Renal Interstitial Fibrosis Model Research Articles

Bioinformatics analysis reveals CCR7 as a potential biomarker for predicting CKD progression.

Chronic kidney disease (CKD) inevitably progresses to end-stage renal disease if intervention does not occur timely. However, there are limitations in predicting the progression of CKD by solely relying on changes in renal function. A biomarker with high sensitivity and specificity that can predict CKD progression early is required. We used the online Gene Expression Omnibus microarray dataset GSE45980 to identify differentially expressed genes (DEGs) in patients with progressive and stable CKD. We then performed functional enrichment and protein-protein interaction network analysis on DEGs and identified key genes. Finally, the expression patterns of key genes were verified using the GSE60860 dataset, and the receiver operating characteristic curve analysis was performed to clarify their predictive ability of progressive CKD. Ultimately, we verified the expression profiles of these hub genes in an in vitro renal interstitial fibrosis model by real-time PCR and western blot analysis. Differential expression analysis identified 50 upregulated genes and 47 downregulated genes. The results of the functional enrichment analysis revealed that upregulated DEGs were mainly enriched in immune response, inflammatory response, and NF-κB signaling pathways, whereas downregulated DEGs were mainly related to angiogenesis and the extracellular environment. Protein-protein interaction network and key gene analysis identified CCR7 as the most important gene. CCR7 mainly plays a role in immune response, and its only receptors, CCL19 and CCL21, have also been identified as DEGs. The receiver operating characteristic curve analysis of CCR7, CCL19, and CCL21 found that CCR7 and CCL19 present good disease prediction ability. CCR7 may be a stable biomarker for predicting CKD progression, and the CCR7-CCL19/CCL21 axis may be a therapeutic target for end-stage renal disease. However, further experiments are needed to explore the relationship between these genes and CKD.

The ANGPTL4-HIF-1α loop: a critical regulator of renal interstitial fibrosis

BackgroundRenal interstitial fibrosis (RIF) is a progressive, irreversible terminal kidney disease with a poor prognosis and high mortality. Angiopoietin-like 4 (ANGPTL4) is known to be associated with fibrosis in various organs, but its impact on the RIF process remains unclear. This study aimed to elucidate the role and underlying mechanisms of ANGPTL4 in the progression of RIF.MethodsIn vivo, a chronic kidney disease (CKD) rat model of renal interstitial fibrosis was established via intragastric administration of adenine at different time points (4 and 6 weeks). Blood and urine samples were collected to assess renal function and 24-h urinary protein levels. Kidney tissues were subjected to HE and Masson staining for pathological observation. Immunohistochemistry and real-time quantitative PCR (qRT‒PCR) were performed to evaluate the expression of ANGPTL4 and hypoxia-inducible factor-1α (HIF-1α), followed by Pearson correlation analysis. Subsequently, kidney biopsy tissues from 11 CKD patients (6 with RIF and 5 without RIF) were subjected to immunohistochemical staining to validate the expression of ANGPTL4. In vitro, a fibrosis model of human renal tubular epithelial cells (HK2) was established through hypoxic stimulation. Subsequently, an HIF-1α inhibitor (2-MeOE2) was used, and ANGPTL4 was manipulated using siRNA or plasmid overexpression. Changes in ANGPTL4 and fibrosis markers were analyzed through Western blotting, qRT‒PCR, and immunofluorescence.ResultsANGPTL4 was significantly upregulated in the CKD rat model and was significantly positively correlated with renal injury markers, the fibrotic area, and HIF-1α. These results were confirmed by clinical samples, which showed a significant increase in the expression level of ANGPTL4 in CKD patients with RIF, which was positively correlated with HIF-1α. Further in vitro studies indicated that the expression of ANGPTL4 is regulated by HIF-1α, which in turn is subject to negative feedback regulation by ANGPTL4. Moreover, modulation of ANGPTL4 expression influences the progression of fibrosis in HK2 cells.ConclusionOur findings indicate that ANGPTL4 is a key regulatory factor in renal fibrosis, forming a loop with HIF-1α, potentially serving as a novel therapeutic target for RIF.

Attenuating Renal Interstitial Fibrosis by Shenqi Pill viaReducing Inflammation Response Regulated by NF-κBPathway In Vitro and In Vivo.

