Fibrosis In Diabetic Nephropathy Research Articles

Up-regulation of microRNA-93 inhibits TGF-β1-induced EMT and renal fibrogenesis by down-regulation of Orai1

TGF-β1-induced excessive deposition of ECM and EMT process of tubular epithelial cells play critical roles in the development and progression of fibrosis in diabetic nephropathy (DN). Orai1 has been demonstrated to be involved in TGF-β1-induced EMT via TGF-β/Smad3 pathway. We are aimed to explore the effects of miR-93 on TGF-β1-induced EMT process in HK2 cells. In this study, our data showed that miR-93 was dramatically decreased in renal tissues of patients with DN and TGF-β1-stimulated HK2 cells. Moreover, the decreased level of miR-93 was closely associated with the increased expression of Orai1. Overexpression of miR-93 decreased Orai1 expression, and then suppressed TGF-β1-mediated EMT and fibrogenesis. Next, we predicted that the Orai1 was a potential target gene of miR-93, and demonstrated that miR-93 could directly target Orai1. SiRNA targeting Orai1 was sufficient to suppress TGF-β1-induced EMT and fibrogenesis in HK2 cells. Furthermore, Overexpression of Orai1 partially reversed the protective effect of miR-93 overexpression on TGF-β1-mediated EMT and fibrogenesis in HK2 cells. Taken together, Orai1 and miR-93 significantly impact on the progression of TGF-β1-mediated EMT and fibrogenesis in HK2 cells, and they may represent novel targets for the prevention strategies of fibrosis in the context of DN.

High glucose up-regulates microRNA-34a-5p to aggravate fibrosis by targeting SIRT1 in HK-2 cells

Tubulointerstitial fibrosis (TIF) is crucial in the development of renal fibrosis in diabetic nephropathy(DN). Previous data shows that SIRT1 plays an important role on fibrosis, but the effect on TIF in DN and underlying mechanisms remains uncertain. In this study, we evaluated the vital role of SIRT1 and identified SIRT1 as a downstream target gene of microRNA-34a-5p (miR-34a-5p) in TIF of DN. The result revealed that expression of miR-34a-5p, fibronectin(FN),collagen type I (COL1) and transforming growth factor β1 (TGF-β1) were up-regulated accompanied by the corresponding down-regulation of SIRT1 in renal tissues of high fat diet and streptozotocin(HFD/STZ)induced diabetic mice with DN, and that the SIRT1 mRNA level was negatively correlated with miR-34a-5p expression in high glucose stimulated human proximal tubule cell line(HK-2) cells. We then demonstrated that overexpression of SIRT1 reduced, whereas small interfering RNA targeting SIRT1 enhanced the expressions of TGF-β1 and fibrosis-related genes including FN and COL1 in HK-2 cells. Furthermore, we identified that miR-34a-5p directly suppressed SIRT1 to increase the profibrogenic effects of TGFβ1 through targeting the 3'untranslated region of SIRT1. The functional correlation of miR-34a-5p induced SIRT1 decrease was supported by overexpression and inhibition of miR-34a-5p in HK-2 cells. All the results reveal that SIRT1 which is vital in the evolution of renal TIF in DN can be directly suppressed by miR-34a-5p, and suggest that miR-34a-5p is a new target for DN treatment.

Long Non-Coding RNA CYP4B1-PS1-001 Inhibits Proliferation and Fibrosis in Diabetic Nephropathy by Interacting with Nucleolin

Background/Aims: Our previous studies demonstrated that a novel long non-coding RNA, CYP4B1-PS1-001, was significantly downregulated in early diabetic nephropathy in vivo and in vitro, and CYP4B1-PS1-001 overexpression could inhibit the proliferation and fibrosis of mouse mesangial cells (MMCs). However, the underlying mechanism of the CYP4B1-PS1-001-mediated regulation of proliferation and fibrosis in diabetic nephropathy remains undetermined. Methods: RNA-protein pull-down assay, RNA-binding protein immunoprecipitation, and mass spectrometry were used to investigate CYP4B1-PS1-001 interacted with the upregulated protein nucleolin (NCL). siRNA method was applied to knockdown NCL in MMCs, the interaction between CYP4B1-PS1-001 and NCL were determined by Western blot analysis and RT-qPCR. The effect of CYP4B1-PS1-001 in the regulation of NCL was detected by cycloheximide (CHX) and ubiquitination assays. Results: We found that CYP4B1-PS1-001 interacts with NCL, and CYP4B1-PS1-001 inhibits the proliferation and fibrosis of MMCs depending on interaction with NCL. Furthermore, degradation of CYP4B1-PS1-001-associated NCL was mediated by a ubiquitin proteasome-dependent pathway. Conclusion: Our study provides evidence that CYP4B1-PS1-001 regulates the ubiquitination and degradation of NCL and thereby plays a critical role in the proliferation and fibrosis of MMCs, indicating that CYP4B1-PS1-001 and NCL may be promising prognostic biomarkers and molecular targets for the treatment of diabetic nephropathy.

