ribose) polymerase (PARP) and Bax and reduced levels of Bcl2 in LPS-treated mice and HK-2 cells were reversed after Ala administration. In addition, LPS increased the levels of p-PI3K/PI3K, p-Akt/Akt, p-ERK/ERK, p-JNK/JNK, p-p38/p38 and p-FoxO1 in HK-2 cells, and all were reversed after Ala administration. These results indicate that Ala could improve renal function and inhibit inflammation and apoptosis in LPS induced sepsis mouse models. We demonstrated that Ala attenuated LPS induced sepsis by inhibiting the PI3K/Akt and MAPK signaling pathways.

Pharmacologically Inhibiting Glycogen Synthase Kinase-3β Ameliorates Renal Inflammation and Nephrotoxicity in an Animal Model of Cisplatin-Induced Acute Kidney Injury

The adverse effect of cisplatin administration causes acute kidney injury (AKI) following renal inflammation and nephrotoxicity, characterized by proximal tubular cell apoptosis and necrosis. Pro-apoptotic and pro-inflammatory roles of glycogen synthase kinase (GSK)-3β have been reported. This study investigated the therapeutic blockade of GSK-3β in cisplatin-induced AKI. A renal cisplatin nephrotoxicity model showed activation of GSK-3β in vivo, particularly in proximal tubular epithelial cells. Pharmacologically inhibiting GSK-3β abolished cisplatin nephrotoxicity, including proximal tubular injury, cell cytotoxicity, and biochemical dysfunction. Additionally, GSK-3β inhibitor treatment ameliorated renal inflammation by reducing immune cell infiltration, cell adhesion molecule expression, and pro-inflammatory cytokine/chemokine production. Cisplatin treatment caused GSK-3β activation in vitro in the human renal proximal tubular epithelial cell line HK-2, whereas either pharmacological administration of GSK-3β inhibitors or genetic transduction of GSK-3β short-hairpin RNA impeded cisplatin-induced cytotoxicity. These results indicate that cisplatin activates GSK-3β followed by GSK-3β-mediated renal inflammation and nephrotoxicity, contributing to AKI.

Total flavones of Desmodium styracifolium antagonize calcium oxalate monohydrate-triggered IL-2Rβ expression in renal epithelial cells

Purpose: To explore the effect of total flavone of Desmodium styracifolium (TFDS) on calcium oxalate monohydrate (COM)-triggered IL-2Rβ expression in human kidney proximal tubular epithelial cells.&#x0D; Methods: Human kidney proximal tubular epithelial cell line HK-2 was treated with COM, TFDS or both. The expression of IL-2Rβ was evaluated by quantitative polymerase chain reaction (qPCR) or flow cytometry. The responsiveness of HK-2 cells to IL-2 was determined by enzyme-linked immunosorbent assay (ELISA), qPCR and western blot. The signaling mechanism underlying the effect of TFDS was studied using western blot and qPCR. The clinical relevance of IL-2Rβ to renal inflammation was investigated by re-analyzing a Gene Expression Omnibus (GEO) dataset.&#x0D; Results: Total flavones of Desmodium styracifolium (TFDS) antagonize COM-triggered IL-2Rβ expression in HK-2 cells, thus reducing the responsiveness of HK-2 cells to IL-2 stimulation. Mechanistically, TFDS dampens IL-2Rβ expression by preventing the activation of STAT3. The level of IL-2Rβ is positively correlated with the inflammatory status of the kidney.&#x0D; Conclusions: The total flavones of Desmodium styracifolium (TFDS) prevent the upregulation of IL2Rβ in renal epithelial cells upon COM stimulation in a STAT3-dependent manner.

Wedelolactone improves the renal injury induced by lipopolysaccharide in HK-2 cells by upregulation of protein tyrosine phosphatase non-receptor type 2.

