Buyang Huanwu Decoction protects against STZ-induced diabetic nephropathy by inhibiting TGF-β/Smad3 signaling-mediated renal fibrosis and inflammation

Abstract Background Buyang Huanwu Decoction (BHD) is a classical Chinese Medicine formula empirically used for diabetic nephropathy (DN). However, its therapeutic efficacies and the underlying mechanisms remain obscure. In our study, we aim to evaluate the renoprotective effect of BHD on a streptozotocin (STZ)-induced diabetic nephropathy mouse model and explore the potential underlying mechanism in mouse mesangial cells (MCs) treated with high glucose in vitro, followed by screening the active compounds in BHD. Methods Mice were received 50 mg/kg streptozotocin (STZ) or citrate buffer intraperitoneally for 5 consecutive days. BHD was intragastrically administrated for 12 weeks starting from week 4 after the diabetes induction. The quality control and quantitative analysis of BHD were studied by high-performance liquid chromatography (HPLC). Renal function was evaluated by urinary albumin excretion (UAE) using ELISA. The mesangial matrix expansion and renal fibrosis were measured using periodic acid-schiff (PAS) staining and Masson Trichrome staining. Mouse mesangial cells (MCs) were employed to study molecular mechanisms. Results We found that the impaired renal function in diabetic nephropathy was significantly restored by BHD, as indicated by the decreased UAE without affecting the blood glucose level. Consistently, BHD markedly alleviated STZ-induced diabetic glomerulosclerosis and tubulointerstitial injury as shown by PAS staining, accompanied by a reduction of renal inflammation and fibrosis. Mechanistically, BHD inhibited the activation of TGF-β1/Smad3 and NF-κB signaling in diabetic nephropathy while suppressing Arkadia expression and restoring renal Smad7. We further found that calycosin-7-glucoside (CG) was one of the active compounds from BHD, which significantly suppressed high glucose-induced inflammation and fibrosis by inhibiting TGF-β1/Smad3 and NF-κB signaling pathways in mesangial cells. Conclusion BHD could attenuate renal fibrosis and inflammation in STZ-induced diabetic kidneys via inhibiting TGF-β1/Smad3 and NF-κB signaling while suppressing the Arkadia and restoring renal Smad7. CG could be one of the active compounds in BHD to suppress renal inflammation and fibrosis in diabetic nephropathy.

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circRNA_010383 acts as a sponge for miR-135a and its downregulated expression contributes to renal fibrosis in diabetic nephropathy

10.2337/figshare.13177115.v1 ◽

2020 ◽

Author(s):

Ada Admin ◽

Fenfen Peng ◽

Wangqiu Gong ◽

Shuting Li ◽

Bohui Yin ◽

...

Keyword(s):

Diabetic Nephropathy ◽

High Glucose ◽

Renal Fibrosis ◽

Transient Receptor Potential ◽

Mesangial Cells ◽

Receptor Potential ◽

Vascular Complication ◽

Diabetic Patients

Diabetic nephropathy (DN), a vascular complication of diabetes mellitus, is the leading cause of death in diabetic patients. The contribution of aberrantly expressed circRNAs to diabetic nephropathy in vivo is poorly understood. Integrated comparative circRNA microarray profiling was used to examine the expression of circRNAs in diabetic kidney of db/db mice. We found that circRNA_010383 expression was markedly downregulated in diabetic kidneys, mesangial cells and tubular epithelial cells cultured in high-glucose conditions. circRNA_010383 colocalized with microRNA-135a (miR-135a) and inhibited miR-135a function by directly binding to miR-135a. In vitro, the knockdown of circRNA_010383 promoted the accumulation of extracellular matrix (ECM) proteins <a></a><a>and </a>downregulated the expression of transient receptor potential cation channel, subfamily C, member (TRPC1), which is a target protein of miR-135a. Furthermore, <a></a><a>circRNA_010383 overexpression</a> effectively inhibited the high-glucose-induced accumulation of ECM and increased TRPC1 levels in vitro. More importantly, the kidney-target of circRNA_010383 overexpression inhibited <a></a><a>proteinuria</a> and renal fibrosis in db/db mice. Mechanistically, we identified that a loss of circRNA_010383 promoted proteinuria and renal fibrosis in DN by acting as a sponge for miRNA-135a. This study reveals that circRNA_010383 may be a novel therapeutic target for DN in the future.

