Rat Tubular Epithelial Cells Research Articles

Abstract 097: Sexually Dimorphic Transcriptional Phenotypes In Tubular Epithelial Cells Of The Rat Kidney

Sexual dimorphism is a key biological variable in different organ systems. Although a growing body of evidence at single cell-resolution highlights sex differences in the kidneys of humans and mice, information regarding rats is limited. We integrated publicly available datasets to construct a single-cell RNA sequencing atlas of the rat kidney, incorporating data from 3 females and 3 males. Transcriptomes from the tubular epithelial cell clusters, exhibited strong correlation with those from rat-kidney microdissected tubule segments and single-cell RNA sequencing of human kidney biopsies. We hypothesize that transcriptomes of rat tubular epithelial cells present sexual dimorphism. To test this hypothesis, we identified genes that were differentially expressed between sexes (DEGs: log2FC >= 1; adjusted-p <= 0.05) in proximal tubules (PT), thick ascending limbs (TAL), distal convoluted tubules (DCT), principal cells (PC) and intercalated cells A and B (IC-A and IC-B, respectively). Gene enrichments of PT DEGs were conducted using WikiPathways. The number of genes positively enriched in males was as follows: 43 PT, 9 TAL, 16 DCT, 23 PC, 12 I-CA and 19 I-CB. While females presented 44 PT, 4 TAL, 4 DCT, 3 PC, 1 IC-A and 3 IC-B. Four transcripts were upregulated in all male segments, 1) glutathione peroxidase 3 ( Gpx3 ), 2) lysozyme 2 ( Lyz2 ), 3) locus A1 ( RT1-A1 ) which enables beta-2-microglobulin and peptide binding activity, and 4) AABR07060872.1 an Ig-like domain-containing protein. Only one long non-coding RNA ( AABR07039356.2 ) was upregulated in all female segments. The three most enriched pathways in male PT were: 1) Proximal tubule transport (WP4917; p < 1x10 -14 ), 2) Glycolysis and gluconeogenesis (WP534; p < 2x10 -12 ) and 3) Metabolic reprograming in colon cancer (WP4290; p < 8x10 -9 ). In females, the most enriched pathways were: 1) Trans-sulfuration and one-carbon metabolism (WP2525; p < 6x10 -9 ), 2) One-carbon metabolism related pathways (WP3940; p < 1x10 -8 ) and 3) Amino acid metabolism (WP3925; p < 7x10 -8 ). Our results show the presence of sex-related transcriptional differences across the rat nephron, but predominantly in PT. These differences impact key metabolic functions of PT such amino acids and carbohydrates metabolism and transport processes.

#4966 CELLULAR PRION PROTEIN ACTIVATES THE TBK1-IRF3 SIGNALING PATHWAY TO AGGRAVATE RENAL FIBROSIS

Abstract Background and Aims Chronic kidney disease (CKD) is an irreversible degenerative disease characterized by gradual loss of renal function, contributing to high morbidity and mortality as well as heavy economic cost to society. Clinically, there are few available strategies to slow CKD progression. Because the uremic phenotypes include many features of aging, CKD is considered as a premature aging syndrome. Neurodegenerative diseases, such as Alzheimer's disease, and Parkinson's disease are extensively studied aging-related diseases, in which cytotoxic proteins misfolding and aggregation in neural cells is the main characteristic. The misfolding and aggregation of the prion protein is tightly linked to the development of prion diseases. Interestingly, a remarkable elevation of cellular prion protein (PrPC) in the urine and plasma of CKD patients was reported. However, the role of PrPC in the pathogenesis of kidney diseases remains largely unknown. The aim of the current study is to investigate the role of PrPC in renal fibrosis. Methods Immunohistochemistry staining was conducted to evaluate the expression of PrPC in renal biopsies from CKD patients. Enzyme linked immunosorbent assay (ELISA) was performed to detect urinary PrPC of CKD patients. Animal model of renal fibrosis was induced by unilateral ureteral occlusion (UUO). Western blot, real time PCR and immunofluorescence staining were performed to evaluate the expression and distribution of PrPC. Proximal tubule specific Prnp knockout mice (PEPCK-Prnp-KO) was constructed to explore the pathogenic role of PrPC in renal fibrosis. Primary proximal tubular epithelial cells (PTCs) overexpressed PrPC were collected for RNA-seq analysis. TBK1 inhibitor amlexanox, and IRF3 siRNA were used to block TBK1-IRF3 pathway in rat tubular epithelial cells (NRK-52E), respectively. Amlexanox was administered orally to UUO mice. H&E, Masson and Sirius red staining were used to estimate renal pathology. Results The protein expression levels of PrPC both in renal biopsies and urine from CKD patients were elevated and significantly correlated with the severity of interstitial fibrosis and the decline of eGFR. PrPC proteins were significantly increased, and aggregated in proximal renal tubules in fibrotic renal tissues induced by UUO. Compared with wild-type littermates, PEPCK-Prnp-KO mice showed reduced extracellular matrix accumulation and interstitial inflammation at day 7 after UUO. PrPC provokes profibrotic response of renal tubular cells via the TBK1-IRF3 pathway. TBK1 inhibitor amlexanox or silencing endogenous IRF3 significantly inhibited PrPC-induced profibrogenic phenotypic transformation in NRK-52 cells. Amlexanox attenuated interstitial fibrosis induced by UUO. Conclusion Our experimental results indicate that PrPC-TBK1-IRF3 pathway plays a detrimental role in profibrogenic transformation of renal tubular cells and thus contributes to interstitial fibrosis. Blocking TBK1 activation is a plausible strategy for therapeutic intervention of chronic kidney disorders.

