Unilateral Ureteral Obstruction Model Group Research Articles

Catalpol attenuates EMT by inhibiting Wnt/β-catenin and TGF-β/Smads signaling to alleviate kidney fibrosis

Purpose: To determine the anti-fibrosis effect and underlying mechanism of action of catalpol (CAT) in chronic kidney disease (CKD).
 Methods: Forty (40) rats were randomly divided into a sham group (10 rats) and a unilateral ureteral obstruction (UUO) model group (30 rats) which was further randomly subdivided into three groups of ten (10) rats each: the UUO model group, UUO + CAT low-dose group, and UUO + CAT high-dose group. HK-2 cells were stimulated with TGF-β1 for in vitro studies. Renal injury and fibrotic lesions were determined by H&E and Masson’s staining. Key proteins of TGF-β/Smads and Wnt/β-catenin signaling pathways involved in epithelial- mesenchymal transition (EMT) were determined by immunohistochemistry, immunofluorescence staining and Western blotting.
 Results: Catalpol downregulated the expression of α-SMA (p < 0.05) and upregulated the expression of E-cadherin (p < 0.05) stimulated by TGF-β1 and LiCl in HK-2 cells, which is consistent with the role of DKK1 in vitro. CAT ameliorated renal fibrosis and repressed the expression of key proteins of TGFβ/Smads and Wnt/β-catenin pathways in UUO rats.
 Conclusion: Catalpol inhibits EMT and alleviates kidney fibrosis by suppressing the hyperactivation of TGF-β/Smad and Wnt/β-catenin signaling pathways. Therefore, CAT is a promising therapeutic drug for renal fibrosis.

Colonic microflora and plasma metabolite-based comparative analysis of unilateral ureteral obstruction-induced chronic kidney disease after treatment with the Chinese medicine FuZhengHuaYuJiangZhuTongLuo and AST-120

BackgroundMany researchers have investigated the use of Chinese herbs to delay the progression of chronic kidney disease (CKD) through their effects on colonic microflora and microbiota-derived metabolites. However, whether FuZhengHuaYuJiangZhuTongLuo (FZHY) has effects that are similar to those of AST-120 on CKD needs to be elucidated. MethodsIn this study, we compared the effects of FZHY and AST-120 on the colonic microbiota and plasma metabolites in the CKD rat model. We developed a unilateral ureteral obstruction (UUO)-induced CKD rat model and then administered FZHY and AST-120 to these model rats. Non-targeted metabolomic LC-MS analysis, 16S rRNA sequencing, and histopathological staining were performed on plasma, stool, and kidney tissues, respectively, and the joint correlation between biomarkers and metabolites of candidate bacteria was analyzed. ResultsOur results showed that administering FZHY and AST-120 effectively ameliorated UUO-induced abnormal renal function and renal fibrosis and regulated the composition of microbiota and metabolites. Compared to the UUO model group, the p_Firmicutes and o_Peptostreptococcales_Tissierellales were increased, while 14 negative ion metabolites were upregulated and 21 were downregulated after FZHY treatment. Additionally, 40 positive ion metabolites were upregulated and 63 were downregulated. On the other hand, AST-120 treatment resulted in an increase in the levels of g_Prevotellaceae_NK3B31_group and f_Prevotellaceae, as well as 12 upregulated and 23 downregulated negative ion metabolites and 56 upregulated and 63 downregulated positive ion metabolites. Besides, FZHY increased the levels of candidate bacterial biomarkers that were found to be negatively correlated with some poisonous metabolites, such as 4-hydroxyretinoic acid, and positively correlated with beneficial metabolites, such as l-arginine. AST-120 increased the levels of candidate bacterial biomarkers that were negatively correlated with some toxic metabolites, such as glycoursodeoxycholic acid, 4-ethylphenol, and indole-3-acetic acid. ConclusionFZHY and AST-120 effectively reduced kidney damage, in which, the recovery of some dysregulated bacteria and metabolites are probably involved. As their mechanisms of regulation were different, FZHY might play a complementary role to AST-120 in treating CKD.

Shenkang VII Recipe Attenuates Unilateral Ureteral Obstruction-induced Renal Fibrosis via TGF-β/Smad, NF-κB and SHH Signaling Pathway.