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Transcription Factor Egr1 is Involved in High Glucose-Induced Proliferation and Fibrosis in Rat Glomerular Mesangial Cells

Cellular Physiology and Biochemistry ◽

10.1159/000430177 ◽

2015 ◽

Vol 36 (6) ◽

pp. 2093-2107 ◽

Cited By ~ 38

Author(s):

Dan Wang ◽

Mei-Ping Guan ◽

Zong-Ji Zheng ◽

Wen-Qi Li ◽

Fu-Ping Lyv ◽

...

Keyword(s):

Diabetic Nephropathy ◽

High Glucose ◽

Mesangial Cells ◽

Glomerular Mesangial Cells ◽

Protein Levels ◽

Oletf Rats ◽

Type Iv ◽

Regulated Expression ◽

Stage Renal Disease ◽

Tgf Β1

Backgroud: Diabetic nephropathy is one of the most frequent causes of end-stage renal disease and is associated with proliferation of glomerular mesangial cells (MCs) and excessive production of the extracellular matrix (ECM). Several studies have shown that early growth response factor 1 (Egr1) plays a key role in renal fibrosis by regulating the expression of genes encoding ECM components. However, whether Egr1 also contributes to diabetic nephropathy is unclear. Methods: In the present study, we compared the expression of Egr1 in kidneys from OLETF rats with spontaneous type 2 diabetes and healthy LETO rats. We also examined whether high glucose and TGF-β1 signaling up-regulated Egr1 expression in cultured MCs, and whether Egr1 expression influenced MC proliferation and expression of ECM genes. Results: We found that higher expression of Egr1 and TGF-β1, at both the mRNA and protein levels, the kidneys from OLETF rats vs. LETO rats. High glucose or TGF-β1 signaling rapidly up-regulated expression of Egr1 mRNA and protein in cultured MCs. Overexpressing Egr1 in MCs by transfection with M61-Egr1 plasmid or treatment with high glucose up-regulated expression of fibronectin, type IV collagen and TGF-β1, and promoted MC proliferation. Conversely, siRNA-mediated silencing of Egr1 expression down-regulated these genes and inhibited MC proliferation. Chromatin immunoprecipitation (ChIP) assays revealed that Egr1 bound to the TGF-β1 promoter. Conclusion: Our results provide strong evidence that Egr1 contributes to diabetic nephropathy by enhancing MC proliferation and ECM production, in part by interacting with TGF-β1.

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Transcriptional Suppression of Diabetic Nephropathy with Novel Gene Silencer Pyrrole-Imidazole Polyamides Preventing USF1 Binding to the TGF-β1 Promoter

International Journal of Molecular Sciences ◽

10.3390/ijms22094741 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4741

Author(s):

Makiyo Okamura ◽

Noboru Fukuda ◽

Shu Horikoshi ◽

Hiroki Kobayashi ◽

Akiko Tsunemi ◽

...

Keyword(s):

