Yanbin Gao

Effect of miR-21 on Renal Fibrosis by Regulating MMP-9 and TIMP1 in kk-ay Diabetic Nephropathy Mice

MicroRNAs (miRs) play important roles in initiation and progression of many pathologic processes. However, the roles of miRs in diabetic nephropathy remain unclear. This study was to determine whether miR-21 was involved in diabetic nephropathy and to explore the relationship between miR-21 and MMP9/TIMP1 expression in diabetic nephropathy. In situ hybridization studies showed that miR-21 was primarily localized and distributed in cortical glomerular and renal tubular cells in diabetic kk-ay kidney. Real-time quantitative RT-PCR demonstrated that the expression of miR-21 was significantly increased in kk-ay mice, compared with control C57BL mice. Interestingly, miR-21 expression positively correlated with urine albumin creatine ratio (ACR), TIMP1, collagen IV (ColIV), and fibronectin (FN); while negatively correlated with creatine clearance ratio (Ccr) and MMP-9 protein. Importantly, antagomir-21 not only ameliorated Ccr and ACR but also decreased TIMP1, ColIV, and FN proteins. In conclusion, our data demonstrate that miR-21 contributes to renal fibrosis by mediating MMP9/TIMP1 and that inhibition of miR-21 may be a novel target for diabetic nephropathy.

miR-21 overexpression enhances TGF-β1-induced epithelial-to-mesenchymal transition by target smad7 and aggravates renal damage in diabetic nephropathy

Epithelial-to-mesenchymal transition (EMT) plays an important role in renal interstitial fibrosis (RIF) with diabetic nephropathy (DN). Smad7 (a inhibitory smad), a downstream signaling molecules of TGF-β1, represses the EMT. The physiological function of miR-21 is closely linked to EMT and RIF. However, it remained unclear whether miR-21 over-expression affected TGF-β1-induced EMT by regulating smad7 in DN. In this study, real-time RT-PCR, cell transfection, luciferase reporter gene assays, western blot and confocal microscope were used, respectively. Here, we found that miR-21 expression was upregulated by TGF-β1 in time- and concentration -dependent manner. Moreover, miR-21 over-expression enhanced TGF-β1-induced EMT(upregulation of a-SMA and downregulation of E-cadherin) by directly down-regulating smad7/p-smad7 and indirectly up-regulating smad3/p-smad3, accompanied by the decrease of Ccr and the increase of col-IV, FN, the content of collagen fibers, RTBM, RTIAW and ACR. Meantime, the siRNA experiment showed that smad7 can directly regulate a-SMA and E-cadherin expression. More importantly, miR-21 inhibitor can not only inhibit EMT and fibrosis but also ameliorate renal structure and function. In conclusion, our results demonstrated that miR-21 overexpression can contribute to TGF-β1-induced EMT by inhibiting target smad7, and that targeting miR-21 may be a better alternative to directly suppress TGF-β1-mediated fibrosis in DN.

Tongxinluo ameliorates renal structure and function by regulating miR-21-induced epithelial-to-mesenchymal transition in diabetic nephropathy

Diabetic nephropathy (DN) is one of the most important diabetic microangiopathies. The epithelial-to-mesenchymal transition (EMT) plays an important role in DN. The physiological role of microRNA-21 (miR-21) was closely linked to EMT. However, it remained elusive whether tongxinluo (TXL) ameliorated renal structure and function by regulating miR-21-induced EMT in DN. This study aimed to determine the effect of TXL on miR-21-induced renal tubular EMT and to explore the relationship between miR-21 and TGF-β1/smads signals. Real-time RT-PCR, cell transfection, in situ hybridization (ISH), and laser confocal microscopy were used, respectively. Here, we revealed that TXL dose dependently lowered miR-21 expression in tissue, serum, and cells. Overexpression of miR-21 can enhance α-smooth muscle actin (SMA) expression and decrease E-cadherin expression by upregulating smad3/p-smad3 expression and downregulating smad7 expression. Interestingly, TXL also increased E-cadherin expression and decreased α-SMA expression by regulating miR-21 expression. More importantly, TXL decreased collagen IV, fibronectin, glomerular basement membrane, glomerular area, and the albumin/creatinine ratio, whereas it increased the creatinine clearance ratio. The results demonstrated that TXL ameliorated renal structure and function by regulating miR-21-induced EMT, which was one of the mechanisms to protect against DN, and that miR-21 may be one of the therapeutic targets for TXL in DN.