Complement C5a inhibition moderates lipid metabolism and reduces tubulointerstitial fibrosis in diabetic nephropathy.

Complement C5 mediates pro-inflammatory responses in many immune-related renal diseases. Given that the C5a level is elevated in diabetes, we investigated whether activation of C5a/C5aR signalling plays a pathogenic role in diabetic nephropathy (DN) and the therapeutic potential of C5a inhibition for renal fibrosis. Human renal biopsies from patients with DN and control subjects were used for immunohistochemical staining of complement C5 components. Renal function and tubulointerstitial injury were compared between db/m mice, vehicle-treated mice and C5a inhibitor-treated db/db mice. A cell culture model of tubule epithelial cells (HK-2) was used to demonstrate the effect of C5a on the renal fibrotic pathway. Increased levels of C5a, but not of its receptor C5aR, were detected in renal tubules from patients with DN. The intensity of C5a staining was positively correlated with the progression of the disease. In db/db mice, administration of a novel C5a inhibitor, NOX-D21, reduced the serum triglyceride level and attenuated the upregulation of diacylglycerolacyltransferase-1 and sterol-regulatory element binding protein-1 expression and lipid accumulation in diabetic kidney. NOX-D21-treated diabetic mice also had reduced serum blood urea nitrogen and creatinine levels with less glomerular and tubulointerstitial damage. Renal transforming growth factor beta 1 (TGF-β1), fibronectin and collagen type I expressions were reduced by NOX-D21. In HK-2 cells, C5a stimulated TGF-β production through the activation of the PI3K/Akt signalling pathway. Blockade of C5a signalling by NOX-D21 moderates altered lipid metabolism in diabetes and improved tubulointerstitial fibrosis by reduction of lipid accumulation and TGF-β-driven fibrosis in diabetic kidney.

An integrin antagonist (MK‐0429) decreases proteinuria and renal fibrosis in the ZSF1 rat diabetic nephropathy model

Multiple integrins have been implicated in modulating renal function. Modulation of integrin function can lead to pathophysiological processes associated with diabetic nephropathy such as alterations in the glomerular filtration barrier and kidney fibrosis. The complexity of these pathophysiological changes implies that multiple integrin subtypes might need to be targeted to ameliorate the progression of renal disease. To address this hypothesis, we investigated the effects of MK‐0429, a compound that was originally developed as an αvβ3 inhibitor for the treatment of osteoporosis, on renal function and fibrosis. We demonstrated that MK‐0429 is an equipotent pan‐inhibitor of multiple av integrins. MK‐0429 dose‐dependently inhibited podocyte motility and also suppressed TGF‐β‐induced fibrosis marker gene expression in kidney fibroblasts. Moreover, in the obese ZSF1 rat model of diabetic nephropathy, chronic treatment with MK‐0429 resulted in significant reduction in proteinuria, kidney fibrosis, and collagen accumulation. In summary, our results suggest that inhibition of multiple integrin subtypes might lead to meaningful impact on proteinuria and renal fibrosis in diabetic nephropathy.

