Treatment Of Renal Interstitial Fibrosis Research Articles

The efficacy and safety of tonifying-kidney and invigorating-blood circulation (Bushen-Huoxue) formula on renal fibrosis in patients with chronic kidney disease: A protocol for systematic review and meta-analysis

Background: The incidence of chronic kidney disease (CKD) is increasing and the progression of renal interstitial fibrosis (RIF) in end-stage renal disease is attributed to the development of various CKD. One of the treatments for RIF is an oral treatment known as Bushen-Huoxue (BSHX), which stimulates blood circulation and tonifies the kidney. Randomized controlled trials of BSHX treatment of RIF have been reported, but their efficacy and safety have not been comprehensively examined. Therefore, our study aims to describe the efficacy and safety of oral application of the traditional Chinese medicine (TCM) formula of BSHX on treating RIF in CKD by systematic analysis and meta-analysis. Methods: A total of 13 databases, including PubMed, EMBASE, MEDLINE, Web of Science, Cochrane Library, SpringerLink, WHO International Clinical Trials Registry Platform, Wanfang China database, China National Knowledge Infrastructure, Chinese Biomedical Literature Database, Chinese Scientific Journal Database, and China’s Conference Papers Database and China Dissertation database will be searched from inception to July 2023. In addition, other suitable search strategies will be used as well to complete this review. All randomized controlled trials that use the BSHX formula for RIF to apply TCM treatments externally for CKD will be included. The eligible studies will be selected to enroll by 2 independent researchers following a predetermined protocol. The review manager software (Rev-Man 5.3) will be used to assess the risk of bias, reporting bias, and data synthesis. Result: We will compile and discuss the recent examples of the TCM BSHX formula treatment for RIF in CKD. Conclusion: We will provide strong evidences for evaluating the efficacy and safety of the TCM BSHX formula treatment for RIF in CKD.

Irisin ameliorates UUO-induced renal interstitial fibrosis through TGF-β1/periostin/MMP-2 signaling pathway.

Renal fibrosis is the most common pathway in progressive kidney diseases. The unilateral ureteral obstruction (UUO) model is used to induce progressive renal fibrosis. We evaluated the effects of irisin on renal interstitial fibrosis in UUO mice. The GSE121190, GSE36496, GSE42303, and GSE96101 datasets were downloaded from the Gene Expression Omnibus (GEO) database. In total, 656 differentially expressed genes (DEGs) were identified in normal and UUO mouse renal samples. Periostin and matrix metalloproteinase-2 (MMP-2) were selected to evaluate the effect of irisin on renal fibrosis in UUO mice. In UUO mice, irisin ameliorated renal function, decreased the expression of periostin and MMP-2, and attenuated epithelial-mesenchymal transition and extracellular matrix deposition in renal tissues. In HK-2 cells, irisin treatment markedly attenuated TGF-β1-induced expression of periostin and MMP-2. Irisin treatment also inhibited TGF-β1-induced epithelial-mesenchymal transition, extracellular matrix formation, and inflammatory responses. These protective effects of irisin were abolished by the overexpression of periostin and MMP-2. In summary, irisin treatment can improve UUO-induced renal interstitial fibrosis through the TGF-β1/periostin/MMP-2 signaling pathway, suggesting that irisin may be used for the treatment of renal interstitial fibrosis.

The Expression and Molecular Mechanisms of Matrix Metalloproteinase- 9 and Vascular Endothelial Growth Factor in Renal Interstitial Fibrosis in Rats.

