miR-124-3p improves mitochondrial function of renal tubular epithelial cells in db/db mice.

Diabetic kidney disease (DKD) is one of the most serious complications of diabetes mellitus (DM) and the main cause of end-stage renal failure. However, the pathogenesis of DKD is complicated. In this study, we found that miR-124-3p plays a key role in regulating renal mitochondrial function and explored its possible mechanism in DKD progression by performing a series of in vitro and in vivo experiments. Decreased expression of miR-124-3p was found in db/db mice compared to db/m mice. Moreover, miR-124-3p down-regulated FOXQ1 by targeting FOXQ1 mRNA 3'-UTR in NRK-52E cells. Also, an increase in FOXQ1 and down-regulation of Sirt4 were found in db/db mouse kidney and renal tubular epithelial cells cultured with high glucose and high lipid. Overexpression of FOXQ1 could further down-regulate the expression of Sirt4 and aggravate the damage of mitochondria. Conversely, the knockdown of the FOXQ1 gene induced Sirt4 expression and partially restored mitochondrial function. To verify the effects of miR-124-3p on Sirt4 and mitochondria, we found that miR-124-3p mimics could up-regulate Sirt4 and inhibit ROS production and MitoSOX, thus restoring the number and morphology of mitochondria. These results showed that under high-glucose and high-lipid conditions, the down-regulation of miR-124-3p induces FOXQ1 in renal tubular epithelial cells, which in turn suppresses Sirt4 and leads to mitochondrial dysfunction, promoting the development of DKD.

Alpha lipoamide inhibits diabetic kidney fibrosis via improving mitochondrial function and regulating RXRα expression and activation.

Previous studies have shown mitochondrial dysfunction in various acute kidney injuries and chronic kidney diseases. Lipoic acid exerts potent effects on oxidant stress and modulation of mitochondrial function in damaged organ. In this study we investigated whether alpha lipoamide (ALM), a derivative of lipoic acid, exerted a renal protective effect in a type 2 diabetes mellitus mouse model. 9-week-old db/db mice were treated with ALM (50 mg·kg-1·d-1, i.g) for 8 weeks. We showed that ALM administration did not affect blood glucose levels in db/db mice, but restored renal function and significantly improved fibrosis of kidneys. We demonstrated that ALM administration significantly ameliorated mitochondrial dysfunction and tubulointerstitial fibrotic lesions, along with increased expression of CDX2 and CFTR and decreased expression of ß-catenin and Snail in kidneys of db/db mice. Similar protective effects were observed in rat renal tubular epithelial cell line NRK-52E cultured in high-glucose medium following treatment with ALM (200 µM). The protective mechanisms of ALM in diabetic kidney disease (DKD) were further explored: Autodock Vina software predicted that ALM could activate RXRα protein by forming stable hydrogen bonds. PROMO Database predicted that RXRα could bind the promoter sequences of CDX2 gene. Knockdown of RXRα expression in NRK-52E cells under normal glucose condition suppressed CDX2 expression and promoted phenotypic changes in renal tubular epithelial cells. However, RXRα overexpression increased CDX2 expression which in turn inhibited high glucose-mediated renal tubular epithelial cell injury. Therefore, we reveal the protective effect of ALM on DKD and its possible potential targets: ALM ameliorates mitochondrial dysfunction and regulates the CDX2/CFTR/ß-catenin signaling axis through upregulation and activation of RXRα. Schematic figure illustrating that ALM alleviates diabetic kidney disease by improving mitochondrial function and upregulation and activation of RXRα, which in turn upregulated CDX2 to exert an inhibitory effect on ß-catenin activation and nuclear translocation. RTEC renal tubular epithelial cell. ROS Reactive oxygen species. RXRα Retinoid X receptor-α. Mfn1 Mitofusin 1. Drp1 dynamic-related protein 1. MDA malondialdehyde. 4-HNE 4-hydroxynonenal. T-SOD Total-superoxide dismutase. CDX2 Caudal-type homeobox transcription factor 2. CFTR Cystic fibrosis transmembrane conductance regulator. EMT epithelial mesenchymal transition. α-SMA Alpha-smooth muscle actin. ECM extracellular matrix. DKD diabetic kidney disease. Schematic figure was drawn by Figdraw ( www.figdraw.com ).