Periostin-binding DNA aptamer treatment attenuates renal fibrosis under diabetic conditions

Diabetic nephropathy, the major cause of chronic kidney disease, is associated with progressive renal fibrosis. Recently, accumulation of periostin, an extracellular matrix protein, was shown to augment renal fibrosis. Aptamers have higher binding affinities without developing the common side effects of antibodies. Thus, we evaluated the effect of periostin inhibition by an aptamer-based inhibitor on renal fibrosis under diabetic conditions. In vitro, transforming growth factor-β1 (TGF-β1) treatment significantly upregulated periostin, fibronectin, and type I collagen mRNA and protein expressions in inner medullary collecting duct (IMCD) cells. These increases were attenuated significantly in periostin-binding DNA aptamer (PA)-treated IMCD cells exposed to TGF-β1. In vivo, PA treatment attenuated the increased blood urea nitrogen levels in the diabetic mice significantly. Fibronectin and type I collagen mRNA and protein expressions increased significantly in the kidneys of diabetic mice: PA administration abrogated these increases significantly. Immunohistochemistry and Sirius Red staining also revealed that fibronectin expression was significantly higher and tubulointersititial fibrosis was significantly worse in diabetic mice kidneys compared with control mice. These changes were ameliorated by PA treatment. These findings suggested that inhibition of periostin using a DNA aptamer could be a potential therapeutic strategy against renal fibrosis in diabetic nephropathy.

O20 Low molecular weight fucoidan inhibits fibrotic processes in diabetic nephropathy

Introduction The aim of this study was to determine whether low-molecular-weight fucoidan (LMWF) can reduce TGF-β-mediated pathogenesis of tubulointerstitial fibrosis in diabetic nephropathy(DN) using in vitro and in vivo models. Methods LMWF was administered to diabetic rats at a daily dose of 100 mg/kg body weight by intragastric administration. Following 12 weeks of treatment, we measured body and kidney weight, blood glucose, blood pressure, creatinine and urea concentration in serum and urine, urine output, urinary osmolality, and urinary protein excretion. α-SMA, E-cadherin, fibronectin, TGF-β1, and CTGF were measured by Western blot to evaluate the level of epithelial-to-mesenchymal transition (EMT). Results 10 ng/ml TGF-β1 induced EMT and activated downstream pathways in HK2 cells. Treatment with LMWF significantly reversed EMT and dose-dependently inhibited accumulation of extracellular matrix proteins, including connective tissue growth factor and fibronectin. In type 1 and type 2 diabetic rat models, it was found that diabetes resulted in TGF-β signalling activation and increased expression of TGF-β downstream signals in the kidneys. LMWF significantly reduced blood urea nitrogen (BUN) and blood creatinine compared with the untreated type 1 diabetic group. LMWF also significantly reduced BUN and blood creatinine compared with the untreated type 2 diabetic group. Conclusion Our results demonstrate that LMWF treatment attenuated renal dysfunction and fibrogenesis in the kidneys of type 1 and type 2 diabetic rats. The present study suggests that LMWF may be developed as a candidate drug to prevent or inhibit fibrotic progression in DN.

Visualization of kidney fibrosis in diabetic nephropathy by long diffusion tensor imaging MRI with spin-echo sequence

Renal fibrosis (RF) is an indicator for progression of chronic kidney disease (CKD). Although diabetic nephropathy (DN) is the leading cause of CKD and end-stage renal disease in Western populations, the ability of MRI to evaluate RF in DN patients has not been determined. As a first step to identify possible MRI methods for RF evaluation, we examined the use of diffusion tensor imaging (DTI) MRI to evaluate RF in a rat model of DN (SHR/NDmcr-cp(cp/cp): SHR/ND). The signal-to-noise ratio in DTI MRI was enhanced using a spin-echo sequence, and a special kidney attachment was developed for long-term stabilization. The changes in renal temperature and blood flow during measurement were minimal, suggesting the feasibility of this method. At 38 weeks of age, RF had aggressively accumulated in the outer stripe (OS) of the outer medulla. FA maps showed that this method was successful in visualizing and evaluating fibrosis in the OS of the SHR/ND rat kidney (r = 0.7697, P = 0.0126). Interestingly, in the FA color maps, the directions of water molecule diffusion in RF were random, but distinct from conventional water diffusion in brain neuron fibers. These findings indicate that DTI MRI may be able to evaluate RF in CKD by DN.