To explore a new approach for the treatment of renal interstitial fibrosis (RIF), we detected the expression of matrix metalloproteinase-9 (MMP9) and vascular endothelial growth factor (VEGF). Twenty-four male Sprague Dawley (SD) rats were randomly divided into 2- week normal control (2NC) group, 4-week NC (4NC) group, 2-week unilateral ureteral obstruction (2UUO) group, and 4-week UUO (4UUO) group. We performed left ureteral ligation on UUO groups. Then, we sacrificed the rats of the 2NC group and 2UUO group at 2 weeks and the other groups at 4 weeks after the surgery. Immunohistochemistry and western blot were applied to detect the expression of MMP9, VEGF, fibronectin (FN), type IV collagen (Col-IV), and transforming growth factor-β1 (TGF-β1). MMP9 levels reduced after UUO surgery. Its expression was less in the 4UUO group than in the 2UUO group (P<0.05). The expression of VEGF, TGF- β1, FN, and Col-IV was higher in UUO groups than in NC groups (P<0.05). The expression of these indicators was higher in the 4UUO group than in the 2UUO group (P<0.05). In the correlation analysis, MMP9 levels in UUO groups had a negative correlation with the expression of TGF-β1, VEGF, Col-IV, FN, and RIF index (all P<0.05). In UUO groups, VEGF levels had a positive correlation with the expression of TGF-β1, Col-IV, FN, and RIF index (all P<0.05). In conclusion, with the aggravation of RIF lesions, MMP9 levels decreased, and VEGF levels increased. Whether there is a mutual inhibition relationship between them remains to be confirmed by further experiments.

Mitophagy in renal interstitial fibrosis.

As a high energy consumption organ, kidney relies on a large number of mitochondria to ensure normal physiological activities. Under specific stimulation, mitophagy and mitochondrial dynamics (fission, fusion) cooperatively regulate mitochondrial quality and participate in many life activities such as energy metabolism, inflammatory response, oxidative stress, cell senescence and death. Mitophagy plays a key role in the progression of acute kidney injury and chronic kidney disease. The early induction of oxidative stress in renal parenchyma, the activation of pro-inflammatory cytokines and TGF-β signal pathway are closely related to renal interstitial fibrosis. Macrophage reprogramming is also considered to be an important participant in the progression of kidney fibrosis. This review summarizes the molecular mechanism of mitochondrial autophagy and its relationship with the pathway of promoting fibrosis, and discusses the possibility of restoring mitophagy balance as a pharmacological target for the treatment of renal interstitial fibrosis, so as to provide new ideas for more efficient anti-fibrosis and delay the progress of chronic kidney disease.

Aristolocholic acid I promotes renal tubular epithelial fibrosis by upregulating matrix metalloproteinase-9 expression via activating the C3a/C3aR axis of macrophages

Aristolochic acid I (AAI) can cause nephrotoxicity and is characterized by interstitial fibrosis. The C3a/C3aR axis of macrophages and matrix metalloproteinase-9 (MMP-9) play important roles in fibrosis, but whether they are involved in AAI-induced renal interstitial fibrosis and are related remains to be elucidated. In this study, we investigated whether C3a/C3aR axis of macrophages promotes renal interstitial fibrosis by regulating MMP-9 in aristolochic acid nephropathy (AAN). Intraperitoneal injection of AAI for 28 days successfully induced AAN in C57bl/6 mice. The content of C3a in the kidney of AAN mice was increased, and there was a significant distribution of macrophages in the renal tubules. The same results were observed in the in vitro experiment. We also explored the role and mechanism of macrophages after AAI administration in the epithelial–mesenchymal transformation (EMT) of renal tubular epithelial cells (RTECs) and found that AAI could activate the C3a/C3aR axis of macrophages to upregulate p65 expression in macrophages. p65 upregulated MMP-9 expression in macrophages not only directly but also by promoting the secretion if interleukin-6 by macrophages and then activating STAT3 in RTECs. The upregulation of MMP-9 expression could promote the EMT of RTECs. Taken together, our study demonstrated that the AAI-activated the C3a/C3aR axis of macrophages, which induced MMP-9 production, was one of the causes of renal interstitial fibrosis. Therefore, targeting the C3a/C3aR axis of macrophages is an effective therapeutic strategy for the prevention and treatment of renal interstitial fibrosis in AAN.

Mechanism of dioscin ameliorating renal fibrosis through NF‑κB signaling pathway‑mediated inflammatory response.