Diabetic Nephropathy ◽

High Glucose ◽

Transforming Growth Factor ◽

Mesangial Cells ◽

Diabetic Rats ◽

Glomerular Injury ◽

Upstream Stimulatory Factor ◽

Injury Score ◽

Upstream Stimulatory Factor 1 ◽

Tgf Β1

Upstream stimulatory factor 1 (USF1) is a transcription factor that is increased in high-glucose conditions and activates the transforming growth factor (TGF)-β1 promoter. We examined the effects of synthetic pyrrole-imidazole (PI) polyamides in preventing USF1 binding on the TGF-β1 promoter in Wistar rats in which diabetic nephropathy was established by intravenous administration of streptozotocin (STZ). High glucose induced nuclear localization of USF1 in cultured mesangial cells (MCs). In MCs with high glucose, USF1 PI polyamide significantly inhibited increases in promoter activity of TGF-β1 and expression of TGF-β1 mRNA and protein, whereas it significantly decreased the expression of osteopontin and increased that of h-caldesmon mRNA. We also examined the effects of USF1 PI polyamide on diabetic nephropathy. Intraperitoneal injection of USF1 PI polyamide significantly suppressed urinary albumin excretion and decreased serum urea nitrogen in the STZ-diabetic rats. USF1 PI polyamide significantly decreased the glomerular injury score and tubular injury score in the STZ-diabetic rats. It also suppressed the immunostaining of TGF-β1 in the glomerulus and proximal tubules and significantly decreased the expression of TGF-β1 protein from kidney in these rats. These findings indicate that synthetic USF1 PI polyamide could potentially be a practical medicine for diabetic nephropathy.

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High glucose-induced RhoA activation requires caveolae and PKCβ1-mediated ROS generation

AJP Renal Physiology ◽

10.1152/ajprenal.00749.2010 ◽

2012 ◽

Vol 302 (1) ◽

pp. F159-F172 ◽

Cited By ~ 24

Author(s):

Y. Zhang ◽

F. Peng ◽

B. Gao ◽

A. J. Ingram ◽

J. C. Krepinsky

Keyword(s):

Diabetic Nephropathy ◽

Nadph Oxidase ◽

High Glucose ◽

Transforming Growth Factor ◽

Mesangial Cells ◽

Ros Generation ◽

Caveolin 1 ◽

Rhoa Activation ◽

Rhoa Signaling ◽

Tgf Β1

Glomerular matrix accumulation is a hallmark of diabetic nephropathy. We previously showed that RhoA activation by high glucose in mesangial cells (MC) leads to matrix upregulation (Peng F, Wu D, Gao B, Ingram AJ, Zhang B, Chorneyko K, McKenzie R, Krepinsky JC. Diabetes 57: 1683–1692, 2008). Here, we study the mechanism whereby RhoA is activated. In primary rat MC, RhoA activation required glucose entry and metabolism. Broad PKC inhibitors (PMA, bisindolylmaleimide, Gö6976), as well as specific PKCβ blockade with an inhibitor and small interfering RNA (siRNA), prevented RhoA activation by glucose. PKCβ inhibition also abrogated reactive oxygen species (ROS) generation by glucose. The ROS scavenger N-acetylcysteine (NAC) or NADPH oxidase inhibitors apocynin and DPI prevented glucose-induced RhoA activation. RhoA and some PKC isoforms localize to caveolae. Chemical disruption of these microdomains prevented RhoA and PKCβ1 activation by glucose. In caveolin-1 knockout cells, glucose did not induce RhoA and PKCβ1 activation; these responses were rescued by caveolin-1 reexpression. Furthermore, glucose-induced ROS generation was significantly attenuated by chemical disruption of caveolae and in knockout cells. Downstream of RhoA signaling, activator protein-1 (AP-1) activation was also inhibited by disrupting caveolae, was absent in caveolin-1 knockout MC and rescued by caveolin-1 reexpression. Finally, transforming growth factor (TGF)-β1 upregulation, mediated by AP-1, was prevented by RhoA signaling inhibition and by disruption or absence of caveolae. In conclusion, RhoA activation by glucose is dependent on PKCβ1-induced ROS generation, most likely through NADPH oxidase. The activation of PKCβ1 and its downstream effects, including upregulation of TGF-β1, requires caveolae. These microdomains are thus important mediators of the profibrogenic process associated with diabetic nephropathy.

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Beyond proteinuria: VDR activation reduces renal inflammation in experimental diabetic nephropathy

AJP Renal Physiology ◽

10.1152/ajprenal.00090.2011 ◽

2012 ◽

Vol 302 (6) ◽

pp. F647-F657 ◽

Cited By ~ 100

Author(s):

Maria-Dolores Sanchez-Niño ◽

Milica Bozic ◽

Elizabeth Córdoba-Lanús ◽

Petya Valcheva ◽

Olga Gracia ◽

...

