Rat Proximal Tubular Epithelial Cells Research Articles

Farnesoid X receptor activation protects against renal fibrosis via modulation of β-catenin signaling

ObjectiveActivation of farnesoid X receptor (FXR), a bile acid nuclear receptor, may be implicated in the pathophysiology of diabetic nephropathy. We explored a possible role for FXR activation in preventing renal fibrosis in high fat diet (HFD)-fed mice. MethodsWe investigated the effects of HFD on mouse kidney and renal tubular epithelial cells both in vivo and in vitro, and observed the changes of FXR and β-catenin pathway. FXR agonist was also used to alleviate this HFD-induced effect, and the interaction between FXR and β-catenin was further verified. ResultsMice were fed by a 60% kcal fat diet for 20 weeks developed the typical traits of metabolic syndrome with subsequent renal lipid accumulation and renal injury. Treatment with the FXR agonist CDCA or GW4064 decreased body weight, renal lipid accumulation, as well as renal injury. Moreover, renal β-catenin signaling was activated and improved with FXR-agonist treatment in HFD-fed mice. To examine whether FXR affected β-catenin signaling, and was involved in tubulo-interstitial fibrosis, we explored the FXR expression and function in ox-LDL induced-renal tubular injury. In rat proximal tubular epithelial cells (NRK-52E) stimulated by ox-LDL, FXR protein was decreased compared to control group, and phosphorylated (Ser675) β-catenin was activated by ox-LDL in a dose- and time-dependent manner. Ox-LDL enhanced α-SMA and fibronectin expressions and reduced E-cadherin levels, whereas FXR agonism or FXR overexpression inhibited fibronectin and α-SMA expressions and restored E-cadherin. Moreover, FXR agonist treatment also decreased phosphorylated (Ser675) β-catenin, nuclear translocation and β-catenin-mediated transcription induced by ox-LDL in NRK-52E cells. We showed that FXR could bind with β-catenin via the AF1 domain, and disrupt the assembly of the core β-catenin/TCF4 complex. ConclusionThese experimental data suggest that FXR activation, via modulating β-catenin signaling, may contribute to attenuating the development of lipid-mediated tubulo-interstitial fibrosis.

Human umbilical cord mesenchymal stem cell exosomes alleviate acute kidney injury by inhibiting pyroptosis in rats and NRK-52E cells

Human umbilical cord mesenchymal stem cells (hucMSCs) have been shown to improve kidney injury. Exosomes have been indicated to be important mediators of renal protection in MSC therapy. In spite of this, the mechanism remains unclear. Our study investigated how exosomes derived from hucMSCs (hucMSC-Ex) improve acute kidney injury (AKI). Exosomes were extracted by using an ultracentrifugation technique and identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot. Twenty-four male SD rats were randomly divided into four groups: sham group, sham + hucMSC-Ex group, ischemia–reperfusion injury (IRI) group, and IRI + hucMSC-Ex group. In vitro, we treated rat proximal renal tubular epithelial cell line (NRK-52E) with cisplatin to mimic in vivo models of AKI. The NRK-52E cells were treated with or without 160 μg/mL hucMSC-Ex, and 1 µg/mL cisplatin was added after 9 h. Cells were harvested after 24 h. In the IRI group, the levels of serum creatinine (Scr) and blood urea nitrogen (BUN) were increased; renal tubules were dilated, epithelial cells were vacuolated, and collagen fibers were deposited in the renal interstitium. After treatment with cisplatin, the NRK-52E cells displayed pyroptotic morphology characterized by pyroptotic bodies. The protein expression levels of fibronectin, α-smooth muscle actin (α-SMA), vimentin, gasdermin D (GSDMD), caspase-1, interleukin-1 (IL-1β) and NLRP3 in IRI tissues and in cisplatin treatment NRK-52E cells were significantly upregulated. However, after the hucMSC-Ex intervention, kidney injury was effectively improved in vivo and in vitro. The current study shows that pyroptosis is involved in AKI and that hucMSC-Ex improves AKI by inhibiting pyroptosis.

