Renal Tubule Epithelial Cells Research Articles

CD44-targeted hyaluronic acid-curcumin prodrug protects renal tubular epithelial cell survival from oxidative stress damage

Based on the abnormally increased expression of CD44 receptors on renal tubule epithelial cells during ischemia/reperfusion-induced acute kidney injury (AKI), we developed a hyaluronic acid-curcumin (HA-CUR) polymeric prodrug targeting to epithelial cells and then relieving oxidative stress damages. The water solubility of HA-CUR was significantly enhanced and approximately 27-fold higher than that of CUR. Cellular uptake test showed HA-CUR was preferably internalized by H2O2-pretreated tubular epithelial (HK-2) cells compared with free CUR benefiting from the specific binding between HA and CD44 receptors. Biodistribution results further demonstrated the increased accumulation of HA-CUR in kidneys with 13.9-fold higher than that of free CUR. Pharmacodynamic studies indicated HA-CUR effectively ameliorated AKI, and the exact mechanism was that HA-CUR protected renal tubule epithelial cells from oxidative stress damage via inhibiting PtdIns3K-AKT-mTOR signaling pathway. Taken together, this study provides a new therapeutic strategy for the treatment of AKI based on the pathogenesis of the disease.

Albumin and palmitate overload increases osteopontin production in renal tubule epithelial cells

Osteopontin (OPN) is one of the proinflammatory cytokines upregulated in the kidneys of diabetic animals and patients with diabetic nephropathy (DN). An increase in albuminuria and albumin‐bound fatty acids presents a proinflammatory environment to the proximal tubules in proteinuric kidney diseases including DN. This study was designed to examine if fatty acid overload or albumin itself could stimulate OPN production by renal tubular epithelial cells. Renal tubular OPN expression and secretion were examined in Zucker diabetic (ZD) rats and NRK‐52E cell line exposure to bovine serum albumin (BSA) or BSA overloaded with saturated fatty acid palmitate. Gene microarray and real‐time PCR confirmed an albuminuria‐associated upregulation of OPN transcripts in kidney cortex of ZD rats at the age of 7–20 weeks. Immunofluorescence staining of kidney sections revealed an increase in OPN protein in damaged proximal tubules of diabetic rats. Western blot analysis detected a progressive increase in urinary OPN levels in the ZD rats, which was prevented by antiproteinuric treatment with losartan, an angiotensin II receptor blocker. When exposed to fatty acid‐free bovine serum albumin (BSA, 10 mg/ml), NRK‐52E cells exhibited an increase in OPN protein expression. Palmitate (250 μM) further enhanced albumin‐induced OPN in NRK‐52E cells. These results demonstrate a stimulatory effect of albumin and its conjugated fatty acids on OPN expression in renal tubule epithelial cells. Thus, besides lowering albuminuria/proteinuria, mitigating circulating fatty acid levels may be an important factor in preventing renal inflammation in proteinuric diseases.Support or Funding InformationNIH SC1DK112151, NIH/NCRR/RCMI 8G12MD007602 & 8U54MD007588This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MP81-04 3D RENAL TUBULAR ORGANOIDS GENERATED FROM URINE-DERIVED STEM CELLS