BMP-7 ameliorates partial epithelial-mesenchymal transition by restoring SnoN protein level via Smad1/5 pathway in diabetic kidney disease.

Tubulointerstitial fibrosis (TIF) is involved in the development of diabetic kidney disease (DKD). Transforming growth factor ß1 (TGF-ß1) is involved in the extensive fibrosis of renal tissue by facilitating the partial epithelial-mesenchymal transition (EMT), increasing the synthesis of extracellular matrix (ECM), inhibiting degradation, inducing apoptosis of renal parenchyma cells, and activating renal interstitial fibroblasts and inflammatory cells. Recent studies indicated that bone morphogenetic protein-7 (BMP-7) upregulated the expression of endogenous SnoN against renal TIF induced by TGF-ß1 or hyperglycemia. Nevertheless, the mechanisms underlying the BMP-7-mediated restoration of SnoN protein level remains elusive. The present study demonstrated the increased expression of BMP-7 in diabetic mellitus (DM) mice by hydrodynamic tail vein injection of overexpressed BMP-7 plasmid, which attenuated the effects of DM on kidney in mice. Partial tubular EMT and the accumulation of Collagen-III were resisted in DM mice that received overexpressed BMP-7 plasmid. Similar in vivo results showed that BMP-7 was competent to alleviate NRK-52E cells undergoing partial EMT in a high-glucose milieu. Furthermore, exogenous BMP-7 activated the Smad1/5 pathway to promote gene transcription of SnoN and intervened ubiquitination of SnoN; both effects repaired the SnoN protein level in renal tubular cells and kidney tissues of DM mice. Therefore, these findings suggested that BMP-7 could upregulate SnoN mRNA and protein levels by activating the classical Smad1/5 pathway to refrain from the partial EMT of renal tubular epithelial cells and the deposition of ECM in DKD-induced renal fibrosis.

Atorvastatin Restores PPARα Inhibition of Lipid Metabolism Disorders by Downregulating miR-21 Expression to Improve Mitochondrial Function and Alleviate Diabetic Nephropathy Progression.

Atorvastatin is a classical lipid-lowering drug. It has been reported to have renoprotective effects, such as reducing urinary protein excretion and extracellular matrix aggregation. The present study aimed to investigate the specific mechanism of action of Atorvastatin in type 1 diabetic mice (T1DM) in inhibiting renal tubular epithelial cell injury following treatment with high glucose and high fat. The anti-injury mechanism of Atorvastatin involved the inhibition of miR-21 expression and the upregulation of the transcription and expression of its downstream gene Peroxisome proliferator-activated receptors-α(PPARα). An increase in blood glucose and lipid levels was noted in the T1DM model, which was associated with renal fibrosis and inflammation. These changes were accompanied by increased miR-21 levels, downregulation of PPARα and Mfn1 expressions, and upregulation of Drp1 and IL6 expressions in renal tissues. These phenomena were reversed following the administration of Atorvastatin. miR-21 targeted PPARα by inhibiting its mRNA translation. Inhibition of miR-21 expression or Fenofibrate (PPARα agonist) administration prevented the decrease of PPARα in renal tubular epithelial cells under high glucose (HG) and high fat (Palmitic acid, PA) conditions, alleviating lipid metabolism disorders and reducing mitochondrial dynamics and inflammation. Consistent with the in vivo results, the in vitro findings also demonstrated that mRTECs administered with Atorvastatin in HG + PA increased PPARα expression and restored the normal expression of Mfn1 and Drp1, and effectively increasing the number of biologically active mitochondria and ATP content, reducing ROS production, and restoring mitochondrial membrane potential following Atorvastatin intervention. In addition, these effects were noted to the inhibition of FN expression and tubular cell inflammatory response; however, in the presence of miR-21mimics, the aforementioned effects of Atorvastatin were significantly diminished. Based on these observations, we conclude that Atorvastatin inhibits tubular epithelial cell injury in T1DM with concomitant induction of lipid metabolism disorders by a mechanism involving inhibition of miR-21 expression and consequent upregulation of PPARα expression. Moreover, Atorvastatin regulated lipid metabolism homeostasis and PPARα to restore mitochondrial function. The results emphasize the potential of Atorvastatin to exhibit lipid-regulating functions and non-lipid effects that balance mitochondrial dynamics.