Keyword(s):

Renal Function ◽

Diabetic Nephropathy ◽

Mrna Expression ◽

Experimental Model ◽

High Glucose ◽

Glomerular Cell ◽

Renal Inflammation ◽

Matrix Deposition ◽

Mediators Of Inflammation ◽

Anti Inflammatory

Local inflammation is thought to contribute to the progression of diabetic nephropathy. The vitamin D receptor (VDR) activator paricalcitol has an antiproteinuric effect in human diabetic nephropathy at high doses. We have explored potential anti-inflammatory effects of VDR activator doses that do not modulate proteinuria in an experimental model of diabetic nephropathy to gain insights into potential benefits of VDR activators in those patients whose proteinuria is not decreased by this therapy. The effect of calcitriol and paricalcitol on renal function, albuminuria, and renal inflammation was explored in a rat experimental model of diabetes induced by streptozotocin. Modulation of the expression of mediators of inflammation by these drugs was explored in cultured podocytes. At the doses used, neither calcitriol nor paricalcitol significantly modified renal function or reduced albuminuria in experimental diabetes. However, both drugs reduced the total kidney mRNA expression of IL-6, monocyte chemoattractant protein (MCP)-1, and IL-18. Immunohistochemistry showed that calcitriol and paricalcitol reduced MCP-1 and IL-6 in podocytes and tubular cells as well as glomerular infiltration by macrophages, glomerular cell NF-κB activation, apoptosis, and extracellular matrix deposition. In cultured podocytes, paricalcitol and calcitriol at concentrations in the physiological and clinically significant range prevented the increase in MCP-1, IL-6, renin, and fibronectin mRNA expression and the secretion of MCP-1 to the culture media induced by high glucose. In conclusion, in experimental diabetic nephropathy VDR activation has local renal anti-inflammatory effects that can be observed even when proteinuria is not decreased. This may be ascribed to decreased inflammatory responses of intrinsic renal cells, including podocytes, to high glucose.

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circRNA_010383 acts as a sponge for miR-135a and its downregulated expression contributes to renal fibrosis in diabetic nephropathy

10.2337/figshare.13177115 ◽

2020 ◽

Author(s):

Ada Admin ◽

Fenfen Peng ◽

Wangqiu Gong ◽

Shuting Li ◽

Bohui Yin ◽

...

Keyword(s):

Diabetic Nephropathy ◽

High Glucose ◽

Renal Fibrosis ◽

Transient Receptor Potential ◽

Mesangial Cells ◽

Receptor Potential ◽

Vascular Complication ◽

Diabetic Patients

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Suppression of Gremlin by RNA interference attenuates glucose-induced fibrogenesis in rat mesangial cells

10.21203/rs.2.21574/v1 ◽

2020 ◽

Author(s):

Xiujuan Zhang ◽

Li Zhang ◽

Xinzhe Wang ◽

Yuwen Song ◽

Kai Lou ◽

...

Keyword(s):

Diabetic Nephropathy ◽

Rna Interference ◽

High Glucose ◽

Renal Fibrosis ◽

Mesangial Cells ◽

Lentiviral Vector ◽

Type Iv ◽

Key Factor ◽

Vector Targeting ◽

End Stage

Abstract Background: diabetic nephropathy (DN) is the most common cause of end-stage of renal disease. It is beneficial for us to find effective way to treat the disease. Gremlin is deemed as a key factor in the development of diabetic nephropathy at present. We hypothesized that the pathological changes might be prevented by eliminating Gremlin function in high glucose-induced renal fibrosis. Methods: lentiviral vector targeting Gremlin was applied to inhibit Gremlin expression at a high glucose concentration which simulated diabetic nephropathy in rat mesangial cells. Results: the shRNA vector, designated shGremlin, significantly inhibited Gremlin expression in rat mesangial cells cultured under high glucose conditions. Increase in BMP-7 as well as its downstream genes or proteins including phospholated Smad 1/5/8, type IV collagen and fibronectin was observed. Conclusions: our work provides a valuable method to prevent glomerular/renal fibrosis with RNA interference, and also enable development of new therapies that target Gremlin.