You have accessJournal of UrologyStem Cell Research: Stem Cell Research II1 Apr 2018MP81-04 3D RENAL TUBULAR ORGANOIDS GENERATED FROM URINE-DERIVED STEM CELLS Fangpong Shu, Haibin Guo, Xiong Geng, Anthony Atala, and Yuanyuan Zhang Fangpong ShuFangpong Shu More articles by this author , Haibin GuoHaibin Guo More articles by this author , Xiong GengXiong Geng More articles by this author , Anthony AtalaAnthony Atala More articles by this author , and Yuanyuan ZhangYuanyuan Zhang More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2712AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES In vitro 3D organoids provide an alternative model for studying of renal toxicology. However, to obtain human renal tubular epithelial cells for these propose, a kidney biopsy is required, with potential risks and complications due to this invasive procedure. Development of in vitro renal tissue model has been limited due to the challenges of differentiating stem cells into various functional renal cells. Our previous studies have demonstrated that human urine-derived stem cells (USCs) originate from glomerular parietal stem cells, possess self-renewal and multipotent capacity2. The goal of this study is to induce human USCs to differentiate into renal tubule epithelial cells when grown on kidney extracellular matrix (k-ECM) to generate in vitro 3D renal tubular organoids for potential use as biological models for nephrotoxic drug development and for renal repair. METHODS USCs obtained from 4 healthy individuals were isolated and expanded in vitro. Normal human renal cells were used as control. Porcine-derived k-ECM hydrogen was produced (k-ECM gel) when mixed with hydraulic acid gel (1:9). To generate the renal tubular organoids, USCs were induced within 1% k-ECM gel for one week, compared to USC aggregates without k-ECM gel. The organoids of USCs+k-ECM gel were cultured in Hanging Drop 96-well plates for 4 days and then transferred to ultra-low attachment plates. To optimize the protocols and measure cell viability, different cell concentrations were assessed by ATPase and LIVE/DEAD Kit. The size of organoids and the renal tubule structures were assessed by histology, immunofluorescence staining and Western blot using renal tubular epithelial cell markers. Renal tubular cell function was measured by ?-Glutamyltransferase (GGT) activity after Ethanol as nephrotoxic agent was added into the culture medium. RESULTS 3D organoids (about 350μm at diameter) were generated by 4,000 USCs cultured for 7 days. ATPase and LIVE/DEAD assays showed that majority of USCs (>95%) remained viable up to 4 weeks. H.E. staining demonstrated the renal tubular-like structures formed within the USCs+k-ECM gel, but no within the USC aggregates without k-ECM gel. Induced USCs expressed proximal renal tubule epithelial cells marker (Aquaporin 1, i.e. AQP1) within whole mount staining. In addition, induced USCs expressing GGT was similar to normal renal cells after ethanol treatment. CONCLUSIONS Human USCs induced by k-ECM formed renal tubular organoids, which could provide an alternative tool for nephrotoxic drug screening, renal disease research and kidney tissue repair. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e1098 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Fangpong Shu More articles by this author Haibin Guo More articles by this author Xiong Geng More articles by this author Anthony Atala More articles by this author Yuanyuan Zhang More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Application of Optical Tweezers to Understand the Effect of Renal Ciliary Length Alterations on Ciliary Biomechanics

A renal primary cilium act as a cellular mechanosensor. Fluid flow causes bending of renal cilia resulting in the increased intracellular concentration calcium ion, a second messenger which initiates downstream cell signaling pathways. Better understanding of ciliary biomechanics can help to interpret how ciliary mechanotransduction may be impaired in ciliopathies, such as polycystic kidney disease. Fluid flow in kidney tubules can generate three separate forces: shear and stretch acting on renal tubule wall or epithelial cell layers, and hydrodynamic drag which acts on protruding structures i.e., primary cilia. As compared to direct fluid flow experiments, optical trapping method has the advantage that it can isolate and evaluate only the effects of hydrodynamic drag on ciliary biomechanics, avoiding any background effects of shear and stretch forces acting on cellular layers. Optical tweezers technique is non‐invasive and non‐contact measurement and allow accurate measurement due to the ability to control a trapped primary cilium at a time. Thus, using optical tweezers biomechanical properties of an individual cilium can be correlated with bio‐physiological features of the cilium, such as length and spatial distribution.As reported previously by other research groups, kidney transplantation and ischemia‐reperfusion injury can affect renal ciliary lengths. Previous cell culture studies have shown presence or absence of fluid flow affects ciliary lengths of mouse kidney cortical duct epithelial cells. Stabilization of hypoxia inducible factor (HIF) 1α by cobalt chloride (CoCl2) can affect renal ciliary lengths [1] and can also alter bending modulus of the cilia [2]. We aim to understand the significance of altered ciliary length on the kidney cellular function and diseases. In this study, using optical trapping method we aim to understand if ciliary lengths are related to ciliary biomechanics and flow sensing. We used Mardin‐Darby Canine Kidney (MDCK) epithelial cells which were differentiated by serum starvation to generate primary cilia. We determined the mean square displacement (MSD) values of optically trapped cilia and correlated with ciliary lengths. The MSD value measures the displacement of a particle (i.e., tip of a cilium) from the initial position and long‐time section of MSD relies on the spring constant. MSD values can represent ability of cilia to deflect in response to fluid flow. Currently, we are trying to manipulate ciliary lengths using HIF stabilizer CoCl2 in the presence or absence of HIF inhibitors: chetomin and echinomycin. We anticipate that chetomin or echinomycin may restrict alterations of ciliary length by CoCl2 and may maintain the MSD value of cilia to a base level i.e., without any HIF stabilization. These findings can establish a direct connection between the ciliary length and ciliary flow‐sensing. Thus, this study aims to test whether renal epithelial ciliary lengths are altered in order to adjust the ciliary biomechanical properties or flow‐sensing function as a cellular response to adapt adverse physiological conditions, such as lack of fluid flow and localized hypoxia.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Quantitative Cellular Anatomy of the Rat Kidney