Senescent renal tubular epithelial cells activate fibroblasts by secreting Shh to promote the progression of diabetic kidney disease.

Introduction: Diabetic kidney disease (DKD) is one of the complications of diabetes; however, the pathogenesis is not yet clear. A recent study has shown that senescence is associated with the course of DKD. In the present study, we explored whether senescent renal tubular cells promote renal tubulointerstitial fibrosis by secreting Sonic hedgehog (Shh) which mediates fibroblast activation and proliferation in DKD. Methods: A 36-week-old db/db mice model and the renal tubular epithelial cells were cultured in high glucose (HG, 60 mmol/L) medium for in vivo and in vitro experiments. Results: Compared to db/m mice, blood glucose, microalbuminuria, serum creatinine, urea nitrogen, and UACR (microalbuminuria/urine creatinine) were markedly increased in db/db mice. Collagen III, monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor-alpha (TNF-α) were also increased in db/db mice kidneys, suggesting fibrosis and inflammation in the organ. Moreover, the detection of SA-ß-galactosidase (SA-ß-Gal) showed that the activity of SA-ß-Gal in the cytoplasm of renal tubular epithelial cells increased, and the cell cycle inhibition of the expression of senescence-related gene cell cycle inhibitor p16 INK4A protein and p21 protein increased, indicating that renal fibrosis in db/db mice was accompanied by cell senescence. Furthermore, Shh is highly expressed in the injured renal tubules and in the kidney tissue of db/db mice, as detected by enzyme-linked immunosorbent assay (ELISA). The results of immunofluorescence staining showed increased positive staining for Shh in renal tubular epithelial cells of db/db mice and decreased positive staining for Lamin B1, but increased positive staining for γH2A.X in cells with high Shh expression; similar results were obtained in vitro. In addition, HG stimulated renal tubular epithelial cells to secrete Shh in the supernatant of the medium. D-gal treatment of renal tubular epithelial cells increased the protein levels of Shh and p21. We also found enhanced activation and proliferation of fibroblasts cultured with the supernatant of renal tubular epithelial cells stimulated by HG medium but the proliferative effect was significantly diminished when co-cultured with cyclopamine (CPN), an inhibitor of the Shh pathway. Discussion: In conclusion, HG induces renal tubular epithelial cell senescence, and the secretion of senescence-associated proteins and Shh mediates inflammatory responses and fibroblast activation and proliferation, ultimately leading to renal fibrosis.

Blood glucose control contributes to protein stability of Ski-related novel protein N in a rat model of diabetes.