Exosomes from high glucose-treated glomerular endothelial cells activate mesangial cells to promote renal fibrosis

ABSTRACT The interaction between glomerular endothelial cells (GECs) and glomerular mesangial cells (GMCs) is an essential aspect of diabetic nephropathy (DN). Therefore, understanding how GECs communicate with GMCs in the diabetic environment is crucial for the development of new targets for the prevention and treatment of DN. Exosomes, nanometer-sized extracellular membrane vesicles secreted by various cell types, play important roles in cell-to-cell communication via the transfer of mRNA, microRNA and protein. In this study, we demonstrate that high glucose (HG)-treated GECs secrete a higher number of exosomes highly enriched in TGF-β1 mRNA compared with normal glucose (NG)-treated GECs. Exosomes released by HG-treated GECs can promote α-smooth muscle actin (α-SMA) expression, proliferation and extracellular matrix protein overproduction in GMCs through the TGF-β1/Smad3 signaling pathway. Thus, we provide new insights into the pathogenesis of DN that involves intercellular transfer of TGF-β1 mRNA in the GEC-to-GMC direction via exosomes. Summary: In this study, we demonstrate that TGF-β1-containing exosomes from high glucose-treated glomerular endothelial cells can activate glomerular mesangial cells to promote renal fibrosis.

Downregulation of miR-30c promotes renal fibrosis by target CTGF in diabetic nephropathy

MicroRNAs (miRs) play important roles in initiation and progression of many pathologic processes. However, the role of miR-30c in diabetic nephropathy (DN) remains unclear. This study was to determine whether miR-30c was involved in the mechanism of renal fibrosis by inhibiting target CTGF expression in DN. In this study, In Situ Hybridization(ISH), RT-PCR, cell transfection, western blotting and laser confocal telescope were used, respectively. ISH showed that miR-30c, concentrated in cytoplasmic foci in the proximity of the nucleus, was mainly localized in glomerular and renal tubular epithelial cells within the cortex. RT-PCR showed that miR-30c expression was significantly decreased in DN (p<0.05), consistent with of the results of ISH. Luciferase reporter gene assays showed that CTGF was a validated target of miR-30c. Furthermore, miR-30c overexpression directly decreased CTGF mRNA and protein. Conversely, miR-30c inhibitor enhanced CTGF expression. Interestingly, miR-30c expression was negatively correlated with ACR (r=-0.870, P=0.003) and positively correlated with Ccr (r=0.8230, P=0.01), whereas it was uncorrelated with KW/BW, SBP, HbA1C, HOMR-IR and T-Cho (p>0.05). More importantly, miR-30c mimics significantly decreased col-IV, FN, GSI, GBM, GA, MRA/CLA and ACR (p<0.05) and, in contrast, slightly but significantly increased Ccr (p<0.05). In conclusion, our results suggested that loss of miR-30c may contribute to the pathogenesis of DN by inhibiting target CTGF expression; replenishing miR-30c may ameliorate renal structure and function by reducing renal fibrosis in DN.

Traditional Chinese Medicine Tang-Luo-Ning Ameliorates Sciatic Nerve Injuries in Streptozotocin-Induced Diabetic Rats

Diabetic peripheral neuropathy (DPN) is a common microvascular complication of diabetes associated with high disability rate and low quality of life. Tang-Luo-Ning (TLN) is an effective traditional Chinese medicine for the treatment of DPN. To illustrate the underlying neural protection mechanisms of TLN, the effect of TLN on electrophysiology and sciatic nerve morphology was investigated in a model of streptozotocin-induced DPN, as well as the underlying mechanism. Sciatic motor nerve conduction velocity and digital sensory nerve conduction velocity were reduced in DPN and were significantly improved by TLN or α-lipoic acid at 10 and 20 weeks after streptozotocin injection. It was demonstrated that TLN intervention for 20 weeks significantly alleviated pathological injury as well as increased the phosphorylation of ErbB2, Erk, Bad (Ser112), and the mRNA expression of neuregulin 1 (Nrg1), GRB2-associated binding protein 1 (Gab1), and mammalian target of rapamycin (Mtor) in injured sciatic nerve. These novel therapeutic properties of TLN to promote Schwann cell survival may offer a promising alternative medicine for the patients to delay the progression of DPN. The underlying mechanism may be that TLN exerts neural protection effect after sciatic nerve injury through Nrg1/ErbB2→Erk/Bad Schwann cell survival signaling pathway.

Serum miR-21 may be a Potential Diagnostic Biomarker for Diabetic Nephropathy.