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P0969PARKIN INHIBITS THE PROGRESSION OF DIABETIC NEPHROPATHY BY PROMOTING GATA4 DEGRADATION TO REDUCE THE SENESCENCE OF RENAL TUBULAR CELLS

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfaa144.p0969 ◽

2020 ◽

Vol 35 (Supplement_3) ◽

Author(s):

Kehong Chen ◽

Jia Chen ◽

Yani He

Keyword(s):

Renal Function ◽

Diabetic Nephropathy ◽

High Glucose ◽

Specific Gene ◽

Protective Mechanism ◽

Premature Senescence ◽

Renal Tubular Cells ◽

Renal Tubular ◽

Tgf Β1

Abstract Background and Aims Diabetic nephropathy (DN) can cause end-stage renal disease. During the progression of DN, hyperglycemia accelerates the senescence of renal tubular epithelial cells (RTECs), leading to the tubulointerstitial inflammation and fibrosis. E3-ubiquitin ligase Parkin can regulate several pathophysiological processes. This study explored the role and mechanisms of Parkin in the progression of diabetic nephropathy (DN). Method 149 cases of patients with DN diagnosed by renal biopsy were recruited in our study. 32 normal kidney samples were obtained from renal carcinoma as control. Renal Parkin expression was detected by immunohistochemistry. The correlation between Parkin expression and renal pathological injury scores, renal function injury parameters were analyzed. In vivo, we generated Parkin overexpressed streptozotocin-induced DN mice using ultrasound- mediated adenovirus transfection. In vitro, knockdown and overexpression experiments were performed by parkin siRNA or Parkin overexpressed adenovirus in high glucose (HG) stimulated mouse primary RTEC. Moreover, we used co-immunoprecipitation and pull down experiments to evaluate the interaction of GATA4 with parkin. Results We found that Parkin expression gradually decreased and was inversely associated with IL-6, TGF-β1 and GATA4 expression in the kidney during the progression of DN. Parkin over-expression (OE) reduced inflammation, fibrosis, premature senescence of RTECs and improved renal function while Parkin knockout (KO) had opposite effects in DN mice. Parkin-OE decreased GATA4 protein, but not its mRNA transcripts in the kidney of DN mice and high glucose (HG)-treated RTECs. Immunoprecipitation indicated that Parkin directly interacted with GATA4 in DN kidney. Parkin-OE enhanced GATA4 ubiquitination. Furthermore, Parkin-KO up-regulated growth arrest specific gene 1 (GAS1) expression in renal tubular tissues of DN mice and GATA4-OE enhanced the HG-up-regulated GAS1 expression in RTECs. Conversely, GAS1-OE mitigated the effect of Parkin-OE on HG-induced P21, IL-6 and TGF-β1 expression in RTECs. These results indicate that Parkin inhibits the progression of DN by promoting GATA4 ubiquitination and downregulating the GATA4/GAS1 signaling to inhibit premature senescence, inflammation and fibrosis in DN mice. Thus, these findings uncover new mechanisms underlying the action of Parkin during the process of DN. Conclusion Parkin inhibited the progression of DN by promoting GATA4 ubiquitination and downregulating the GATA4/GAS1 signaling to inhibit premature senescence, inflammation and fibrosis. Our findings uncover a novel endogenous protective mechanism by which Parkin regulates the senescence of RTECs, inflammation and fibrosis during the pathogenesis of DN.

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Circ-ACTR2 aggravates the high glucose-induced cell dysfunction of human renal mesangial cells through mediating the miR-205-5p/HMGA2 axis in diabetic nephropathy

Diabetology & Metabolic Syndrome ◽

10.1186/s13098-021-00692-x ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Jie Yun ◽

Jinyu Ren ◽

Yufei Liu ◽

Lijuan Dai ◽

Liqun Song ◽

...

