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Introduction This study aims to explore the effect and related molecular mechanism of miR-153-3p on high glucose-stimulated human glomerular mesangial cells. Materials and methods The quantitative real-time polymerase chain reaction (qPCR) assay was employed to check miR-153-3p and PAQR3 expression levels in diabetic nephropathy patients. (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) MTT assay was applied to investigate the effects of miR-153-3p transfection or PAQR3 administration on mesangial cell (MC) activity. ELISA assays were used to check the expression levels of extracellular matrix (ECM) related proteins. The bioinformatics method and dual-luciferase reporter assay were employed together to anticipate and check the targeting relationship between miR-153-3p and PAQR3. Western blot assays were applied to check the PAQR3, PI3K and AKT expression after miR-153-3p transfection or PAQR3 administration. Results The expression level of miR-153-3p was lower in diabetic nephropathy patients, while the expression of PAQR3 was concomitantly higher. Upregulation of miR-153-3p can reduce MC proliferation and ECM accumulation. Further research indicated that miR-153-3p directly regulated PAQR3 expression via coupling with the 3’-UTR of PAQR3. Finally, the fact that miR-153-3p regulates the PI3K/AKT pathway by PAQR3 was confirmed. Conclusion MiR-153-3p regulates the PI3K/AKT pathway through PAQR3, thereby playing a role in regulating cell proliferation and ECM accumulation in high glucose-stimulated MCs.

Catalpol is an iridoid glycoside obtained from Rehmannia glutinosa, which in previous studies showed various pharmacological properties, including anti-inflammatory, antioxidant, antidiabetic, antitumor, and dopaminergic neurons protecting effects. Here, we examined the effect of catalpol on renal injury induced by angiotensin II (Ang II) and further to explore its latent molecular mechanisms. We used an in vivo model of Ang II-induced renal injury mice; catalpol (25, 50, and 100 mg/kg) was administered for 28 days. Mouse glomerular mesangial cells (SV40 MES 13), rat kidney interstitial fibroblasts cells (NRK-49F), and human proximal tubular epithelial cells (HK-2) were induced by Ang II (10 µM) in the presence or absence of catalpol (1, 5, and 10 µM) and incubated for 48 hours in vitro. In our study, periodic acid-Schiff and Masson staining of renal tissue showed that catalpol reduced Ang II-induced renal injury in a concentration-dependent manner. The positive expressions of collagen IV and TGF-β1 were observed to decrease sharply after catalpol treatment. In renal tissue, the levels of pro-inflammatory cytokines tumor necrosis factor α and interleukin 6 were evidently decreased after catalpol intervention. Catalpol can relieve Ang II-induced renal injury by inactivating NF-κB and TGF-β1/Smads signaling pathways. Therefore, catalpol may act as a potential drug to treat Ang II-induced renal injury.

This study was to explore the effective components, potential targets, and pathways of Jianpi Qushi Huayu Formula (JQHF) for the treatment of chronic glomerulonephritics (CGN). First, the Chinese Medicine System Pharmacology Database and Analysis Platform (TCMSP), GeneCards, and OMIM databases were used to collect the major active components of JQHF and potential therapeutic targets of CGN. Then, functional enrichment analysis was performed to clarify the mechanisms of the JQHF on CGN. Subsequently, molecular docking was simulated to assess the binding ability of key targets and major active components. Finally, quantitative real-time PCR and western blot were performed for experimental verification of cells in vitro. A total of 55 active ingredients contained and 220 putative identified targets were screened from JQHF, of which 112 overlapped with the targets of CGN and were considered potential therapeutic targets. Then, we found quercetin and kaempferol are two key ingredients of JQHF, which may act on the top 10 screened targets of PPI, affecting CGN through related signal transduction pathways. Subsequently, molecular docking predicted that quercetin and kaempferol bind firm with the top 10 core targets of PPI. Further experiment verified some results and showed that JQHF has protected glomerular mesangial cells from lipopolysaccharide-induced inflammation by inhibiting expressions of IL6, TNF-α, and AKT1, and activating expressions of VEGFA. Based on network pharmacology, we explored the multi-component, multi-target, and multi-pathway characteristics of JQHF in treating CGN, and found that JQHF could act on IL6, TNF-α, VEGFA, and AKT1 to exert the effect of anti-CGN, which provided new ideas and methods for further research on the mechanism of JQHF in treating CGN.