This study aimed to explore the protective effects of the traditional Chinese Medicine formula Shenkang VII recipe (SK-7) on renal fibrosis and the mechanisms. Renal fibrosis was induced by unilateral ureteral obstruction (UUO) in rats. The rats were then divided into 5 groups: control group (Sham operation), UUO model group, UUO model plus low to high doses of SK-7 (0.5, 1.0, or 2.0 g/kg/day, for 14 days) groups. The animals were sacrificed on the 7th or 14th day. Kidney tissues were collected for histopathological examinations (hematoxylin and eosin and Masson's trichrome staining). Immunohistochemistry was used to detect the expression of collagen type III (Col III), fibronectin (FN), α-smooth muscle actin (α-SMA), TIMP metallopeptidase inhibitor 2 (TIMP2), matrix metallopeptidase 2 (MMP2), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and monocyte chemotactic protein-1 (MCP-1). The TGF-β1/Smad, NF-kB and Sonic hedgehog signaling proteins were detected by Western blotting. Our results showed that SK-7 prevented UUO-induced renal injury and accumulation of collagen fibrils. Renal fibrosis biomarkers Col III, FN, α-SMA and TIMP2 were increased in the rats after UUO and decreased by SK-7, while MMP2 was upregulated after treatment. SK-7 also suppressed the levels of TNF-α, IL-1β and MCP-1 in UUO rats. In addition, SK-7 inhibited activation of the TGF-β/Smad, NF-κB and sonic hedgehog signaling (SHH) pathways. Taken together, these findings suggest that SK-7 may regulate the synthesis and degradation of extracellular matrix, reduce inflammation and suppress the proliferation of fibroblasts, by blocking the TGF-β1/Smad, NF-κB and SHH signaling pathways to exert its anti-renal fibrosis effect in UUO rats.

Ribes diacanthum Pall (RDP) ameliorates UUO-induced renal fibrosis via both canonical and non-canonical TGF-β signaling pathways in mice

Ethnopharmacological relevanceRibes diacanthum Pall (RDP), a folk medicine, has been widely used in Mongolia to treat urinary system diseases. Aim of the studyTo investigate the effectiveness of RDP on unilateral ureteral obstruction (UUO)-induced renal interstitial fibrosis and the underlying mechanisms. Materials and methodsA total of 60 mice were randomly divided into six groups: sham group, sham plus RDP (40 mg/kg) group, UUO model group, and UUO model plus RDP (10, 20 or 40 mg/kg) groups. After surgery, aqueous extract of RDP were administrated intragastrically (i.g) daily for a week and ipsilateral kidneys were collected seven days after surgery. Levels of blood urea nitrogen (BUN) and serum creatinine (Scr) were detected to reflect the kidney injury. Hematoxylin & eosin and Masson's trichrome staining were used to evaluate the kidney morphological changes and fibrosis, respectively. ELISA was used to examine the levels of pro-inflammatory cytokines. Immunohistochemistry, western blot and PCR were used to examine the expression levels of key proteins involved in transforming growth factor (TGF-β)/Smad and mitogen-activated protein kinase (MAPK) signaling pathways. ResultsRDP treatment attenuates the level of BUN and kidney fibrosis in UUO mice, decreases the expressions of interleukin-6, tumor necrosis factor-α, Interleukin-1α, TGF-β1, monocyte chemotactic protein-1, α-smooth muscle actin, collagen I, fibronectin, and vimentin, while increases the expressions of E-cadherin and hepatocyte growth factor. Moreover, RDP administration significantly decreases the levels of p-Smad2/3, p-ERK1/2, p-p38 and p-JNK, while increases the expression level of Smad7 in UUO models. ConclusionThese data demonstrate that RDP ameliorates renal fibrosis through TGF-β/Smad and MAPK pathways in a UUO mouse model.

