Oleanolic acid attenuates TGF-ß1-induced epithelial-mesenchymal transition in NRK-52E cells.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) plays an important role in the progression of renal interstitial fibrosis, which finally leads to renal failure. Oleanolic acid (OA), an activator of NF-E2-related factor 2 (Nrf2), is reported to attenuate renal fibrosis in mice with unilateral ureteral obstruction. However, the role of OA in the regulation of EMT and the underlying mechanisms remain to be investigated. This study aimed to evaluate the effects of OA on EMT of renal proximal tubular epithelial cell line (NRK-52E) induced by TGF-ß1, and to elucidate its underlying mechanism. METHODS: Cells were incubated with TGF-ß1 in the presence or absence of OA. The epithelial marker E-cadherin, the mesenchymal markers, α-smooth muscle actin (α-SMA), fibronectin, Nrf2, klotho, the signal transducer (p-Smad2/3), EMT initiator (Snail), and ILK were assayed by western blotting. RESULTS: Our results showed that the NRK-52E cells incubated with TGF-ß1 induced EMT with transition to the spindle-like morphology, down-regulated the expression of E-cadherin but up-regulated the expression of α-SMA and fibronectin. However, the treatment with OA reversed all EMT markers in a dose-dependent manner. OA also restored the expression of Nrf2 and klotho, decreased the phosphorylation of Smad2/3, ILK, and Snail in cells which was initiated by TGF-ß1. CONCLUSION: OA can attenuate TGF-ß1 mediate EMT in renal tubular epithelial cells and may be a promising therapeutic agent in the treatment of renal fibrosis.

JiaWeiDangGui Decoction Ameliorates Proteinuria and Kidney Injury in Adriamycin-Induced Rat by Blockade of TGF-ß/Smad Signaling.

JiaWeiDangGui (JWDG) decoction has anti-inflammatory and antifibrotic effects, which is used widely for the treatment of various kidney diseases. In previous studies, we have found that JWDG decoction can reduce the quantity of proteinuria, but the mechanism was unknown. Here, we studied the protective effect of JWDG decoction in adriamycin-induced nephropathy on rat. JWDG decoction, at 10 mL/kg/d, 20 mL/kg/d, and 40 mL/kg/d, was orally administered daily for 12 weeks. Therapeutic effects and mechanisms were further examined. The kidney function related biochemical indexes were measured by automatic biochemistry analyzer. The pathomorphological changes were observed using light and transmission electron microcopies. The proteins expressions of podocin, nephrin, collagen IV, and fibronectin (FN) were examined by immunohistochemical staining, and key proteins involved in TGF-ß/Smad signaling were evaluated by RT-PCR and western blotting. Compared with vehicle-treated controls, JWDG decoction decreased the quantity of proteinuria; reduced glomerulosclerotic lesions induced by ADR; and preserved the expression of podocin and nephrin. JWDG decoction also inhibited the expression of the collagen IV, FN, and fibrogenic TGF-ß. Further studies revealed that inhibition of renal fibrosis was associated with the blockade of TGF-ß/Smad signaling and downregulation of snail expression dose dependently. JWDG decoction prevents proteinuria production, podocyte dysfunction, and kidney injury in adriamycin nephropathy by inhibiting TGF-ß/Smad signaling.

Triptolide Attenuates Podocyte Injury by Regulating Expression of miRNA-344b-3p and miRNA-30b-3p in Rats with Adriamycin-Induced Nephropathy.

Objectives. We investigated the action of triptolide in rats with adriamycin-induced nephropathy and evaluated the possible mechanisms underlying its protective effect against podocyte injury. Methods. In total, 30 healthy male Sprague-Dawley rats were randomized into three groups (normal group, model group, and triptolide group). On days 7, 28, 42, and 56, 24 h urine samples were collected. All rats were sacrificed on day 56, and their blood and renal tissues were collected for determination of biochemical and molecular biological parameters. Expression of miRNAs in the renal cortex was analyzed by a biochip assay and RT-PCR was used to confirm observed differences in miRNA levels. Results. Triptolide decreased proteinuria, improved renal function without apparent adverse effects on the liver, and alleviated renal pathological lesions. Triptolide also elevated the nephrin protein level. Furthermore, levels of miR-344b-3p and miR-30b-3p were elevated in rats with adriamycin-induced nephropathy, while triptolide treatment reversed the increase in the expression of these two miRNAs. Conclusions. These results suggest that triptolide may attenuate podocyte injury in rats with adriamycin-induced nephropathy by regulating expression of miRNA-344b-3p and miRNA-30b-3p.

Ferulic Acid Attenuates TGF-ß1-Induced Renal Cellular Fibrosis in NRK-52E Cells by Inhibiting Smad/ILK/Snail Pathway.