We have shown that complement 3 (C3) is upregulated in cardiovascular and renal organs, which induces the synthetic phenotype and exaggerates the growth of mesenchymal cells from spontaneously hypertensive rats (SHRs). However, the mechanisms of the upregulation of C3 have remained unclear. In the present study, we investigated the role of TWIST1, a transcription factor that regulates mesodermal embryogenesis, in the upregulation of C3 in glomerular mesangial cells (GMCs) from SHRs and Wistar-Kyoto (WKY) rats. Immunocytochemical staining and western blot analysis showed that the expression of TWIST1 in GMCs from SHRs was higher than that in GMCs from WKY rats in vivo and in vitro. Real-time PCR analysis showed increases in the expression of Twist1 mRNA with attenuated expression of miR-151-3p in GMCs from SHRs compared to that in cells from WKY rats. Chromatin immunoprecipitation assays showed increases in TWIST1 binding to the C3 promoter in GMCs from SHRs compared to that in cells from WKY rats. Transfection of Twist1 cDNA by a lentiviral vector increased the expression of C3 mRNA in GMCs from WKY rats. TWIST1 siRNA significantly decreased the mRNA expression of C3 and osteopontin in GMCs from SHRs. These results indicate that the increases in TWIST1 expression, attenuation of miR-151-3p, and strong binding of TWIST1 upregulate C3 gene expression in GMCs from SHRs. The enhanced TWIST1-C3 system induces the synthetic phenotype of mesenchymal tissue that may be associated with cardiovascular and renal remodeling in hypertension.

This research studied the effect of long non-coding RNA X-inactive-specific transcript (XIST) on DN. The effect of high glucose (HG) on the expression of XIST and miR-423-5p was detected by quantitative real-time PCR (qRT-PCR) in human kidney (HK) cells (human glomerular mesangial cells (HMCs) and human kidney-2 (HK-2) cells). The effect of XIST depletion and miR-423-5p inhibition or overexpression on high mobility group protein A2 (HMGA2) protein level was examined by western blot in HG-induced HK cells. The impacts of XIST depletion on viability and apoptosis were assessed by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) and flow cytometry assays in HG-induced HK cells. We found the expression of XIST and HMGA2 protein was significantly upregulated in DN tissues and cells. Moreover, HG treatment induced the upregulation of XIST and HMGA2 protein level in HK cells. Besides, both XIST depletion and HMGA2 depletion decreased cell proliferation but increased apoptosis in HG-treated HK cells. Furthermore, HMGA2 upregulation or miR-423-5p inhibition partly eliminated the effects of XIST depletion on cell proliferation, apoptosis of HG-treated HK cells. Interestingly, HMGA2 upregulation partly reversed miR-423-5p overexpression-mediated suppression on viability and promotion on apoptosis in HG-treated HK cells. Mechanistically, XIST sponged miR-423-5p to regulate HMGA2 expression in DN cells. Taken together, XIST depletion suppressed proliferation and promoted apoptosis via miR-423-5p/HMGA2 axis in HG-treated HK cells, which may provide a potential therapeutic target for DN.

Background Diabetic nephropathy (DN) is a common complication of diabetes. Long non-coding RNA (lncRNA) cancer susceptibility candidate 2 (CASC2) is reported to exert a protective role in DN by a previous study. The working mechanism underlying the protective role of CASC2 in DN progression was further explored in this study. Methods The expression of CASC2 and microRNA-135a-5p (miR-135a-5p) was determined by real-time quantitative polymerase chain reaction (RT-qPCR). Cell proliferation ability was assessed by Cell Counting Kit-8 (CCK8) assay and 5-ethynyl-29-deoxyuridine (EDU) assay. Enzyme-linked immunosorbent assay (ELISA) was conducted to analyze the production of inflammatory cytokines in the supernatant. Western blot assay was performed to analyze protein expression. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were performed to verify the target relationship between miR-135a-5p and CASC2 or tissue inhibitors of metalloproteinase 3 (TIMP3). Results High glucose (HG) treatment reduced the expression of CASC2 in human glomerular mesangial cells (HMCs) in a time-dependent manner. CASC2 overexpression suppressed HG-induced proliferation, inflammation and fibrosis in HMCs. miR-135a-5p was validated as a target of CASC2, and CASC2 restrained HG-induced influences in HMCs partly by down-regulating miR-135a-5p. miR-135a-5p bound to the 3ʹ untranslated region (3ʹUTR) of TIMP3, and CASC2 positively regulated TIMP3 expression by sponging miR-135a-5p in HMCs. miR-135a-5p silencing inhibited HG-induced effects in HMCs partly by up-regulating its target TIMP3. CASC2 overexpression suppressed HG-induced activation of Jun N-terminal Kinase (JNK) signaling partly through mediating miR-135a-5p/TIMP3 signaling. Conclusions In conclusion, CASC2 alleviated proliferation, inflammation and fibrosis in DN cell model by sponging miR-135a-5p to induce TIMP3 expression.