Interleukin-22 ameliorated renal injury and fibrosis in diabetic nephropathy through inhibition of NLRP3 inflammasome activation

Diabetic nephropathy (DN) is one of the most lethal complications of diabetes mellitus with metabolic disorders and chronic inflammation. Although the cytokine IL-22 was initially implicated in the pathogenesis of chronic inflammatory diseases, recent studies suggested that IL-22 could suppress inflammatory responses and alleviate tissue injury. Herein, we examined the role of IL-22 in DN. We found that serum levels of IL-22 were significantly downregulated in both patients and mice with DN. The expression of IL-22 was further decreased with the progression of DN, whereas IL-22 gene therapy significantly ameliorated renal injury and mesangial matrix expansion in mice with established nephropathy. IL-22 could also markedly reduce high glucose-induced and TGF-β1-induced overexpression of fibronectin and collagen IV in mouse renal glomerular mesangial cells in a dose-dependent manner, suggesting the potential role of IL-22 to inhibit the overproduction of ECM in vitro. Simultaneously, IL-22 gene therapy drastically alleviated renal fibrosis and proteinuria excretion in DN. In addition, IL-22 gene therapy markedly attenuated hyperglycemia and metabolic disorders in streptozotocin-induced experimental diabetic mice. Notably, IL-22 drastically reversed renal activation of NLRP3, cleavage of caspase-1, and the maturation of IL-1β in DN, suggesting unexpected anti-inflammatory function of IL-22 via suppressing the activation of NLRP3 inflammasome in vivo. Moreover, IL-22 markedly downregulated high glucose-induced activation of NLRP3 inflammasome in renal mesangial cells in a dose-dependent manner, indicating that the effects of IL-22 on NLRP3 inflammasome activation was independent of improved glycemic control. These results suggested that nephroprotection by IL-22 in DN was most likely associated with reduced activation of NLRP3 inflammasome. In conclusion, our finding demonstrated that IL-22 could exert favorable effects on DN via simultaneously alleviating systemic metabolic syndrome and downregulating renal NLRP3/caspase-1/IL-1β pathway, suggesting that IL-22 might have therapeutic potential for the treatment of DN.

PDGF receptor-β uses Akt/mTORC1 signaling node to promote high glucose-induced renal proximal tubular cell collagen I (α2) expression.

Increased expression of PDGF receptor-β (PDGFRβ) has been shown in renal proximal tubules in mice with diabetes. The core molecular network used by high glucose to induce proximal tubular epithelial cell collagen I (α2) expression is poorly understood. We hypothesized that activation of PDGFRβ by high glucose increases collagen I (α2) production via the Akt/mTORC1 signaling pathway in proximal tubular epithelial cells. Using biochemical and molecular biological techniques, we investigated this hypothesis. We show that high glucose increases activating phosphorylation of the PDGFRβ, resulting in phosphorylation of phosphatidylinositol 3-kinase. A specific inhibitor, JNJ-10198409, and small interfering RNAs targeting PDGFRβ blocked this phosphorylation without having any effect on MEK/Erk1/2 activation. We also found that PDGFRβ regulates high glucose-induced Akt activation, its targets tuberin and PRAS40 phosphorylation, and finally, mTORC1 activation. Furthermore, inhibition of PDGFRβ suppressed high glucose-induced expression of collagen I (α2) in proximal tubular cells. Importantly, expression of constitutively active Akt or mTORC1 reversed these processes. As a mechanism, we found that JNJ and PDGFRβ knockdown inhibited high glucose-stimulated Hif1α expression. Furthermore, overexpression of Hif1α restored expression of collagen I (α2) that was inhibited by PDGFRβ knockdown in high glucose-stimulated cells. Finally, we show increased phosphorylation of PDGFRβ and its association with Akt/mTORC1 activation, Hif1α expression, and elevated collagen I (α2) levels in the renal cortex of mice with diabetes. Our results identify PDGFRβ as a driver in activating Akt/mTORC1 nexus for high glucose-mediated expression of collagen I (α2) in proximal tubular epithelial cells, which contributes to tubulointerstitial fibrosis in diabetic nephropathy.