One of the most significant clinical features of chronic kidney disease is renal interstitial fibrosis (RIF). This study aimed to investigate the role and mechanism of Shenqi Pill (SQP) on RIF. RIF model was established by conducting unilateral ureteral obstruction (UUO) surgery on rat or stimulating human kidney-2 (HK-2) cell with transforming growth factor β1 (TGFβ1). After modeling, the rats in the SQP low dose group (SQP-L), SQP middle dose group (SQP-M) and SQP high dose group (SQP-H) were treated with SQP at 1.5, 3 or 6 g/kg/d, and the cells in the TGFβ1+SQP-L/M/H were treated with 2.5%, 5%, 10% SQP-containing serum. In in vivo assays, serum creatinine (SCr) and blood urea nitrogen (BUN) content were measured, kidney histopathology was evaluated., and α-smooth muscle actin (α-SMA) expression was detected by immunohistochemistry. Interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) content, inhibitor of kappa B alpha (IKBα) and P65 phosphorylation were assessed. Meanwhile, cell viability, inflammatory cytokines content, α-SMA expression, IKBα and P65 phosphorylation were detected in vitro experiment. Results. SQP exhibited reno-protective effect by decreasing SCr and BUN content, improving renal interstitial damage, blunting fibronectin (FN) and α-SMA expression in RIF rats. Similarly, after the treatment with SQP-containing serum, viability and α-SMA expression were remarkably decreased in TGFβ1-stimulated HK-2 cell. Furthermore, SQP markedly down-regulated IL-1β, IL-6, and TNF-α content, IKBα and RelA (P65) phosphorylation both in vivo and in vitro. Conclusion. SQP has a reno-protective effect against RIF in vivo and in vitro, and the effect is partly linked to nuclear factor-kappa B (NF-κB) pathway related inflammatory response, which indicates that SQP may be a candidate drug for RIF. DOI: 10.52547/ijkd.7546.

SNHG16/miR-205/HDAC5 is involved in the progression of renal fibrosis.

Renal interstitial fibrosis (RIF) represents an irreversible and progressive pathological manifestation of chronic renal disease, which ultimately leads to end-stage renal disease. Long noncoding RNAs (lncRNAs) have been suggested to be involved in the progression of RIF. Small nucleolar RNA host gene 16 (SNHG16), a member of lncRNAs, has been found to be involved in the progression of pulmonary fibrosis. This paper first researched the effect of SNHG16 on renal fibrosis. We established a unilateral ureteral obstruction (UUO)-induced mouse RIF model by ligation of the left ureter to evaluate the biological function of SNHG16 in RIF. As a result, SNHG16 was upregulated in UUO-induced renal fibrotic tissues. Knockdown of SNHG16 inhibited RIF and reduced alpha-smooth muscle actin(α-SMA), fibronectin, and college IV expression. miR-205 was a target of SNHG16, and downregulated in UUO-induced renal fibrotic tissues. Inhibition of miR-205 promoted RIF and increased the expression of α-SMA, college IV, and fibronectin. Overexpression of SNHG16 promoted the UUO-induced RIF, but miR-205 abrogated this effect of SNHG16. Histone deacetylase 5 (HDAC5) showed high expression in UUO-induced renal fibrotic tissues. Knockdown of HDAC5 significantly reduced α-SMA, fibronectin, and college IV expression in renal tissues of UUO-induced mice. Inhibition of miR-205 promoted HDAC5 expression, but knockdown of SNHG16 inhibited HDAC5 expression in renal tissues of UUO-induced mice. In conclusion, SHNG16 is highly expressed in renal fibrotic tissues of UUO-induced mice. Knockdown of SHNG16 may prevent UUO-induced RIF by indirectly upregulating HDAC5 via targeting miR-205. SHNG16 may be novel target for treating renal fibrosis.

Curcumin Interferes with TGF- β 1-Induced Fibrosis in NRK-49F Cells by Reversing ADAMTS18 Gene Methylation.

To explore the molecular mechanism by which curcumin affects renal interstitial fibrosis (RIF) progression by regulating ADAM metallopeptidase with thrombospondin type 1 motif 18 (ADAMTS18) methylation. NRK-49F cells RIF model were induced with transforming growth factor β 1 (TGF- β 1). Effects of different concentrations of curcumin (0, 10, 20, and 30 μmol/L) on cell proliferation, cell cycle, cell apoptosis as well as cyclin D1 expression were analyzed by cell counting kit-8, flow cytometry and Western blot, respectively. ADAMTS18 methylation levels were determined by methylation-specific polymerase chain reaction. ADAMTS18, fibronectin (FN), type I collagen (Col- I) and alpha-smooth muscle actin (α -SMA) mRNA and protein expressions were analyzed by real-time PCR (RT-PCR) and Western blot, respectively. Meanwhile, cells were treated with 50 mmol/L 5-aza-2'-deoxycytidine (5-aza-dC, demethylation agent) for 72 h. Effect of curcumin on extracellular matrix (ECM) deposition was evaluated by immunochemical staining and Western blot. NRK-49F cells were transfected with ADAMTS18 small interfering RNA and grouped into a normal control, ADAMTS18-knock-out (KO), and ADAMTS18-KO+ 30 μmol/L curcumin groups, and whether curcumin can reverse the effect of ADAMTS18 knockdown on RIF was evaluated. Compared with the control group, TGF-β 1 significantly inhibited the proliferation of NRK-49F cells, blocked the G1/G0 phase, promoted cell apoptosis and inhibited cyclin D1 expression (P<0.01). Among the different concentrations of curcumin, 30 μmol/L curcumin significantly reversed these processes (P<0.01). Immunochemical staining and Western blot results showed that curcumin significantly inhibited the deposition of FN, Col- I and α-SMA (P<0.01). Curcumin and 5-zaz-dC had synergistic effects, promoting ADAMTS18 expression, removing ADAMTS18 methylation, and reducing ECM deposition. ADAMTS18 knockdown promoted ECM accumulation, and curcumin reversed this process (P<0.01). TGF-β 1-induced fibrosis in NRK-49F cells. Curcumin promoted ADAMTS18 expression, reduced ECM accumulation, and alleviated RIF progression by inhibiting ADAMTS18 methylation.