The understanding of renal physiology at a cellular level is progressing rapidly as a result of the recent introduction of techniques for measurement of mRNA levels (RNA‐Seq) and protein levels (protein mass spectrometry) in very small samples consisting of single tubules and single cells. An important question is: How can we use single‐cell and single‐tubule data to predict results of analyses carried out at a whole kidney level, e.g. western blotting? The purpose of this work is to estimate the number of each cell type present in renal tubules of a kidney from a typical 250‐g adult rat (from proximal tubule [PT] to inner medullary collecting duct [IMCD]). For this, we used quantitative structural data gleaned from various 20th century publications. These studies identify the length of each tubule segment, the number of nephrons/collecting ducts, the number of cells per mm and the protein content per mm of each renal tubule segment. The calculations account for differing lengths of loops of Henle, junctions in the connecting tubule (CNT) and IMCD, and different populations of nephrons (short‐looped [71%] and long‐looped [29%]). The calculations reveal that the 38,000 nephrons and their collecting ducts have an aggregate length of 840 meters. The total number of cells from PT to IMCD is 207 million of which 52% are PT cells, 17% are thick ascending limb (TAL) cells, 10% are distal convoluted tubule (DCT) cells, 5% are collecting duct intercalated cells (ICs) and 10% are aquaporin‐2‐expressing cells (PCs). The calculations reveal that the total protein mass of all renal tubule epithelial cells is 119 mg or 40% of the dry weight of the kidney. In terms of protein mass, percentage of individual cell types are: PT, 72%; TAL, 10%; DCT, 7%; ICs, 2%; and PCs, 5%. The average protein masses per cell (pg/cell) are: PT, 804; medullary TAL, 402; cortical TAL, 287; DCT, 425; CNT, 288, cortical collecting duct PC, 212 and IMCD, 300. These calculations provide an illustration of how old data can take on a new significance in light of modern systems biology.Support or Funding InformationThis work was supported by the APS 2017 Frontiers Research Community Leader Fellowship to TR.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of insulin resistance by PGE1 via autophagy-dependent FGF21 pathway in diabetic nephropathy

Insulin resistance is a critical process in the initiation and progression of diabetic nephropathy (DN). Alprostadil (Prostaglandin E1, PGE1) had protective effects on renal function. However, it is unknown whether PGE1 inhibited insulin resistance in renal tubule epithelial cells via autophagy, which plays a protective role in DN against insulin resistance. Insulin resistance was induced by palmitic acid (PA) in human HK-2 cells, shown as the decrease of insulin-stimulated AKT phosphorylation, glucose transporter-4 (GLUT4), glucose uptake and enhanced phosphorylation of insulin receptor substrate 1(IRS-1) at site serine 307 (pIRS-1ser307) and downregulated expression of IRS-1. Along with less abundance of p62, autophagy markers LC3B and Beclin-1 significantly increased in HK-2 cells exposed to PA. Such abnormal changes were significantly reversed by PGE1, which mimicked the role of autophagy gene 7 small interfering RNA (ATG7 siRNA). Furthermore, PGE1 promoted the protein expression of autophagy-related fibroblast growth factor-21 (FGF21), which alleviated insulin resistance. Results from western blotting and immunohistochemistry indicated that PGE1 remarkably restored autophagy, insulin resistance and the FGF21 expression in rat kidney of type 2 diabetes mellitus (T2DM). Collectively, we demonstrated the potential protection of PGE1 on insulin resistance in renal tubules via autophagy-dependent FGF21 pathway in preventing the progression of DN.

The effect of cholesterol overload on mouse kidney and kidney-derived cells

Introduction: Dyslipidemia is one of the onset and risk factors of chronic kidney disease and renal function drop is seen in lipoprotein abnormal animal models. However, the detailed molecular mechanism of renal lipotoxicity has not been clarified. Therefore, the present study aimed to investigate the influence of cholesterol overload using mouse kidney tissue and kidney-derived cultured cells.Methods: C57BL/6 mice were fed normal diet (ND) or 1.25% cholesterol-containing high-cholesterol diet (HCD) for 11 weeks, and we used megalin as a proximal tubule marker for immunohistology. We added beta-very low density lipoprotein (βVLDL) to kidney-derived cells and examined the effect of cholesterol overload on megalin protein and mRNA expression level, cell proliferation and cholesterol content in cells.Results: In the kidney of HCD mice, the gap between glomerulus and the surrounding Bowman’s capsule decreased and the expression level of megalin decreased. After βVLDL treatment to the cells, the protein expression and mRNA expression level of megalin decreased and cell proliferation was restrained. We also observed an increase in cholesterol accumulation in the cell and free cholesterol/phospholipid ratios increased.Conclusions: These findings suggest that the increased cholesterol load on kidney contribute to the decrease of megalin and the overloaded cholesterol is taken into the renal tubule epithelial cells, causing suppression on cell proliferation, which may be the cause of kidney damage.