Dioscin (DIS) is a natural compound derived from Chinese herbal medicine. In recent years, multiple studies have reported that DIS has immunoregulation, anti‑fibrosis, anti‑inflammation, anti‑viral and anti‑tumor effects. However, the mechanism by which DIS ameliorates renal fibrosis and inflammation remains to be elucidated. The aim of the present study was to investigate the role of DIS in renal fibrosis and inflammation and to explore its underlying mechanism. It used network pharmacology to predict the targets of DIS for the treatment of renal interstitial fibrosis. The present study was performed using unilateral ureteral obstruction mice and HK‑2 cells invivo and invitro. The mice were treated with different doses of DIS. Kidney tissues were collected for histopathology staining, western blotting, immunohistochemistry staining and reverse transcription‑quantitative (RT‑q) PCR. TGF‑β1 (2ng/ml) was used to induce renal fibrosis in the cells. Then, cells were respectively treated with DIS (3.125, 6.25, 12.5µM) and Bay11‑7082 (an inhibitor of NF‑κB p65 nuclear transcription, 1µM) for another 24h. The expressions of inflammatory factors and NF‑κB pathway proteins were detected by immunofluorescence, ELISA, western blotting and RT‑qPCR. DIS alleviated renal injury in the UUO mice. Mechanistically, DIS not only decreased the expressions of inflammatory factors including IL‑1β, NOD‑like receptor thermal protein domain associated protein 3, monocyte chemotactic protein 1, IL‑6, TNF‑α and IL‑18 but also reduced the level of phosphorylation of NF‑κB p65 invivo and invitro, which was similar to the impact of Bay11‑7082. DIS ameliorated renal fibrosis by inhibiting the NF‑κB signaling pathway‑mediated inflammatory response, which may be a therapeutic pathway for delaying chronic kidney disease.

Long non-coding RNA lnc-CHAF1B-3 promotes renal interstitial fibrosis by regulating EMT-related genes in renal proximal tubular cells

Renal interstitial fibrosis (RIF) is a common pathological manifestation of chronic kidney diseases. Epithelial-mesenchymal transition (EMT) of tubular epithelial cells is considered a major cause of RIF. Although long non-coding RNAs (lncRNAs) are reportedly involved in various pathophysiological processes, the roles and underlying molecular mechanisms of lncRNAs in the progression of RIF are poorly understood. In this study, we investigated the function of lncRNAs in RIF. Microarray assays showed that expression of the lncRNA lnc-CHAF1B-3 (also called claudin 14 antisense RNA 1) was significantly upregulated in human renal proximal tubular cells by both transforming growth factor-β1 (TGF-β1) and hypoxic stimulation, accompanied with increased expression of EMT-related genes. Knockdown of lnc-CHAF1B-3 significantly suppressed TGF-β1-induced upregulated expression of collagen type I alpha 1, cadherin-2, plasminogen activator inhibitor-1, snail family transcriptional repressor I (SNAI1) and SNAI2. Quantitative reverse transcriptase PCR analyses of paraffin-embedded kidney biopsy samples from IgA nephropathy patients revealed lnc-CHAF1B-3 expression was correlated positively with urinary protein levels and correlated negatively with estimated glomerular filtration rate. In situ hybridization demonstrated that lnc-CHAF1B-3 is expressed only in proximal tubules. These findings suggest lnc-CHAF1B-3 affects the progression of RIF by regulating EMT-related signaling. Thus, lnc-CHAF1B-3 is a potential target in the treatment of RIF.

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.

Research Progress of Chinese Herbal Medicine Intervention in Renal Interstitial Fibrosis.

Chronic kidney diseases usually cause renal interstitial fibrosis, the prevention, delay, and treatment of which is a global research hotspot. However, no definite treatment options are available in modern medicine. Chinese herbal medicine has a long history, rich varieties, and accurate treatment effects. Hitherto, many Chinese herbal medicine studies have emerged to improve renal interstitial fibrosis. This paper reviews the mechanisms of renal interstitial fibrosis and recent studies on the disease intervention with Chinese herbal medicine through literature search, intend to reveal the importance of Chinese herbal medicine in renal interstitial fibrosis. The results show that Chinese herbal medicine can improve renal interstitial fibrosis, and the effects of Chinese herbal medicine on specific pathological mechanisms underlying renal interstitial fibrosis have been explored. Additionally, the limitations and advantages of Chinese herbal medicine in the treatment of renal interstitial fibrosis, possible research directions, and new targets of Chinese herbal medicine are discussed to provide a basis for studies of renal interstitial fibrosis.