Research on the intervention of Yishen Huoxue prescription to renal fibrosis through the signal regulation by microRNA-126 to VEGF-Notch

To explore the molecular mechanism of Yishen Huoxue prescription in delaying the development of renal fibrosis by regulating the microRNA-126/vascular endothelial growth factor-Notch (miR-126/VEGF-Notch) signaling pathway. Ninety male Sprague-Dawley (SD) rats were randomly divided into sham operation group (sham group), unilateral ureteral obstruction (UUO) model group, losartan group (50 mg×kg-1×d-1) and high, medium and low doses Yishen Huoxue prescription group (25.2, 12.6, 6.3 mL/kg). Each treatment group began to administer drugs by gavage on the day when UUO modeling was finished until the end of the experiment. Left renal tissues of rats were harvested after 7, 14 and 21 days postoperatively. The pathological changes of renal tissue were observed after hematoxylin and eosin (HE) and Masson staining under the microscope, and the renal fibrosis score was calculated. The mRNA expressions of renal tissues miR-126, VEGFA, vascular endothelial growth factor receptor-2 (VEGFR-2), Notch1 were detected by reverse transcription-polymerase chain reaction (RT-PCR). (1) Pathology results: the kidney tissue of sham group was normal. In UUO model group, renal tubules expanded and inflammatory cells in renal interstitium increased; renal tubulointerstitial fibrosis could be seen 7 days after operation, and the degree of fibrosis was gradually increased with time. The renal fibrosis score at each time point after operation in UUO model group was significantly higher than that in sham group. Compared with UUO model group, each treatment group were improved the degree of renal swelling and atrophy of renal parenchyma; the score of renal fibrosis were significantly decreased in the middle and high doses Yishen Huoxue prescription group and losartan group at the 7th day after operation (1.00±1.00, 0.91±0.58, 1.01±0.58 vs. 2.00±0.00, all P < 0.01); but there was no significant difference between low dose Yishen Huoxue prescription group and UUO model group. (2) RT-PCR results: Compared with sham group, the mRNA expressions of miR-126, VEGFA, VEGFR-2 and Notch1 in renal tissues were significantly increased at each time point after operation in UUO model group. Compared with the UUO model group, the mRNA expressions of miR-126, VEGFA, VEGFR-2 and Notch1 in renal tissue of 7 days postoperatively in the middle and high doses Yishen Huoxue prescription group and the losartan group were significantly increased [miR-126 (2-ΔΔCt): 0.465±0.067, 0.639±0.092, 0.404±0.069 vs. 0.132±0.021; VEGFA (2-ΔΔCt): 0.024±0.005, 0.027±0.007, 0.023±0.006 vs. 0.014±0.006; VEGFR-2 (2-ΔΔCt): 0.021±0.007, 0.023±0.008, 0.019±0.007 vs. 0.012±0.004; Notch1 (2-ΔΔCt): 0.017±0.004, 0.020±0.005, 0.018±0.005 vs. 0.007±0.004; all P < 0.05]; compared with the losartan group, the mRNA expressions of each index in the middle and high doses Yishen Huoxue prescription group were increased at all the time points, but there was no significant difference between them (all P > 0.05). There was no significant difference in mRNA expression of each index between low dose Yishen Huoxue prescription group and UUO model group. The medium and high doses of Yishen Huoxue prescription can effectively antagonize renal fibrosis. Yishen Huoxue prescription may use up-regulation the signaling pathways of miR-126/VEGF-Notch to mediate renal microvascular newly born, eventually to improve renal microvascular damage and delay the development of renal fibrosis progression.

Anti-renal fibrosis effect of asperulosidic acid via TGF-β1/smad2/smad3 and NF-κB signaling pathways in a rat model of unilateral ureteral obstruction