CX3CR1 regulates the development of renal interstitial fibrosis through macrophage polarization.

The binding of CX3C chemokine receptor 1 (CX3CR1) and its unique ligand CX3C chemokine ligand 1 (CX3CL1) can promote the migration of inflammatory cells to the lesion and affect the progression of renal interstitial fibrosis, but the underlying mechanisms remain unclear. This study aims to investigate whether CX3CR1 affects renal interstitial fibrosis by macrophage polarization. A mouse model of renal interstitial fibrosis was established by unilateral ureteral obstruction (UUO). C57/B6 mice were divided into a CX3CR1 inhibitor group (injected with CX3CR1 inhibitor AZD8797) and a model group (injected with physiological saline). After continuous intraperitoneal injection for 5 days, the ligated lateral kidneys of mice were obtained on the 7th day. Hematoxylin and eosin (HE) staining and Masson staining were used to observe the infiltration of inflammatory cells and the collagen fiber deposition in renal interstitium, respectively. The mRNA and protein expressions of CX3CR1, alpha-smooth muscle actin (α-SMA) and fibronectin (FN) in the kidneys were detected by reverse transcription PCR (RT-PCR) and Western blotting, respectively. Differentially expressed genes in kidney of the 2 groups were identified by whole genome sequencing and the differential expression of arginase-1 (Arg-1) was verified by RT-PCR. Flow cytometry was used to detect the proportion of M2 type macrophages in kidneys of the 2 groups. The infiltration of inflammatory cells and the collagen fiber deposition in renal interstitium were significantly reduced in the CX3CR1 inhibitor group. The mRNA and protein levels of CX3CR1 and the mRNA levels of α-SMA and FN in the CX3CR1 inhibitor group were significantly lower than those of the model group (all P<0.05). Whole genome sequencing showed that the top 5 differentially expressed genes in kidney of the 2 groups were Ugt1a6b, Serpina1c, Arg-1, Retnla, and Nup62. RT-PCR verified that the expression level of Arg-1 in kidney of the CX3CR1 inhibitor group was significantly higher than that of the model group (P<0.001). Flow cytometry showed that the proportion of Arg1+CD206+M2 macrophages in kidney of the CX3CR1 inhibitor group was significantly higher than that of the model group (P<0.01). Inhibiting CX3CR1 can effectively prevent the progression of renal interstitial fibrosis. The mechanism may be related to macrophage polarization towards M2 type and upregulation of Arg-1 expression.

Gypenosides suppress fibrosis of the renal NRK-49F cells by targeting miR-378a-5p through the PI3K/AKT signaling pathway.