Atorvastatin reduces contrast media-induced pyroptosis of renal tubular epithelial cells by inhibiting the TLR4/MyD88/NF-κB signaling pathway

BackgroundContrast-induced acute kidney injury (CI-AKI) is the third most common cause of hospital-acquired renal failure. However, there is no effective treatment of CI-AKI, and its mechanism is unknown. Interestingly, atorvastatin has been reported to be effective in renal injury. Therefore, the aim of this study was to explore the effect and possible molecular mechanism of atorvastatin in CI-AKI.MethodsOn the CI-AKI in vitro model, rat tubular epithelial cells (NRK-52E) were treated with 18 mg I/ml meglumine diatrizoate (MEG) and then pretreated with atorvastatin. pcDNA3.1-TLR4 treatment was performed to overexpress toll-like receptor 4 (TLR4) in NRK-52E cells. Cell Counting Kit-8 (CCK-8) and lactate dehydrogenase (LDH) kits were used to detect NRK-52E cell viability as well as LDH release in each group, respectively; qRT-PCR to determine mRNA expression of TLR4 in cells; western blot to detect protein expression levels of pyroptosis-related proteins (NLRP3, caspase-1, ASC, and GSDMD) and TLR4/MyD88/NF-κB signaling pathway-related proteins (TLR4, MyD88, NF-κBp65, and p-NF-κB p65) in cells.ResultsMEG treatment significantly inhibited the viability of NRK-52E cells, increased pro-inflammatory factor levels and promoted pyroptosis, representing successful establishment of a rat tubular epithelial cell (NRK-52E) CI-AKI in vitro model. Notably, atorvastatin increased the activity of MEG-treated NRK-52E cells and alleviated cell injury in a concentration-dependent manner. In addition, atorvastatin significantly down-regulated the expression of TLR4 in MEG-treated NRK-52E cells. However, overexpression of TLR4 inhibited the effects of atorvastatin on increasing cell viability, alleviating cell injury, reducing pro-inflammatory factors (IL-1β, IL-6, and TNF-α) levels, and inhibiting apoptosis (by down-regulating the expression of NLRP3, caspase-1, ASC, and GSDMD). Furthermore, atorvastatin also inhibited the expression of TLR4/MyD88/NF-κB pathway-related proteins (TLR4, MyD88, and p-NF-κB p65).ConclusionAtorvastatin can attenuate CI-AKI through increasing the activity of MEG-treated renal tubular epithelial cells, relieving cell injury, as well as inhibiting pyroptosis and inflammation. More importantly, the mechanism was achieved by inhibiting the TLR4//MyD88/NF-κB signaling pathway.

DBP and BaP co-exposure induces kidney injury via promoting pyroptosis of renal tubular epithelial cells in rats

Dibutyl phthalate (DBP) and benzo(a)pyrene (BaP) are widespread environmental and foodborne contaminants that have detrimental effects on human health. Although people are often simultaneously exposed to DBP and BaP via the intake of polluted food and water, the combined effects on the kidney and potential mechanisms remain unclear. Hence, we treated rats with DBP and BaP for 90 days to investigate their effects on kidney histopathology and function. We also investigated the levels of paramount proteins and genes involved in pyroptosis and TLR4/NF-κB p65 signaling in the kidney. Our research showed that combined exposure to DBP and BaP triggered more severe histopathological and renal function abnormalities than in those exposed to DBP or BaP alone. Simultaneously, combined exposure to DBP and BaP enhanced the excretion of IL-1β and IL-18, along with the release of LDH in rat renal tubular epithelial cells (RTECs). Moreover, combined exposure to DBP and BaP increased the expression of pyroptosis marker molecules, including NLRP3, ASC, cleaved-Caspase-1, and GSDMD. Meanwhile, the combination of DBP and BaP activated TLR4/NF-κB signaling in the kidney. Taken together, the combined exposure to DBP and BaP causes more severe kidney injury than that caused by DBP or BaP exposure separately. In addition, pyroptosis of RTECs regulated by TLR4/NF-κB signaling may add to the kidney damage triggered by combined exposure to DBP and BaP.

