Clinical And Basic Research Of The Effect Of Yishenjiangzhou Granules On Tubular Impairment In Chronic Renal Failure

Background:Spleen-kidney Qi deficiency intermingling with damp turbidity in TCM was the most common syndrome patterns in CKD of stages3to4. TGF-β1-induced apoptosis in tubular epithelial cells is a central stimulus of the events leading to renal interstitial fibrosis, which contributes to chronic progressive kidney disease. Mitochondrial dysfunction, abnormal mitochondrial regeneration and dynamics has been proposed to play an important role in TGF-β1-induced apoptosis. Yishenjiangzhuo Granules was produced by People Hospital Affiliated to Fujian University of Traditional Chinese Medicine. It has been successfully used in clinical treatment of chronic renal failure (CRF) for20years.Objective:(1) To observe the clinical effect of Yishenjiangzhuo Granules (YSJZG) on CKD (stage3-4) which were diagnosed as spleen-kidney Qi deficiency intermingling with damp turbidity in TCM and its amelioration of renal tubular injury.(2) To investigate the mechanism of its therapeutic intervention in chronic renal failure from views of molecular pathology, gene, protein levels and relative-pathway through basic research.Methods:(1) Clinical investigation:According to the dignosis standard, patients who were diagnosed as chronic glomerulonephritis (CKD of stages3to4) with spleen-kidney Qi deficiency intermingling with damp turbidity in TCM. They were randomly divided into treatment group with31cases and control group with30cases. The treatment group were assigned to receive YSJZG (30g/d) and the control group were assigned to receive Niaoduqing Granules (NDQG)(25g/d) for12weeks. The24hours urinary protein and Blood Urea Nitrogen(BUN), Serum Creatinine (Scr), glomerular filtration rate (GFR) and clinical symptoms in TCM were used to evaluate of the therapeutic efficacy. The biomarkers of renal tubular injury including urinary neutrophil gelatinase-associated lipocalin (NGAL), Beta-2-microglobulin (β2-MG), a1-microglobulin (a1-MG) and urinary transforming growth factor-β1(TGF-β1) were also examined.(2) Basic research:in animal experiments, the CRF model was established by5/6subtotal nephrectomy. The Sprague-Dawley (SD) rats were randomly divided into six groups:sham operation group (SOG), model group (MG),5g/kg/d NDQG, low (3g/kg/d), moderate (6g/kg/d) and high concentration (9g/kg/d) YSJZG group (L-YSJZG, M-YSJZG, H-YSJZG)(n=10). After treated for10weeks, the24-hour urinary protein excretion and renal function were examined. HE, PAS and Masson staining, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, electron microscopy were used to assess the histological structure of kidney, tubular epithelial cell apoptosis and changes of mitochondrial structure respectively. Superoxide dismutase (SOD), glutathione (GSH), Malondialdehyde (MDA), and the expression of TGF-β1was quantitated by colorimetric method and immunohistochemistry. The protein expression of pro-and anti-apoptotic genes (Bax/Bcl-2), mitochondrial biogenesis-relative genes (PGC-1α、Tfam) and signaling molecules (pp38MAPK、 pERKl/2) was analysed by Western Blot. In cell experiments, different concentration of containing drug-serum was made. The Sprague-Dawley (SD) rats were randomly divided into five groups:normal control group (NG), Niaoduqing Granules group (NDQG), low, moderate and high concentration YSJZG group (L-YSJZG, M-YSJZG, H-YSJZG). They were gastric perfused daily with normal saline,5g/kg NDQG,3g/kg、6g/kg、9g/kg YSJZG respectively. Blood drawn from abdominal aortic1h aftern ending perfusion on the7th day, and the serum separated was taken for testing. Cultured NRK-52E cells in vitro were divided into six groups in random:Control group, lOng/ml TGF-β1group, TGF-β1+NDQG group, TGF-β1+L-YSJZG, TGF-β1+M-YSJZG, TGF-β1+H-YSJZG. After cells were stimulated with different factors for48h, morphological changes, cell viability, cellular apoptosis, the injury of mitochondrion including mitochondrial dysfunction, abnormal mitochondrial regeneration