Ski-related novel protein N (SnoN) negatively regulates the transforming growth factor-ß1 (TGF-ß1)/Smads signaling pathway and is present at a low level during diabetic nephropathy (DN), but its underlying regulatory mechanism is currently unknown. The present study aimed to assess the effects of insulin-controlled blood glucose on renal SnoN expression and fibrosis in rats with diabetes mellitus (DM). Streptozotocin-induced DM rats were treated with insulin glargine (INS group) following successful model establishment. Blood samples were collected and centrifuged for biochemical indexes and the kidneys were collected for morphological analysis. In vitro, rat renal proximal tubular epithelial cells were treated with high-glucose medium for 24 h and transferred to normal glucose medium for 24 h. The expression levels of TGF-ß1, SnoN, Smad ubiquitin regulatory factor 2 (Smurf2), Arkadia, Smads, E-cadherin, α-smooth muscle actin and collagen III were assessed by western blotting and immunohistochemistry. The ubiquitylation of SnoN was detected by immunoprecipitation, and the expression levels of SnoN mRNA were evaluated by reverse transcription-quantitative PCR. The biochemical parameters and morphology indicated that renal fibrosis was notable in the DM group and mitigated in the INS group. Compared with the control group, TGF-ß1, phosphor (p)-Smad2, p-Smad3, Smurf2 and Arkadia levels were enhanced in the DM group, and the levels of SnoN protein were decreased, whereas the levels of SnoN mRNA and ubiquitylation were increased in renal tissues. Notably, treatment with insulin reversed this trend. Furthermore, changing the glucose levels in the medium from high to normal glucose suppressed the epithelial-mesenchymal transition of NRK-52E cells by restoring the SnoN protein levels, and this phenomenon was impaired by the knockout of SnoN. SnoN protein levels were likely reduced through a mechanism enhanced by the ubiquitin proteasome system, which reversed the transcriptional activation of SnoN during DN progression. In addition, controlling blood glucose may delay DN fibrosis by rescuing the protein stability of SnoN.

The role of CDX2 in renal tubular lesions during diabetic kidney disease.

Renal tubules are vulnerable targets of various factors causing kidney injury in diabetic kidney disease (DKD), and the degree of tubular lesions is closely related to renal function. Abnormal renal tubular epithelial cells (RTECs) differentiation and depletion of cell junction proteins are important in DKD pathogenesis. Caudal-type homeobox transcription factor 2 (CDX2), represents a key nuclear transcription factor that maintains normal proliferation and differentiation of the intestinal epithelium. The present study aimed to evaluate the effects of CDX2 on RTECs differentiation and cell junction proteins in DKD. The results demonstrated that CDX2 was mainly localized in renal tubules, and downregulated in various DKD models. CDX2 upregulated E-cadherin and suppressed partial epithelial-mesenchymal transition (EMT), which can alleviate hyperglycemia-associated RTECs injury. Cystic fibrosis transmembrane conductance regulator (CFTR) was regulated by CDX2 in NRK-52E cells, and CFTR interfered with ß-catenin activation by binding to Dvl2, which is an essential component of Wnt/ß-catenin signaling. CFTR knockdown abolished the suppressive effects of CDX2 on Wnt/ß-catenin signaling, thereby upregulating cell junction proteins and inhibiting partial EMT in RTECs. In summary, CDX2 can improve renal tubular lesions during DKD by increasing CFTR amounts to suppress the Wnt/ß-catenin signaling pathway.

EI24 alleviates renal interstitial fibrosis through inhibition of epithelial-mesenchymal transition and fibroblast activation.

Etoposide-induced 2.4 (EI24) exerts tumor suppressor activity through participating in cell apoptosis, autophagy, and inflammation. However, its role in renal diseases has not been elucidated. This study showed that the EI24 level decreased gradually in the kidneys of mice with unilateral ureteral obstruction (UUO) and in another fibrosis model induced by diabetic kidney disease. The overexpression of EI24 was used to investigate the possible role both in vivo and in vitro. The overexpression 1 day after UUO through tail vein injection alleviated the progression of renal interstitial fibrosis (RIF). EI24 inhibited epithelial-mesenchymal transition, excessive deposition of the extracellular matrix, and activation of fibroblasts. Furthermore, administration of EI24-overexpressing plasmids restrained the phosphorylation of nuclear factor-κB (NF-κB) and c-Jun kinase (JNK) through regulating the proteasome-dependent degradation of TRAF2, and then, inhibited the expression of downstream inflammation-associated cytokines (interleukin-6, tumor necrosis factor-α, and monocyte chemotactic protein-1) and infiltration of macrophages and neutrophils in mouse kidney after UUO. In conclusion, the data indicated that EI24, a novel anti-fibrosis regulator, was important in the progression of RIF.