Oxidative stress and Cx43-mediated apoptosis are involved in PFOS-induced nephrotoxicity

Perfluorooctane sulfonate (PFOS) is a persistent organic pollutant that can cause nephrotoxicity. However, the underlying mechanisms are not fully understood and require further investigation. In the present study, we established a PFOS-exposed Sprague-Dawley (SD) rat kidney injury model by intraperitoneal injection of PFOS (1 mg/kg and 10 mg/kg body weight) every alternate day for 15 days and cytotoxicity models of normal rat kidney epithelial (NRK52E) and human renal proximal tubular (HK2) cells exposed to PFOS (20 μM and 60 μM) for 24 h to reveal the mechanisms underlying PFOS-induced nephrotoxicity. Data showed that PFOS increased the kidney index and induced nephrotoxicity in rats. Furthermore, PFOS significantly increased malondialdehyde (MDA) levels, decreased GSH peroxidase (GSH-PX) activity in kidney tissues, and increased intracellular reactive oxygen species (ROS) levels in NRK52E and HK2 cells. Following PFOS treatment, mitochondrial damage in the renal tubular epithelial cells of rats was observed and the mitochondrial membrane potential (ΔΨm) was decreased in NRK52E cells. PFOS upregulated apoptosis of tubular epithelial cells and expression of Connexin 43 (Cx43) in vitro and in vivo. The Cx43 inhibitor gap26 attenuated the apoptosis of tubular epithelial cells. In conclusion, our findings reveal that PFOS may trigger renal tubular cell apoptosis through oxidative stress and upregulation of Cx43, resulting in PFOS-induced nephrotoxicity.

Pharmacological inhibition of MyD88 suppresses inflammation in tubular epithelial cells and prevents diabetic nephropathy in experimental mice.

Emerging evidence shows that chronic inflammation mediated by toll-like receptors (TLRs) contributes to diabetic nephropathy. Myeloid differentiation primary-response protein-88 (MyD88) is an essential adapter protein of all TLRs except TLR3 in innate immunity. It is unclear whether MyD88 could be a therapeutic target for diabetic nephropathy. Here, we used a new small-molecule MyD88 inhibitor, LM8, to examine the pharmacological inhibition of MyD88 in protecting kidneys from inflammatory injury in diabetes. We showed that MyD88 was significantly activated in the kidney of STZ-induced type 1 diabetic mice in tubular epithelial cells as well as in high glucose-treated rat tubular epithelial cells NRK-52E. In cultured tubular epithelial cells, we show that LM8 (2.5-10 μM) or MyD88 siRNA attenuated high-concentration glucose-induced inflammatory and fibrogenic responses through inhibition of MyD88-TLR4 interaction and downstream NF-κB activation. Treatment with LM8 (5, 10 mg/kg, i.g.) significantly reduced renal inflammation and fibrosis and preserved renal function in both type 1 and type 2 diabetic mice. These renoprotective effects were associated with reduced MyD88-TLR4 complex formation, suppressed NF-κB signaling, and prevention of inflammatory factor expression. Collectively, our results show that hyperglycemia activates MyD88 signaling cascade to induce renal inflammation, fibrosis, and dysfunction. Pharmacological inhibition of MyD88 may be a therapeutic approach to mitigate diabetic nephropathy and the inhibitor LM8 could be a potential candidate for such therapy.

RETRACTED ARTICLE: Mesenchymal stem cells ameliorate renal fibrosis by galectin-3/Akt/GSK3β/Snail signaling pathway in adenine-induced nephropathy rat

BackgroundTubulointerstitial fibrosis (TIF) is one of the main pathological features of various progressive renal damages and chronic kidney diseases. Mesenchymal stromal cells (MSCs) have been verified with significant improvement in the therapy of fibrosis diseases, but the mechanism is still unclear. We attempted to explore the new mechanism and therapeutic target of MSCs against renal fibrosis based on renal proteomics.MethodsTIF model was induced by adenine gavage. Bone marrow-derived MSCs was injected by tail vein after modeling. Renal function and fibrosis related parameters were assessed by Masson, Sirius red, immunohistochemistry, and western blot. Renal proteomics was analyzed using iTRAQ-based mass spectrometry. Further possible mechanism was explored by transfected galectin-3 gene for knockdown (Gal-3 KD) and overexpression (Gal-3 OE) in HK-2 cells with lentiviral vector.ResultsMSCs treatment clearly decreased the expression of α-SMA, collagen type I, II, III, TGF-β1, Kim-1, p-Smad2/3, IL-6, IL-1β, and TNFα compared with model rats, while p38 MAPK increased. Proteomics showed that only 40 proteins exhibited significant differences (30 upregulated, 10 downregulated) compared MSCs group with the model group. Galectin-3 was downregulated significantly in renal tissues and TGF-β1-induced rat tubular epithelial cells and interstitial fibroblasts, consistent with the iTRAQ results. Gal-3 KD notably inhibited the expression of p-Akt, p-GSK3β and snail in TGF-β1-induced HK-2 cells fibrosis. On the contrary, Gal-3 OE obviously increased the expression of p-Akt, p-GSK3β and snail.ConclusionThe mechanism of MSCs anti-renal fibrosis was probably mediated by galectin-3/Akt/GSK3β/Snail signaling pathway. Galectin-3 may be a valuable target for treating renal fibrosis.

Deletion of Smad3 protects against C-reactive protein-induced renal fibrosis and inflammation in obstructive nephropathy.