The Repaire Mechanism of Mesenchymal Stem Cell microvesicle miR‐451a in Diabetic Nephropathy

ObjectiveMescenchymal stem cells (MSCs) play a central role in the repair of cell and tissue damage. Microvesicles (MVs) derived from MSCs have been described as a new mechanism of cell‐to‐cell communication. Furthermore, MVs shuttle a specific subset of cellular miRNA. miR‐451a was highly expressed in MVs. miR‐451a regulates the F‐box protein family invoved in Diabetic Nephropathy (DN) focused on signal transduction and cell cycle. Epithelial to mesenchymal transition (EMT) was critical for the renal fibrosis in Diabetic Nephropathy (DN). We studied the protective effects and mechanisms of miR‐451a in fibrosis of DN. In this study, the effects of MVs‐miR‐451a on EMT and fibrosis of DN were evaluated.MethodsMVs were obtained from MSCs supernatant by gradient centrifugation and ultracentrifugation. MVs were identified by scanning electron microscope, particle size analyzer, and flow cytometry. RNAs was extracted from MSC‐MVs. miR‐451a level in MSCs and MVs were tested through fluorogenic quantitative PCR (qPCR). HK‐2 damage model is established by the high sugar (30 mM) and uric acid (10 mg/dL) in vitro. MVs, miR‐451a mimic, miR‐451a inhibitor were added respectively to study change of miR‐451a's level, p15INK4b (p15) and p19INK4d (p19)'s level (represented cell cycle), E‐cadherin and α‐SMA (represented fibrosis) by qPCR. The corresponding protein expression levels of the markers were determined by WB. The changes of cell cycle were analyzed by flow cytometry. The cell proliferation ability and viability were tested by BrdU and CCK8. The mannitol hypertonic and fibroblasts‐micro capsule (HFF‐MV) control groups were set up.Constructed plasmids target genes p19INK4d 3 ′UTR region wild type plasmid and mutation plasmid. Determined p19INK4d was one of the miR‐451a target genes and the binding sites by dual luciferase reaction. The MVs and miR‐451a agomir were used to treat renal injury of fibrosis induced by high glucose and uric acid in vivo. The markers were the same to study in vitro. Furthermore, the renal function of mice was tested.ResultsMVs showed the presence of several markers known to be expressed on MSCs. The size of MVs ranged from 10 nm to 1μm. MVs enriched miR‐451a (4.87 fold change of MSCs). The expression level of miR‐451a in injured HK2 cell and kidney were decreased to 0.28 and 0.067 (compared with the control). Infusion of MVs and miR‐451a reduced HK2 cell and mice renal genes and proteins expression of p15, p19 and the factors associate with fibrosis. And they up‐regulated the proportion of S and G2, and stimulated the proliferation and viability of HK2 cell. p19 was identified as one of target gene of miR451a. After transplantation of MVs and agomir into DN mice with high uric acid, improvements in renal function and histopathological change of kidney were observed. Engrafted MVs were localized to areas of injured kidney.ConclusionOur results indicate that infused MVs may improve the recovery from fibrosis in DN mice with high uric acid by increasing miR‐451a expression to re‐start cell proliferation and regulate EMT.

Biomarkers of kidney injury and indicators of renal inflammation in African American men with diabetes and diabetic nephropathy

Diabetic nephropathy (DN) is a progressive kidney disease caused by damage to the capillaries in the kidneys' glomeruli due to changes in glomerular hemodynamics, causing renal lesions and fibrosis. Oxidative stress and inflammatory responses represent underlying causes for developing chronic kidney damage. Treatment outcomes for DN are poor because many patients with DN are diagnosed late due to lack of clinical tests based on sensitive biomarkers. Monocyte chemoattractant protein‐1 (MCP‐1) is a major promoter of inflammation, renal injury, and fibrosis in diabetic nephropathy. In the United States, minority ethnic communities bear a disproportionate burden of complications of diabetes and are at a higher risk of developing end stage renal disease. Participation in biomedical research by African American men is especially low due to historical mistrust with the research institutions. The objective of this study is to assess correlations between biomarkers of kidney injury and indicators of inflammation among three groups of African American men: 1) diabetic patients without DN, 2) diabetic patients with diagnosed DN, and 3) non‐diabetic controls. Fasting blood and urine samples were collected from patients recruited through a community health clinic. Surveys were administered to establish patient profiles and family history. Enzyme‐linked immunosorbent assays (ELISA) were used to determine the levels of plasma MCP‐1 and the levels of three biomarkers of kidney injury, namely, cystatin C, neutrophil gelatinase‐associated lipocalin (NGAL), and kidney injury molecule 1 (KIM‐1). The data were analyzed by 2‐way ANOVA. The data showed that the levels of serum cystatin C were significantly higher in patients with diagnosed DN compared to non‐diabetic controls (p<0.01). Additionally, diabetic patients showed a significant increase in serum cystatin C levels relative to the non‐diabetic controls (p<0.05). In contrast, urine cystatin C levels were significantly lower in patients with DN compared to the other two groups. Plasma NGAL levels were significantly higher in patients with DN compared to both non‐diabetic and diabetic patients (p<0.01). However, there was no significant difference in the levels of urine KIM‐1 among the three groups. In conclusion, serum MCP‐1 exhibited higher levels in patients with DN compared to both diabetic patients and non‐diabetic controls (p<0.0001). The significant increase in serum MCP‐1 levels suggests that inflammation is an underlying cause of kidney injury in African American men who have DN. Furthermore, the data suggest that a significant number of diabetic African American men may have undiagnosed kidney injury as shown by elevated cystatin C levels.Support or Funding InformationThis study was supported by the Minority Men's Health Initiative (MMHI) through NIH/NIMHD Center Award # U54MD008621 (CFDA 93.307), Sub‐Award # HU140004 and sub‐award #HU150006.