Astragaloside IV attenuated TGF-β1- induced epithelial-mesenchymal transition of renal tubular epithelial cells via connexin 43 and Akt/mTOR signaling pathway

IntroductionThe objective of the study was to observe whether connexin 43 (Cx43) could regulate epithelial mesenchymal transformation (EMT) of renal tubular epithelial cells (RTECs) by influencing Akt/mTOR signaling pathway, and whether ASV could inhibit the development of renal interstitial fibrosis by regulating Cx43. MethodsLentivirus infection was transfected into RTECs with the final concentration of 50 ×PFU/ cell to regulate the expression of Cx43. And RTECs were intervened by different doses of Astragaloside IV (ASV). After synchronous culture of RTECs in each group，the expression levels of EMT-related indicators and Cx43 were detected by fluorescence microscope and Western-Blotting (WB), even the protein expressions and phosphorylation levels of AKT and mTOR in different groups were detected by WB. ResultsWhen the expression of Cx43 in RTECs was regulated by lentivirus infection, the degree of EMT induced by TGF‑β1 and the phosphorylation level of Akt and mTOR were changed accordingly, indicating that Akt/mTOR pathway might be a downstream molecular mechanism by which Cx43 could regulate EMT. After intervention with different doses of ASV, the expression level of Cx43 increased with obvious concentration dependence, and the expression levels of p-Akt and p- mTOR were significantly altered, suggesting that ASV could effectively increase the protein expressions of TGF‑β1-induced Cx43 in RTECs and inhibit the phosphorylation levels of Akt and mTOR. ConclusionCx43 were the main material basis of RTECs’ injury, and ASV could inhibit TGF-β1- induced RTECs’ transdifferentiation. In-depth study of the mechanism might provide a broad application prospect for the treatment of renal interstitial fibrosis.

POS-402 UNCOUPLING PROTEIN 1 (UCP1) INHIBITS EPITHELIAL MESENCHYMAL TRANSFORMATION AND EXTRACELLULAR MATRIX AGGREGATION IN RENAL INTERSTITIAL FIBROSIS THROUGH SIRT3 DEPENDENT REACTIVE OXYGEN SPECIES (ROS) PATHWAY

Renal interstitial fibrosis is a common pathological process of all chronic kidney diseases (CKD) developing to end-stage renal disease (ESRD). It is characterized by inflammatory cell infiltration, massive accumulation of extracellular matrix (ECM), disappearance of renal proper cells and reduction of microvessels. At present, although the research on renal interstitial fibrosis is gradually in-depth, its diagnosis and treatment methods are still very scarce. Therefore, to clarify the new pathogenesis of renal interstitial fibrosis and develop new methods for early diagnosis and treatment of renal interstitial fibrosis has become the focus and difficulty of current renal diseases research. Uncoupling protein-1 (UCP1) is the most important marker of brown adipose tissue (BAT), located in the inner membrane of the mitochondria. UCP1 can promote mitochondrial inner membrane proton conduction and decouple ATP synthesis from cellular respiration. The current research on UCP1 mainly focuses on fat metabolism, but its function in CKD has not been reported yet. Western blot, immunoblot analysis and immunohistochemistry were used to detect the expression of UCP1. UCP1 over expression lentivirus and UCP1 agonist CL316243 were used to upregulate UCP1. EMT related markers, collagen I, fibronectin, antioxidant enzyme SOD2 and CAT were detected by western blot and immunofluorescence. ROS detection kit was used to show the ROS production. SIRT3 knockdown was achieved by siRNA. This study firstly discovers that UCP1 is significant downregulated in patients with renal fibrosis and unilateral ureteral obstruction (UUO) animal model. Further research finds that upregulation of UCP1 can reverse the epithelial mesenchymal transformation (EMT) and ECM accumulation in renal fibrosis model both in vivo and in vitro. Meanwhile, UCP1 reduces the production of reactive oxygen species (ROS) by increasing the stability of SIRT3, which is weakened when SIRT3 is knocked down. Accordingly, this study proposes a scientific hypothesis in which UCP1 relieve EMT, ECM accumulation and renal interstitial fibrosis by increasing the stability of SIRT3 and decreasing the production of ROS. It will provide new ideas and targets for the treatment of CKD.