Introduction and Aims: Elevated plasma levels of C-reactive protein (CRP) are closely associated with progressive renal injury in patients with chronic kidney disease (CKD). Here, we tested a hypothesis that CRP may promote renal fibrosis and inflammation via a TGF-β/Smad3-dependent mechanism.Methods: Role and mechanisms of TGF-β/Smad3 in CRP-induced renal fibrosis and inflammation were examined in a mouse model of unilateral ureteral obstruction (UUO) induced in CRP Tg/Smad3 KO mice and in a rat tubular epithelial cell line in which Smad3 gene is stably knocked down (S3KD-NRK52E).Results: We found that mice overexpressing the human CRP gene were largely promoted renal inflammation and fibrosis as evidenced by increasing IL-1β, TNF-α, MCP-1 expression, F4/80+ macrophages infiltration, and marked accumulation of α-smooth muscle actin (α-SMA), collagen I and fibronectin in the UUO kidney, which were blunted when Smad3 gene was deleted in CRPtg-Smad3KO. Mechanistically, we found that the protection of renal inflammation and fibrosis in the UUO kidney of CRPtg-Smad3KO mice was associated with the inactivation of CD32-NF-κB and TGF-β/Smad3 signaling.Conclusion: In conclusion, Smad3 deficiency protects against CRP-mediated renal inflammation and fibrosis in the UUO kidney by inactivating CD32-NF-κB and TGF-β/Smad3 signaling.

The PAR-1 antagonist vorapaxar ameliorates kidney injury and tubulointerstitial fibrosis.

Protease-activated receptor (PAR)-1 has emerged as a key profibrotic player in various organs including kidney. PAR-1 activation leads to deposition of extracellular matrix (ECM) proteins in the tubulointerstitium and induction of epithelial-mesenchymal transition (EMT) during renal fibrosis. We tested the anti-fibrotic potential of vorapaxar, a clinically approved PAR-1 antagonist for cardiovascular protection, in an experimental kidney fibrosis model of unilateral ureteral obstruction (UUO) and an AKI-to-chronic kidney disease (CKD) transition model of unilateral ischemia-reperfusion injury (UIRI), and dissected the underlying renoprotective mechanisms using rat tubular epithelial cells. PAR-1 is activated mostly in the renal tubules in both the UUO and UIRI models of renal fibrosis. Vorapaxar significantly reduced kidney injury and ameliorated morphologic changes in both models. Amelioration of kidney fibrosis was evident from down-regulation of fibronectin (Fn), collagen and α-smooth muscle actin (αSMA) in the injured kidney. Mechanistically, inhibition of PAR-1 inhibited MAPK ERK1/2 and transforming growth factor-β (TGF-β)-mediated Smad signaling, and suppressed oxidative stress, overexpression of pro-inflammatory cytokines and macrophage infiltration into the kidney. These beneficial effects were recapitulated in cultured tubular epithelial cells in which vorapaxar ameliorated thrombin- and hypoxia-induced TGF-β expression and ECM accumulation. In addition, vorapaxar mitigated capillary loss and the expression of adhesion molecules on the vascular endothelium during AKI-to-CKD transition. The PAR-1 antagonist vorapaxar protects against kidney fibrosis during UUO and UIRI. Its efficacy in human CKD in addition to CV protection warrants further investigation.

Nrp-1 Mediated Plasmatic Ago2 Binding miR-21a-3p Internalization: A Novel Mechanism for miR-21a-3p Accumulation in Renal Tubular Epithelial Cells during Sepsis.

The mechanism underlying sepsis-associated acute kidney injury (SAKI), which is an independent risk factor for sepsis-associated death, is unclear. A previous study indicates that during sepsis miR-21a-3p accumulates in renal tubular epithelial cells (TECs) as the mediator of inflammation and mediates TEC malfunction by manipulating its metabolism. However, the specific mechanism responsible for the accumulation of miR-21a-3p in TECs during sepsis is unrevealed. In this study, a cecal ligation and puncture- (CLP-) induced sepsis rat model and rat TEC line were used to elucidate the mechanism. Firstly, miR-21a-3p and Ago2 levels were found out to increase in both plasma and TECs during sepsis, and the increase of intracellular Ago2 and miR-21a-3p could be mitigated when Ago2 was either inactivated or downregulated in septic plasma. Moreover, membrane Nrp-1 expression of TECs was increased significantly during sepsis and Nrp-1 knockdown also mitigated the rise of both the intracellular Ago2 and miR-21a-3p levels in TECs incubated with septic plasma. Furthermore, it was revealed that Ago2 can be internalized by TECs mediated with Nrp-1 and this process had no effect on the intracellular content of miR-21a-3p. Both Ago2 and miR-21a-3p could bind to TECs derived Nrp-1 directly. Finally, it was determined that miR-21a-3p was internalized by TECs via Nrp-1 and Ago2 facilitated this process. Taken together, it can be concluded from our results that Ago2 binding miR-21a-3p from septic plasma can be actively internalized by TECs via Nrp-1 mediated cell internalization, and this mechanism is crucial for the rise of intracellular miR-21a-3p content of TECs during sepsis. These findings will improve our understanding of the mechanisms underlying SAKI and aid in developing novel therapeutic strategies.