Ubiquitin-specific peptidases7 (USP7) participates in the regulation of various metabolic and immune disorders. However, the role of USP7 in lupus nephritis (LN) remains unknown. The current study set out to elucidate the regulatory role of USP7 in LN together with JMJD3 and NF-κB. SLE MRL/LPR mice and mouse glomerular mesangial cells SV40 MES 13 cells were employed for in vivo or vitro experiments. USP7, JMJD3 and NF-κB expression in MRL/LPR mice were detected, followed by investigation of their functions in the proliferation of mesangial cells and mesangial matrix. Subsequently, the interaction among USP7, JMJD3 and NF-κB was determined by means of ChIP and co-immunoprecipitation assay. The results indicated that USP7, JMJD3, p-NF-κB p65 were all highly-expressed in MRL/LPR mice. USP7 promoted the proliferation of mesangial cells and mesangial matrix, and stabilized the JMJD3 protein via deubiquitination in SV40 MES 13 cells. Meanwhile, silencing of JMJD3 inhibited the promotive effect of USP7 on the proliferation of mesangial cells and mesangial matrix. Furthermore, JMJD3 increased the expression of NF-κB p65 through demethylation, whereas silencing JMJD3 alleviated the proliferation of mesangial cells and mesangial matrix. Lastly, NF-κB p65 was proved to aggravate LN pathogenesis. Altogether, our findings highlighted that USP7 promoted the occurrence of LN by regulating the NF-κB p65 signaling pathway via stabilization of JMJD3.

Glomerular mesangial cell (GMC) proliferation is a histopathological alteration in human mesangioproliferative glomerulonephritis (MsPGN) or in animal models of MsPGN, e.g., the rat Thy‐1 nephritis (Thy-1N) model. Although sublytic C5b-9 assembly on the GMC membrane can trigger cell proliferation, the mechanisms are still undefined. We found that sublytic C5b-9-induced rat GMC proliferation was driven by extracellular signal‐regulated kinase 1/2 (ERK1/2), sry-related HMG-box 9 (SOX9), and Cyclin D1. Here, ERK1/2 phosphorylation was a result of the calcium influx-PKC-α-Raf-MEK1/2 axis activated by sublytic C5b-9, and Cyclin D1 gene transcription was enhanced by ERK1/2-dependent SOX9 binding to the Cyclin D1 promoter (−582 to −238 nt). In addition, ERK1/2 not only interacted with SOX9 in the cell nucleus to mediate its phosphorylation at serine residues 64 (a new site identified by mass spectrometry) and 181 (a known site), but also indirectly induced SOX9 acetylation by elevating the expression of general control non-repressed protein 5 (GCN5), which together resulted in Cyclin D1 synthesis and GMC proliferation. Moreover, our in vivo experiments confirmed that silencing these genes ameliorated the lesions of Thy‐1N rats and reduced SOX9 phosphorylation, acetylation and Cyclin D1 expression. Furthermore, the renal tissue sections of MsPGN patients also showed higher phosphorylation or expression of ERK1/2, SOX9, and Cyclin D1. In summary, these findings suggest that sublytic C5b-9-induced GMC proliferation in rat Thy-1N requires SOX9 phosphorylation and acetylation via enhanced Cyclin D1 gene transcription, which may provide a new insight into human MsPGN pathogenesis., Kidney disease: Understanding why cells overproliferate Researchers have clarified the mechanism behind a kidney disease called mesangioproliferative glomerulonephritis (MsPGN), which causes certain kidney cells to overproliferate. In MsPGN, cells called glomerular mesangial cells (GMCs) overproliferate, causing kidney lesions, decreased function, and kidney failure. The mechanism at the molecular level has been unclear. A team led by Yingwei Wang at Nanjing Medical University in China investigated what leads GMCs to proliferate, using a rat animal model. They found that the transcription factor protein SOX9, often implicated in cell proliferation, is both phosphorylated and acetylated, enhancing its transcriptional activities and permitting it to activate other molecules that control the cell cycle, leading to overproliferation of GMCs. Elucidating the disease mechanism in this animal model may help to understand the disease in humans, and identify therapeutic targets.

We aimed to assess the effects of bradykinin (BK) on the proliferation, apoptosis, and cycle of glomerular mesangial cells via the transforming growth factor-β 1 (TGF-β1)/Smad signaling pathway. Rat glomerular mesangial cells, HBZY-1, were divided into normal group (untreated), model group (5 ng/L TGF-β1), BK group (5 ng/L TGF-β1 + 1 ng/L BK), and inhibitor group [5 ng/L TGF-β1 + 1 ng/L LY2109761 (TGF-β1-specific inhibitor)]. The cell proliferation, cycle, apoptosis, expression of type I collagen (Col-1), and protein expressions of Col-1, TGF-β1, and phosphorylated Smad2 (p-Smad2) were detected by EdU labeling, flow cytometry, acridine orange/ethidium bromide (AO/EB) dual staining, immunofluorescence assay, and Western blotting, respectively. Compared with the normal group, the cell proliferation rate (P = 0.02) and protein expression levels of Col-1 (P = 0.02), TGF-β1 (P = 0.01), p-Smad2 (P = 0.02), and p-Smad7 (P = 0.00) in the model group significantly increased, and apoptosis rate (P = 0.01) significantly decreased. Compared with the model group, the BK and inhibitor groups significantly decreased in proliferation rate (P = 0.01) and protein expression levels of Col-1 (P = 0.01), TGF-β1 (P = 0.01), and p-Smad2 (P = 0.00). Also, they were significantly elevated in apoptosis rate (P = 0.02) and p-Smad7 protein expression (P = 0.02). BK regulates the proliferation, apoptosis, and the cycle of glomerular mesangial cells by inhibiting the TGF-β1/Smad signaling pathway.