A Review of Traditional Chinese Medicine in Treating Renal Interstitial Fibrosis via Endoplasmic Reticulum Stress-Mediated Apoptosis.

Renal interstitial fibrosis (RIF) is the main pathological manifestation of end-stage renal disease. Recent studies have shown that endoplasmic reticulum (ER) stress is involved in the pathogenesis and development of RIF. Traditional Chinese medicine (TCM), as an effective treatment for kidney diseases, can improve kidney damage by affecting the apoptotic signaling pathway mediated by ER stress. This article reviews the apoptotic pathways mediated by ER stress, including the three major signaling pathways of unfolded protein response, the main functions of the transcription factor C/EBP homologous protein. We also present current research on TCM treatment of RIF, focusing on medicines that regulate ER stress. A new understanding of using TCM to treat kidney disease by regulating ER stress will promote clinical application of Chinese medicine and discovery of new drugs for the treatment of RIF.

Regulation of connective tissue growth factor expression by miR-133b for the treatment of renal interstitial fibrosis in aged mice with unilateral ureteral obstruction

IntroductionRenal interstitial fibrosis, an important pathological feature of kidney aging and chronic renal failure, is regulated by mesenchymal stem cells (MSCs). We have previously demonstrated low expression of miR-133b in MSC-derived extracellular vesicles (MSC-EVs) in aged rats. However, miR-133b can mediate the inhibition of epithelial-mesenchymal transition (EMT) of renal tubules induced by transforming growth factor-β1 (TGF-β1). We investigated the effect of miR-133b for the treatment of geriatric renal interstitial fibrosis and evaluated its target genes.MethodsWe performed real-time polymerase chain reaction to detect miR-133b expression induced during EMT of HK2 cells by TGF-β1 at different concentrations (0, 6, 8, and 10 ng/mL) and at different time points (0, 24, 48, and 72 h). The target genes of miR-133b were validated using the dual-luciferase reporter assay. In vitro experiments were performed to evaluate mRNA and protein expression of miR-133b targets, E-cadherin, α-smooth muscle actin (SMA), fibronectin, and collagen 3A1 (Col3A1), in HK2 cells transfected with miR-133b under TGF-β1 stimulation. A 24-month-old unilateral ureteral obstruction (UUO) mouse model was established and injected with transfection reagent and miR-133b into the caudal vein. The target gene of miR-133b and other parameters mentioned above such as mRNA and protein expression levels and renal interstitial fibrosis were detected at 7 and 14 days.ResultsmiR-133b expression gradually decreased with an increase in TGF-β1 concentration and treatment time, and the miR-133b mimic downregulated connective tissue growth factor (CTGF) expression. The dual-luciferase reporter assay confirmed CTGF as a direct target of miR-133b. Transfection of the miR-133b mimic inhibited TGF-β1-induced EMT of HK2 cells; this effect was reversed by CTGF overexpression. miRNA-133b expression significantly increased (approximately 70–100 times) in mouse kidney tissues after injection of the miRNA-133b overexpression complex, which significantly alleviated renal interstitial fibrosis in mice with UUO.ConclusionmiR-133b exerted targeted inhibitory effects on CTGF expression, which consequently reduced TGF-β1-induced EMT of HK2 cells and renal interstitial fibrosis in aged mice with UUO.

NCTD Prevents Renal Interstitial Fibrosis via Targeting Sp1/lncRNA Gm26669 Axis.

In our previous study, we demonstrated that norcantharidin (NCTD) is a potential therapeutic agent for renal interstitial fibrosis (RIF). Recently, we found that lncRNA Gm26669 (Gm26669) contributed to the development of RIF and could be regulated by NCTD. However, the upstream mechanisms of Gm26669 and whether the anti-RIF effects of NCTD are related to its regulatory action on Gm26669 remain unclear. Our bioinformatics analysis indicated that special protein1 (Sp1), a transcription factor, may bind to the promoter of Gm26669. In the present study, we observed a significant increase in the nuclear translocation of Sp1 using both in vivo and in vitro models of RIF. Furthermore, the knockdown of Sp1 inhibited the expression of collagen type I (CoL-I) and fibronectin (Fn). Mechanistically, Sp1 promoted the expression levels of CoL-I and Fn by directly binding to the promoter of Gm26669 to elevate its expression level. Moreover, we found that NCTD alleviated RIF by inhibiting Gm26669 and the nuclear translocation of Sp1. Collectively, above results suggested that NCTD might prevent RIF via targeting the Sp1/Gm26669 axis, thus providing a new theoretical basis for the clinical application of NCTD in the treatment of RIF.