BackgroundRenal fibrosis is the most common pathway leading to end-stage renal disease. It is characterized by excess extracellular matrix (ECM) accumulation and renal tissue damage, subsequently leading to kidney failure. Asperulosidic acid (ASPA), a bioactive iridoid glycoside, exerts anti-tumor, anti-oxidant, and anti-inflammatory activities, but its effects on renal fibrosis induced by unilateral ureteral obstruction (UUO) have not yet been investigated. PurposeThis study aimed to investigate the protective effect of ASPA on renal fibrosis induced by UUO, and to explore its pharmacological mechanism. MethodsThirty-six Sprague-Dawley (SD) rats were randomly divided into six groups: sham group, UUO model group, three ASPA treatment groups (10, 20, and 40 mg/kg), and captopril group (20 mg/kg). Rats were administered vehicle, ASPA or captopril intraperitoneally once a day for 14 consecutive days. Urea nitrogen (BUN), uric acid (UA) and inflammatory factors in serum samples were evaluated on the 7th, 10th, and 14th day after renal fibrosis induction. In addition, the 12 h urine was collected to test the content of urinary protein (upro) on the 14th day. The obstructive renal tissues were collected for pathological analysis (hematoxylin and eosion (H&E) staining and Masson's Trichrome staining) and immunohistochemical analysis on the 14th day after renal fibrosis induction. The mRNA expression of related factors and the protein levels of smad2, smad3, and smad4 were measured in UUO-induced rats by real time PCR and Western blot, respectively. ResultsThe levels of BUN, UA, and upro were elevated in UUO-induced rats, but ASPA treatment improved renal function by reducing the levels of BUN, UA, and upro. The protein levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6, as well as the mRNA levels of TNF-α, IL-1β, IL-6, monocyte chemoattractant protein-1 (MCP-1) and interferon-γ (IFN-γ), were decreased after ASPA administration (10, 20 and 40 mg/kg) in a dose-dependent manner. The ASPA exerted an alleviation effect on the inflammatory response through inhibition of nuclear factor-kappa B (NF-κB) pathway. In addition, reductions in α-smooth muscle actin (α-SMA), collagen III, and fibronectin expression were observed after ASPA administration at doses of 20 and 40 mg/kg. Furthermore, the renal expression of transforming growth factor-β1 (TGF-β1), smad2, smad3, and smad4 was down-regulated by ASPA treatment at doses of 20 and 40 mg/kg. ConclusionASPA possessed protective effects on renal interstitial fibrosis in UUO-induced rats. These effects may be through inhibition of the activation of NF-κB and TGF-β1/smad2/smad3 signaling pathways.

Effects of Qingshen granules on Janus Kinase/signal transducer and activator of transcription signaling pathway in rats with unilateral ureteral obstruction

OBJECTIVETo investigate the effects of Qingshen granules (QSG) on janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway in a rat model of unilateral ureteral obstruction (UUO). METHODSSixty male Sprague-Dawley rats were randomly divided into six groups, with 10 animals in each group: the untreated sham-operated normal control group; the untreated UUO model control group, the high dose QSG-treated (16 g•kg−1•d−1) UUO group; the medium dose QSG-treated (8 g•kg−1•d−1) UUO group; the low dose QSG-treated (4 g•kg−1•d−1) UUO group; and the valsartan-treated group (20 mg•kg−1•d−1). The two untreated control groups received physiological saline (1 mL/100 g per day). All the rats were sacrificed after a 4-week course of treatment. Serum creatinine and leptin; protein expressions of leptin receptor (OB-R), p-JAK2, p-STAT3, nuclear factors-κBp6 (NF-kBp65), and monocytechemotatic protein-1 (MCP-1); mRNA of JAK2, STAT3, calcium-dependent adhesion (E-cadherin), alphasmooth muscle actin (α-SMA) in the kidney tissues; and the expressions of type IV collagen (Col-IV and fibronectin (FN) and the pathomorphology in kidney tissues were treated. RESULTSCompared with the normal group, the BUN, Scr, and serum leptin levels and the expressions of MCP-1, p-JAK2, p-STAT3, NF-kBp65 and OB-R in renal tissues, and the mRNA expressions of leptin, JAK2 protein, STAT3 protein, α-SMA protein in model group were significantly higher (P < 0.01) in the UUO model group. These parameters were significantly reduced in all the QSG-treated groups and the valsartan-treated group than the UUO model group (P < 0.05 or P < 0.01), with the lowest levels found in the medium dose QSG-treated group (P < 0.05). However, the expression levels of E-cadherin, FN, and Col-IV in the renal tissues were contrary to the expressions described above. Severe pathological injury was evident in the renal tissues of UUO model rats, which was alleviated in the QSG-treated and valsartan-treated groups, with the least damage found in the medium dose QSG-treated group. CONCLUSIONOur data suggest that the leptin-mediated JAK/STAT signaling pathway is involved in the process of renal interstitial fibrosis in UUO rats. QSG inhibited the activity of the signaling pathway, reduced the activity of NF-kB and inflammatory effect, and the transdifferentiation in the renal tubular epithelial cells. Treatment with QSG may delay the renal fibrosis and protect the renal function from damage following UUO in rats.