Polyploidy and Mitotic Cell Death Are Two Distinct HIV-1 Vpr-Driven Outcomes in Renal Tubule Epithelial Cells.

Prior studies have found that HIV, through the Vpr protein, promotes genome reduplication (polyploidy) in infection-surviving epithelial cells within renal tissue. However, the temporal progression and molecular regulation through which Vpr promotes polyploidy have remained unclear. Here we define a sequential progression to Vpr-mediated polyploidy in human renal tubule epithelial cells (RTECs). We found that as in many cell types, Vpr first initiates G2 cell cycle arrest in RTECs. We then identified a previously unreported cascade of Vpr-dependent events that lead to renal cell survival and polyploidy. Specifically, we found that a fraction of G2-arrested RTECs reenter the cell cycle. Following this cell cycle reentry, two distinct outcomes occur. Cells that enter complete mitosis undergo mitotic cell death due to extra centrosomes and aberrant division. Conversely, cells that abort mitosis undergo endoreplication to become polyploid. We further show that multiple small-molecule inhibitors of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, including those that target ATR, ATM, and mTOR, indirectly prevent Vpr-mediated polyploidy by preventing G2 arrest. In contrast, an inhibitor that targets DNA-dependent protein kinase (DNA-PK) specifically blocks the Vpr-mediated transition from G2 arrest to polyploidy. These findings outline a temporal, molecularly regulated path to polyploidy in HIV-positive renal cells.IMPORTANCE Current cure-focused efforts in HIV research aim to elucidate the mechanisms of long-term persistence of HIV in compartments. The kidney is recognized as one such compartment, since viral DNA and mRNA persist in the renal tissues of HIV-positive patients. Further, renal disease is a long-term comorbidity in the setting of HIV. Thus, understanding the regulation and impact of HIV infection on renal cell biology will provide important insights into this unique HIV compartment. Our work identifies mechanisms that distinguish between HIV-positive cell survival and death in a known HIV compartment, as well as pharmacological agents that alter these outcomes.

IL-1β increases urinary corin in patients with primary proteinuric kidney diseases and in 293 cells.

Corin is a serine protease that is important for the regulation of blood pressure and water balance. Corin was initially discovered in the heart, however, it has also been detected in kidney cells, though its function in the kidneys is unclear. To further investigate the function of corin in the kidney, the present study analyzed the levels of corin in urine and blood samples collected from normal individuals and patients with primary proteinuric diseases. The associations between the levels of corin, and the cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were then assessed. The results demonstrated that corin was detectable in the urine and plasma following an enzyme-linked immunosorbent assay; the level of corin in the urine was associated with the level of urinary β2-microglobulin (P=0.01), which was indicative of renal tubular injury. When compared with normal individuals, the levels of urinary corin in proteinuric patients were markedly increased (P=0.02), and were also associated with IL-1β (P=0.03). This correlation between corin and IL-1β was confirmed in vitro using 293 cells. As the IL-1β concentrations increased (0, 0.1, 1, 10 ng/ml), an elevation in the level of corin was observed in the culture medium (P<0.01); however, the amount of corin was not markedly altered in the cell lysate (P>0.05). In addition, when TNF-α reached 10 ng/ml, the level of corin in the medium increased significantly when compared with the control group (0 ng/ml; P=0.02), however, no significant difference in corin levels was detected in the cell lysate. The results suggest that the cytokines IL-1β and TNF-α may increase urinary corin in patients with primary proteinuric kidney diseases. Cytokines may accelerate corin shedding from the cell membrane of renal tubule epithelial cells. These findings indicate that corin may be associated with kidney inflammation and injury.