The incidence of renal fibrosis caused by chronic kidney disease is increasing year by year. Preventing the activation and conversion of kidney-intrinsic fibroblasts to a myofibroblast phenotype is an important target for blocking the development of renal interstitial fibrosis. Our team established a stable renal interstitial fibrosis cell model in the early stage, and the screening results showed that GPs has good anti-fibrosis potential. At this stage, only a few literatures have reported its anti-fibrosis effect, and the mechanism of action is still unclear. The massive synthesis and secretion of extracellular-matrix (ECM) components by activated fibroblasts in the kidneys causes irreversible renal interstitial fibrosis. Gypenosides (GPs) have been shown to decelerate this process, in which micro RNAs (miRNAs) play an important regulatory role. This study aimed to evaluate the mechanism underlying the suppressive effect of GPs on renal fibrosis. This study used TGF-β1-stimulated NRK-49F renal cells as an in-vitro model of renal interstitial fibrosis. First, the concentration range of GPs that significantly affects the cytoactive was determined. Then, the anti-fibrotic effects of various concentrations of GPs in the in-vitro model were assessed via immunofluorescence, western blotting, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Non-coding-RNA sequencing combined with bioinformatics was used to predict the mechanistic basis of the anti-fibrotic effect of GPs, and qRT-PCR was used to verify the sequencing results and bioinformatic predictions. The identified relationships of the anti-fibrotic effect of GPs with miR-378a-5p and the PI3K/AKT signaling were evaluated using a miR-NC mimic and the PI3K inhibitor LY294002 as controls, respectively. TGF-β1 stimulation up-regulated α-SMA, COL1, and COL3 in NRK-49F cells, and this effect was suppressed by GPs. Additionally, TGF-β1 stimulation significantly changed the expression levels of 151 miRNAs, and GPs significantly suppressed the effect of TGF-β1 on the levels of 18 of these miRNAs. Among them, miR-3588 and miR-378a-5p were down-regulated, and miR-135b-5p and miR-3068-5p were up-regulated upon TGF-β1 induction. Of these miRNAs, miR-378a-5p was predicted to target the mRNAs of numerous proteins mainly enriched in the PI3K/AKT signaling pathway. The miRNA transfection experiments with the miR-NC mimic and PI3K inhibitor as controls showed that miR-378a-5p overexpression could suppress the TGF-β1-induced up-regulation of α-SMA, COL1, PI3K, and AKT, including the phosphorylated form (p-AKT). GPs inhibit the PI3K/AKT signaling by up-regulating miR-378a-5p in TGF-β1-stimulated NRK-49F cells and thereby reduce their massive secretion of ECM components. Given that this in-vitro model of renal interstitial fibrosis closely mimics the in-vivo pathogenesis, our results most likely apply to the in-vivo conditions.

Prevention of Renal Interstitial Fibrosis by Suplatast in a Mouse Model.

Suplatast is a T helper 2 (Th2) cytokine inhibitor. Here, we tested its therapeutic effects using a mouse model of renal interstitial fibrosis caused by unilateral ureteral obstruction (UUO). In this model, suplatast was found to prevent the induced fibrosis in the obstructed kidney when given in the drinking water at 100 mg/kg/d. Mechanistically, suplaplast inhibited the phosphorylation of extracellular signal-regulated kinase (ERK) that was otherwise increased by UUO. Similarly, suplaplast reduced the increased accumulation of KIM-1, transforming growth factor β (TGF-β), type I collagen, interleukin-4 (IL-4), janus kinase (JAK)1 and signal transducer and activator of transcription (STAT)3 mRNA seen in the kidneys of UUO-treated mice. Furthermore, STAT3 phosphorylation, which was stimulated by UUO, was also significantly decreased by suplatast. Collectively, these data show that suplatast reduces UUO-induced renal interstitial fibrosis via mechanisms including a reduction of phosphorylation of ERK and JAK/STAT pathway signaling.

Baicalin protects against renal interstitial fibrosis in mice by inhibiting the TGF-β/Smad signalling pathway

Context Baicalin, a flavonoid extracted from radix scutellariae, possesses various pharmacological effects, including protective effects on renal interstitial fibrosis (RIF), but its possible role and mechanisms have not been fully elucidated. Objective This study explores the protective effects and mechanisms of baicalin on RIF. Materials and methods C57BL/6 male mice were divided into six groups: sham, model, low baicalin, middle baicalin, high baicalin and positive drug groups. The unilateral ureteral obstruction (UUO) model of RIF was constructed and treated with baicalin doses (10, 20 and 40 mg/kg) and a positive control drug (valsartan, 8 mg/kg). H&E staining was used to observe the pathological changes in renal tissues, Masson staining was performed to evaluate collagen deposition in renal tissues, and immunohistochemical examination was adopted to determine α-SMA and extracellular matrix (ECM) expression. Primary mouse fibroblasts were isolated, extracted and treated with baicalin and/or TGF-β. qRT-PCR and enzyme-linked immunosorbent assay (ELISA) were applied to detect the inflammatory responses. Moreover, ECM and TGF-β/Smad expression levels were evaluated by western blot assay. Results Baicalin ameliorated RIF in UUO mice by inhibiting fibrosis and inflammatory responses. The TGF-β/Smad pathway was significantly suppressed in the UUO mouse model. Additionally, baicalin significantly inhibited ECM expression and inflammatory factors in fibroblasts treated with TGF-β. TGF-β/Smad pathway activation was significantly decreased in fibroblasts. Discussion and conclusions These findings support the use of baicalin as a potential therapeutic option for the treatment of RIF by possibly inhibiting the TGF-β/Smad signalling pathway.

The antifibrotic and anti-inflammatory effects of FZHY prescription on the kidney in rats after unilateral ureteral obstruction.