PD31-06 THE EFFECT OF KIDNEY PRESERVATION AT 10°C WITH HEMOPURE AND HYDROGEN SULFIDE DONOR, SODIUM THIOSULFATE, IN A SYNGENEIC RAT RENAL TRANSPLANTATION MODEL

You have accessJournal of UrologyCME1 Apr 2023PD31-06 THE EFFECT OF KIDNEY PRESERVATION AT 10°C WITH HEMOPURE AND HYDROGEN SULFIDE DONOR, SODIUM THIOSULFATE, IN A SYNGENEIC RAT RENAL TRANSPLANTATION MODEL Maria Abou Taka and Alp Sener Maria Abou TakaMaria Abou Taka More articles by this author and Alp SenerAlp Sener More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003324.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Renal transplantation is the preferred treatment for patients with end-stage renal disease. Yet, donor kidney demand outweighs supply. Static cold storage (SCS) at 4°C is the current gold standard for renal preservation. SCS contributes to kidney damage through renal ischemia-reperfusion injury (IRI) that involves cellular death and inflammation. In porcine models of renal transplantation, we added non-FDA-approved hydrogen sulfide (H2S) donor, AP39, to a bovine-based blood substitute, Hemopure, at 21°C and 37°C, which improved renal graft quality; though, the experimental setup was costly to meet renal metabolic demand. In rats, we investigated sodium thiosulfate (STS), an FDA-approved H2S donor, at 4°C and observed similar benefits. However, there is still a risk of 4°C cold IRI. Recent studies showed that 10°C human organ graft preservation enhanced patient survival without requiring extensive oxygen during preservation. Therefore, we hypothesize that preservation solutions with STS and Hemopure at 10°C will reduce renal IRI compared to SCS. METHODS: Using an in vitro model of rat renal IRI, we evaluated STS use at 4°C, 10°C, 21°C, and 37°C. We treated rat proximal tubular epithelial cells with 150 µM STS for 24 hours in hypoxic conditions to mimic ischemia, and 24 hours in normoxic conditions to mimic reperfusion. To assess cellular viability, we used flow cytometry and stained cells with FITC-Annexin-V and PerCP-Propidium Iodide to determine apoptosis and necrosis levels, respectively. RESULTS: STS treatment significantly enhanced cellular viability at 10°C compared to 4°C, 21°C, and 37°C, as determined by the percentage of cells negatively stained with FITC-Annexin-V and PerCP-Propidium Iodide. STS treatment also significantly decreased apoptotic and necrotic cells at 10°C compared to 4°C, 21°C, and 37°C, as shown by the percentage of cells positively stained with FITC-Annexin-V and PerCP-Propidium Iodide. CONCLUSIONS: Overall, 10°C STS treatment significantly protects rat proximal tubular epithelial cells from IRI, suggesting that 10°C kidney preservation may enhance renal graft survival, especially in a cost-effective manner, compared to other preservation temperatures currently used in transplantation. Source of Funding: Ontario Graduate Scholarship, Department of Surgery Internal Research Funds, Lawson Internal Research Fund © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e902 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Maria Abou Taka More articles by this author Alp Sener More articles by this author Expand All Advertisement PDF downloadLoading ...

Effect of High-altitude Hypoxia Environment on Uric Acid Excretion, Desmin Protein Level in Podocytes, and Na+-K+- ATPase Activity.

To investigate the oxidative stress and adaptive compensation of kidneys in rats in high-altitude hypoxia environments, 20 Wistar rats (3 months) were randomly and equally grouped. The rats in the test group were fed in a low-pressure oxygen chamber, and those in the control group (controls) were fed in a normal environment. On the 5th, 10th, 20th, and 30th day, the excretion of uric acid in rats was detected by a biochemical analyzer, the level of desmin protein in rat podocytes was detected by immunohistochemistry, and the activity of Na+-K+- ATPase in rat proximal tubular epithelial cells was measured by liquid scintillation method. The results showed that with the increased time, the level of uric acid in the blood of rats in the test group increased dramatically (P<0.05). On the 30th day, the blood uric acid content of the test group was 52.33μmol/L, and that of the control group was 38.43μmol/L. The blood uric acid content in the test group was dramatically increased relative to the control group. Immunohistochemistry showed that the desmin protein in podocytes of the test group (0.14) was considerably higher than that in the control group (P<0.05). The Na+-K+- ATPase activity of proximal renal tubular epithelial cells in the test group was 611.2 pmol pi/mg protein/h, which was considerably lower than the versus control group (P<0.05). In summary, in high altitude hypoxia environment, uric acid accumulated in the body, and renal filtration and excretion ability was limited.