SnoN upregulation ameliorates renal fibrosis in diabetic nephropathy

Progressive reduction of SnoN is associated with gradual elevation of TGF-β1 during diabetic nephropathy progression, suggesting SnoN to be a possible mediator of TGF-β1 signaling, with potential therapeutic benefits against TGF- β1 –induced renal fibrosis. To characterize SnoN for its role in renal fibrosis, we assessed SnoN expression patterns in response to high glucose stress, and evaluated the effects of upregulating SnoN on renal fibrosis. High glucose stress induced significantly elevated SnoN, TGF-β1, and Arkadia transcription; however, significantly reduced SnoN protein levels were observed under these conditions. Upregulating the SnoN protein was achieved by Arkadia knockdown, which resulted in inhibited high glucose-induced epithelial-mesenchymal transition (EMT) in renal tubular cells, the onset phase of renal fibrosis. Alternatively, EMT was suppressed by dominantly expressed exogenous SnoN without interfering with TGF-β1. Overall, renal SnoN upregulation ameliorates renal fibrosis by relieving high glucose-induced EMT; these findings support a translational approach targeting SnoN for the treatment of diabetic nephropathy.

Profibrotic IHG-1 complexes with renal disease associated HSPA5 and TRAP1 in mitochondria

The highly conserved mitochondrial protein induced in high glucose-1 (IHG-1) functions to maintain mitochondrial quality and is associated with the development of fibrosis in diabetic nephropathy. Towards identifying novel approaches to treating diabetic kidney disease, IHG-1-protein-protein interactions were investigated using epitope-tagged immunoprecipitation analyses followed by mass spectrometry. Here we show that IHG-1 is solely expressed in mitochondria and localised to the inner mitochondrial membrane, the region where mitochondrial reactive oxygen species are generated. Chaperones HSPA5 and TRAP1 and cold shock protein YBX1 were identified as IHG-1 binding partners. All three proteins are important in the cellular response to oxidative stress and play important roles in mitochondrial transcription and DNA repair. Both redox imbalance and IHG-1 stimulate TGF-β signalling. IHG-1, HSPA5 and YBX1 all show increased expression in diabetic nephropathy, chronic kidney disease and in the Unilateral Ureteral Obstruction model of kidney fibrosis. Increased IHG-1 expression in UUO correlated with loss of TRAP1 expression. IHG-1 may target TRAP1 for degradation. When IHG-1 is no longer localised to mitochondria, it retains the ability to interact with the cold shock protein YBX1, facilitating anti-fibrotic actions in the nucleus. Targeting these proteins may offer alternative treatments for fibrotic kidney disease.