Keyword(s):

Oxidative Stress ◽

Diabetic Nephropathy ◽

High Glucose ◽

Cell Injury ◽

Mesangial Cells ◽

Protein Detection ◽

Cell Counting ◽

Luciferase Reporter ◽

Enzyme Linked Immunosorbent Assay ◽

Cell Dysfunction

Abstract Background Circular RNAs (circRNAs) have been considered as pivotal biomarkers in Diabetic nephropathy (DN). CircRNA ARP2 actin-related protein 2 homolog (circ-ACTR2) could promote the HG-induced cell injury in DN. However, how circ-ACTR2 acts in DN is still unclear. This study aimed to explore the molecular mechanism of circ-ACTR2 in DN progression, intending to provide support for the diagnostic and therapeutic potentials of circ-ACTR2 in DN. Methods RNA expression analysis was conducted by the quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Cell growth was measured via Cell Counting Kit-8 and EdU assays. Inflammatory response was assessed by Enzyme-linked immunosorbent assay. The protein detection was performed via western blot. Oxidative stress was evaluated by the commercial kits. The molecular interaction was affirmed through dual-luciferase reporter and RNA immunoprecipitation assays. Results Circ-ACTR2 level was upregulated in DN samples and high glucose (HG)-treated human renal mesangial cells (HRMCs). Silencing the circ-ACTR2 expression partly abolished the HG-induced cell proliferation, inflammation and extracellular matrix accumulation and oxidative stress in HRMCs. Circ-ACTR2 was confirmed as a sponge for miR-205-5p. Circ-ACTR2 regulated the effects of HG on HRMCs by targeting miR-205-5p. MiR-205-5p directly targeted high-mobility group AT-hook 2 (HMGA2), and HMGA2 downregulation also protected against cell injury in HG-treated HRMCs. HG-mediated cell dysfunction was repressed by miR-205-5p/HMGA2 axis. Moreover, circ-ACTR2 increased the expression of HMGA2 through the sponge effect on miR-205-5p in HG-treated HRMCs. Conclusion All data have manifested that circ-ACTR2 contributed to the HG-induced DN progression in HRMCs by the mediation of miR-205-5p/HMGA2 axis.

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Protective Effects of Ethanolic Extract from Rhizome of Polygoni avicularis against Renal Fibrosis and Inflammation in a Diabetic Nephropathy Model

International Journal of Molecular Sciences ◽

10.3390/ijms22137230 ◽

2021 ◽

Vol 22 (13) ◽

pp. 7230

Author(s):

Jung-Joo Yoon ◽

Ji-Hun Park ◽

Yun-Jung Lee ◽

Hye-Yoom Kim ◽

Byung-Hyuk Han ◽

...

Keyword(s):

Insulin Resistance ◽

Diabetic Nephropathy ◽

Renal Dysfunction ◽

Renal Fibrosis ◽

Mesangial Cells ◽

Resistance Index ◽

Ethanolic Extract ◽

Glomerular Sclerosis ◽

Oral Glucose ◽

Inflammatory Factor

Progressive diabetic nephropathy (DN) in diabetes leads to major morbidity and mortality. The major pathological alterations of DN include mesangial expansion, extracellular matrix alterations, tubulointerstitial fibrosis, and glomerular sclerosis. Polygoni avicularis is widely used in traditional oriental medicine and has long been used as a diuretic, astringent, insecticide and antihypertensive. However, to the best of the authors’ knowledge, the effects of the ethanolic extract from rhizome of Polygoni avicularis (ER-PA) on DN have not yet been assessed. The present study aimed to identify the effect of ER-PA on renal dysfunction, which has been implicated in DN in human renal mesangial cells and db/db mice and investigate its mechanism of action. The in vivo experiment was performed using Polygoni avicularis-ethanol soluble fraction (ER-PA) and was administrated to db/db mice at 10 and 50 mg/kg dose. For the in vitro experiments, the human renal mesangial cells were induced by high glucose (HG, 25 mM). The ER-PA group showed significant amelioration in oral glucose tolerance, and insulin resistance index. ER-PA significantly improved the albumin excretion and markedly reduced plasma creatinine, kidney injury molecule-1 and C-reactive protein. In addition, ER-PA significantly suppressed inflammatory cytokines. Histopathologically, ER-PA attenuated glomerular expansion and tubular fibrosis in db/db mice. Furthermore, ER-PA suppressed the expression of renal fibrosis biomarkers (TGF and Collagen IV). ER-PA also reduced the NLR family pyrin domain containing 3 inflammatory factor level. These results suggest that ER-PA has a protective effect against renal dysfunction through improved insulin resistance as well as the inhibition of nephritis and fibrosis in DN.

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