Ovarian Hormone‐Dependent miR‐146b‐5p Regulation of Plasminogen Activator Inhibitor‐1 in Renal Epithelial Cells

Chronic kidney disease (CKD) progression differs in males and females and the influence of gonadal hormones on pathology are incompletely understood. We utilized the 5/6 nephrectomy (5/6Nx) rat model, which recapitulates hallmarks of human CKD, to study differences in regulation of CKD pathology between sexes. We previously found that genomic deletion of microRNA 146b‐5p (miR‐146b) results in a sex‐dependent phenotype in 5/6Nx rats. Male miR‐146b‐/‐ rats develop renal pathology similar to their WT counterparts. However, female miR‐146b‐/‐ rats experience markedly exacerbated renal pathology relative to WT, 5/6Nx, females as well as WT and miR‐146b‐/‐, male, 5/6Nx rats. These sex differences are abolished by ovary removal, suggesting that renoprotective effects of female gonadal hormones are miR‐146b dependent. Our objective is to identify a molecular mechanism behind these effects. PAI‐1 has previously been shown to be regulated by miR‐146b‐5p isomiR, miR‐146a‐5p, which shares a common seed sequence. PAI‐1 expression is also known to be ovarian hormone dependent, its renal expression is low in health, but elevated in many disease settings, where it may contribute to remodeling. We hypothesized that PAI‐1 expression in rat proximal tubular epithelial cells (NRK‐52E) will be dependent upon (1) the presence of miR‐146b, (2) ovarian hormones, and (3) transforming growth factor beta (TGFβ), which is elevated in our 5/6Nx rats and known to contribute to renal pathology. To aid in testing our hypothesis we used CRISPR‐Cas9 technology to generate miR‐146b knockout rat proximal tubule epithelial (NRK‐52E) cells. Results. PAI‐1 mRNA was highly elevated in untreated miR‐146b KO rat NRK‐52E cells relative to WT NRK‐52E cell controls (11.25 ± 5.3 vs. 1.00 ± 0.68, P&lt;0.05). Conversely, WT NRK‐52E cells treated with pre‐miR‐146b had significantly reduced PAI‐1 mRNA expression (0.137 ± 0.14 vs. 1.00 ± 0.45, P&lt;0.05). Treatment of WT NRK‐52E cells with β‐estradiol did not significantly alter PAI‐1 mRNA expression relative to untreated controls, whereas treatment with progesterone (P4) significantly increased it. Treatment of WT NRK‐52E cells with TGFβ resulted in a significant increase of PAI‐1 mRNA expression. Lastly, treatment of WT NRK‐52E cells with P4 and TGFβ combined resulted in a significant increase in PAI‐1 mRNA expression, and this expression was significantly reduced by transfection with pre‐miR‐146b. Collectively, these observations demonstrate that miR‐146b, P4, and TGFβ regulate PAI‐1 expression in NRK‐52E cells and thereby suggest PAI‐1 as a mechanistic candidate behind the sex‐specific effects of miR‐146b in CKD.

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.

Renal protective effects and mechanisms of the angiotensin receptor-neprilysin inhibitor LCZ696 in mice with cardiorenal syndrome

AimsThis study investigated the renal protective effects and mechanisms of angiotensin receptor-neprilysin inhibitor LCZ696 in mice with cardiorenal syndrome. Materials and methodsMice were divided into abdominal aortic ligation alone, or treatment with LCZ696 or valsartan, whilst those undergoing sham surgery served as controls. Rat proximal renal tubular epithelial cells from the NRK-52E line were treated with control solution, LCZ696 or valsartan, in the presence or absence of Ang II for 24 h. Key findingsCompared to controls, abdominal aortic ligation significantly increased plasma NT-proBNP and urine neutrophil gelatinase-associated lipocalin (NGAL), which were associated with reduced renal length and velocity time integral on ultrasonography. Histology revealed wrinkling of the glomerular capillary wall and sclerosis of the glomerulus, dilatation of the Bowman's capsule, accompanied by diffuse renal tubular atrophy and fibrosis, accompanied by lower kidney index and higher percentage area of fibrosis. Increases in NGAL and decreased ANP protein and mRNA expression levels were observed. These abnormalities were significantly prevented by LCZ696 and to a lesser extent by valsartan. Cellular experiments demonstrated a central role of Ang II/transforming growth factor-β1/Smad2/3/connective tissue growth factor-dependent signaling leading to type IV collagen deposition. This upregulation was reversed by LCZ696 in a greater extent than valsartan treatment alone, accompanied by a significant improvement in NGAL. SignificanceLCZ696 can reduce kidney injury to a level beyond valsartan therapy alone in mice with cardiorenal syndrome, which can be speculated by effects on epithelial-mesenchymal transition and fibrosis through downregulating the TGF-β1/Smad2/3/CTGF/Collagen IV pathway.

