Artificially Cultivated Ophiocordyceps sinensis Alleviates Diabetic Nephropathy and Its Podocyte Injury via Inhibiting P2X7R Expression and NLRP3 Inflammasome Activation.

BACKGROUND/AIMS: It is known that chronic low-grade inflammation contributes to the initiation and development of both diabetes and diabetic nephropathy (DN), so we designed this study to investigate the role of P2X7R and NLRP3 inflammasome in DN pathogenesis and the antagonistic effects of artificially cultivated Ophiocordyceps sinensis (ACOS). METHODS: A rat model of DN caused by high-fat-diet feeding and low-dose streptozotocin injection and a mouse podocyte injury model induced by high-glucose (HG) stimulation were established, and the intervention effects of ACOS on them were observed. The biological parameters of serum and urine and the pathological manifestations of kidney tissue were examined. The expression of mRNA and protein of P2X7R and NLRP3 inflammasome (NLRP3, ASC, and caspase-1) and downstream effectors (IL-1ß and IL-18), as well as podocyte-associated molecules, was determined by real-time quantitative PCR and Western blot assay, respectively. RESULTS: The DN rats showed to have developed insulin resistance, elevated fasting blood glucose, increased urinary protein excretion, and serum creatinine level as well as corresponding glomerular pathological alterations including podocyte damages. ACOS significantly antagonized the above changes. The experiments in vivo and in vitro both displayed that the mRNA and protein expression of P2X7R, NLRP3, ASC, caspase1 (procaspase-1 mRNA in the gene level and active caspase-1 subunit P10 in the protein level), IL-1ß, and IL-18 was significantly upregulated and the mRNA and protein expression of podocyte-associated molecules was significantly changed (downregulation of nephrin, podocin, and WT-1 expression and upregulation of desmin expression) indicating podocyte injury in the kidney tissue of DN rats and in the HG-stressed mouse podocytes, respectively. ACOS also significantly antagonized all the above changes. CONCLUSION: Our research work suggests that P2X7R and NLRP3 inflammasome are involved in the pathogenesis of DN, and ACOS can effectively inhibit the high expression of P2X7R and the activation of NLRP3 inflammasome, which may contribute to the therapeutic effects of Ophiocordyceps sinensis.

Aristolochic Acid-Induced Autophagy Promotes Epithelial-to-Myofibroblast Transition in Human Renal Proximal Tubule Epithelial Cells.

Autophagy plays an essential role in cellular homeostasis in kidney. Previous studies have found that aristolochic acid (AA) can induce autophagy of renal tubular epithelial cells and epithelial-to-myofibroblast transition (EMT). However, the relationship between AA-induced autophagy and EMT is unclear. Our results showed that, after AA stimulation, the appearance of autophagy preceded EMT. Autophagy of HKC cells began to increase gradually from the 3rd hour, reached the peak at 12th hour, and then weakened gradually until 36th hour; the EMT process of HKC continued to increase from 6th hour to 36th hour after AA stimulation. The enhancement of autophagy using autophagy inducers, rapamycin or serum-free medium, led to an aggravation of EMT and upregulated expression of fibronectin, a component of extracellular matrix, in AA-treated HKC cells. In contrast, the inhibition of autophagy by autophagy inhibitor, 3-methyladenine, or by knockdown of Beclin 1 led to an attenuation of EMT and downregulated expression of fibronectin in AA-treated HKC cells. Taken together, our study suggests that, after AA stimulation, two types of cell responses of HKC cells, autophagy and EMT, will successively appear, and autophagy can promote EMT of HKC.

The Protective Effects of Curcumin on Obesity-Related Glomerulopathy Are Associated with Inhibition of Wnt/ß-Catenin Signaling Activation in Podocytes.