The Mechanism of Pkcb-P66Shc-Nadph Oxidase Pathway in High Glucose Induced-Oxidative Stress in Renal Tubular Epithelial Cells

Background P66Shc is closely related to oxidative stress, but the exact mechanism of its involvement in diabetic nephro?pathy is poorly understood. Methods In this study, molecular biological methods including western blot analysis, RNA interference, oxygen production measurement and apoptosis assay were applied to explore expressions and functions of P66Shc in damaged renal tubule epithelial cells (HK-2 cells) caused by high glucose levels. Results Increased oxidative stress and up-regulated expression of P66Shc-NADPH oxidase and PKCβ were found in high glucose stimulated HK-2 cells. P66Shc-siRNA alleviated oxidative stress and apoptosis in damaged HK-2 cells. P66Shc-siRNA could significantly down-regulate the expression of NADPH oxidase, but had no effect on the high expression of PKCβ. As a PKCβ inhibitor, enzastaurin inhibited the expressions of p66Shc and NADPH oxidase. Conclusion The study clarified a novel mechanism of oxidative stress damaging HK-2 cells via the modulation of the PKCβ-P66Shc-NADPH oxidase pathway. Understanding the molecular pathway of oxidative stress could translate into the development of therapeutic strategies for diabetic nephropathy. Acknowledgements This work was supported by the National Natural Science Foundation of China [No. 81400300] and Natural Science Foundation of Jiangsu Province [No. BK20130642].

Endocytosis of Albumin Induces Matrix Metalloproteinase-9 by Activating the ERK Signaling Pathway in Renal Tubule Epithelial Cells.

Matrix metalloproteinase-9 (MMP-9) is dysregulated in chronic kidney diseases including diabetic nephropathy. This study was performed to examine the expression of MMP-9 in renal tubule epithelial cells (TECs) under diabetic conditions and its regulatory mechanisms. We characterized MMP-9 protein in diabetic animals and primary cultured rat TECs exposed to exogenous albumin and high glucose. We also used specific inhibitors to determine if internalization of albumin and/or extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation were required for MMP-9 secretion. Immunostaining of kidney sections revealed enhanced MMP-9 signal in the damaged proximal tubules in Zucker diabetic fatty (ZDF) rats. ZDF rats also exhibited an albuminuria-related and age-dependent increase in MMP-9 excretion, which was prevented by rosiglitazone. In primary cultured rat TECs, high glucose exposure did not increase MMP-9 secretion. In contrast, administration of rat serum albumin (RSA, 0.1–0.5 mg/mL) resulted in a dose-dependent increase in MMP-9 expression and secretion by TECs, which was abolished in the presence of an ERK1/2-specific inhibitor, U0126. Simvastatin, an inhibitor of albumin endocytosis, also prevented MMP-9 secretion. Taken together, these results demonstrate that endocytosis of albumin stimulates MMP-9 secretion by TECs through the ERK signaling pathway.

Antimicrobial agents, triclosan, chloroxylenol, methylisothiazolinone and borax, used in cleaning had genotoxic and histopathologic effects on rainbow trout

Triclosan (TRC), chloroxylenol (PCMX) and methylisothiazolinone (MIT) have been commonly used as an antimicrobial in soaps while borax (BRX) is used in household cleaning. After using these chemicals, they are washed down drains and getting into the aquatic ecosystem in which they may affect aquatic living organisms. In the present study, the chronic effects of TRC, PCMX, MIT and BRX on genotoxicity, gene expression and histopathology of rainbow trout (Oncorhynchus mykiss) were evaluated for 40 days under semi static condition. The comet assay results indicated that MIT, TRC and PCMX caused significant DNA damage to erythrocytes of the fish. Transcription of SOD, GPX1, GPX2, GSTA, HSP90BB, HSP90BA, CAT, and HSC70A genes were significantly regulated as a result of TRC, PCMX, MIT, and BRX exposure except PCMX exposed GSTA gene. Histological lesions were detected in gills, spleen liver, and trunk kidney of the fish. Lamellar fusion, hyperplasia and epithelial necrosis in gills, melanomacrophage centers and splenic necrosis in spleen, pyknotic nucleus, fat vacuoles, necrotic hepatocytes in liver, cloudy swelling in the tubules, renal tubule epithelial cells degeneration, glomerular capillaries dilation and glomerulus degeneration in kidney, were observed. Our study demonstrates the chronic toxic effect of TRC, PCMX, MIT, and BRX is high in rainbow trout. Therefore, we should be more careful when using these chemicals for cleaning in order to protect aquatic environment.

Sclt1 deficiency causes cystic kidney by activating ERK and STAT3 signaling.