MicroRNA-29b prevents renal fibrosis by attenuating renal tubular epithelial cell\u2013mesenchymal transition through targeting the PI3K/AKT pathway

PurposeThis study aimed to investigate the effects of miR-29b on renal interstitial fibrosis in the obstructed kidney of mouse with unilateral ureteral obstruction (UUO) via inhibiting phosphatidylinositol 3-kinase/protein kinaseB (PI3K/AKT) signaling pathway.MethodsAdult male CD-1 mice were intraperitoneally injected with vehicle or PI3K inhibitor LY294002 (3 mg/kg, 30 mg/kg) daily for 1 or 2 weeks after performing UUO or sham operation. The mice were sacrificed on days 7 and 14 after surgery. The rat proximal tubular epithelial cell (TEC) line NRK-52E was cultured in DMEM and treated with various concentrations angiotensin II (AngII). Obstructed and sham mouse kidneys were analyzed via HE, Masson and immunohistochemistry to assess the degree of renal fibrosis. Real-time quantitative polymerase chain reaction assays (RT-PCR) were performed to investigate changes in the levels of expression of miR-29b and Western blot was used to analyze the activation of PI3K/AKT signaling and expression of E-cadherin, α-smooth muscle actin (α-SMA).ResultsHistologic analyses of obstructed kidney revealed that LY294002 attenuated the degree of renal fibrosis. In this study, loss of miR-29b accompanied with increased epithelial–mesenchymal transition (EMT) was observed in renal tubules of mice after UUO and cultured NRK-52E cells exposed to AngII. LY294002 also prominently decreased phosphorylation of AKT in vivo and vitro. By RT-PCR and Western blot analysis, LY294002 blocked the PI3K/AKT-induced loss of E-cadherin expression and de novo increase of the expression of α-SMA in a time- and dose-dependent manner. The overexpression of miR-29b markedly reversed the phenotype induced by AngII in NRK-52E cells and the downregulation miR-29b expression with an miR-29b inhibitor resulted in enhanced EMT. In addition, the PI3K/AKT signaling pathway was found to be suppressed in the presence of overexpression of miR-29b by direct hybridization with 3′-untranslated region (3′-UTR) of PIK3R2.ConclusionOur findings suggested that miR-29b significantly prevented tubulointerstitial injury in mouse model of UUO by attenuating renal tubular epithelial cell–mesenchymal transition via repressing PI3K/AKT signaling pathway.

Saturated fatty acid stimulates production of extracellular vesicles by renal tubular epithelial cells.

Lipotoxicity, an accumulation of intracellular lipid metabolites, has been proposed as an important pathogenic mechanism contributing to kidney dysfunction in the context of metabolic disease. Palmitic acid, a predominant lipid derivative, can cause lipoapoptosis and the release of inflammatory extracellular vesicles (EVs) in hepatocytes, but the effect of lipids on EV production in chronic kidney disease remains vaguely explored. This study was aimed to investigate whether palmitic acid would stimulate EV release from renal proximal tubular epithelial cells. Human and rat proximal tubular epithelial cells, HK-2 and NRK-52E, were incubated with 1% bovine serum albumin (BSA), BSA-conjugated palmitic acid (PA), and BSA-conjugated oleic acid (OA) for 24-48h. The EVs released into conditioned media were isolated by ultracentrifugation and quantified by nanoparticle-tracking analysis (NTA). According to NTA, the size distribution of EVs was 30-150nm with similar mode sizes in all experimental groups. Moreover, BSA-induced EV release was significantly enhanced in the presence of PA, whereas EV release was not altered by the addition of OA. In NRK-52E cells, PA-enhanced EV release was associated with an induction of cell apoptosis reflected by an increase in cleaved caspase-3 protein by Western blot and Annexin V positive cells analyzed by flow cytometry. Additionally, confocal microscopy confirmed the uptake of lipid-induced EVs by recipient renal proximal tubular cells. Collectively, our results indicate that PA stimulates EV release from cultured proximal tubular epithelial cells. Thus, extended characterization of lipid-induced EVs may constitute new signaling paradigms contributing to chronic kidney disease pathology.