ObjectiveTo explore the effects of wedelolactone (WEL) on sepsis-induced renal injury in the human renal proximal tubular epithelial cell line HK-2.MethodsHK-2 cells were stimulated by 1 µg/ml lipopolysaccharide (LPS) to trigger renal injury in vitro. HK-2 cells were pretreated with or without WEL (0.1, 1 and 10 µM) before LPS stimulation. Protein and mRNA analyses were performed using enzyme-linked immunosorbent assays, Western blot analysis and quantitative reverse transcription–polymerase chain reaction. The MTT assay and flow cytometry were used to measure cell viability and the rate of cell apoptosis. Protein tyrosine phosphatase non-receptor type 2 (PTPN2) knockdown was induced by the transection of HK-2 cells with short hairpin RNA.ResultsCell viability was significantly increased in a dose-dependent manner by WEL in LPS-induced HK-2 cells. WEL also decreased the levels of four inflammatory cytokines and cell apoptosis in LPS-induced HK-2 cells. The level of PTPN2 was increased after WEL treatment. PTPN2 knockdown partly abolished the inhibitory effects of WEL on cell apoptosis, the levels of inflammatory cytokines and on p38 mitogen-activated protein kinase/nuclear factor-kappaB signalling in LPS-induced HK-2 cells.ConclusionWEL improved renal injury by suppressing inflammation and cell apoptosis through upregulating PTPN2 in HK-2 cells. PTPN2 might be used as a potential therapeutic target for LPS-induced sepsis.

POS-373 Advanced oxidation protein products activate oxidative stress in vitro via soluble (pro)renin receptor mediated intrarenal renin angiotensin system and Nox4/H2O2 pathway

Advanced oxidation protein products (AOPPs) are newly identified as a novel oxidative stress biomarkers. Previous research has indicated potential associations between AOPPs and activation intrarenal local renin angiotensin system (RAS). However, no known empirical research has focused on exploring relationships between AOPPs and the new member of RAS, (pro) renin receptor (PRR). Aim of this study was to test the role of soluble PRR (sPRR) in AOPPs-induced oxidative stress responses in cultured human renal proximal tubular cell line HK-2 cells. The HK-2 cells were incubated for 24h in the presence of different concentrations of AOPPs, or incubated for different time lengths in the presence of 100 μg/ml of AOPPs. In HK-2 cells treated with PRO20, furin inhibitors, GM6001, PF-429242, sPRR-His, or transfected with PRR siRNA, site-1 protease (S1P) siRNA, Nox4 siRNA prior to exposure to AOPPs, the changes in the protein expressions of PRR/sPRR and Nox4 were examined with Western blotting, mRNA lelvels of PRR, angiotensin converting enzyme (ACE), angiotensin II type 1 receptor (AT1R), angiotensinogen (AGT), intercellular adhesion molecule (ICAM1), tumor necrosis factor-α (TNF-α) by using real-time quantitative PCR, the cell medium for renin activity, ACE activity, sPRR, Ang II, H2O2, thiobarbituric acid reactive substances (TBARS) concentrations by using ELISA Assay kits. AOPPs showed time-dependent and dose-independent effects on PRR cleavage produces sPRR. AOPP-albumin treatment induced a >4-times increase in medium sPRR due to enhanced cleavage of PRR but unmodified albumin has no effect. Inhibition of S1P with PF-429242 or siRNA remarkably suppressed AOPPs-induced sPRR production. Surprisingly, this cleavage event was unaffected by inhibition of furin or ADAM19. AOPPs (100 μg/ml) treatment for 24 h induced renin activity and oxidative stress, both of which were attenuated by a PRR decoy inhibitor PRO20 and S1P inhibitor PF429242. What’s more, PF-429242 significantly decreased AOPPs stimulated Nox4 and H2O2 expression and administration of a recombinant sPRR, termed sPRR-His, reversed the effects of S1P inhibition. Taken together, these findings suggest that AOPPs induce oxidative stress via S1P-derived soluble (pro)renin receptor activation intrarenal renin angiotensin system and Nox4/H2O2 pathway in vitro.

Circular RNA circ_0068,888 protects against lipopolysaccharide-induced HK-2 cell injury via sponging microRNA-21–5p

Our previous findings revealed that hsa_circ_0068,888 was markedly down-regulated in the plasma of patients with sepsis-associated acute kidney injury (AKI). However, its molecular mechanism in AKI remains unclear. Herein, we explored the role of hsa_circ_0068,888 in AKI. Human renal proximal tubular cell line HK-2 was stimulated with lipopolysaccharide (LPS) to mimic AKI in vitro. Decreased hsa_circ_0068,888 expression was observed in AKI cell model. The overexpression of hsa_circ_0068,888 significantly increased the viability of LPS-stimulated HK-2 cells, whereas hsa_circ_0068,888 downregulation showed the opposite effect. Furthermore, LPS triggered inflammatory response and oxidative stress, which was inhibited by hsa_circ_0068,888 overexpression and enhanced by hsa_circ_0068,888 down-regulation. Hsa_circ_0068,888 overexpression suppressed the activation of nuclear factor-κB (NF-κB) pathway triggered by LPS as evidenced by decreased p-p65 protein level and nuclear translocation of p65 in hsa_circ_0068,888 overexpressed cells. Additionally, we proved that hsa_circ_0068,888 targeted microRNA-21–5p (miR-21–5p). The expression of miR-21–5p was markedly increased and was negatively regulated by hsa_circ_0068,888 in LPS-stimulated HK-2 cells. Furthermore, we demonstrated that miR-21–5p overexpression reversed the effects on cell viability, inflammatory response, oxidative stress, and NF-κB pathway induced by hsa_circ_0068,888 overexpression in LPS-stimulated HK-2 cells. Overall, these results implied that hsa_circ_0068,888 shows a protective effect on AKI by sponging miR-21–5p. Hence, up-regulation of hsa_circ_0068,888 might be a potential strategy in treatment for AKI.