To explore the potential impact of traditional Chinese herb FuZhengHuaYuJiangZhuTongLuo recipe (FZHY) on renal interstitial fibrosis (RIF) in chronic kidney disease (CKD) at cellular and molecular levels. Unilateral ureteral obstruction (UUO) rats were established as the RIF model in vivo. The rats were given intragastric administration with FZHY once a day for consecutive 7, 14 and 21 days, respectively. The renal function parameters and inflammation indicators in kidney tissues were measured using enzyme-linked immunosorbent assay, the CD4+/CD8+ T cells in peripheral blood was detected using flow cytometry, the renal fibrosis degree was estimated using Masson's staining, and the fibrosis-related genes' expression was detected using quantitative polymerase chain reaction, western blotting, and immunohistochemistry analyses. FZHY prescription reduced the serum creatinine and blood urea nitrogen, decreased the levels of c-reactive protein, interleukin-1, interleukin-6 and tumor necrosis factor-α in kidney tissues, and increased the ratio of CD4+/CD8+ T cells in peripheral blood. FZHY prescription suppressed the renal tissue fibrosis and reduced the levels of laminin, fibronectin, collagen I and collagen III. FZHY prescription suppressed the renal fibrosis and improved the condition of "Healthy Qi Deficiency and Evil Qi Excess" in rats with UUO, which may provide an effective method for CKD treatment.

Abnormally decreased renal Klotho is linked to endoplasmic reticulum-associated degradation in mice.

Aim: Endoplasmic reticulum-associated degradation (ERAD), which involves degradation of improperly folded proteins retained in the ER, is implicated in various diseases including chronic kidney disease. This study is aimed to determine the role of ERAD in Klotho deficiency of mice and human kidney tubular epithelial cells (HK-2) with renal interstitial fibrosis (RIF).Methods: Following establishment of a mouse RIF model by unilateral ureteral obstruction (UUO), a specific ERAD inhibitor, Eeyarestatin I (EerI), was administered to experimental animals by intraperitoneal injection. Serum and kidney samples were collected for analysis 10 days after operation. Soluble Klotho levels were measured by enzyme-linked immunosorbent assay, while the degree of kidney injury was assessed by renal histopathology. Renal Klotho expression was determined by quantitative real-time PCR, immunohistochemical and western blotting analyses. ERAD and unfolded protein response (UPR) were evaluated by detecting associated components such as Derlin-1, glucose-regulated protein 78 (GRP78), activating transcription factor 4 (ATF4) and protein disulfide isomerase (PDI). HK-2 cells were exposed to transforming growth factor (TGF)-β1 with or without EerI, and expressions of related proteins including Klotho, Derlin-1, GRP78, ATF4 and PDI were determined by western blotting analyses.Results: UUO induced severe kidney injuries and RIF. Klotho expression in both serum and kidney tissue was obviously downregulated, while Derlin-1 was notably upregulated, indicating that ERAD was activated to potentially degrade improperly folded Klotho protein in this model. Intriguingly, treatment with EerI led to significantly increased Klotho expression, especially soluble (functional) Klotho. Furthermore, specific inhibition of ERAD increased expression of GRP78, ATF4 and PDI compared with the UUO group. The consistent results in vitro were also obtained in TGF-β1-treated HK-2 cells exposed to EerI. These observations suggest that UPR was remarkably enhanced in the presence of ERAD inhibition and compensated for excess improperly folded proteins, subsequently contributing to the additional production of mature Klotho protein.Conclusion: ERAD is involved in Klotho deficiency in RIF and its specific inhibition significantly promoted Klotho expression, possibly through enhanced UPR. This may represent a novel regulatory mechanism and new therapeutic target for reversing Klotho deficiency.

Hirudin, a thrombin inhibitor, attenuates TGF-β-induced fibrosis in renal proximal tubular epithelial cells by inhibition of protease-activated receptor 1 expression via S1P/S1PR2/S1PR3 signaling.

Renal interstitial fibrosis (RIF) is the final common outcome of numerous chronic kidney diseases, contributing to end-stage renal disease. Hirudin, a thrombin inhibitor, has attracted increased attention as a potential treatment approach for renal fibrosis. The present study aimed to investigate the molecular mechanism underlying the effect of hirudin on fibrosis in renal proximal tubular epithelial cells. An in vivo mouse RIF model established using unilateral ureteral obstruction (UUO) and an in vitro of RIF using the renal tubular epithelial cell line HK-2 treated with TGF-β were used. Expressions of sphingosine-1-phosphate (S1P) receptors (S1PR)1-4 and protease-activated receptor 1 (PAR1) were measured by reverse transcription-quantitative PCR and western blotting in mice with UUO and TGF-β induced HK-2 cells. Western blotting was used to detect the expression of N-cadherin, Slug, E-cadherin, Collagen IV, fibronectin, MMP9 and monocyte chemoattractant protein-1. Immunofluorescence staining was conducted to measure α-SMA level expression. The results demonstrated that the expression levels of S1PR1, S1PR2, S1PR3, S1PR4 and PAR1 were upregulated in both TGF-β-induced HK-2 cells and renal tissues from mice with unilateral ureteral ligation. Notably, hirudin inhibited TGF-β-induced PAR1, S1PR2 and S1PR3 upregulation in both HK-2 cells and renal tissues. Additionally, the inhibition of S1PR2 and S1PR3 resulted in PAR1 downregulation. Furthermore, treatment with S1P and PAR1 agonists abolished the effect of hirudin on the expression of EMT, fibrosis-related proteins and monocyte chemoattractant protein 1. In conclusion, hirudin attenuated TGF-β-induced fibrosis in proximal renal tubular epithelial HK-2 cells by inhibiting PAR1 expression via the S1P/S1PR2/S1PR3 signaling pathway. Therefore, hirudin may be considered as a promising therapeutic agent for RIF.