Telmisartan attenuates diabetic nephropathy progression by inhibiting the dimerization of angiotensin type-1 receptor and adiponectin receptor-1

AimsThe heterodimerization of angiotensin II receptors (AT1R and AT2R) with adiponectin receptor AdipoR1 and AdipoR2 may instigate high glucose (HG)-induced renal tubulointerstitial injury. This study examined the effect of telmisartan on diabetic nephropathy (DN) and its underlying mechanism. Main methodsDiabetes was induced in rats through a single intraperitoneal injection of streptozotocin. Diabetic rats treated with or without the intravenous injection of AdipoR1 siRNA were intragastrically administered with 5 mg/kg/d telmisartan or a vehicle for 12 weeks. The rat proximal tubular epithelial cell line NRK-52E was treated with HG (30 mmol/L) with or without telmisartan (10 μM) for 48 h. Key findingsIn streptozotocin-induced diabetic rats, telmisartan treatment could decrease the inulin clearance rate, restore the glomerular surface area and mesangial area, alleviate renal fibrosis, and decrease urinary albumin excretion. Furthermore, diabetic rats exhibited increased AT1R-AdipoR1 heterodimers in the renal tubular compartment, which could be attenuated by telmisartan treatment, accompanied by a decrease in the expression level of cytokines MIP-1α, ICAM-1 and MCP-1. In vitro, HG promoted the dimerization formation of AT1R-AdipoR1 in cultured NRK-52E cells, but this effect was not found in NRK-52E cells transfected with the AdipoR1-G269E,G273E mutant. Telmisartan could inhibit HG-induced AT1R-AdipoR1 dimerization, downregulate the expression levels of inflammatory cytokines, and alleviate cell apoptosis in NRK-52E cells. Furthermore, AdipoR1 knockdown could abate the renoprotective benefits of telmisartan. SignificanceThe heterodimerization of AT1R-AdipoR1 probably contributes to the renal injury of DN, and provides an additional mechanistic insight into how telmisartan prevents the development and progression of DN.

Microvesicles containing miR-34a induce apoptosis of proximal tubular epithelial cells and participate in renal interstitial fibrosis.

Function and potential mechanism of microvesicles (MVs) containing microRNA34a in renal interstitial fibrosis were investigated. A rat model of renal interstitial fibrosis was established by unilateral ureteral ligation (UUO). Rat proximal tubular epithelial cell line (NRK-52E) was used to explore the effect of MVs containing microRNA-34a on tubular epithelial cells during fibrosis, which were secreted by tubulointerstitial fibroblasts. Regardless of the UUO renal interstitial fibrosis model, or the TGF-β1-treated renal tubular epithelial cells, microRNA-34a was increased in the MVs secreted by tubulointerstitial fibroblasts. miR-34a could be transmitted through the damaged tubule basement membrane to proximal tubular epithelial cells, where it induced apoptosis of renal tubular epithelial cells by inhibiting the expression of Bcl-2, further aggravating renal interstitial fibrosis. MicroRNA-34a secreted by damaged renal interstitial fibroblasts can promote renal tubular epithelial cell apoptosis and participate in renal interstitial fibrosis by inhibiting Bcl-2.

Regulation of PTEN/AKT/FAK pathways by PPARγ impacts on fibrosis in diabetic nephropathy.

Renal tubular epithelial-to-mesenchymal transition (EMT) and tubulointerstitial fibrosis (TIF) are important pathological features of diabetic nephropathy (DN). However, the regulatory mechanism underlying EMT and TIF are still unclear. Previous studies showed that the decrease in the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) was closely related to the aggravation of DN, but no published study showed how PTEN participated in the regulation of EMT and TIF. In this study, the rat proximal tubular epithelial cells (NRK52E) and C57BL mice and human kidney tissues were used as the research objects to investigate the mechanism underlying the regulatory effect of peroxisome proliferator-activated receptors γ (PPARγ) on PTEN and its influence on EMT and TIF, the regulation of PTEN's dual activity of lipid phosphatase/protein phosphatase by the serine threonine protein kinase B(AKT)/focal adhesion kinase (FAK) signaling pathway, and the role of PTEN in EMT and TIF. The results showed that PPARγ regulated the expression of PTEN at a transcriptional level and further regulated EMT and TIF. This dual activity could regulate the phosphorylation level of AKT and FAK and also affect FAK transcription. However, the 129 mutant of PTEN (PTEN-G129E) lost the lipid phosphatase activity, and its protein phosphatase activity was involved only in EMT and renal fibrosis through regulating FAK phosphorylation. This study systematically elucidated the role of PPARγ/PTEN/AKT/FAK signaling pathway in EMT and TIF during the pathogenesis of DN.