Effects of Qingshen Granules on the Oxidative Stress-NF/kB Signal Pathway in Unilateral Ureteral Obstruction Rats.

Background. The activation of NF-kappa B (NF/kB) signaling pathway plays an important role in the process of epithelial-mesenchymal transition (EMT) and renal interstitial fibrosis (RIF) in renal tubules. The process of oxidative stress reaction in kidney is via excessive reactive oxygen species (ROS) production to activate NF/kB signaling pathway. Qingshen Granule (QSG) is an effective Chinese formula utilized to treat chronic renal failure. Previous studies confirmed that QSG could inhibit RIF in unilateral ureteral obstruction (UUO) rats. In this study, we used UUO rats to investigate the effects of QSG on oxidative stress and the activation of NF/kB signaling. Seventy male Sprague-Dawley (SD) rats were randomly divided into a sham group, UUO model group, Qingshen Granules (QSG) high-dose, medium-dose, and low-dose groups, PDTC group, and candesartan group (10 rats in each group). Our study demonstrated that oxidative stress-NF/kB signal pathway contributed to the formation of UUO renal interstitial fibrosis. QSG may protect against RIF by inhibiting the oxidative stress-NF/kB signal pathway, reducing inflammation, and improving renal tubular EMT.

Demethylzeylasteral ameliorates inflammation in a rat model of unilateral ureteral obstruction through inhibiting activation of the NF‑κB pathway.

The present study investigated the pharmacodynamic role and therapeutic mechanism of demethylzeylasteral in the suppression of inflammation in a rat model of unilateral ureteral obstruction and reduction in nuclear factor (NF)‑κB pathway activity. The rats in the unilateral ureteral obstruction model were treated with 30‑120mg/kg demethylzeylasteral for 8weeks. The activities of tumor necrosis factor (TNF)‑α, interleukin (IL)‑6 and caspase‑3/9, and the protein expression levels of cyclooxygenase (COX)‑2 and intercellular adhesion molecule‑1 (ICAM‑1) and NF‑κB p65 were analyzed using ELISA kits and western blot analyses, respectively. Compared with the rats in the unilateral ureteral obstruction model group, demethylzeylasteral treatment markedly inhibited the increased concentrations of serum creatinine and blood urea nitrogen, urinary protein/creatinine ratio, and concentrations of high‑density lipoprotein and low‑density lipoprotein cholesterol, and prevented kidney damage. In addition, demethylzeylasteral inhibited the levels of TNF‑α andIL‑6 and suppressed the protein expression levels of COX‑2 and ICAM‑1 in the kidneys of the rats in the unilateral ureteral obstruction model. Demethylzeylasteral also significantly suppressed the protein expression of NF‑κB p65. The results of the present study suggested that demethylzeylasteral unilateral ureteral obstruction and inhibited inflammation via inhibiting the activation of COX‑2, ICAM‑1 and NF‑κB p65, and suppressing the activities of caspase‑3/9 in rats with unilateral ureteral obstruction.

Effect of Quyu Chencuo Formula () on Renal Fibrosis in Obstructive Nephropathy Rats.

To observe the effect of Quyu Chencuo Formula (, QCF) on renal fibrosis in rats with obstructive nephropathy. Twenty-four rats were randomly divided into three groups, 4 for sham operation as the control group, 10 for unilateral ureteral obstruction (UUO) model group, and the rest 10 for QCF treating UUO model group. All rats were sacrificed under 3% pentobarbital (50 mg/kg) anesthesia on the 14th day after surgery, then the right kidney samples of rats were harvested for hematoxylin eosin (HE) staining and Masson staining to observe the renal pathological changes. Immunohistochemistry and Western blotting were used to examine the expression of transforming growth factor β1 (TGF-β1), and real-time polymerase chain reaction (RT-PCR) was employed to examine the expressions of TGF-β1, α-smooth muscle actin (α-SMA) and E-cadherin mRNA. HE and Masson staining showed that the renal interstitial of the rats in the control group had no significant fibrotic lesion; in the model group, there were obvious interstitial fibrosis; for the QCF group, there were epithelial cell necrosis, infiltration of lymphocytes and mononuclear cells, aggravated interstitial fibrosis in varied degrees, but the pathological changes were less in the QCF group than in the model group. The immunohistochemistry and Western blotting results showed that the TGF-β1 expression was increased significantly in the model group, while decreased significantly in the QCF group (P<0.05); RT-PCR showed that the mRNA expression of α-SMA and TGF-β1 increased significantly in the model group, while both were significantly decreased in the QCF group compared with the model group (P<0.05). The mRNA expression of E-cadherin was decreased significantly in the model group, and it was significantly increased in the QCF group as compared with the model group (P<0.05). QCF may improve renal fibrosis by regulating the expressions of TGF-β1, α-SMA and E-cadherin, and prevent the progress of kidney fibrosis.