Huangkui capsule in combination with metformin ameliorates diabetic nephropathy via the Klotho/TGF-β1/p38MAPK signaling pathway

Ethnopharmacological relevanceHuangkui capsule (HKC), extracted from Abelmoschus manihot (L.) medic (AM), as a patent proprietary Chinese medicine on the market for approximately 20 years, has been clinically used to treat chronic glomerulonephritis. Renal fibrosis has been implicated in the onset and development of diabetic nephropathy (DN). However, the potential application of HKC for preventing DN has not been evaluated. Aim of the studyThis study was designed to investigate the efficacy and underlying mechanisms of HKC combined with metformin (MET), the first-line medication for treating type 2 diabetes, in the treatment of renal interstitial fibrosis. Materials and methodsA rat model of diabetes-associated renal fibrosis was established by intraperitoneal injection of streptozotocin (STZ, 65 mg/kg) combined with a high-fat and high-glucose diet. The rats were randomly divided into five groups: normal control, DN, HKC (1.0 g/kg/day), MET (100 mg/kg/d), and HKC plus MET (1.0 g/kg/day + 100 mg/kg/d). Following drug administration for 8 weeks, we collected blood, urine, and kidney tissue for analysis. Biochemical markers and metabolic parameters were detected using commercial kits. Histopathological staining was performed to monitor morphological changes in the rat kidney. High-glucose-induced human kidney HK-2 cells were used to evaluate the renal protective effects of HKC combined with MET (100 μg/mL+10 mmol/L). MTT assay and acridine orange/ethidium bromide were used to examine cell proliferation inhibition rates and apoptosis. Immunofluorescence assay and Western blot analysis were performed to detect renal fibrosis-related proteins including Klotho, TGF-β1, and phosphorylated (p)-p38. ResultsCombination therapy (HKC plus MET) significantly improved the weight, reduced blood glucose (BG), blood urea nitrogen (BUN), total cholesterol (T-CHO), triglycerides (TG), low-density lipoprotein (LDL) and increased the level of high-density lipoprotein (HDL) of DN rats. Combination therapy also significantly reduced urine serum creatinine (SCR) and urine protein (UP) levels as well as reduced the degrees of renal tubule damage and glomerulopathy in DN rats. Combination therapy ameliorated renal fibrosis, as evidenced by reduced levels of alpha-smooth muscle actin and fibronectin and increased expression of E-cadherin in the kidneys. Moreover, HKC plus MET alleviated the degree of DN in part via the Klotho/TGF-β1/p38MAPK signaling pathway. In vitro experiments showed that combination therapy significantly inhibited cell proliferation and apoptosis and regulated fibrosis-related proteins in high-glucose (HG)-induced HK-2 cells. Further studies revealed that combination therapy suppressed cell proliferation and fibrosis by inhibiting the Klotho-dependent TGF-β1/p38MAPK pathway. ConclusionsHKC plus MET in combination suppressed abnormal renal cell proliferation and fibrosis by inhibiting the Klotho-dependent TGF-β1/p38MAPK pathway. Collectively, HKC combined with MET effectively improved DN by inhibiting renal fibrosis-associated proteins and blocking the Klotho/TGF-β1/p38MAPK signaling pathway. These findings improve the understanding of the pathogenesis of diabetes-associated complications and support that HKC plus MET combination therapy is a promising strategy for preventing DN.