Renal fibrosis is a common cause of renal dysfunction with chronic kidney disease. Central to this process is epithelial-mesenchymal transformation (EMT) of proximal tubular epithelial cells driven by transforming growth factor-ß1 (TGF-ß1) signaling. The present study aimed to investigate the effect of Ferulic acid (FA) on EMT of renal proximal tubular epithelial cell line (NRK-52E) induced by TGF-ß1 and to elucidate its underlying mechanism against EMT related to TGF-ß1/Smads pathway. The NRK-52E cells were treated for 48 h with TGF-ß1 (5 ng/mL) in different concentrations of FA (0 to 200 µM). Fibronectin, a mesenchymal marker, was assessed by western blotting. Western blotting was also used to examine the EMT markers (E-cadherin, and α-smooth muscle actin (α-SMA)), signal transducer (p-Smad2/3), and EMT initiator (Snail). ILK was also assayed by western blotting. The results showed that TGF-ß1 induced spindle-like morphological transition in NRK-52E cells. Smad2/3 signaling pathway activation, increased fibronectin, α-SMA, ILK, and Snail expression, and decreased E-cadherin expression in TGF-ß1-treated NRK-52E cells. FA efficiently blocked P-Smad2/3 activation and attenuated all these EMT changes induced by TGF-ß1. These findings suggest that FA may serve as a potential fibrosis antagonist for renal proximal tubule cells by inhibiting EMT process.

Profiling the metabolism of astragaloside IV by ultra performance liquid chromatography coupled with quadrupole/time-of-flight mass spectrometry.

Astragaloside IV is a compound isolated from the Traditional Chinese Medicine Astragalus membranaceus, that has been reported to have bioactivities against cardiovascular disease and kidney disease. There is limited information on the metabolism of astragaloside IV, which impedes comprehension of its biological actions and pharmacology. In the present study, an ultra-performance liquid chromatography coupled with quadrupole/time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS)-based approach was developed to profile the metabolites of astragaloside IV in rat plasma, bile, urine and feces samples. Twenty-two major metabolites were detected. The major components found in plasma, bile, urine and feces included the parent chemical and phases I and II metabolites. The major metabolic reactions of astragaloside IV were hydrolysis, glucuronidation, sulfation and dehydrogenation. These results will help to improve understanding the metabolism and reveal the biotransformation profiling of astragaloside IV in vivo. The metabolic information obtained from our study will guide studies into the pharmacological activity and clinical safety of astragaloside IV.

[Treatment of chronic primary glomerulopathy patients of Shen deficiency and dampness heat syndrome by yishen qingli granule combined low-dose Tripterygium wilfordii multiglycoside tablet: a clinical efficacy observation].

OBJECTIVE: To evaluate the clinical efficacy and safety of treatment of chronic primary glomerulopathy (CPG) patients of Shen deficiency and dampness heat syndrome (SDDHS) by Yishen Qingli Granule (YQG) combined with low-dose Tripterygium Wilfordii multiglycoside Tablet (TWT). METHODS: Totally 231 CPG patients of SDDHS were enrolled in this study (including 60 patients from First Affiliated Hospital of Nanjing University of Chinese Medicine, 58 from First Affiliated Hospital of Nanjing Medical University, 46 from Xinqiao Hospital of Third Military Medical University, 35 from First Affiliated Hospital of Guangzhou University of Chinese Medicine, 14 from First Affiliated Hospital of Soochow University, and 18 from Wuxi Affiliated Hospital of Nanjing University of Chinese Medicine). They were randomly assigned to the control group (116 cases) and the trial group (115 cases) according to block group method. There were 217 cases in the safety analysis set (109 cases in the trial group vs 108 cases in the control group), and 203 cases in the full analysis set (99 cases in the trial group vs 104 cases in the control group). All patients received basic treatment such as ACEI/ARB. Furthermore, YQG (consisting of raw astragalus 10 g, prepared Polygonum Multiflorum 10 g, Pyrrosia 10 g, 1.5 g each package, containing 10 g of crude drugs) was additionally given to patients in the trial group, each package, twice daily. The TWT (10 mg) was given, twice a day. The TWT dose was adjusted according to 24 h urinary total protein (UTP). The placebos of YQG and TWT were administered to those in the control group. The treatment course consisted of 24 weeks and the follow-up visit lasted for 24 weeks. The biochemical indices were observed before and after treatment including 24 h UTP, urine red cell count (U(RBC)), renal functions (BUN, SCr), blood routine test (WBC), and liver functions (SGPT, SGOT). Reverse reactions such as gastrointestinal discomfort, skin rash, and irregular menstruation were also observed. RESULTS: Compared with the control group, the total effective rate was better in the trial group (82.83% vs 61.54%, P < 0.01). Results of stratified comparison of UTP showed better efficacy in the trial group (0.8-3.0 g/24 h, P < 0.01). The UTP decline occurred in the trial group after 8 weeks of treatment, with stable action, showing statistical difference when compared with the control group (P < 0.01). In the trial group, U(RBC) level decreased after treatment but changed more significantly. But there was no statistical difference in the changes when compared with the control group (P > 0.05). After treatment, there were no statistical difference in safety indicators such as WBC, SGPT, and SGOT between the two groups after treatment (P > 0.05). CONCLUSION: On the basis of basic treatment such as ACEI/ARB, application of YQG combined with low-dose TWT had better effect in controlling proteinuria of CPG patients, and could help stabilizing their conditions with less adverse reactions.