Curcumin mediates repulsive guidance molecule B (RGMb) in the treatment mechanism of renal fibrosis induced by unilateral ureteral obstruction

In this study, we explored the role and mechanism of repulsive guidance molecule B (RGMb, also known as Dragon) in the protective effects of curcumin against renal fibrosis and verified Dragon’s effect on renal tubular epithelial cell apoptosis and cell programmability. Unilateral ureteral obstruction (UUO) was surgically induced in rats to establish a model of renal interstitial fibrosis (RIF). The rats were then treated with curcumin. Curcumin prominently decreased the serum creatinine (SCr) and blood urea nitrogen (BUN) levels, and also improved the tubular injury in the UUO-induced rats. Curcumin significantly downregulated the TGF-β1, P-Smad2/3, cleaved caspase-3, cleaved caspase-8 and Dragon levels. Dragon knockdown also markedly reduced the TGF-β1, P-Smad2/3, Smad2/3, cleaved caspase-3, cleaved caspase-8, fibronectin, collagen I, collagen IV, vimentin, and α-SMA expression levels. Conversely, Dragon overexpression caused higher expression levels of these proteins, and curcumin reversed this effect. Furthermore, Dragon knockdown increased the E-cadherin levels, whereas Dragon overexpression decreased these levels. Overexpressing Dragon significantly decreased the cell viability, and curcumin reversed this effect. In conclusion, curcumin acted on Dragon and attenuated RIF in UUO rat models. Curcumin downregulated the TGF-β1/Smad signaling pathway and inhibited Dragon and fibrogenic molecules in both rats and HK-2 cells.

The mushroom, Cordyceps cicadae, ameliorates renal interstitial fibrosis via TLR2-mediated pathways

Purpose: To evaluate the mushroom, Cordyceps cicadae, for its ability to suppress tissue fibrosis and Toll-like receptors 2 (TLR 2) pathway activation in a mouse model of renal interstitial fibrosis (RIF).Methods: Cordyceps cicadae powder was obtained from BioAsia Group (Shanghai, China). RIF was induced via unilateral ureteral obstruction (UUO) in male C57Bl/6 mice. Animals were treated via the intragastric administration of Cordyceps cicadae powder (0.1g, 0.3 g/ml/100 g/day), beginning 24 h prior to UUO, and the treatment was continued for the following 14 days. Changes in tissue histology were then assessed via hematoxylin and eosin, and Sirius red stainings. Tissue macrophages were characterized based upon their expression of inducible nitric oxide synthase (iNOS) and interleukin-10 (IL-10), while Western blotting technique was used to measure the levels of TLR2, Myeloid differentiation factor 88 (MyD88), and nuclear factor-κB (NF-κB)/p-NF-κB in samples from these animals.Results: Treatment with Cordyceps cicadae powder is associated with a shift in macrophage phenotype that in turn decreased the production of extracellular matrix and alleviated RIF occurrence in mice model.Conclusion: This mechanistic study highlights the novel potential approach for treating and preventing RIF using Cordyceps cicadae powder.&#x0D; Keywords: Renal interstitial fibrosis, TLR2-mediated pathway, Cordyceps cicadae

Naringin attenuates renal interstitial fibrosis by regulating the TGF-β/Smad signaling pathway and inflammation.