&lt;p&gt;Ultrasound Assisted a Peroxisome Proliferator-Activated Receptor (PPAR)γ Agonist-Loaded Nanoparticle-Microbubble Complex to Attenuate Renal Interstitial Fibrosis&lt;/p&gt;

ObjectiveTo investigate the antifibrotic effect of the combination of a PPARγ agonist-loaded nanoparticle-microbubble complex with ultrasound (US) exposure on renal interstitial fibrosis (RIF).Materials and MethodsPolylactide-co-glycolide (PLGA) nanoparticles were used to load PPARγ agonist (rosiglitazone, RSG) and prepare PLGA-RSG nanoparticles (PLNPs-RSG); then, a novel complex between PLNPs-RSG and SonoVue microbubbles (MBs) (PLNPs-RSG-MBs) was prepared. The size distribution, zeta potentials, RSG-loading capacity and entrapment efficiency were measured, and the release of RSG was assessed using a UV-vis spectrophotometer. The in vitro cytotoxicity and in vivo systemic toxicity assays were performed. The cellular uptake assessment was performed using a confocal laser scanning microscope (CLSM). The in vivo biodistribution assessment was performed using fluorescence imaging with a near-infrared (NIR) imaging system. Furthermore, this complex was administered to a unilateral ureteral obstruction (UUO) rat model with the assistance of US exposure to investigate the antifibrotic effect.ResultsThis PLNPs-RSG-MBs complex had a size of 2199.5± 988.1 nm and a drug-loading efficiency of 28.5%. In vitro cytotoxicity and in vivo systemic toxicity assays indicated that the PLNPs-RSG-MBs complex displayed excellent biocompatibility. In addition, the complex showed high cellular uptake efficiency in vitro and kidney-targeting ability in vivo. In a UUO rat model, the combination of the PLNPs-RSG-MBs complex with US exposure significantly reduced collagen deposition and successfully attenuated renal fibrosis.ConclusionThe combination of the PLNPs-RSG-MBs complex with US exposure may be a promising approach for the treatment of RIF.

Anti-renal interstitial fibrosis effect of norcantharidin is exerted through inhibition of PP2Ac-mediated C-terminal phosphorylation of Smad3.

Norcantharidin (NCTD), the demethylated analog of cantharidin isolated from Mylabris, is known to inhibit renal fibrosis. However, the underlying mechanism is largely unknown. The present study investigates whether NCTD exerts this effect through regulation of the protein phosphatase 2A catalytic subunit (PP2Ac)-Smad3 pathway. HK-2 human renal proximal tubule cells exposed to transforming growth factor (TGF)-β1 were used as an in vitro model of renal fibrosis. The levels of total Smad3, C-terminal-phosphorylated Smad3 (p-Smad3), PP2Ac, and fibronectin (Fn) were evaluated by Western blotting. A PP2Ac overexpression plasmid and the PP2Ac inhibitor okadaic acid (OA) were used for functional analyses. The subcellular localization of Smad3 was visualized by immunofluorescence labeling. The results showed that PP2Ac overexpression increased Smad3 phosphorylation and nuclear translocation in HK-2 cells, while pharmacologic inhibition of PP2Ac with OA had the opposite effect. NCTD suppressed Fn and p-Smad3 expression and TGF-β1-induced nuclear entry of Smad3, but these effects were abrogated by inhibition of PP2Ac. Thus, the anti-renal interstitial fibrosis effect of NCTD is exerted through inhibition of PP2Ac-mediated C-terminal phosphorylation of Smad3. These findings highlight the therapeutic potential of NCTD for the treatment of renal interstitial fibrosis.

A synthetic retinoic acid receptor agonist Am80 ameliorates renal fibrosis via inducing the production of alpha-1-acid glycoprotein

Renal fibrosis is a major factor in the progression of chronic kidney disease and the final common pathway of kidney injury. Therefore, the effective therapies against renal fibrosis are urgently needed. The objective of this study was to investigate the effect of Am80, a synthetic retinoic acid receptor (RAR) agonist, in the treatment of renal interstitial fibrosis using unilateral ureteral obstruction (UUO) mice. The findings indicate that Am80 treatment suppressed renal fibrosis and inflammation to the same degree as the naturally-occuring retinoic acid, all-trans retinoic acid (atRA). But the adverse effect of body weight loss in Am80-treated mice was lower compared to the atRA treatment. The hepatic mRNA levels of alpha-1-acid glycoprotein (AGP), a downstream molecule of RAR agonist, was increased following administration of Am80 to healthy mice. In addition, increased AGP mRNA expression was also observed in HepG2 cells and THP-1-derived macrophages that had been treated with Am80. AGP-knockout mice exacerbated renal fibrosis, inflammation and macrophage infiltration in UUO mice, indicating endogenous AGP played an anti-fibrotic and anti-inflammatory role during the development of renal fibrosis. We also found that no anti-fibrotic effect of Am80 was observed in UUO-treated AGP-knockout mice whereas atRA treatment tended to show a partial anti-fibrotic effect. These collective findings suggest that Am80 protects against renal fibrosis via being involved in AGP function.