24-LB: RIPK1 Activation Induces Necroptosis, Proinflammation, and Profibrosis in Kidney of STZ/HFD-Induced Diabetic Mice

Diabetic kidney disease (DKD) is attributed largely to renal tubular interstitial fibrosis. However, the complex mechanism of renal tubular interstitial fibrosis in DKD pathogenesis remains unclear. Recently, necroptosis has emerged as an important cell death model in many pathological conditions, which is regulated through RIPK1/RIPK3 pathway. In this present study, we aimed to evaluate whether necroptosis is involved in the development of diabetic kidney disease, and to investigate the mechanisms involved. In the current study, the results of immunohistochemistry and Western blot showed that the ratio of p-RIPK1/RIPK1, p-RIPK3/RIPK3, p-MLKL/MLKL were significantly increased in the kidney of STZ/HFD induced diabetic mice. Additionally, the levels of serum MCP-1, and the expression of IL-1 β and TGF - β in kidney were increased in the diabetic mice. Moreover, Necrostatin-1, an inhibitor of RIPK1, can effectively decrease the ratio of p-RIPK1/RIPK1, p-RIPK3/RIPK3, p-MLKL/MLKL, the levels of serum MCP-1, and the expression of TGF-β and IL-1 β in kidney in the diabetic mice. Necrostatin-1 treatment also resulted in delayed progression of renal structural lesions and significantly improved the function of kidney. High glucose stimulation, which induced excessive ROS production, led to necrosis and increased phosphorylation of RIPK1, RIPK3 and MLKL in rat tubular epithelial cell NRK-52E cells. Moreover, NAC treatment, a ROS inhibitor, could partly reverse the effect of glucose on RIPK1, RIPK3, and MLKL. In conclusion, RIPK1 activation may induce necroptosis, pro-inflammation and pro-fibrosis in kidney of STZ/HFD induced diabetic mice; High glucose induces RIPK1 -dependent necroptosis in NRK-52E cells; excessive ROS production mediate high glucose induced necroptosis. Disclosure Q. Chen: None. Y. Long: None. M. Guo: None. X.M. Ma: None. Y. Pu: None. Y. Xu: None. Funding Science and Technology Bureau of Luzhou (2016LZXNYD-G02)

The association between the diesel exhaust particle exposure from bus emission and the tubular epithelial cell deformation of rats.

The diesel vehicle emissions regarding particles have become a problem due to human health adversely. Especially ultrafine particles (diameter ≤ 100nm) can deeply penetrate the human body leading to cell deformation. Investigation of the diesel ultrafine particle exposure to the cell deformation has become a challenge to build up understanding the impacts of ultrafine particles on human health. Moreover, the relationship between high exposure to diesel ultrafine particles and the deformation of the rat's tubular epithelial cells is not clear. In this study, we investigated the impact of the diesel ultrafine particle exposure to the rat's tubular cells. Three diesel busses were used as the sources of the particles, while 50 rats were used as the experimental animals. The diesel emission was filtered using an N95 particulate filter and a suction pump. The rats were exposed to the diesel ultrafine particle emission for 100s with three different concentrations C1, C2, and C3 for eight consecutive days. All rats were sacrificed on the day after exposures to examine the histological images. The results showed that the deformation level of the tubular epithelial cells was positively associated with the concentration of the ultrafine particles.

The role of activated NLRP3 inflammatory body in acute kidney injury in rats caused by sepsis and NLRP3-TXNIP signaling pathway.

ObjectiveThe model of acute renal injury (AKI) induced by sepsis in rats was established by abdominal resection through surgical suture. The activation mechanism of nod-like receptor with pyrin domain containing 3 (NLRP3) inflammatory corpuscle in AKI induced by sepsis was analyzed.MethodsHere, 60 male rats were selected and divided into two groups, including sham-operated group (NO-OPs group, n = 15) and sepsis group (CELP group, n = 45). In order to examine each index of CELP group, four time points (10, 20, 30, and 40 h) were set as control. In NO-OPs group, only abdominal resection through surgical suture was carried out. The expression levels of NLRP3, apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), caspase-1, and the expression level of NLRP3-TXNIP signaling pathway were measured by immunohistochemistry, Western blotting, immunoprecipitation, and mito-TEMPO (a mitochondria-targeted antioxidant) 40 h after operation and 10, 20, 30, and 40 h post-operation in CELP group. Herein, 40 h post-operation in NO-OPs group and 10, 20, 30, and 40 h post-operation in CELP group, peripheral blood samples were collected.ResultsCompared with NO-OPs group, the levels of serum creatinine (Scr) and blood urea nitrogen (BUN) in CELP group were increased (P < 0.05). Compared with NO-OPs group, the expression levels of interleukin-1β (IL-1β), NLRP3, ASC, and caspase-1 in CELP group were increased (P < 0.05). The expression level of TXNIP in renal tubular epithelial cells in rats was up-regulated. There was a positive correlation between TXNIP and NLRP3. The binding of NLRP3-TXNIP signaling pathway could be inhibited by siRNA transfection or mito-TMPO, and the activity of NLRP3 inflammatory bodies could be inhibited as well.ConclusionActivation of NLRP3 inflammatory corpuscles could promote AKI induced by sepsis. Simultaneously, renal injury may lead to the production of mitochondrial reactive oxygen species (mROS), which may induce the binding of TXNIP to NLRP3.