and dynamics, and ROS-MAPK signaling molecules were assessed by phase contrast microscopy, Trypan Blue Staining Cell Viability Assay, TUNEL assay, electron microscopy, JC-1probe,2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA), RT-PCR and Western Blot respectively.Results:(1) Clinical investigation:after12weeks, the total therapeutic efficacy of teratment group with YSJZG is much better than that of control group (P<0.05). YSJZG can markedly alleviate TCM symptoms compared with NDQG (P<0.05). Serum BUN, Scr, urinary protein, urinary NGAL,β2-MG, a1-MG and TGF-β1were significantly downregulated in treatment group with YSJZG, while GFR were markedly increased (P<0.05). These effects of YSJZG was better than that of NDQG (P<0.05, YSJZG vs. NDQG). Scr, urinary NGAL, β2-MG, a1-MG and TGF-β1were inversely, independently, and closely related to GFR. The levels of urinary TGF-β1were positively correlated with three other urinary biomarkers.(2) Basic experiment:in animal experiments, compared with sham group, BUN, Scr and24-hour urinary protein excretion were significantly increased in model group (P<0.01). YSJZG significantly lowered the lever of the above indexes in a dose-dependent manner YSJZG also significantly alleviated tubulointerstitial injury and dereased the tubulointerstitial injury index (TⅡ) in the remnant kidney in a dose-dependent manner (P<0.01). TUNEL-positive cells were significantly reduced and the mitochondrial changes were more markedly ameliorated after YSJZG treatment (P<0.01). YSJZG could significantly supress the expression of Bax protein, upregulate the expression of Bcl-2protein, and also markely restored the expression of Tfam and PGC-1α protein (P<0.01). YSJZG significantly downregulated TGF-β1expression, markers of oxidative stress and phosphorylation of p38MAPK and ERK1/2in a dose-dependent manner (P<0.01, vs. MG). The efficacy of YSJZG was better than that of NDQG (P<0.01, M-YSJZG or H-YSJZG vs. NDQG). In cell experiment, incubation with YSJZG containing drug-serum increased in cell viability, supressed TGF-β1-induced apoptosis, recovered the normal structure, function of mitochondrion in a dose-dependent manner. YSJZG dramatically increased Opal mRNA expression, supressed Fisl and Drpl mRNA expression, and markely restored the expression of Tfam and PGC-1a protein induced by TGF-131in a dose-dependent manner. YSJZG could significantly block the expression of Bax protein, upregulate the expression of Bcl-2protein and inhibit the phosphorylation of both redox-sensitive p38MAPK and ERK1/2induced by10ng/ml TGF-β1in a dose-dependent manner (P<0.01vs. TGF-β1group; P<0.01, M-YSJZG or H-YSJZG vs. NDQG).Conclusions:(1) Clinical investigation:Yishenjiangzhuo Granules can alleviate the clinical Symptoms of TCM and improve renal function of CRF. Yishenjiangzhuo Granules can also inhibit the injury of renal tubule and restrain the secretion and expression of TGF-β1. This might be one of the mechanism of its delaying the progress of CRF.(2) Basic experiment:Yishenjiangzhuo Granules can inhibit the injury of renal tubular interstitium in the remnant kidney, then improve renal function of the rats of CRF and its efficacy is better than Niaoduqing Granules. Yishenjiangzhuo Granules could block the expression of TGF-β1, suppress the oxdative stress and the activation of MAPK pathway, alleviate the injury of mitochondrion through recovering the mitochondrial biogenesis, and then inhibited the apoptosis of renal tubular epithelial cells of renal tubular interstitium in the remnant kidney. TGF-β1could unbalance the mitochondrial dynamics, inhibit the mitochondrial biogenesis and decrease mitochondrial membrane potential, then promote apoptosis in renal tubular epithelial cells through activation of ROS-MAPK pathway. YSJZG containing drug-serum could recover the mitochondrial dynamics and biogenesis and repair mitochondrial function, and then inhibited the apoptosis of renal tubular epithelial cells by suppression of ROS-MAPK pathway, and its efficacy is better than Niaoduqing Granules.