Ciliopathies form a group of inherited disorders sharing several clinical manifestations because of abnormal cilia formation or function, and few treatments have been successful against these disorders. Here, we report a mouse model with mutated Sclt1 gene, which encodes a centriole distal appendage protein important for ciliogenesis. Sodium channel and clathrin linker 1 (SCLT1) mutations were associated with the oral-facial-digital syndrome (OFD), an autosomal recessive ciliopathy. The Sclt1-/- mice exhibit typical ciliopathy phenotypes, including cystic kidney, cleft palate and polydactyly. Sclt1-loss decreases the number of cilia in kidney; increases proliferation and apoptosis of renal tubule epithelial cells; elevates protein kinase A, extracellular signal-regulated kinases, SMAD and signal transducer and activator of transcription 3 (STAT3) pathways; and enhances pro-inflammation and pro-fibrosis pathways with disease progression. Embryonic kidney cyst formation of Sclt1-/- mice was effectively reduced by an anti-STAT3 treatment using pyrimethamine. Overall, we reported a new mouse model for the OFD; and our data suggest that STAT3 inhibition may be a promising treatment for SCLT1-associated cystic kidney.

AM251 Suppresses Epithelial-Mesenchymal Transition of Renal Tubular Epithelial Cells.

Epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells is one of the causative mechanisms of kidney fibrosis. In our study, we screened lipophilic compounds using a lipid library including approximately 200 lipids to identify those that suppressed EMT induced by a transforming growth factor (TGF)-β1 stimulus. Initial screening was performed with the immortalized HK-2 renal tubule epithelial cell line. The most promising compounds were further tested in RPTEC primary renal tubule epithelial cells. We found that the synthetic lipid AM251 suppressed two hallmark events associated with EMT, the upregulation of collagen 1A1 (COL1A1) and downregulation of E-cadherin. Though AM251 is known to act as an antagonist for the cannabinoid receptor type 1 (CB1) and an agonist for the G protein-coupled receptor 55 (GRP55), the suppression of EMT by AM251 was not mediated through either receptor. Microarray analyses revealed that AM251 inhibited induction of several EMT transcription factors such as SNAIL1, which is the key inducer of EMT, and the AP-1 transcription factors FOSB and JUNB. Activation of SMAD2/3 and p38 mitogen-activated protein kinase (MAPK) was inhibited by AM251, with greater inhibition of the latter, indicating that AM251 acted upstream of SMAD/p38 MAPK in the TGF-β signaling pathway. Our findings regarding the effects of AM251 on the TGF-β signaling pathway may inform development of a novel therapeutic agent suppressing EMT, thus preventing kidney fibrosis.

HIV infection-induced transcriptional program in renal tubular epithelial cells activates a CXCR2-driven CD4+ T-cell chemotactic response.

Viral replication and interstitial inflammation play important roles in the pathogenesis of HIV-associated nephropathy. Cell-cell interactions between renal tubule epithelial cells (RTECs) and HIV-infected T cells can trigger efficient virus internalization and viral gene expression by RTEC. To understand how HIV replication initiates HIV-associated nephropathy, we studied the cellular response of RTECs to HIV, examining the transcriptional profiles of primary RTECs exposed to cell-free HIV or HIV-infected T cells. HIV-induced gene expression in hRTECs was examined in vitro by Illumina RNA deep sequencing and revealed an innate response to HIV, which was subclassified by gene ontology biological process terms. Chemokine responses were examined by CD4 T-cell chemotaxis assays. As compared with cell-free virus infection, exposure to HIV-infected T cells elicited a stronger upregulation of inflammatory and immune response genes. A major category of upregulated genes are chemokine/cytokine families involved in inflammation and immune response, including inflammatory cytokines CCL20, IL6 and IL8-related chemokines: IL8, CXCL1, CXCL2, CXCL3, CXCL5 and CXCL6. Supernatants from virus-exposed RTECs contained strong chemoattractant activity on primary CD4 T cells, which was potently blocked by a CXCR2 antagonist that antagonizes IL8-related chemokines. We observed a preferential migration of CXCR2-expressing, central memory CD4 T cells in response to HIV infection of RTECs. Interactions between primary RTECs and HIV-infected T cells result in potent induction of inflammatory response genes and release of cytokines/chemokines from RTECs that can attract additional T cells. Activation of these genes reflects an innate response to HIV by nonimmune cells.

Effect of propofol on human renal tubule epithelial cell fibrosis induced by ATP depletion/recovery: the role of TAK1