MiR-135a regulates renal fibrosis in rats with diabetic kidney disease through the Notch pathway.

To explore the influence of micro ribonucleic acid (miR)-135a on the renal fibrosis in rats with diabetic kidney disease (DKD) through the Notch signaling pathway. A total of 30 male Wistar rats weighing 200-220 g were selected and randomly divided into Control group (n=10), diabetes mellitus (DM) group (n=10), and miR-13a inhibitor group (n=10). Streptozotocin (STZ) was intraperitoneally injected daily to establish the DM model in rats of both DM group and miR-135a group, while normal saline was given daily through intraperitoneal injection in rats of Control group. After 4 weeks, the rats in miR-135a inhibitor group were intraperitoneally injected with miR-135a inhibitor, and those in Control and DM groups were administrated with an equal amount of normal saline. Changes in the blood glucose (BG), glycated hemoglobin (GHb), serum creatinine (Scr), triglyceride (TG), and total cholesterol (TC) of rats were evaluated, and the pathological changes in the renal tissues of DM rats were observed via hematoxylin-eosin (HE) staining. Sirius red staining was performed to observe the changes in collagen fibers in the kidney of all groups of rats. The expressions of Notch and Hes1 in the renal tissues of rats in each group were detected using immunohistochemistry. Immunofluorescence assay was employed to detect the positive expression of Notch in the renal tissues of rats. The mRNA expressions of Notch and miR-135a were detected via quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). Finally, Western blotting was conducted to detect the protein expressions of Notch, Notch intracellular domain (NICD) and Hes1. Compared with Control group, rats in DM group had substantially raised levels of BG, GHb, Scr, TG, and TC (p<0.05). HE staining showed that the rats in Control group had renal tubular cells with normal morphology and well-defined structure, while those in DM group exhibited evident cavitation in the renal tubular epithelium. Sirius red staining results manifested that the red collagen fibers were evenly distributed with light staining in the glomeruli and renal tubules of rats in Control group. In contrast, the collagen fibers of the glomeruli and renal tubules of rats in DM group exhibited deep and evident red staining. Moreover, compared with DM group, rats in miR-135a inhibitor group had notably faded red staining in the glomeruli and renal tubules of rats, evenly distributed collagen and remarkably decreased fibrotic nodules. According to immunohistochemistry detection results, the protein levels of Notch and Hes1 in the renal tubulointerstitial cells and renal tubular epithelial cells of rats in DM group were markedly higher than those in Control group. Compared with those in DM group, their protein levels were remarkably lowered in miR-135a inhibitor group (p<0.05). Immunofluorescence assay results revealed that the protein level of Notch in the renal tissues of rats in DM group was considerably higher than that in Control group (p<0.05), while its protein level in miR-135a inhibitor group was significantly lower than that in DM group. According to qRT-PCR results, compared with those in Control group, mRNA expressions of Notch mRNA and miR-135a in the rat kidney tissues were substantially raised in DM group (p<0.05), and they were notably lowered in miR-13a inhibitor group compared with those in DM group (p<0.05). Finally, Western blotting results manifested that the protein levels of Notch, NIC, and Hes1 in the renal tissues of rats in DM group were considerably higher than those in Control group (p<0.05), and that their protein expression levels in miR-135a inhibitor group were markedly lower than those in DM group (p<0.05). Inhibition of miR-135a can reduce the renal fibrosis in DKD rats through the Notch pathway.

Therapeutic Effects of Human Urine-Derived Stem Cells in a Rat Model of Cisplatin-Induced Acute Kidney Injury In Vivo and In Vitro.

Acute kidney injury (AKI) is an extremely dangerous clinical syndrome with high morbidity and mortality. Stem cell-based therapies have shown great promise for AKI treatment. Urine-derived stem cells (USCs) are a novel cell source in tissue engineering and cell therapy which provide advantages of simple, noninvasive, and low-cost harvest methods, efficient proliferation, and multi-differentiation potential. Here, we described the therapeutic effects of USCs in a rat model of cisplatin-induced AKI as a novel therapy. In vivo, the intravenous administration of USCs alleviated the renal functional damage in AKI rats, for the levels of blood urea nitrogen (BUN) and serum creatinine (SCr) were significantly decreased. The USCs-treated group also exhibited improved histological and ultrastructural changes, promoted proliferation, and inhibited apoptosis in renal tissues. After the USC therapy, the expression levels of proinflammatory cytokines (TNF-α and IL-6) and apoptosis-related proteins (BAX and cleaved caspase-3) were downregulated. In addition, the presence of a few GFP-labeled USCs was confirmed in rat renal tissues. In vitro, rat tubular epithelial (NRK-52E) cells were incubated with cisplatin to induce cell damage and then cocultured with USCs. After coculture with USCs, the cisplatin-induced NRK-52E cells showed higher cell viability and a lower apoptosis ratio than those of the control group, and cell cycle arrest was improved. In conclusion, our results demonstrated that USC therapy significantly improved the renal function and histological damage, inhibited the inflammation and apoptosis processes in the kidney, and promoted tubular epithelial proliferation. Our study exhibited the potential of USCs in the treatment of AKI, representing a new clinical therapeutic strategy.