Over-expression of angiotensin II (Ang II) is an important reason for the development of chronic kidney disease. Calycosin is the active component of traditional Chinese medicine astragali radix. The present work aims to explore whether calycosin could affect the growth and apoptosis ability of the Ang II treated glomerular mesangial cells and the underlying mechanism. Human glomerular mesangial cells (GMCs) were cultured and treated by Ang II and 0, 0.1, 1, or 10 µM calycosin, and the viability and proliferation of the cells were determined by methyl thiazolyl tetrazolium (MTT) and 5-ethynil-2'-deoxyuridine (EdU) staining; moreover, the apoptosis of the cells was examined by flow cytometry assay; furthermore, the expression levels of extracellular signal-regulated kinase (ERK), p-ERK, anti-apoptotic factor Bcl-2, as well as pro-apoptotic factor Bax have been examined by Western blot (WB) methods; finally, the expression of autophagic markers in each group was examined by WB and immunocytochemistry methods. We found that Ang II increased viability and proliferation, meanwhile inhibited apoptosis of the GMCs; furthermore, 1 and 10 µM calycosin significantly inhibited the growth and promoted the apoptosis of the GMCs treated by Ang II; moreover, calycosin also inhibited ERK signaling in mesangial cells activated by Ang II treatment; Finally, calycosin could inhibit Ang II induced autophagy of GMCs in a dose-dependent manner. In conclusion, calycosin may alleviate Ang II-induced pro-proliferative and anti-apoptotic effects on glomerular mesangial cells at least partially via inhibiting autophagy and ERK signaling pathway, suggesting that calycosin may function as a potential alternative medication for the management of chronic kidney diseases.

Introduction Diabetic nephropathy (DN) has become an increasing threat to health, and inflammation and fibrosis play important roles in its progression. Wogonin, a flavonoid, has been proven to suppress inflammation and fibrosis in various diseases, including acute kidney injury. This study aimed at investigating the effect of wogonin on diabetes-induced renal inflammation and fibrosis. Materials and methods Streptozotocin (STZ)-induced diabetic mouse models received gavage doses of wogonin (10, 20, and 40 mg/kg) for 12 weeks. Metabolic indices from blood and urine and pathological damage of glomerulus in the diabetic model were assessed. Glomerular mesangial cells SV40 were cultured in high glucose (HG) medium containing wogonin at concentrations of 1.5825, 3.125, and 6.25 μg/mL for 24 h. Inflammation and fibrosis indices were evaluated by histopathological, Western blotting, and PCR analyses. Results Wogonin treatment ameliorated albuminuria and histopathological lesions in diabetic mice. Inflammatory cytokines, such as monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and related signaling pathway NF-κB were downregulated after the administration of wogonin in vivo and in vitro. Furthermore, wogonin reduced the expression of extracellular matrix (ECM), including fibronectin (FN), collagen IV (Col-IV), α-smooth muscle actin (α-SMA), and transforming growth factor-β1 (TGF-β1) in the kidneys of diabetic mice and HG-induced mesangial cells. Moreover, the inhibition of TGF-β1/Smad3 pathway might be responsible for these changes. Conclusion Wogonin may ameliorate renal inflammation and fibrosis in diabetic nephropathy by inhibiting the NF-κB and TGF-β1/Smad3 signaling pathways.

Circular RNAs (circRNAs) have crucial roles in various diseases; however, the mechanisms of action underlying circRNAs in the occurrence and development of diabetic nephropathy (DN) remains largely unknown. The present study investigated the differentially expressed circRNAs in the DN mice kidney cortex using circRNA sequencing and elucidated the role of circRNAs in mesangial cells. It was revealed that 40 circRNAs were unconventionally expressed, including 18 upregulated circRNAs and 22 downregulated circRNAs. Furthermore, circ_0000491 levels were significantly augmented in both DN mice and high glucose (HG, 30 mM)-induced mouse mesangial cells (MES13 cells). Knockdown of circ_0000491 significantly suppressed the increase of vimentin, fibronectin and α-smooth muscle actin, as well as collagen type I, III and IV, whilst reversing the decrease of E-cadherin in HG-induced MES13 cells. It was further revealed that circRNA_0000491 sponged miR-101b and that miR-101b directly targets TGFβRI. In addition, the expression levels of miR-101b were negatively associated with the transcriptional level of circRNA_0000491 and miR-101b inhibitors reversed the suppression of extracellular matrix (ECM)-associated protein synthesis mediated by knocking-down circRNA_0000491. In conclusion, the present study investigated the circRNA_0000491/miR-101b/TGFβRI axis in ECM accumulation and fibrosis-associated protein expression levels of mesangial cells, which suggested that circRNA_0000491 may be beneficial for the development of an effective therapeutic target for DN.