High glucose down-regulates microRNA-181a-5p to increase pro-fibrotic gene expression by targeting early growth response factor 1 in HK-2 cells

Tubulointerstitial fibrosis (TIF) plays an important role in the progression of renal fibrosis in diabetic nephropathy (DN). Accumulating evidence supports a crucial effect of early growth response factor 1 (Egr1) on renal fibrosis in DN, but the underlying mechanisms are not entirely clear. Here, we explored the aggravating role of Egr1 and identified microRNA-181a-5p (miR-181a-5p) as an upstream regulator of Egr1 in TIF of DN. We demonstrated that overexpression of Egr1 enhanced, whereas small interfering RNA targeting Egr1 decreased the expressions of transforming growth factor β1 (TGF-β1) and fibrosis-related genes including fibronectin and collagen I in human proximal tubule cell line (HK-2) cells. We then found that miR-181a-5p expression was down-regulated, accompanied by the corresponding up-regulation of Egr1, TGF-β1, fibronectin and collagen I in renal tissues of type 2 diabetic Otsuka–Long–Evans–Tokushima–Fatty rats with DN, and that the expression of miR-181a-5p was negatively correlated with the level of Egr1 in HK-2 cells treated with high glucose. Furthermore, we identified that miR-181a-5p directly suppressed Egr1 to decrease the expressions of TGF-β1, fibronectin and collagen I in HK-2 cells through targeting the 3′ untranslated region of Egr1. The functional relevance of miR-181a-5p-induced Egr1 decrease was supported by inhibition and overexpression of miR-181a-5p in HK-2 cells. Thus, we concluded that aberrant Egr1 expression, which can be suppressed by miR-181a-5p directly, plays a crucial role in the progression of renal TIF in DN. This study indicates that targeting miR-181a-5p may be a novel therapeutic approach of DN.

Tongxinluo Inhibits Renal Fibrosis in Diabetic Nephropathy: Involvement of the Suppression of Intercellular Transfer of TGF-[Formula: see text]1-Containing Exosomes from GECs to GMCs.

Glomerular mesangial cells (GMCs) activation is implicated in the pathogenesis of diabetic nephropathy (DN). Our previous study revealed that high glucose (HG)-treated glomerular endothelial cells (GECs) produce an increased number of TGF-[Formula: see text]1-containing exosomes to activate GMCs through the TGF-[Formula: see text]1/Smad3 signaling pathway. We also identified that Tongxinluo (TXL), a traditional Chinese medicine, has beneficial effects on the treatment of DN in DN patients and type 2 diabetic mice. However, it remained elusive whether TXL could ameliorate renal structure and function through suppression of intercellular transfer of TGF-[Formula: see text]1-containing exosomes from GECs to GMCs. In this study, we demonstrate that TXL can inhibit the secretion of TGF-[Formula: see text]1-containing exosomes from HG-treated GECs. Furthermore, exosomes produced by HG induced-GECs treated with TXL cannot trigger GMC activation, proliferation and extracellular matrix (ECM) overproduction both in vitro and in vivo. These results suggest that TXL can prevent the transfer of TGF-[Formula: see text]1 from GECs to GMCs via exosomes, which may be one of the mechanisms of TXL in the treatment of DN.

Lysine 63 ubiquitination is involved in the progression of tubular damage in diabetic nephropathy

The purpose of our study was to evaluate how hyperglycemia (HG) influences Lys63 protein ubiquitination and its involvement in tubular damage and fibrosis in diabetic nephropathy (DN). Gene and protein expression of UBE2v1, a ubiquitin-conjugating E2-enzyme variant that mediates Lys63-linked ubiquitination, and Lys63-ubiquitinated proteins increased in HK2 tubular cells under HG. Matrix-assisted laser desorption/ionization-time of flight/tandem mass spectrometry identified 30 Lys63-ubiquitinated proteins, mainly involved in cellular organization, such as β-actin, whose Lys63 ubiquitination increased under HG, leading to cytoskeleton disorganization. This effect was reversed by the inhibitor of the Ubc13/UBE2v1 complex NSC697923. Western blot analysis confirmed that UBE2v1 silencing in HK2 under HG, restored Lys63-β-actin ubiquitination levels to the basal condition. Immunohistochemistry on patients with type 2 diabetic (T2D) revealed an increase in UBE2v1- and Lys63-ubiquitinated proteins, particularly in kidneys of patients with DN compared with control kidneys and other nondiabetic renal diseases, such as membranous nephropathy. Increased Lys63 ubiquitination both in vivo in patients with DN and in vitro, correlated with α-SMA expression, whereas UBE2v1 silencing reduced HG-induced α-SMA protein levels, returning them to basal expression. In conclusion, UBE2v1- and Lys63-ubiquitinated proteins increase in vitro under HG, as well as in vivo in T2D, is augmented in patients with DN, and may affect cytoskeleton organization and influence epithelial-to-mesenchymal transition. This process may drive the progression of tubular damage and interstitial fibrosis in patients with DN.-Pontrelli, P., Conserva, F., Papale, M., Oranger, A., Barozzino, M., Vocino, G., Rochetti, M. T., Gigante, M., Castellano, G., Rossini, M., Simone, S., Laviola, L., Giorgino, F., Grandaliano, G., Di Paolo, S., Gesualdo, L. Lysine 63 ubiquitination is involved in the progression of tubular damage in diabetic nephropathy.