Interstitial fibrosis is a typical feature of all progressive renal diseases. The process of fibrosis is frequently coupled with the presence of pro-fibrotic factors and inflammation. Naringin is a dihydroflavone compound that has been previously reported to exhibit anti-fibrotic effects in the liver, where it prevents oxidative damage. In the present study, a rat model of renal interstitial fibrosis and fibrosis cell model were established to evaluate the effects of naringin on inflammatory proteins and fibrosis markers in kidney of rats and NRK-52E cells, and to elucidate the role of the TGF-β/Smad signaling pathway in this mechanism. Compared with those in fibrotic NRK-52E cells that were stimulated by transforming growth factor-β (TGF-β), gene expression levels of α-smooth muscle actin (α-SMA), collagen 1 (COL1A1), collagen 3 (COL3A1), interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) were all found to be significantly decreased in fibrotic NRK-52E cells following treatment with naringin (50, 100 and 200 ng/ml). Results from the histopathological studies showed that naringin treatment preserved the renal tissue structure and reduced the degree of fibrosis in the kidney tissues of rats that underwent unilateral ureteral obstruction (UUO). In addition, naringin administration reduced the expression of α-SMA, COL1A1, COL3A1, IL-1β, IL-6 and TNF-α in the kidneys of rats following UUO. The current study, using western blot analysis, indicated that naringin also downregulated the activation of Smad2/3 and the expression of Smad4, high-mobility group protein B1, activator protein-1, NF-κB and cyclooxygenase-2 whilst upregulating the expression of Smad7 in fibrotic NRK-52E cells and rats in the UUO group. In conclusion, naringin could antagonize renal interstitial fibrosis by regulating the TGF-β/Smad pathway and the expression of inflammatory factors.

Quantitative Real-Time PCR Evaluation of microRNA Expressions in Mouse Kidney with Unilateral Ureteral Obstruction.

MicroRNAs (miRNAs) are single stranded, non-coding RNA molecules that typically regulate gene expression at the post-transcriptional level by binding to partially complementary target sites in the 3' untranslated region (UTR) of messenger RNA (mRNA), which reduces the mRNA's translation and stability. The miRNA expression profiles in various organs and tissues of mice have been investigated, but standard methods for the purification and quantification of miRNA in mouse kidney have not been available. We have established an effective and reliable method for extracting and evaluating miRNA expression in mouse kidney with renal interstitial fibrosis by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The protocol required five steps: (1) creation of sham and unilateral ureteral obstruction (UUO) mice; (2) extraction of kidney samples from the UUO mice; (3) extraction of total RNA, which includes miRNA, from the kidney samples; (4) complementary DNA (cDNA) synthesis with reverse transcription from miRNA; and (5) qRT-PCR using the cDNA. Using this protocol, we successfully confirmed that compared to the controls, the expression of miRNA-3070-3p was significantly increased and those of miRNA-7218-5p and miRNA-7219-5p were significantly decreased in the kidneys of a mouse model of renal interstitial fibrosis. This protocol can be used to determine the miRNA expression in the kidneys of mice with UUO.

Transcriptomic analysis of the mechanisms of alleviating renal interstitial fibrosis using the traditional Chinese medicine Kangxianling in a rat model

Renal interstitial fibrosis (RIF) is currently recognized as a crucial mechanism of the pathogenesis of chronic kidney disease (CKD). Kangxianling (KXL, anti-fibrin) is a traditional Chinese medicine that has been proven to significantly reduce the levels of ECM deposition and inhibit renal fibrosis.To characterize the mechanisms and drug targets of KXL, we established a RIF rat model and treated the rats with KXL and losartan. Histological analyses validated the establishment of the RIF model and the treatment effect of KXL. Multiple levels of transcriptomic datasets were generated using lncRNA, mRNA and microRNA sequencing of kidney tissues. Functional annotations and pathway analyses were performed to unravel the therapeutic mechanisms.A multi-level transcriptomic regulatory network was built to illustrate the core factors in fibrosis pathogenesis and therapeutic regulation. KXL and losartan significantly reduced the progression of RIF, and a better therapeutic effect was shown with higher concentrations of KXL. According to the cluster analysis results of the RNA-seq data, the normal control (NC) and high concentration of KXL (HK) treatment groups were the closest in terms of differentially expressed genes. The WNT, TGF-β and MAPK pathways were enriched and dominated the pathogenesis and therapy of RIF. miR-15b, miR-21, and miR-6216 were upregulated and miR-107 was downregulated in the fibrosis model. These small RNAs were shown to play critical roles in the regulation of the above fibrosis-related genes and could be inhibited by KXL treatment. Finally, based on the lncRNA datasets, we constructed a mRNA-lncRNA-miRNA coexpression ceRNA network, which identified key regulatory factors in the pathogenesis of kidney fibrosis and therapeutic mechanisms of KXL. Our work revealed the potential mechanism of the Chinese medicine Kangxianling in inhibiting renal interstitial fibrosis and supported the clinical use of KXL in the treatment of kidney fibrosis.