Effect of erythropoietin on the expression of dynamin-related protein-1 in rat renal interstitial fibrosis

This study aimed to observe the expression of dynamin-related protein-1 (Drp-1) in the renal interstitium in a rat model of renal interstitial fibrosis induced by unilateral ureteral obstruction (UUO). In addition, the renoprotective effect of erythropoietin in this model was investigated. A total of 81 rats were randomly assigned to sham surgery, UUO model and treatment groups. Following surgery, the rats in the treatment group were subcutaneously administered erythropoietin at a dose of 3,000 IU/kg once a week until the time of sacrifice. Rats in the sham surgery and UUO model groups were administered an identical volume of normal saline. In each group, nine rats were chosen randomly for sacrifice on days 7, 14 and 21 after surgery for histological examination of renal tissue. Renal tissue specimens were examined by hematoxylin and eosin and Masson's trichrome staining. Immunohistochemical analysis was performed to determine the expression of Drp-1 in the renal interstitium. Renal function damage, as evaluated by the measurement of serum creatinine (Cr) and blood urea nitrogen (BUN) levels, was less severe in the treatment group compared with that in the model group at day 21 (P<0.01). Compared with the UUO model group, the renal interstitial injury score and fibrotic area of the treatment group were decreased markedly at the three time points (P<0.05). The expression level of Drp-1 in the treatment group was decreased markedly at the three time points compared with that in the model group (P<0.05). In conclusion, the expression of Drp-1 is increased in rat renal interstitial fibrosis, and erythropoietin may alleviate the degree of renal interstitial fibrosis by downregulating the expression of Drp-1.

Effect of herba centellae on the expression of HGF and MCP-1

The aim of this study was to explore the effects of herba centellae on protein and mRNA expression of hepatocyte growth factor (HGF), and monocyte chemotactic protein-1 (MCP-1) in renal tubulointerstitial fibrosis (TIF). A unilateral ureteral obstruction (UUO) model was established in 50 male Sprague Dawley (SD) rats. Blood samples were collected and the blood urea nitrogen (BUN) levels, serum creatinine (Scr), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured. Immunohistochemistry (IHC) detected the localization and expression levels of HGF and MCP-1. In addition, quantitative polymerase chain reaction (qPCR) detected the mRNA expression of HGF and MCP-1. Thirty rats were used to prepare the rat serum containing drug by cell culture, and qPCR and immunocytochemistry (ICC) were performed to examine the mRNA and protein expression of HGF and MCP-1. MCP-1 and its mRNA expression was significantly higher in rat renal interstitium of the UUO group and cells of the transforming growth factor-β1 (TGF-β1) stimulation group compared with that of the control group (P<0.01). MCP-1 and its mRNA expression in the drug intervention group were significantly reduced compared with that of the UUO model group (P<0.01). However, MCP-1 and its mRNA expression in the high-dose herba centellae group was significantly lower compared with that of the low-dose herba centellae group (P<0.01). Furthermore, HGF and its mRNA expression significantly increased in the drug intervention group (P<0.01), and expression in the high-dose group was significantly higher compared with that of the low-dose group (P<0.01), but similar to that of the fosinopril group (P>0.05). The levels of BUN decreased in the drug intervention group; however, no significant differences were determined in Scr, ALT and AST levels. Herba centellae may have inhibited MCP-1 and its mRNA expression through the upregulation of HGF and its mRNA expression, in order to achieve resistance to TIF without showing evident hepatorenal toxicity.