Glomerular mesangial cell (MC) proliferation is one of the main pathological changes in diabetic nephropathy (DN), but its mechanism needs further elaboration. The Hippo and PI3K/Akt signalling pathways are involved in the regulation of MC proliferation, but their relationship in hyperglycaemia-induced MC proliferation has not been reported. We used db/db mice and high-glucose-cultured mesangial cells to generate a diabetic nephropathy model. An MST1-knockdown plasmid was used to identify whether the PI3K/Akt pathway is linked to the Hippo pathway through MST1. LY294002 and SC79 were used to verify the role of the PI3K/Akt signalling pathway in MC cells. RNA silencing and overexpression were performed by using YAP and PTEN-expression/knockdown plasmids to investigate the function of YAP and PTEN, respectively, in the Hippo and PI3K/Akt signalling pathways. By examining a potential feedback loop, we found decreased phosphorylation of MST1 and Lats1 and increased PI3K/Akt activation in db/db mice and high glucose-treated MCs, along with increased MC proliferation. The results of our gene silencing experiment proved PI3K/Akt-mediated intervention in the Hippo pathway and the regulatory effect of YAP on PI3K/Akt through PTEN. The Hippo pathway is inhibited under diabetic conditions, leading to YAP activation and promoting MC proliferation. The PI3K/Akt pathway is activated through the inhibitory effect of YAP on its repressor, PTEN. Finally, activation of the PI3K/Akt pathway inhibits the Hippo pathway, resulting in nuclear YAP accumulation and accelerating MC proliferation and DN formation.

Rat Thy-1 nephritis (Thy-1N) is an experimental mesangial proliferative glomerulonephritis (MsPGN) for studying human MsPGN. Although sublytic C5b-9 complex formation on glomerular mesangial cells (GMCs) and renal MCP-1 and RANTES production in rats with Thy-1N have been proved, the role and mechanism of MCP-1 or RANTES synthesis in GMCs induced by sublytic C5b-9 are poorly elucidated. In this study, we first found the expression of transcription factor (KLF6), co-activator (KAT7) and chemokines (MCP-1 and RANTES) was all up-regulated both in renal tissue of Thy-1N rats (in vivo) and in sublytic C5b-9-induced GMCs (in vitro). Further in vitro experiments revealed that KLF6 bound to MCP-1 promoter (-297 to -123 nt) and RANTES promoter (-343 to -191 nt), leading to MCP-1 and RANTES gene transcription. Meanwhile, KAT7 also bound to the same region of MCP-1 and RANTES promoter in a KLF6-dependent manner, and KLF6 was acetylated by KAT7 at lysine residue 100, which finally promoted MCP-1 and RANTES expression. Moreover, our in vivo experiments discovered that knockdown of renal KAT7 or KLF6 gene obviously reduced MCP-1 and RANTES production, GMCs proliferation, ECM accumulation, and proteinuria secretion in Thy-1N rats. Collectively, our study indicates that sublytic C5b-9-induced MCP-1 and RANTES synthesis is associated with KAT7-mediated KLF6 acetylation and elevated KLF6 transcriptional activity, which might provide a new insight into the pathogenesis of rat Thy-1N and human MsPGN.

Activation of immunological pathways and disturbances of extracellular matrix (ECM) dynamics are important contributors to the pathogenesis of chronic kidney diseases. Glomerular mesangial cells (MCs) are critical for homeostasis of glomerular ECM dynamics. Interleukin-6 (IL-6) can act as a pro/anti-inflammatory agent relative to cell types and conditions. This study investigated whether IL-6 influences ECM protein production by MCs and the regulatory pathways involved. Experiments were carried out in cultured human MCs (HMCs) and in mice. We found that overexpression of IL-6 and its receptor decreased the abundance of fibronectin and collagen type IV in MCs. ELISA and immunoblot analysis demonstrated that thapsigargin [an activator of store-operated Ca2+ entry (SOCE)], but not the endoplasmic reticulum stress inducer tunicamycin, significantly increased IL-6 content. This thapsigargin effect was abolished by GSK-7975A, a selective inhibitor of SOCE, and by silencing Orai1 (the channel protein mediating SOCE). Furthermore, inhibition of NF-κB pharmacologically and genetically significantly reduced SOCE-induced IL-6 production. Thapsigargin also stimulated nuclear translocation of the p65 subunit of NF-κB. Moreover, MCs overexpressing IL-6 and its receptor in HMCs increased the content of the glucagon-like peptide-1 receptor (GLP-1R), and IL-6 inhibition of fibronectin was attenuated by the GLP-1R antagonist exendin 9–39. In agreement with the HMC data, specific knockdown of Orai1 in MCs using the targeted nanoparticle delivery system in mice significantly reduced glomerular GLP-1R levels. Taken together, our results suggest a novel SOCE/NF-κB/IL-6/GLP-1R signaling pathway that inhibits ECM protein production by MCs.