Chinese herbal decoction astragalus and angelica exerts its therapeutic effect on renal interstitial fibrosis through the inhibition of MAPK, PI3K-Akt and TNF signaling pathways

Astragalus and Angelica decoction (A&A) has been clinically used as a classical traditional Chinese medicine (TCM) formula in China for many years for the treatment of kidney diseases, especially renal interstitial fibrosis (RIF). However, the mechanisms underlying the therapeutic effects of A&A on RIF remains poorly understood. In the present study, systematic network pharmacology and effective experimental verification were utilized for the first time to elucidate the pharmacological efficacy and potential mechanism. The outcomes indicated that 22 active components and 87 target genes of A&A were identified and cross-referenced with RIF-associated genes, contributing to confirmation of 74 target genes of A&A for RIF. Pathway and functional enrichment analyses revealed that A&A had substantial effects on MAPK, PI3K-Akt and TNF signaling pathways. In addition, seven core targets with relatively higher betweenness and degree were identified in the constructed Chinese medicine material-chemical component-target-signal pathway network. Moreover, we verified the potential therapeutic effect of A&A in vivo (using a mouse model of RIF), confirming that A&A could effectively protect the kidney by regulating these target genes. The therapeutic effect of A&A on RIF could be attributed to its role in regulating the cell cycle, limiting the apoptosis, and inhibiting the inflammation.

FoxM1 inhibition ameliorates renal interstitial fibrosis by decreasing extracellular matrix and epithelial–mesenchymal transition

FoxM1 is a transcriptional regulator involved in tumor development, pulmonary fibrosis, and cardiac fibrosis. However, its role in renal interstitial fibrosis (RIF) has yet to be elucidated. We established a TGF-β1-stimulated human proximal tubular epithelial cell (HK-2) model in vitro and a unilateral ureteral obstruction (UUO)-induced rat RIF model in vivo. FoxM1 inhibition was achieved by siRNA interference in vitro and by injecting thiostrepton into UUO-induced RIF rats in vivo. The degree of renal damage and fibrosis were determined by histological assessment via hematoxylin and eosin (H&E) staining. Immunohistochemistry, western blots, and qPCR were used to determine the expression levels of FoxM1, Collagen I, E-cadherin, α-SMA, and Snail1. Our results showed that FoxM1 inhibition could ameliorate RIF and reduce the deposition of Collagen I. H&E staining revealed that renal structural damage, inflammatory cell infiltration, and ECM deposition were significantly attenuated by thiostrepton treatment in the UUO rats. Furthermore, FoxM1 downregulation significantly suppressed epithelial-to-mesenchymal transition, as evidenced by decreased protein and mRNA expression levels of α-SMA and Snail1 and a significant increase in protein and mRNA expression levels of E-cadherin. Collectively, these results suggested that FoxM1 inhibition could be a novel therapeutic strategy for the treatment of RIF.

Niao Du Kang Mixture Increases the Expression of mir-129-5p to Relieve Renal Fibrosis.

Objective To investigate the efficacy of Niao Du Kang (NDK) mixture in renal fibrosis of rats and to explore the mechanism underlying the effect of NDK on renal fibrosis. Methods Unilateral ureteral obstruction (UUO) was used to replicate a rat renal interstitial fibrosis model. The drug-administered groups were given 20 ml/kg (NDK-H), 10 ml/kg (NDK-M), and 5 ml/kg (NDK-L) NDK mixture once a day for 21 days beginning 48 hours after surgery. The 24-hour urine protein and serum creatinine (CR) levels in the sham group rats, UUO rats, and NDK mixture-treated rats were measured after the last administration. The pathological changes of rat kidney tissue were observed by HE staining. The degree of fibrosis was observed by Masson's staining and scored. The expression levels of TGF-β, α-SMA mRNA, and mir-129-5p in kidney were detected by qRT-PCR. HK-2 cells were treated with 5 ng/ml TGF-β to induce HK-2 cell fibrosis. The expression levels of TGF-β, α-SMA mRNA, and mir-129-5p in HK-2 cells were detected by qRT-PCR. TargetScan predicted the target gene of mir-129-5p, HK-2 cells were transfected with mir-129-5p mimic, and an overexpressed mir-129-5p HK-2 cell model was constructed. qRT-PCR was used to detect the expression of PDPK1 mRNA. Western blot was used to detect the expression of PDPK1, AKT, and p-AKT in HK-2 cells induced by TGF-β and in UUO rats. Results NDK mixture significantly reduced the 24-hour urine protein and CR levels of UUO rats. HE staining showed that the NDK mixture group exhibited a significantly reduced degree of renal interstitial fibrosis. NDK mixture also reduced the expression of TGF-β and α-SMA, and the middle-dose group showed a better therapeutic effect. In vitro studies showed that NDK mixture-containing serum increased the expression of mir-129-5p to reduce renal fibrosis. In addition, NDK mixture increased the expression of mir-129-5p in vivo. Further studies indicated that mir-129-5p could target PDPKl to reduce its expression. The NDK-containing serum group also exhibited reduced expression of PDPK1. Conclusion NDK mixture can significantly improve renal function and improve renal fibrosis in UUO model rats. Furthermore, NDK mixture can inhibit the expression of PDPK1 by upregulating the expression of mir-129-5p and then inhibiting the PI3K/AKT pathway to improve renal fibrosis.