Formin mDia1 contributes to migration and epithelial-mesenchymal transition of tubular epithelial cells exposed to TGF-β1.

Renal tubular epithelial cells may undergo epithelial-mesenchymal transition (EMT) in response to stimuli, such as transforming growth factor (TGF)-β1, leading to myofibroblast activation and renal fibrosis. The formin mDia1 is required for nucleation and polymerization of actin and the microtubule cytoskeleton. The present study sought to explore the role of mDia1 in EMT of tubular epithelial cells. A rat model of unilateral ureteral obstruction (UUO) was established. The expression of TGF-β1, collagen I, collagen III, and mDia1 in the kidneys was examined at day 7 after surgery. The effect of mDia1 on EMT was explored in NRK-52E cells by exposing them to TGF-β1. Increased expression of TGF-β1, collagen I, collagen III, and mDia1 was found in obstructive kidneys of UUO model rats. Exposing rat tubular epithelial cells to TGF-β1 promoted collagen I and collagen III expression but had no effect on mDia1 expression. Silencing mDia1 expression impeded epithelial cell migration as well as reduced TGF-β1, collagen, and Profilin1 expression, whereas mDia1 overexpression exerted an opposite effect. Furthermore, mDia1 regulated the expression of vimentin, α-smooth muscle actin, and E-cadherin and focal adhesion-kinase (FAK)/Src activation through Profilin1. Inhibition of the mDia1 activator RhoA by fasudil reversed EMT, and FAK/Src activation induced by mDia1. In conclusion, mDia1 regulated tubular epithelial cell migration, collagen expression, and EMT in NRK-52E cells exposed to TGF-β1. Thus, suppression of mDia1 activation might be a strategy to counteract renal fibrosis.

WNT1-inducible signaling protein-1 mediates TGF-β1-induced renal fibrosis in tubular epithelial cells and unilateral ureteral obstruction mouse models via autophagy.

Renal fibrosis is a common pathway for the progression of all chronic kidney diseases to end-stage kidney disease. Studies show that WNT1-inducible signaling pathway protein-1 (WISP-1) is involved in the fibrosis of various organs. The aim of the study was to explore the functional role and potential mechanism of WISP-1 in renal fibrosis. We observed that overexpression of WISP-1 in rat tubular epithelial cells (TECs) enhanced transforming growth factor-β1 (TGF-β1)-induced production of fibrotic markers, including collagen I (Col I), fibronectin (FN) and TGF-β1, while inhibition of WISP-1 suppressed such production. In vivo, the messenger RNA and protein levels of Col I, FN, and α-smooth muscle actin were significantly inhibited after anti-WISP-1 antibody treatment for 7 days in unilateral ureteral obstruction mouse models. Moreover, blockade of WISP-1 by anti-WISP-1 antibody significantly reduced autophagy-related markers, including anti-microtubule-associated protein-1 light chain 3 (LC3) and beclin 1, while increasing sequestosome 1. In addition, overexpression of WISP-1 in TECs increased autophagy as evidenced by greater numbers of GFP-LC3 puncta and increased expression of LC3 and beclin 1 in response to TGF-β1. In contrast, knockdown of WISP-1 by small interfering RNA decreased the number of GFP-LC3 puncta and the expression of LC3 and beclin 1 in TGF-β1-treated TECs. Collectively, these data suggest that WISP-1, as a profibrotic protein, may mediate renal fibrosis by inducing autophagy in both obstructive nephropathy and TGF-β1-treated TECs. WISP-1 may serve as an effective therapeutic target for the treatment of renal fibrosis.

Upregulation of Tissue Transglutaminase in Transforming Growth Factor-b Induced Epithelial Mesenchymal TG2Transdifferentiation In Rat Tubular Epithelial Cells

AbstractBackground: Epithelial-Mesenchymal Transdifferentiation (EMT) is believed to be a key step in the course of glomerular and tubulointerstitial fibrosis. Many of the regulators of this process have been identified and Transforming Growth Factor-b (TGF-b) plays a central role. Tissue transglutaminase (TG2) has a crucial role in extracellular matrix formation and stabi-lization.Aim of Study: This study was designed to explore whether TG2 is essential in EMT induced by TGF-b in renal tubular epithelial cells.Material and Methods: EMT was achieved by treating NRK52-E renal tubular epithelial cells with high concentration of rhTGF-b (10ng/ml) for 24, 48 and 72h. Development of mesenchymal cell markers Fibroblast Specific Protein (FSP-1) and a Smooth Muscle Actin (a-SMA) and decrease in tubular epithelial cell surface marker E-Cadherin were used to confirm EMT. The NTU283; TG2 inhibitor was used for the inhibition assays. TG2 activity was quantified using a 14C putrescine incorporation assay. Protein expression and localisation of different markers were assessed by western blot and immunofluorescent dual immunofluorescent staining. Phase analysis was used to quantify the amount of staining for each of the markers used.Results: A significant increase in TG2 activity and protein expression were both observed with TGF-b induced transdif-ferentiation at 48h. This observation was associated with loss of epithelial cell marker E-Cadherin and appearance of intra-cellular a-SMA and FSP-1, characteristic of myofibroblasts. Dual staining showed high TG2 levels in cells (0.254±0.07), compared to the control (0.02±0.008). This also showed loss of epithelial phenotype, decrease of E-cadherin and acquisition of myofibroblast characteristics a-SMA (0.671±0.13) com-pared to the control (0.076±0.016) and FSP-1 (0.492±0.017) compared to control (0.03±0.017). Cells also demonstrated high degree of co-localization of Collagen I (ColI) and a-SMA with TG2. The NTU283; TG2 inhibitor ameliorated TGF-b induced EMT. This was also associated with changed pattern of collagen I and a-SMA co-localization.Conclusion: This data demonstrated upregulation of TG2 during the process of TGF-b induced EMT. TG2 inhibitor showed non-significant reduction of both TG2 and mesenchy-mal markers but demonstrated changes in the molecular configuration of mesenchymal markers a-SMA and Col I, raising the possibility of a role of TG2 in EMT.