Hsa_circ_0010957 knockdown attenuates lipopolysaccharide-induced HK2 cell injury by regulating the miR-1224-5p/IRAK1 axis.

Circular RNAs (circRNAs) are involved in the progression of various diseases, including lupus nephritis. Hsa_circ_0010957 is reported to be dysregulated in lupus nephritis, but the exact function of this circRNA is unknown. This research aims to study the function and mechanism of circRNA hsa_circ_0010957 in a lipopolysaccharide (LPS)-induced cellular model of lupus nephritis. Human renal proximal tubular cell line HK2 cells were challenged by LPS. Hsa_circ_0010957, microRNA-1224-5p (miR-1224-5p), and interleukin-1 receptor-associated kinase 1 (IRAK1) abundances were examined by quantitative reverse transcription polymerase chain reaction or western blot. LPS-induced damage was evaluated via cell viability, apoptosis, inflammatory response and oxidative injury. The target interaction was analyzed by dual-luciferase reporter analysis and RNA immunoprecipitation. Hsa_circ_0010957 abundance was enhanced in LPS-challenged HK2 cells. Hsa_circ_0010957 knockdown alleviated LPS-induced apoptosis, the inflammatory response and oxidative injury in HK2 cells. MiR-1224-5p was targeted by hsa_circ_0010957, and miR-1224-5p knockdown reversed the influence of hsa_circ_0010957 silence on LPS-induced injury. IRAK1 was targeted via miR-1224-5p, and hsa_circ_0010957 could regulate IRAK1 by miR-1224-5p. MiR-1224-5p overexpression could mitigate LPS-induced apoptosis, the inflammatory response and oxidative injury, and this effect was abolished by IRAK1. Hsa_circ_0010957 silence weakened LPS-induced HK2 cell apoptosis, the inflammatory response and oxidative injury via regulating the miR-1224-5p/IRAK1 axis.

Dysregulated ion channels and transporters activate endoplasmic reticulum stress in rat kidney of fetal growth restriction

AimsThe mechanisms underlying the fetal origin of renal disease remains unknown. This study aimed to investigate the profiles of ion channel and transporter proteins in the fetal kidney in fetal growth restriction (FGR)rats, and to explore their association with the fetal origin of renal disease. Main methodsAn FGR rat model was developed by administration of a low-protein diet. Then 367 differentially expressed proteins (DEPs) from quantitative proteome analysis were subjected to Ingenuity Pathway Analysis. 22 DEPs associated with ion channels/transporters were evaluated in the fetal kidney. Na+/H+ exchanger1(NHE1) and its downstream unfolded protein response (UPR) pathway were investigated. Furthermore, overexpression of NHE1 were achieved via plasmid transfection to evaluate the potential influence on the UPR pathway and cell apoptosis in human proximal tubular epithelial cell line HK2 cells. Key findingsFindings were as follows: 1) In the FGR fetal kidney, aquaporin 2/4, solute carrier (SLC) 8a1, 33a1, etc. were downregulated, whereas other transporters including SLC 2a1, 4a1, 9a1, 29a3, etc. were upregulated. 2) NHE1 mRNA levels were markedly elevated in the FGR fetus. Further investigation revealed an increase in the UPR pathway regulators. 3) In vitro study showed that NHE1 overexpression in HK2 cells significantly induced expression of the endoplasmic reticulum stress (ERS) regulators and led to a decrease in the anti-apoptotic potential. SignificanceWe speculate that maternal protein malnutrition causes dysregulation of ion channels/transporters in the fetal kidney. Upregulated NHE1 may activate the UPR pathway and induce cell apoptosis thus leading to impairment of kidney function.