Diabetic nephropathy (DN) is a chronic loss of kidney function that frequently occurs in patients with diabetes mellitus and is characterized by abnormal glomerular mesangial cell (GMC) proliferation and apoptosis. By using microarray analysis, microRNA (miR)-181a-5p has previously been identified to be dysregulated in DN. The present study aimed to determine the underlying molecular mechanisms and function of miR-181a-5p in GMCs under DN conditions. First, reverse transcription-quantitative PCR was performed to detect miR-181a-5p and kruppel-like factor 6 (KLF6) expression in GMCs following high-glucose treatment. Subsequently, MTT and flow cytometric assays were performed in order to determine the effect of miR-181a-5p and KLF6 on high-glucose-driven GMC proliferation and apoptosis. After confirming that KLF6 was a target gene of miR-181a-5p via a bioinformatics analysis and luciferase reporter assay, the mRNA and protein expression levels of associated factors in different treatment groups were measured. The results demonstrated that miR-181a-5p was significantly downregulated, while KLF6 was significantly upregulated in GMCs following treatment with high glucose. Furthermore, overexpression of miR-181a led to suppression of cell proliferation and promoted apoptosis of GMCs induced by high glucose, while these effects were inhibited by co-transfection with KLF6. Finally, miR-181-5p was demonstrated to inhibit the expression of KLF6, Bcl-2, Wnt1 and β-catenin, while increasing the expression levels of Bax and caspase-3. In conclusion, the expression levels of miR-181a-5p were downregulated in GMCs following treatment with high glucose and overexpression of miR-181a-5p may inhibit GMC proliferation and promote apoptosis, at least partially through targeting KLF6 via the Wnt/β-catenin signaling pathway. Overall, the results of the present study suggest that miR-181a-5p may have a crucial role in the occurrence and development of DN and may be a valuable diagnostic marker and therapeutic target for DN.

The aim of this study was to determine whether interleukin-1β (IL-1β) promotes oxidised low-density lipoprotein (Ox-LDL) uptake by human glomerular mesangial cells (HMCs) and its effect on the expression of lectin-like Ox-LDL receptor 1 (LOX-1) and to identify pathways through which IL-1β affects lipid uptake. Confocal laser scanning microscopy and flow cytometry were used to observe the effect of IL-1β on lipid uptake by HMCs and the pathway by which IL-1β may mediate lipid uptake. Real-time polymerase chain reaction (PCR) and western blotting were used to evaluate the effect of IL-1β on LOX-1 expression. Confocal laser scanning microscopy and flow cytometry revealed that IL-1β promoted uptake of fluorescent Dil-labelled Ox-LDL(Dil-Ox-LDL) by HMCs and the enhanced uptake of Dil-Ox-LDL was partially inhibited by an anti-LOX-1 antibody evaluated by flow cytometry. Further, IL-1β promoted LOX-1 mRNA and protein expression of HMCs in a dose- and time-dependent manner. Thus, Ox-LDL is ingested by HMCs under basic conditions. Inflammatory cytokine IL-1β promotes Ox-LDL uptake by HMCs. Furthermore, IL-1β promotes the mRNA and protein expression of LOX-1, a specific receptor of Ox-LDL, suggesting that the enhancement of Ox-LDL uptake may be mediated by LOX-1 pathway. Anti-LOX-1 therapy may be a promising option for treatment of glomerulosclerosis.

C1qTNF-related protein 6 (CTRP6) is a member of the CTRP family and exerts a key role in the progression of diabetes mellitus. However, the role of CTRP6 in diabetic nephropathy remains unknown. The present study was designed to examine the roles of CTRP6 in diabetic nephropathy and explore the potential molecular mechanisms. Our results showed that the expression level of CTRP6 was significantly increased in high glucose (HG)-stimulated glomerular mesangial cells (MCs). The following loss/gain-of-function assays demonstrated that CTRP6 knockdown significantly inhibited HG-induced reactive oxygen species (ROS) production in MCs. CTRP6 knockdown caused significant decreases in tumour necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 production levels in HG-induced MCs. Moreover, knockdown of CTRP6 inhibited HG-stimulated extracellular matrix (ECM) accumulation in MCs characterized by decreased expression and production levels of fibronectin (FN) and collagen IV (Col IV). Furthermore, CTRP6 knockdown suppressed HG-induced the activation of Akt/NF-κB pathway in MCs, while overexpression of CTRP6 exhibited the opposite effects. Treatment with LY294002, an inhibitor of Akt, reversed the induction effects of CTRP6 overexpression on ROS production, inflammation and ECM accumulation in MCs. In conclusion, these findings demonstrated that CTRP6 knockdown inhibits HG-induced ROS production, inflammation and ECM accumulation in MCs, which were mediated by the inactivation of the Akt/NF-κB pathway. The roles of CTRP6 in diabetic nephropathy provided evidence for its therapeutic potential for the treatment of diabetic nephropathy.