The Protective Effect of Fluorofenidone against Cyclosporine A-Induced Nephrotoxicity

Background/Aims: Cyclosporine A (CsA) is an immunosuppressant drug that is used during organ transplants. However, its utility is limited by its nephrotoxic potential. This study aimed to investigate whether fluorofenidone (AKF-PD) could provide protection against CsA-induced nephrotoxicity. Methods: Eighty-five male Sprague-Dawley rats were divided into 5 groups: drug solvent, CsA, CsA with AKF-PD (250, 500 mg/kg/day), and CsA with pirfenidone (PFD, 250 mg/kg/day). Tubulointerstitial injury index, extracellular matrix (ECM) deposition, expression of type I and IV collagen, transforming growth factor (TGF)-β1, platelet-derived growth factor (PDGF), Fas ligand (FASL), cleaved-caspase-3, cleaved-poly(ADP-ribose) polymerase (PARP)-1, and the number of transferase-mediated nick end-labeling (TUNEL)-positive renal tubule cells were determined. In addition, levels of TGF-β1, FASL, cleaved-caspase-3, cleaved-PARP-1, and number of annexin V-positive cells were determined in rat proximal tubular epithelial cells (NRK-52E) treated with CsA (20 μmol/L), AKF-PD (400 μg/mL), PFD (400 μg/mL), and GW788388 (5 μmol/L). Results: AKF-PD (250, 500 mg/kg/day) significantly reduced tubulointerstitial injury, ECM deposition, expression of type I and IV collagen, TGF-β1, PDGF, FASL, cleaved-caspase-3, cleaved-PARP-1, and number of TUNEL-positive renal tubule cells in the CsA-treated kidneys. In addition, AKF-PD (400 μg/mL) significantly decreased TGF-β1, FASL, cleaved-caspase-3, and PARP-1 expression in NRK-52E cells and further reduced the number of annexin V-positive cells. Conclusion: AKF-PD protect kidney from fibrosis and apoptosis in CsA-induced kidney injury.

3,2-Hydroxypyridinone-Grafted Chitosan Oligosaccharide Nanoparticles as Efficient Decorporation Agents for Simultaneous Removal of Uranium and Radiation-Induced Reactive Oxygen Species in Vivo.

Most of the key radionuclides in the nuclear fuel cycle, such as actinides, possess a combination of heavy metal chemotoxicity and radiotoxicity and therefore represent a severe threat to the ecological environment and public safety. The radiotoxicity originates from direct radiation-induced organ damage and indirect damage, mostly through radiation-induced reactive oxygen species (ROS). Although effective chelating agents that can accelerate the excretion of actinides, such as uranium, have been developed in the past several decades, very few of them can reduce radiation-induced damage from internal contamination. In fact, the strategy of simultaneous removal of actinides and their induced-ROS in vivo has scarcely been considered. Here, we report a 3,2-hydroxypyridinone-grafted chitosan oligosaccharide nanoparticle (COS-HOPO) as a new type of decorporation agent that is effective for the removal of both uranium and ROS in vivo. The cytotoxicity and decorporation assays indicate that the marriage of chitosan oligosaccharide (COS) and hydroxypyridinone (HOPO) gives rise to a remarkable decrease in toxicity and promotion of the uranium removal capability from both kidneys and femurs. The decorporation efficacy can reach up to 43% in rat proximal tubular epithelial cells (NRK-52E), 44% in kidneys, and 32% in femurs. Moreover, the ROS levels of the cells treated with COS-HOPO are significantly lower than those of the control group, implying a promising radiation protection effect. The detoxification mechanism of COS-HOPO is closely related to both chelating U(VI)- and scavenging U(VI)-induced intracellular ROS.

MiR-29b regulates Ang II-induced EMT of rat renal tubular epithelial cells via targeting PI3K/AKT signaling pathway.