Effect of zinc deficiency on mouse renal interstitial fibrosis in diabetic nephropathy.

There is emerging evidence that tubulointerstitial fibrosis is the final common pathway of the majority of chronic progressive renal diseases, including diabetic nephropathy (DN). Zinc, an essential dietary element, has been suggested to be important for a number of protein functions during fibrosis invivo and invitro. However, the effect of zinc deficiency (ZnD) on renal interstitial fibrosis in DN remains unclear. The present study investigated the effect and the underlying mechanisms of ZnD on renal interstitial fibrosis during DN using an streptozotocin‑induced model of diabetes with immunofluorescence staining and western blot analysis. The present study identified that dietary zinc restriction significantly decreased zinc concentrations in the plasma and mouse kidney. ZnD enhanced albuminuria and extracellular matrix protein expression, associated with diabetic renal interstitial fibrosis by activation of renal interstitial fibroblasts and regulation of the expression of fibrosis‑associated factors, which may be mediated by the activation of fibroblasts via the TGF‑β/Smad signaling pathway. The data indicates that ZnD serves an important role in the pathogenic mechanisms of renal interstitial fibrosis during the development of DN.

CCL18 synergises with high concentrations of glucose in stimulating fibronectin production in human renal tubuloepithelial cells

BackgroundDiabetic nephropathy is the leading cause of end stage kidney disease worldwide. The pathogenesis of this disease remains elusive and multiple factors have been implicated. These include the effects of hyperglycaemia, haemodynamic and metabolic factors, and an inflammatory process that stimulates cellular signalling pathways leading to disease progression and severe fibrosis. Fibronectin (Fn) is an important protein of the extracellular matrix that is essential in fibrosis and its presence in increased amounts has been identified in the kidney in diabetic nephropathy.MethodsProximal tubuloepithelial (HK-2) cells were stimulated with high glucose (30 mM D-glucose) or glycated albumin (500 μg/mmol) + 4 mM D-glucose or their controls, Mannitol (26 mM + 4 mM D-glucose) and 4 mM D-glucose, respectively. Following 48 h of stimulation the supernatant was collected and MTT [3-(4,5-dimethylthiazole-2,5-diphenyltetrazolium bromide] assay performed to assess cell viability. HK-2 cells were also stimulated in the above environments with recombinant CCL18 (rCCL18) or MCP-1 (rMCP-1) for 48 h with quantification of Fn levels using ELISA.ResultsCo-stimulation of HK-2 cells with high concentrations of glucose and rCCL18 significantly increased Fn (p < 0.001), in comparison to high concentrations of glucose alone. HK-2 cells stimulated with glycated albumin consistently produced Fn and this did not alter following co-stimulation with rCCL18 or rMCP-1.ConclusionThis study demonstrates how stimulation with a specific chemokine CCL18 in high glucose upregulates the production of Fn from proximal tubuloepithelial cells. This may be relevant to the development of renal fibrosis in diabetic nephropathy

Resveratrol inhibits renal interstitial fibrosis in diabetic nephropathy by regulating AMPK/NOX4/ROS pathway.

Resveratrol inhibits high glucose-induced NRK cell activation by decreasing NOX4-derived ROS. Resveratrol inhibits high glucose-induced NOX4 expression in NRK cells via activation of AMPK. ROS-activated ERK1/2 signaling is involved in high glucose-induced NRK cell activation. Resveratrol attenuated renal fibrosis in db/db mice via regulation of AMPK/NOX4/ROS signaling.