Objective To investigate the effect of propofol on human renal tubule epithelial cell(HK-2 cells)fibrosis induced by ATP depletion/recovery and the role of transforming growth factor β activated kinase 1(TAK1)in it. Methods HK-2 cells were seeded in 96-well plates, and randomly divided into 4 groups(n=36 each)using a random number table: control group(group C), ATP depletion/recovery group(group D/R), propofol group(group P), and TAK1 over-expression group(group T). HK-2 cells were exposed to antimycin A for l h and then returned to normal culture medium to establish the model of ATP depletion/recovery-induced injury.At 1 h before ATP depletion, the cells were incubated for 1 h in the DMEM liquid culture medium containing propofol with the final concentration of 20 μmol/L in group P, and the cells were incubated for 1 h in the DMEM liquid culture medium containing propofol with the final concentration of 20 μmol/L and TAK1 with the titer of 2×107 TU/ml in group T, and the other treatments were similar to those previously described in group D/R.At 12 h after ATP recovery, the cell viability was evaluated by methyl thiazolyl tetrazolium assay, and cell apoptosis was detected using TUNEL and scored.The expression of TAK1 was detected using Western blot at 12, 24 and 48 h after ATP recovery.The expression of α-smooth muscle actin(αSMA), fibronectin(FN), and collagen protein 1(COL1)was measured at 48 h after ATP recovery. Results Compared with group C, the cell viability was significantly decreased, the apoptosis score was increased, and the expression of TAK1, COL1, αSMA and FN was up-regulated after ATP recovery in D/R, P and T groups(P<0.05). Compared with group D/R, the cell viability was significantly increased, the apoptosis score was decreased, and the expression of TAK1, COL1, αSMA and FN was down-regulated after ATP recovery in P and T groups(P<0.05). Compared with group P, the cell viability was significantly decreased, the apoptosis score was increased, and the expression of TAK1, COL1, αSMA and FN was up-regulated after ATP recovery in group T(P<0.05). Conclusion Propofol can reduce HK-2 cell fibrosis induced by ATP depletion/recovery, and the mechanism may be related to down-regulation of TAK1 expression. Key words: Propofol; Reperfusion injury; Kidney; Fibrosis; MAP kinase kinase kinases

Histopathological Evaluation of Dose Dependent Sulfadiazine-Associated Nephrotoxicity and Alteration on Oxidative Stress in Chicken Embryos

Background: Numerous epidemiological and experimental researches indicate that in utero exposure to some environmental chemicals and prescribed drugs during pregnancy can mediate various embryonic abnormalities and complications via reactive oxygen species (ROS) generation, which damages cellular macromolecules. Objectives: The aim of the present study was to evaluate the sulfonamide-associated nephrotoxicity with possible underlying mechanisms in chicken embryo. Materials and Methods: In this experimental study, one hundred fertile eggs were obtained and divided into five groups: 1) control group (without injection), 2) group injected with 2 mg sulfadiazine, 3) group injected with 10 mg sulfadiazine, 4) group injected with 30 mg sulfadiazine and 5) group injected with 70 mg sulfadiazine. After hatching, the renal tissue from the newly hatched chick was harvested for histopathologic investigation and also measurement of oxidative stress parameters [the ferric reducing capacity assay, the glutathione content (GSH) and the situation of lipid peroxidation (LPO)] by spectrophotometer. Results: Histologic examination of the renal tissue revealed that sulfadiazine induces hydropic degeneration, tubular necrosis, glomerular and tubular atrophy, formation of hyaline cast, congestion, hemorrhage, interstitial nephritis and fibrosis. Conclusions: Result showed the dose-dependent administration of sulfadiazine significantly altered the histopathologic structure of renal tissues of chickens. Furthermore, the major histopathologic events in the course of sulfadiazine cytotoxicity are renal tubule epithelial cell necrosis, interstitial nephritis and fibrosis, formation of hyaline cast and congestion and hemorrhage, although sulfadiazine at dose 30 mg and 70 mg caused perturbation in antioxidant defense system by marked increase in LPO, and decrease in GSH.

Autophagy, Innate Immunity and Tissue Repair in Acute Kidney Injury.

Kidney is a vital organ with high energy demands to actively maintain plasma hemodynamics, electrolytes and water homeostasis. Among the nephron segments, the renal tubular epithelium is endowed with high mitochondria density for their function in active transport. Acute kidney injury (AKI) is an important clinical syndrome and a global public health issue with high mortality rate and socioeconomic burden due to lack of effective therapy. AKI results in acute cell death and necrosis of renal tubule epithelial cells accompanied with leakage of tubular fluid and inflammation. The inflammatory immune response triggered by the tubular cell death, mitochondrial damage, associative oxidative stress, and the release of many tissue damage factors have been identified as key elements driving the pathophysiology of AKI. Autophagy, the cellular mechanism that removes damaged organelles via lysosome-mediated degradation, had been proposed to be renoprotective. An in-depth understanding of the intricate interplay between autophagy and innate immune response, and their roles in AKI pathology could lead to novel therapies in AKI. This review addresses the current pathophysiology of AKI in aspects of mitochondrial dysfunction, innate immunity, and molecular mechanisms of autophagy. Recent advances in renal tissue regeneration and potential therapeutic interventions are also discussed.