[Protection mechanisms of modified danggui buxue decoction for podocytes in adriamycin-induced nephropathy rats].

OBJECTIVE: To study the effects of modified danggui buxue decoction (MDBD) on podocytes in adriamycin-induced nephropathy (DN) model rats. METHODS: SD rats were divided into four groups, i.e., the normal control group, the model group, the benazepril group, and the MDBD group. On the 7th, 28th, 42nd, and 56th day of modeling, the urine sample was collected to examine the dynamic changes of urinary albumin quantitation. The renal tissue was processed for the examinations under light microscope and electron microscope. The immunofluorescence of nephrin and podocin were detected. The expressions of the slit membrane expression protein in the renal tissue were further analyzed using RT-PCR and Western blot. RESULTS: (1) Urinary protein (UP): The UP did not obviously decrease in each treatment group on the 7th day, but it decreased so markedly on the 28th, 42nd, and 56th day. There was statistical difference in UP of the benazepril group and the MDBD group when compared with that of the model group (P < 0.05). The decrease was most obvious in the MDBD group. (2) Effects on the podocytes and the renal tissue: RESULTS: under light microscope and electron microscope showed, when compared with the model group, the proliferation of mesangial cells, the renal tubule-interstitial lesion, the podocyte fusion, and the expressions of nephrin and podocin were milder, and the urinary albumin quantitation was more obviously reduced in the benazepril group and the MDBD group. But the renal fibrosis correlated renal pathological progress also existed, indicating the renal lesion degree was milder but could not be reversed. (3) Results of RT-PCR and Western blot: Statistical difference existed in the expressions of nephrin and podocin between the benazepril group and the MDBD group on the 56th day, when compared with the model group (P < 0.05). CONCLUSION: MDBD showed therapeutic effects on adriamycin-induced nephropathy model rats. Its actions could be achieved through reducing albuminuria, inhibiting the proliferation of mesangial cells, attenuating the renal tubule-interstitial lesion, and protecting the integrity of the slit membrane structure.

Antifibrotic effect of the Chinese herbs Modified Danggui Buxue Decoction on adriamycin-induced nephropathy in rats.

OBJECTIVE: To investigate the antifibrotic effect of the Chinese herbs Modified Danggui Buxue Decoction (, MDBD) on adraimycin-induced nephropathy in rats. METHODS: Thirty-two male Sprague Dawley albino rats were randomly divided into 4 groups: the control, model, and two treatment groups, with 8 in each group. Nephropathy was induced in the latter 3 groups by intravenous injection of adriamycin. Rats in the two treatment groups received intragastric administration of benazepri (a positive control) or MDBD, which is composed of extracts of Radix Angelicae sinensis, Astragalus membranaceus (Fisch.) Bge and Rhizoma chuanxiong. Serum albumin, blood lipids, 24-h urine protein and urine N-acetyl-b-D-glucosaminidase (NAG) were measured every 2 weeks. The ratio of kidney to body weight was measured. The expressions of extracellular matrix proteins in the renal cortex, including colleagen IV (Col-IV) and fibronectin (FN), were examined by immunohistochemistry, and the transcription of genes encoding transforming growth factor ß1 (TGF-ß1), the tissue inhibitors of matrix metalloproteinase 1 (TIMP-1) and matrix metalloproteinase 9 (MMP-9) were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) at the end of the 8-week treatment. RESULTS: Compared with the untreated rats in the model group, MDBD significantly increased serum albumin, lowered the blood lipids and decreased the ratio of kidney to body weight. MDBD significantly reduced the excretion levels of urinary protein and NAG as well as the accumulation of extracellular matrix (ECM), including Col-IV and FN, in the renal cortex. Further, MDBD decreased TIMP-1 and TGF-ß1 gene expressions and increased MMP-9 gene expression in the kidney. CONCLUSIONS: MDBD was effective in treating the rat model of nephropathy. The clinical benefit was associated with reduction of renal fibrosis. The antifibrotic effect of MDBD may be mediated through the regulation of TIMP-1, MMP and TGF-ß1 gene expressions.