Autophagy-related protein EI24 delays the development of pulmonary fibrosis by promoting autophagy.

Etoposide-induced protein 2.4 (EI24) is an autophagy-associated protein and acts as a tumor suppressor. However, its role in tissue fibrosis remains unknown. Herein, a downregulation of EI24 levels in the lungs from mouse pulmonary fibrosis (PF) model and lung epithelial cells was observed in response to bleomycin (BLM) or transforming growth factor-ß1 (TGF-ß1). Then, the role of EI24 in PF was investigated through the upregulation of EI24 in vitro and in vivo. EI24 inhibited epithelial-mesenchymal transition (EMT) process and extracellular matrix (ECM) production in EI24-overexpressing cells after stimulation with BLM or TGF-ß1. The overexpression of EI24 at 14 days after the establishment of the PF model through tail vein injection delayed the progression of PF. Moreover, the administration of EI24-overexpressing plasmid promoted the autophagy level in the lungs of the PF mouse model. In addition, the inhibition of autophagy by 3-methyladenine limited the role of EI24 in these processes. Thus, the current data indicated that EI24 attenuates PF through inhibition of EMT process and ECM production by promoting autophagy.

Smad2 and Smad3 play antagonistic roles in high glucose-induced renal tubular fibrosis via the regulation of SnoN.

Diabetic nephropathy (DN) is a serious microvascular complication of diabetes mellitus.The main pathological features of DN include glomerular sclerosis and renal tubular interstitial fibrosis, which results in epithelial mesenchymal transition (EMT) and excessive extracellular matrix (ECM) deposition.Transforming growth factor-ß1(TGF-ß1) is a critical factor that regulates the manifestation of renal fibrosis.Smad2 and Smad3 are the main downstream of the TGF-ß1 pathway. Ski-related novel protein N(SnoN) is a negative regulator of TGF-ß1, and inhibits the activation of the TGF-ß1/Smad2/3 signalling pathway. In this study, the expression of Smad2 and Smad3 proteins, SnoN mRNA, SnoN proteins, and the ubiquitination levels of SnoN were determined in DN rats and renal tubular epithelial cells(NRK52E cells). Knockdown and overexpression of Smad2 or Smad3 in NRK52E cells were used to investigate the specific roles of Smad2 and Smad3 in the development of high glucose-induced renal tubular fibrosis, with a specific focus on their effect on the regulation of SnoN expression. Our study demonstrated that Smad3 could inhibit SnoN expression and increase ECM deposition in NRK52E cells, to promote high glucose-induced renal tubular fibrosis. In contrast, Smad2 could induce SnoN expression and reduce ECM deposition, to inhibit high glucose-induced fibrosis. The underlying mechanism involves regulation of SnoN expression. These findings provide a novel mechanism to understanding the significant role of the TGF-ß1/ Smad2/3 pathway in DN.

BMP-7 inhibits renal fibrosis in diabetic nephropathy via miR-21 downregulation.

Epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) deposition in renal tubular epithelial cells are critical to diabetic nephropathy (DN) pathogenesis, but the underlying mechanisms remain undefined. Bone morphogenetic protein 7 (BMP-7) inhibits EMT and ECM accumulation in renal tubular epithelial cells cultured in presence of high glucose. Meanwhile, miRNA-21 (miR-21) downregulates Smad7, promoting EMT and ECM deposition. However, the association of BMP-7 with miR-21/Smad7 in DN is unknown. Here, NRK-52E cells incubated in presence of high glucose and STZ-induced C57BL diabetic mice were considered in vitro and in vivo models of DN, respectively. In both models, BMP-7 (mRNA/protein) amounts were decreased as well as Smad7 protein expression, while miR-21 expression and TGF-ß1/Smad3 pathway activation were enhanced, accompanied by enhanced EMT and ECM deposition. Further, addition of BMP-7 human recombinant cytokine (rhBMP-7) and injection of the BMP-7 overexpression plasmid in diabetic mice markedly downregulated miR-21 and upregulated Smad7, reduced Smad3 activation without affecting TGF-ß1 amounts, and prevented EMT and ECM accumulation. MiR-21 overexpression in the in vitro model downregulated Smad7, promoted EMT and ECM accumulation without affecting BMP-7 amounts, and miR-21 downregulation reversed it. By interfering with BMP-7 and miR-21 expression in high glucose conditions, miR-21 amounts and Smad3 phosphorylation were further decreased. Smad7 was then upregulated, and EMT and ECM deposition were inhibited; these effects were reversed after miR-21 overexpression. These findings suggest that BMP-7 decreases renal fibrosis in DN by regulating miR-21/Smad7 signaling, providing a theoretical basis for the development of novel and effective therapeutic drugs for DN.

TAK1 may promote the development of diabetic nephropathy by reducing the stability of SnoN protein.

AIMS: This study aimed to investigate the role of transforming growth factor-ß-activated protein kinase 1(TAK1) in the development of diabetic nephropathy (DN) by regulating the protein stability of Ski-related novel protein N(SnoN). MAIN METHODS: A combination of in vivo and in vitro model systems was used to investigate how TAK1 regulated the expression of SnoN protein in DN. The study determined the effects of modulating the expression or activity of TAK1 on the SnoN protein level and its influence on the epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) deposition. KEY FINDINGS: Under the high-glucose condition, the activation of TGF-ß1/TAK1-induced phosphorylation and ubiquitination of SnoN protein resulted in reduced SnoN protein level as a consequence of enhanced SnoN degradation, which promoted EMT and ECM deposition in renal tubular epithelial cells. The study showed that TAK1 impaired SnoN protein level by decreasing the protein stability of SnoN. SIGNIFICANCE: TAK1 mediated the phosphorylation of SnoN, resulting in SnoN ubiquitination and eventual degradation, which enhanced EMT and ECM deposition to promote renal fibrosis during DN.

Ski-related novel protein suppresses the development of diabetic nephropathy by modulating transforming growth factor-ß signaling and microRNA-21 expression.

Unveiling the mechanisms that drive the pathological phenotypes of diabetic nephropathy (DN) could help develop new effective therapeutics for this ailment. Transforming growth factor-ß1 (TGF-ß1)/Smad3 signaling is aberrantly induced in DN, leading to elevated microRNA-21 (miR-21) expression and tissue fibrosis. Ski-related novel protein (SnoN) negatively regulates the TGF-ß pathway, but the relationship between SnoN and miR-21 has not been described in the context of DN. In this study, this association was investigated in vivo (streptozotocin-induced rat model of diabetes) and in vitro (NRK-52E model system under high glucose conditions). In both model systems, we observed reduced amounts of the SnoN protein and elevated miR-21 amounts, indicative of an inverse relationship. These changes in SnoN and miR-21 amounts were accompanied by reduced E-cadherin and elevated α-smooth muscle actin and collagen III levels, consistent with epithelial to mesenchymal transition (EMT). In vitro overexpression of SnoN in NRK-52E cells downregulated miR-21 at the transcriptional and posttranscriptional levels and repressed EMT and extracellular matrix (ECM) deposition. In contrast, knockdown of SnoN resulted in miR-21 upregulation, particularly at the transcriptional level. We further demonstrated that overexpression and inhibition of miR-21 promoted and suppressed EMT and ECM deposition, respectively, without affecting SnoN levels. Our results indicated that SnoN suppresses the development of DN as well as renal fibrosis by downregulating miR-21, and therefore represents a novel and promising therapeutic target for DN.