Quantitative and network pharmacology: A case study of rhein alleviating pathological progress of renal interstitial fibrosis

Ethnopharmacological relevanceThe current network pharmacology model focuses mainly on static and qualitative characterisation between drugs and targets or molecular pathway networks, but it does not reflect the multi-scale, dynamic and quantitative process of drug action. Aim of the studyIn this study, we developed a new model known as quantitative and network pharmacology (QNP) to characterise the dynamic and quantitative interventions of drugs within a multi-scale biological network. Materials and methodsFirstly, we used a systems biology method to construct a molecule-cell dynamic network model to simulate the pathological processes of diseases. Secondly, according to the principles of enzymatic kinetics, we generated a multi-scale drug intervention model to simulate the intervention of drugs in multi-scale networks at different concentrations and pathological stages. Finally, we took rhein treatment of renal interstitial fibrosis (RIF) as an example to illustrate the QNP model. ResultsWe successfully constructed the a QNP model that includes both a multi-scale dynamic network disease model and drug intervention model. The QNP model accurately simulated the pathological process of RIF, and the simulation results were validated by a series of cell and animal experiments. Meanwhile, the QNP model demonstrated that rhein can delay the pathological process at the studied concentrations of 5 nM, 10 nM, and 20 nM, and can also exert a better therapeutic effect on fibrosis before the proliferation stage of RIF. Furthermore, through uncertainty and sensitivity analysis, we identified that FAK and Smad3 may be potential targets for RIF. ConclusionOur QNP model provides a molecular-cellular understanding of the pathological mechanisms of RIF, serving as a new approach and strategy for the construction of dynamic multi-scale network model of diseases and drug intervention.

Chrysophanol ameliorates renal interstitial fibrosis by inhibiting the TGF-β/Smad signaling pathway

Renal interstitial fibrosis (RIF) is a major pathological feature of chronic kidney disease at middle and end stages. Chrysophanol (CP), 1,8-dihydroxy-3-methyl-9,10-anthraquinone, is an anthraquinone isolated from Rheum palmatum L. with a variety of pharmacological activities including the suppression of RIF. However, the effect of CP on renal fibrosis and its potential mechanism have not been elucidated. We conducted a comprehensive study by determining the expression levels of fibrotic markers and proteins including TGF-β1, α-SMA, and Smad3 related to transforming growth factor-beta/Smad (TGF-β/Smad) pathway in unilateral ureteral obstruction (UUO) mice and TGF-β1-stimulated HK-2 cells with the treatment of CP using western blotting and RT-qPCR analyses. Using small interfering RNA and co-immunoprecipitation, we evaluated the influences of CP on the interactions between Smad3 and Smad7 proteins and also on TGF-β RI and TGF-βR II. We found that CP administration significantly ameliorated UUO-induced kidney damage by reversing abnormal serum and urine biochemical parameters and decreasing the production of fibrotic markers including collagen I, collagen III, fibronectin, and α-SMA. Our results showed that TGF-β1 and phospho-Smad3 (p-Smad3) expression was significantly down-regulated and Smad7 expression was up-regulated by CP in UUO mice compared to the model group; however, the expression of Smad2, Smad4, and TGF-β receptors was not affected. Furthermore, CP modulated these fibrotic markers as well as p-Smad3 and Smad7 in TGF-β1-induced HK-2 cells. The inhibitory effect of CP was markedly reduced in TGF-β1-treated HK-2 cells transfected with Smad3 siRNA. Additionally, co-immunoprecipitation analysis indicated that CP blocked the interaction between Smad3 and TGF-β receptor I to suppress p-Smad3 expression. These findings demonstrated that CP alleviated RIF by inhibiting Smad3 phosphorylation, which provides a molecular basis for a new drug candidate for the treatment of RIF.