Bioinformatics analysis of proteomics profiles in senescent human primary proximal tubule epithelial cells

BackgroundDysfunction of renal tubule epithelial cells is associated with renal tubulointerstitial fibrosis. Exploration of the proteomic profiles of senesced tubule epithelial cells is essential to elucidate the mechanism of tubulointerstitium development.MethodsPrimary human proximal tubule epithelial cells from passage 3 (P3) and passage 6 (P6) were selected for evaluation. EdU and SA-β-galactosidase staining were used to detect cell senescence. p53, p21, and p16 were detected by Western blot analysis. Liquid chromatography mass spectrometry (LC-MS) was used to examine differentially expressed proteins (DEPs) between P6 and P3 cells. The expression of DEPs was examined by Western blot analysis. Bioinformatics analysis was performed by protein-protein interaction and gene ontology analyses.ResultsThe majority of tubule cells from passage 6 (P6) stained positive for SA-β-galactosidase, whereas passage 3 (P3) cells were negative. Senescence biomarkers, including p53, p21, and p16, were upregulated in P6 cells relative to P3 cells. EdU staining results showed a lower rate of EdU positive cells in P6 cells than in P3 cells. LC-MS was used to examine DEPs between P6 and P3 cells. These DEPs are involved in glycolysis, response to stress, cytoskeleton regulation, oxidative reduction, ATP binding, and oxidative stress. Using Western blot analysis, we validated the down-regulation of AKR1B1, EEF2, EEF1A1, and HSP90 and the up-regulation of VIM in P6 cells seen in the LC-MS data. More importantly, we built the molecular network based on biological functions and protein-protein interactions and found that the DEPs are involved in translation elongation, stress, and glycolysis, and that they are all associated with cytoskeleton regulation, which regulates senescent cell activities such as apoptosis and EMT in tubule epithelial cells.ConclusionsWe explored proteomic profile changes in cell culture-induced senescent cells and built senescence-associated molecular networks, which will help to elucidate the mechanisms of senescence in human proximal tubule epithelial cells.

The expressions of intercellular adhesion molecule-1 in renal tissue of rats with paraquat poisoning and the effect of melatonin

To study the mechanism of paraquat (PQ) -induced renal injury in rats, the expression changes of ICAM-1 to assess the protective effect of Melatonin in PQ poisoning. Ninety adult healthy Sprague-Dawley (SD) rats were divided into three groups at random. 30 rats; Poisoned group: 30 rats; Melatonin group: 30 rats. Control group and Poisoned group were treated intragastrically with 1 ml of PQ (50 mg/kg) diluted with normal saline. Control groupwere treated with the same dose of normal saline as Poisoned group and Melatonin group. Melatonin group were given 1 ml of Melatonin at a dose of 10 mg/kg diluted with normal saline (once daily, intraperitoneally) Control and Poisoned group were treated with the same dose of normal saline (once daily, intraperitoneally) as Melatonin group. Pathology of renal tissue were oberserved by HE staining, and electron microscope. The histopathological changes and the expression of ICAM-1 were observed with mmunohistochemistry (IHC). (1) There were no obvious pathological changes in Control group. Poisoned group Renal glomerulus had hyperemia and distension.Renal tubule epith elial cell had edema and vacuolar degeneration and renal tubule lumina was narrowing on day 1, There were serious edema exudation and necrosis on day 5,which gradually lessened furthermore; Compared with Poisoned group, the aforementioned pathological lesion was more palliative in Melatonin group. (2) No obvious abnomal changes in ultrastructure of renal tissues in Control group. There were swelling of mitochondrion and rupture of renal tubule epithelial cell and endoplasmic reticulum had extension, lysosome was mult and had much phagocytosis in Poisoned group. (3) There was a very weak expression of ICAM-1 in Control group. while in Poisoned group, there was already a significant higher expression of ICAM-1 of renal tubule on day 1 after PQ poisoning, Immunohistochemistry score (IHS) of Poisoned group on day 1, 3, 5, 7, 14 were (0.1561±0.0295、0.2572±0.0259、0.3028±0.0153、0.2083±0.0227、0.9309±0.0059) , compared with Control group (P<0.01) ; Melatonin group were (0.1259±0.0061、0.2109±0.0280、0.2679±0.0233、0.1771±0.0186、0.0791±0.0135) , compared with Control group (P<0.01) , compared with Poisoned group (P<0.05) ; ICAM-1 was involved in the procedures of renal injury; MT surely had a protective effect, which might be mediated by ICAM-1 in the paraquat-induced renal injury, but its regulation path still need a further exploration.