MiRNAs play important roles in initiation and progress of many pathologic processes. MiR-21 was closely associated with diabetic nephropathy (DN). However, whether serum miR-21 was as a potential diagnostic biomarker for DN and the relationship between serum miR-21 and tissue miR-21 remained unclear. In this study, real-time RT-PCR, cell transfection, luciferase reporter gene assays, western blot and confocal microscope were used, respectively. Here, we found that serum and renal tissue miR-21 was significantly elevated with the progress of DN. Moreover, luciferase reporter gene assays showed that smad7 was a validated miR-21 target, cell transfection showed that miR-21 overexpression downregulated target smad7 expression. Interestingly, serum miR-21 was significantly consistent with the alterations of tissue miR-21 with the development of DN. In addition, serum miR-21 was also positively correlated with GBM, GA, ACR and CCF, while negatively correlated with Ccr. Importantly, antagomiR-21 not only alleviated GBM, GA, ACR and CCF, but also ameliorated Ccr by increasing target smad7. In conclusion, our data demonstrated that serum miR-21 was closely associated with renal structure and function, and serum miR-21 may be regarded as a potential diagnostic biomarker of DN.

Tangluoning, a traditional Chinese medicine, attenuates in vivo and in vitro diabetic peripheral neuropathy through modulation of PERK/Nrf2 pathway

Prolonged hyperglycemia-induced oxidative stress and endoplasmic reticulum stress have been demonstrated to play a key role in progression of diabetic peripheral neuropathy (DPN). PERK/ Nrf2 pathway plays a predominant role in oxidative and endoplasmic reticulum (ER) stress which is associated with cell survival. This study examined the modulation of the PERK/Nrf2 pathway and apoptosis by a traditional Chinese medicine Tangluoning (TLN) in streptozotocin-induced DPN rat models and the effects of serum TLN on the PERK/Nrf2 pathway, apoptosis, intracellular reactive oxygen species and mitochondrial membrane potential in Schwann cells cultured in 150 mM glucose. It is found that TLN attenuated oxidative and ER stress and apoptosis through the PERK/Nrf2 pathway by upregulating p-PERK, Nrf2/ARE pathways and downregulating the CHOP-related apoptosis pathways in the experimental DPN models both in vivo and in vitro.

Exosomes from high glucose-treated glomerular endothelial cells trigger the epithelial-mesenchymal transition and dysfunction of podocytes

New data indicate that abnormal glomerular endothelial cell (GEC)-podocyte crosstalk plays a critical role in diabetic nephropathy (DN). The aim of our study is to investigate the role of exosomes from high glucose (HG)-treated GECs in the epithelial-mesenchymal transition (EMT) and dysfunction of podocytes. In this study, exosomes were extracted from GEC culture supernatants and podocytes were incubated with the GEC-derived exosomes. Here, we demonstrate that HG induces the endothelial-mesenchymal transition (EndoMT) of GECs and HG-treated cells undergoing the EndoMT secrete more exosomes than normal glucose (NG)-treated GECs. We show that GEC-derived exosomes can be internalized by podocytes and exosomes from HG-treated cells undergoing an EndoMT-like process can trigger the podocyte EMT and barrier dysfunction. Our study reveals that TGF-β1 mRNA is enriched in exosomes from HG-treated GECs and probably mediates the EMT and dysfunction of podocytes. In addition, our experimental results illustrate that canonical Wnt/β-catenin signaling is involved in the exosome-induced podocyte EMT. Our findings suggest the importance of paracrine communication via exosomes between cells undergoing the EndoMT and podocytes for renal fibrosis in DN. Thus, protecting GECs from the EndoMT and inhibiting TGF-β1-containing exosomes release from GECs is necessary to manage renal fibrosis in DN.

Metformin ameliorates skeletal muscle insulin resistance by inhibiting miR-21 expression in a high-fat dietary rat model

Insulin resistance (IR) plays a major role in the pathogenesis of abdominal obesity, hypertension, coronary heart disease, atherosclerosis and diabetes. miR-21 and TGF-β/smads is closely related to IR. However, it remained elusive whether metformin improved skeletal muscle insulin resistance (IRSM) by regulating miR-21 and its target signal TGF-β1/smads expression. In this study, high-fat diet rats with IR model and IR-skeletal muscle L6 cells (L6-SMCs) model were established, insulin sensitive index (ISI) and Homeostasis model assessment of IR (HOMA-IR) were applied, miR-21 and TGF-β1/smads mRNA expression were examined by RT-PCR, smad3 and smad7 protein were detected by western-blotting and laser scanning confocal microscopy (LSCM), the valid target of miR-21 was detected by luciferase reporter gene assay. Here, we found that metformin dose-dependently decreased miR-21 expression, accompanied by the decrease of HOMA-IR and the increase of HOMA-ISI. Luciferase report gene assay showed that smad7 was an effective target of miR-21. miR-21 overexpression directly downregulated smad7 and indirectly upregulated smad3 expression. Interestingly, miR-21 expression positively correlated with HOMA-IR and negatively correlated with HOMA-ISI. In conclusion, our results demonstrated that metformin improved IRSM by inhibiting miR-21 expression, and that miR-21 may be one of the therapeutic targets for IR.