Renal interstitial fibrosis is a necessary step in the progression of chronic kidney to end stage renal disease. MicroRNA-29 (miR-29) has been shown to play essential roles in epithelial-mesenchymal transition (EMT), and thus may contribute to the regulation of renal interstitial fibrosis. However, the role of miR-29 in the regulation of EMT during chronic kidney disease and renal transplantation has been a source of intense debate, and the mechanisms underlying this process are incompletely understood. In this study, we investigated the function of miR-29b in the regulation of EMT and to gain a better understanding of the mechanism by which miR-29b modulates EMT by targeting the phosphatidylinositol 3-kinase/protein kinaseB (PI3K/AKT) signaling pathway during the process of renal interstitial fibrosis. The rat proximal tubular epithelial cell line NRK-52E was cultured in DMEM and treated with angiotensinII(AngII) at various concentrations. RT-PCR was performed to investigate changes in the levels of expression of miR-29b in NRK-52E cells and western blotting was used to analyze the expression of PI3K, p-AKT, vimentin and keratin18. The result of the study show that treatment of NRK-52E cells with AngII induced the transition of the cellular phenotype from epithelial to mesenchymal and upregulated the PI3K/AKT signaling pathway; this was also found following treatment with a phosphatase and tensin homolog on chromosome10(PTEN)-specific inhibitor. Increased expression of miR-29b was able to reverse the phenotype induced by AngII in NRK-52E cells and blocking miR-29b activity with an miR-29b inhibitor resulted in enhanced EMT. Additionally, the PI3K/AKT signaling pathway was found to be suppressed in the presence of enhanced expression of miR-29b by direct binding to 3'-untranslated region(3'-UTR) of PIK3R2. We concluded that miR-29b plays an important role in the negative regulation of AngII-induced EMT via PI3K/AKT signaling pathway and propose that enhancing miR-29b level or blocking PI3K/AKT signaling pathway may be a novel therapeutic target in renal interstitial fibrosis.

Calcitriol Ameliorates AngiotensinII-Induced Renal Injury Partly via Upregulating A20.

Inflammation and reactive oxygen species (ROS) play crucial roles in the progression of chronic kidney diseases. Vitamin D has been shown anti-inflammatory effects, but the underlying mechanism is not fully understood. Here, we investigated whether calcitriol exerts protective effects via upregulating A20 in angiotensinII (AngII)-induced renal injury. Male C57BL/6 mice were infused with vehicle or AngII for 10days. Calcitriol reduced infiltration of T lymphocytes and macrophages. This reduction of inflammatory cells was accompanied by elevated A20 and decreased pro-inflammatory cytokines (PICs) and reactive oxygen species (ROS). Calcitriol could inhibit NF-κB activation and necroptotic pathway. Induction of A20 was located primarily to the tubular epithelial cells. In rat proximal tubular epithelial cells (NRK-52E), calcitriol stably upregulated A20 and reduced the PICs and ROS. Inhibitory effect of A20 on PICs and ROS depended on suppressing NF-κB pathway and necroptotic pathway, respectively. A20 knockdown diminished the effect of calcitriol on suppressing NF-κB and necroptotic pathways. However, A20 deficiency could not abrogate the inhibitory effect of calcitriol on NF-κB and necroptotic pathways. Our results established that A20 is involved in the renoprotective effect by calcitriol via negatively modulating the NF-κB pathway and necroptotic pathway in AngII-induced renal injury.

Intermedin attenuates renal fibrosis by induction of heme oxygenase-1 in rats with unilateral ureteral obstruction