Oxidative stress plays an important role in diabetic nephropathy (DN), which is a diabetic complication. Ampelopsin (AMP) is a natural flavonoid that has been found to possess antidiabetic and antioxidative activities. However, the effect of AMP on DN remains unclear. In this study, we aimed to evaluate the protective effect of AMP on glomerular mesangial cells (MCs) exposed to high glucose (HG). We found that AMP improved HG-caused cell viability reduction in MCs. AMP significantly suppressed the intracellular ROS production and expression levels of ROS producing enzymes NADPH oxidase 2 (NOX2) and NOX4. Increased of NOX activity in HG-stimulated MCs was suppressed by AMP. Pretreatment with AMP inhibited extracellular matrix (ECM) accumulation in HG-stimulated MCs with decreased expression levels of fibronectin (FN) and collagen type IV (Col IV). Furthermore, AMP elevated the expression levels of nuclear Nrf2 and heme oxygenase-1 (HO-1), as well as increased the mRNA levels of Nrf2-driven genes NAD(P)H dehydrogenase quinone-1 (NQO-1) and HO-1 in HG-treated MCs. Knockdown of Nrf2 reversed the protective effects of AMP against HG-induced oxidative stress and EMC accumulation in MCs. In conclusion, these findings indicated that AMP protected MCs from HG-induced oxidative damage and ECM accumulation, which might be mediated by Nrf2/HO-1 pathway.

By reducing their metabolism, dipeptidyl peptidase 4 inhibition (DPP4I) enhances the effects of numerous peptides including neuropeptide Y1-36 (NPY1-36), peptide YY1-36 (PYY1-36), and SDF-1α Studies show that separately NPY1-36, PYY1-36 and SDF-1α stimulate proliferation of, and collagen production by, cardiac fibroblasts (CFs), preglomerular vascular smooth muscle cells (PGVSMCs), and glomerular mesangial cells (GMCs), particularly in cells isolated from genetically hypertensive rats. Whether certain combinations of these factors, in the absence or presence of DPP4I, are more profibrotic than others is unknown. Here we contrasted 24 different combinations of conditions (DPP4I, hypertensive genotype and physiologic levels [3 nM] of NPY1-36, PYY1-36, or SDF-1α) on proliferation of, and [3H]-proline incorporation by, CFs, PGVSMCs, and GMCs. In all three cell types, the various treatment conditions differentially increased proliferation and [3H]-proline incorporation, with a hypertensive genotype + DPP4I + NPY1-36 + SDF-1α being the most efficacious combination. Although the effects of this four-way combination were similar in male versus female CFs, physiologic (1 nM) concentrations of 2-methoxyestradiol (2ME; nonestrogenic metabolite of 17β-estradiol), abolished the effects of this combination in both male and female CFs. In conclusion, this study demonstrates that CFs, PGVSMCs, and GMCs are differentially activated by various combinations of NPY1-36, PYY1-36, SDF-1α, a hypertensive genetic background and DPP4I. We hypothesize that as these progrowth conditions accumulate, a tipping point would be reached that manifests in the long term as organ fibrosis and that 2ME would obviate any profibrotic effects of DPP4I, even under the most profibrotic conditions (i.e., hypertensive genotype with high NPY1-36 + SDF-1α levels and low 2ME levels). SIGNIFICANCE STATEMENT: This work elucidates combinations of factors that could contribute to long-term profibrotic effects of dipeptidyl peptidase 4 inhibitors and suggests a novel drug combination that could prevent any potential profibrotic effects of dipeptidyl peptidase 4 inhibitors while augmenting the protective effects of this class of antidiabetic agents.

Multicystic renal dysplasia is a congenital cystic anomaly of the kidney caused by abnormal metanephric differentiation with immature tubules. It is surrounded by mesenchymal collars and islands of immature mesenchyma present between the cysts. The PI3K-AKT-mTOR signaling pathway is a key regulator involved in cell growth, proliferation, motility, survival, and apoptosis. Activation of the PI3K-AKT-mTOR pathway results in the survival and proliferation of tumor cells in many cancers. The aim of this study is to analyze the topographic expression of phospho-AKT, phospho-mTOR, and phospho-70S6K in renal development and in the multicystic dysplastic kidney (MCDK). A total of 17 fetal kidneys of development age from the first to the third trimester and 13 cases of pathological kidneys with MCDK were analyzed by immunohistochemistry in order to evaluate the expression of phospho-AKT (S473), phospho-mTOR, and phospho-70S6K. Phospho-AKT and phospho-mTOR were expressed early in renal development and in an identical manner for every structure derived from the ureteric bud, such as collecting ducts and urothelium. Phospho-p70S6K was expressed early in the urothelium and in glomerular mesangial cells. Later, their expressions differed according to the needs of cell proliferation and differentiation over time by becoming more selective. In MCDK, phospho-AKT, phospho-mTOR, and phospho-70S6K have the same profile: a high cytoplasmic expression in cystic epithelium, loose mesenchyma, and primitive tubes. This study demonstrates the essential and specific role of the PI3K-AKT-mTOR pathway in the formation of cysts in multicystic renal dysplasia.