Effect of resveratrol on mitochondrial function in renal tubular epithelial cells of rats with sepsis-induced acute kidney injury

Objective To evaluate the effect of resveratrol on mitochondrial function in renal tubular epithelial cells of rats with sepsis-induced acute kidney injury. Methods Ninety-six healthy Sprague-Dawley rats of both sexes, aged 5-7 weeks, weighing 180-220 g, were divided into 4 groups (n=24 each) using a random number table method: sham operation group (Sham group), sepsis group (group Sep), sepsis plus vehicle group (Sep+ V group) and sepsis plus resveratrol group (Sep+ R group). Sepsis was induced by cecal ligation and puncture (CLP). Normal saline 0.5 ml, vehicle 0.5 ml and resveratrol 10 mg/kg were intraperitoneally injected at 6, 12 and 18 h after CLP in Sep, Sep+ V and Sep+ R groups, respectively.At 24 h after CLP, serum concentrations of creatinine (Cr) and blood urea nitrogen (BUN) were measured, and kidney tissues were obtained for examination of the pathological changes (using transmission electron microscopy), and the damage to the renal tubules was scored.The renal tubular epithelial cells (RTECs) were isolated from the kidney cortex at 24 h after CLP for determination of mitochondrial transmembrane potential (by flow cytometry with the fluorescent probe JC-1), intracellular ATP content, mitochondrial permeability transition pore (mPTP) opening, lipid peroxide (LPO) content, and lysosomal membrane permeability. Results Compared with group Sham, the serum concentrations of Cr and BUN, renal tubular damage score, mPTP opening, LPO content and lysosomal membrane permeability were significantly increased, and mitochondrial transmembrane potential and ATP content were decreased in Sep and Sep+ V groups (P<0.01). Compared with Sep and Sep+ V groups, the serum concentrations of Cr and BUN, renal tubular damage score, mPTP opening, LPO content and lysosomal membrane permeability were significantly decreased, and mitochondrial transmembrane potential and ATP content were increased in group Sep+ R (P<0.05). Conclusion Resveratrol improves mitochondrial function in renal tubular epithelial cells of rats with sepsis and reduces acute kidney injury, and the mechanism may be related to inhibiting oxidative stress and decreasing the lysosomal membrane permeability. Key words: Phenols; Sepsis; Acute kidney injury; Mitochondria; Kidney tubules; Epithelial cells

MiR-21-3p Plays a Crucial Role in Metabolism Alteration of Renal Tubular Epithelial Cells during Sepsis Associated Acute Kidney Injury via AKT/CDK2-FOXO1 Pathway.

Objective Sepsis and associated acute kidney injury (SAKI) are determined to be closely related to poor prognosis. Because the metabolic alterations of tubular epithelial cells (TECs) are crucial for the occurrence and development of SAKI, we carried out this present study to identify the metabolism changes of TECs during SAKI and relevant mechanisms. Methods Rat SAKI model and rat tubular epithelial cell line were used in our study. ELISA was used to determine the serum cytokines levels. Protein expressions were examined with Western-Blotting and the transcriptions of RNAs were determined with qRT-PCR. ADP/ATP assay and Oil Red O staining were used to examine the energy and lipid metabolism, respectively. Dual-luciferase reporter assay was carried out to determine the interactions between miRNA and specific proteins. Cell cycle arrest and apoptosis were determined with flow cytometry. Results Sepsis and AKI were induced 12 h after CLP. Energy and lipid metabolism reduced significantly while FOXO1 levels increased remarkably in TECs during SAKI. The expressions of both AKT and CDK2 and the transcriptions of relevant mRNAs reduced significantly in TECs during SAKI while miR-21-3p expression increased remarkably. Both AKT and CDK2 were determined as the direct targets of miR-21-3p. Furthermore, by in vitro experiments, it was demonstrated that FOXO1 levels were regulated by miR-21-3p in TECs via AKT/CDK2 and AKT/CDK2-FOXO1 pathway was crucial in the regulations of miR-21-3p on lipid metabolism, cell cycle arrest, and apoptosis of TECs. Conclusions MiR-21-3p mediates metabolism and cell fate alterations of TECs via manipulating AKT/CDK2-FOXO1 pathway, and that is crucial in the regulation of energy metabolism of TECs during SAKI.