BackgroundIntermedin [IMD, adrenomedullin-2 (ADM-2)] attenuates renal fibrosis by inhibition of oxidative stress. However, the precise mechanisms remain unknown. Heme oxygenase-1 (HO-1), an antioxidant agent, is associated with antifibrogenic effects. ADM is known to induce HO-1. Whether IMD has any effect on HO-1 is unclear. Herein, we determined whether the antifibrotic properties of IMD are mediated by induction of HO-1.MethodsRenal fibrosis was induced by unilateral ureteral obstruction (UUO) performed on male Wistar rats. Rat proximal tubular epithelial cell line (NRK-52E) was exposed to rhTGF-β1 (10 ng/ml) to establish an in vitro model of epithelial-mesenchymal transition (EMT). IMD was over-expressed in vivo and in vitro using the vector pcDNA3.1-IMD. Zinc protoporphyrin (ZnPP) was used to block HO-1 enzymatic activity. IMD effects on HO-1 expression in the obstructed kidney of UUO rat and in TGF-β1-stimulated NRK-52E were analyzed by real-time RT-PCR, Western blotting or immunohistochemistry. HO activity in the obstructed kidney, contralateral kidney of UUO rat and NRK-52E was examined by measuring bilirubin production. Renal fibrosis was determined by Masson trichrome staining and collagen I expression. Macrophage infiltration and IL-6 expression were evaluated using immunohistochemical analysis. In vivo and in vitro EMT was assessed by measuring α-smooth muscle actin (α-SMA) and E-cadherin expression using Western blotting or immunofluorescence, respectively.ResultsHO-1 expression and HO activity were increased in IMD-treated UUO kidneys or NRK-52E. The obstructed kidneys of UUO rats demonstrated significant interstitial fibrosis on day 7 after operation. In contrast, kidneys that were treated with IMD gene transfer exhibited minimal interstitial fibrosis. The obstructed kidneys of UUO rats also had greater macrophage infiltration and IL-6 expression. IMD restrained infiltration of macrophages and expression of IL-6 in UUO kidneys. The degree of EMT was extensive in obstructed kidneys of UUO rats as indicated by decreased expression of E-cadherin and increased expression of α-SMA. In vitro studies using NRK-52E confirmed these observations. EMT was suppressed by IMD gene delivery. However, all of the above beneficial effects of IMD were eliminated by ZnPP, an inhibitor of HO enzyme activity.ConclusionThis study demonstrates that IMD attenuates renal fibrosis by induction of HO-1.

The mechanism of blood concentrations of the Shenqi pill repairing injured epithelial cells of renal tubular in vitro.

In the present study, we investigated the best blood concentration of the mechanism of Shenqi pill repairing the injured epithelial cells of renal tubular in vitro. First, the injured hypoxia/reoxygenation model of rat proximal renal tubular epithelial cell strain (NRK-52E) was established. The animals were divided randomly into control, model, low concentration (5 µg/ml), moderate concentration (10 µg/ml) and high concentration (20 µg/ml) groups. The apoptotic rate was measured with flow cytometry and Jag2/Notch2/hes1 mRNA, and the protein expression was measured for 1, 3 and 7 days. It was found that in comparison to the control group, the growth of each group was prolonged with time, the levels of apoptosis, and the Jag2/Notch2/hes1 mRNA and protein expression decreased. Furthermore, the levels of the apoptotic rate, Jag2/Notch2/hes1 mRNA and protein expression of the moderate concentration and high concentration groups were significantly lower than those of the model and low-dose groups at each time-point (P<0.05). In conclusion, the Shenqi Pill alleviates the damage of renal tubular epithelial cells by inhibiting the Jag2/Notch2/hes1 signaling pathway; suitable concentration such as 10–20 µg/ml can exert protective effect.

Role of urotensinII in advanced glycation end product-induced extracellular matrix synthesis in rat proximal tubular epithelial cells.

Urotensin II(UII) was first recognized for its constrictive and natriuretic properties in fish almost 40years ago, and recent studies have suggested that it exerts pro-fibrotic effects in a number of cell lines. In this study, we aimed to evaluate the role of UII in extracellular matrix(ECM) synthesis and secretion in advanced glycation end product(AGE)-stimulated rat proximal tubular epithelial cells(NRK-52E cells). UII promoted the proliferation of the NRK-52E cells in a dose-dependent manner over a concentration range of 10-10-10-8mol/l and this effect was partly inhibited by both nimodipine and EDTA. Furthermore, AGE-BSA promoted the mRNA and protein expression of UII, fibronectin(FN) and collagenIV(ColIV) in the NRK-52E cells in a dose-and time-dependent manner. In addition, UII promoted the mRNA expression and protein secretion of transforming growth factor(TGF)-β1, FN and ColIV by the NRK-52E cells. Our results suggest that UII promotes the proliferation of NRK-52E cells, an effect which is mediated by the influx of extracellular calcium ions. In addition, our data indicate that AGEs promote UII expression in NRK-52E cells, and that TGF-β1 signaling is a candidate pathway mediating the involvement of UII in renal fibrosis. Collectively, our data suggest that the UII-TGF-β1 signaling may be an important factor in tubulointerstitial nephropathy in diabetes.

ChemInform Abstract: Applanatumols A and B, Meroterpenoids with Unprecedented Skeletons from Ganoderma applanatum.

AbstractApplanatumols A (I) and racemic applanatumol B are isolated.