Glomerular mesangial cell (MC) proliferation and extracellular matrix deposition are the main pathological changes in diabetic nephropathy. Hydrogen sulfide (H2S) inhibits the proliferation of MCs. Dopamine 1 receptors (DR1) are expressed in MCs and serve important physiological roles. However, it is unclear whether DR1 activation inhibits MC proliferation by increasing endogenous H2S. The present study found that the production of H2S and the expression of DR1 and cystathionine‑γ‑lyase (CSE) were decreased in the renal tissues of diabetic mice and high glucose (HG)‑induced MCs. SKF38393 (a DR1 agonist) increased the production of H2S and the expression of DR1 and CSE and NaHS (an exogenous H2S donor) only increased H2S production and CSE expression but not DR1 expression. HG increased the thickness of the glomerular basement membrane, cell viability and proliferation, the expression of cyclin D1, PCNA, collagen 1 and α‑smooth muscle actin and the activity of phosphorylated ERK1/2 and decreased the expression of P21 and MMP9. SKF38393 and NaHS reversed the effects of HG. PPG (a CSE inhibitor) abolished the beneficial effects of SKF38393. The beneficial effects of SKF38393 were similar to those of PD98059 (an ERK1/2 inhibitor). Taken together, the findings suggested that the DR1‑CSE/H2S pathway activation attenuated diabetic MC proliferation and extracellular matrix deposition by downregulating the ERK1/2 signaling pathway.

Background LncRNA NNT-AS1 (NNT-AS1) has been extensively studied as the causative agent in propagation and progression of lung and bladder cancers, and cholangiocarcinoma. However, its significance in proliferation and inflammation of diabetic nephropathy is enigmatic. This study focuses on the molecular mechanisms followed by NNT-AS1 to establish diabetic nephropathy (DN) and its potential miRNA target. Methods Bioinformatics analysis to identify potential miRNA target of NNT-AS1 and smad4 transcription factor was conducted using LncBase and TargetScan, and was subsequently confirmed by luciferase reporter assay. Relative quantitative expression of NNT-AS1 in human glomerular mesangial cells (HGMCs) was detected through quantitative real-time PCR and WB analysis. Cell proliferation was detected through CCK-8 assay, whereas, ELISA was conducted to evaluate the expression of inflammatory cytokines. Following this, relative expression of miR-214-5p and smad4 were confirmed through qRT-PCR and western blot analysis. Results Results from the experiments manifested up-regulated levels of NNT-AS1 and smad4 in the blood samples of DN patients as well as in HGMCs, whereas, downregulated levels of miR-214-5p were measured in the HGMCs suggesting the negative correlation between NNT-AS1 and miR-214-5p. Potential binding sites of NNT-AS1 showed miR-214-5p as its direct target and NNT-AS1 as potential absorber for this microRNA, in turn increasing the expression of transcription factor smad4. Conclusion The data suggests that NNT-AS1 can be positively used as a potential biomarker and indicator of DN and causes extracellular matrix (ECM) accumulation and inflammation of human mesangial cells. Supplementary Information The online version contains supplementary material available at 10.1186/s12882-021-02580-y., Highlights 1. Up-regulated levels of NNT-AS1 and smad4 in DN samples and HGMCs was manifested. 2. Potential binding sites of NNT-AS1 showed miR-214-5p as its direct target and NNT-AS1 as potential absorber for this microRNA, in turn increasing the expression of transcription factor smad4. 3. The results manifested that knocking-down NNT-AS1 can significantly decrease the inflammation and progression of DN and hence can be used as a potential therapeutic target. Supplementary Information The online version contains supplementary material available at 10.1186/s12882-021-02580-y.

Up-regulation of thrombin receptor protease-activated receptor 1 (PAR-1) is verified to contribute to chronic kidney diseases, including diabetic nephropathy, however, the mechanisms are still unclear. In this study, we investigated the effect of PAR-1 on high glucose-induced proliferation of human glomerular mesangial cells (HMCs), and explored the mechanism of PAR-1 up-regulation from alteration of microRNAs. We found that high glucose stimulated proliferation of the mesangial cells whereas PAR-1 inhibition with vorapaxar attenuated the cell proliferation. Moreover, high glucose up-regulated PAR-1 in mRNA level and protein expression while did not affect the enzymatic activity of thrombin in HMCs after 48 h culture. Then high glucose induced PAR-1 elevation was likely due to the alteration of the transcription or post-transcriptional processing. It was found that miR-17 family members including miR-17-5p, -20a-5p, and − 93-5p were markedly decreased among the eight detected microRNAs only in high glucose-cultured HMCs, but miR-129-5p, miR-181a-5p, and miR-181b-5p were markedly decreased in both high glucose-cultured HMCs and osmotic press control compared with normal glucose culture. So miR-20a was selected to confirm the role of miR-17 family on PAR-1 up-regulation, finding that miR-20a-5p overexpression reversed the up-regulation of PAR-1 in mRNA and protein levels induced by high glucose in HMCs. In summary, our finding indicated that PAR-1 up-regulation mediated proliferation of glomerular mesangial cells induced by high glucose, and deficiency of miR-17 family resulted in PAR-1 up-regulation.