The present study investigated the effects of curcumin, one of the most important active ingredients of turmeric, on podocyte injury in vitro and obesity-related glomerulopathy (ORG) in vivo. Cellular experiments in vitro showed that curcumin significantly antagonized leptin-induced downregulation of the mRNA and protein expression of podocyte-associated molecules including nephrin, podocin, podoplanin, and podocalyxin. Animal experiments in vivo showed that curcumin significantly reduced the body weight, Lee's index, abdominal fat index, urinary protein excretion, and average glomerular diameter and significantly upregulated the mRNA and protein expressions of the above podocyte-associated molecules in ORG mice. Furthermore, the experiments in vitro and in vivo both displayed that curcumin could downregulate the mRNA and protein expressions of Wnt1, Wnt2b, Wnt6, and ß-catenin and upregulate the phosphorylation level of ß-catenin protein in podocytes and renal tissue. In conclusion, curcumin is able to alleviate the harmful reaction of leptin on podocytes and reduce the severity of ORG. The above protective effects are associated with the inhibition of Wnt/ß-catenin signaling activation in podocytes.

[Effects of Hirsutella sinensis on TGF-beta1 and Snail expressions and transdifferentiation of tubular epithelial-myofibroblast in renal tissue of rats with chronic aristolochic acid nephropathy].

OBJECTIVE: To investigate the antagonizing effect of Hirsutella sinensis (HS) on renal tubular epithelial-myofibroblast transdifferentiation (TEMT) and its possible pathogenic mechanism in rats with chronic aristolochic acid nephropathy (CAAN). METHODS: Eighteen male Sprague-Dawley rats were equally divided into 3 groups, the model (M) group, the intervention (I) group and the control (C) group. The 24 h urinary protein (UP) in rats was measured before intervention and at the end of the 1st, 4th, 8th, and 12th week, and creatinine clearance rate (CCr) was measured before intervention and at the end of the 12th week respectively. All rats were sacrificed at the end of the 12th week, their kidney was taken for examining the degree of fibrosis in renal interstitial with Masson's stain and determining mRNA and protein expressions of transforming growth factor-beta1 (TGF-beta1), Snail, alpha-smooth muscle actin (alpha-SMA) and cytokeratin in renal tissue by Real-time RT-PCR and immunohistochemistry staining, respectively. RESULTS: Compared with the C group, CCr was significantly lower, while 24 h UP was higher; the relative area of interstitial fibrosis was significantly larger in the M group; besides, the mRNA and protein expressions of TGF-beta1, Snail and alpha-SMA were significantly up-regulated (P < 0.01 or P < 0.05), and those of cytokeratin were significantly down-regulated (P < 0.01) in renal tissue of the M group. While in the I group, all the above-mentioned abnormalities were restored to some extent (P < 0.05) and showed significant difference (all P < 0.05) as compared with those in the M group. CONCLUSION: HS can downregulate TGF-beta1 and Snail expressions in renal tissue, antagonize TEMT and renal interstitial fibrosis, and improve renal function in CAAN rats.

[Protective effects of yishen ruanjian power on renal interstitial fribrosis in chronic aristolochic acid induced nephropathy rat model].

OBJECTIVE: To study the protective effects of Yishen Ruanjian Power (YRP) on renal interstitial fibrosis in rats with chronic aristolochic acid induced nephropathy (CAAN). METHODS: Eighteen male SD rats were divided into 3 groups, 6 in each group. Water solution of Caulis Aristolochia Manshuriensis (CAM) Liquid Extract were given to the mice in the model group by gastrogavage to make CAAN animal model. For those in the TCM group, decocted water solution of YRP was given by gastrogavage after the mice being modeled with the above-mentioned method. Tap water was given by gastrogavage to the mice in the control group. Body weight, 24-hr urinary protein excretion and beta2 microglobulin (beta2-MG), and serum creatinine (r) were determined at the end of the 1st, 4th, 8th, 12th and 16th week. At the end of the 16th week, the rats were sacrificed and the pathological figure of their kidneys were observed by Masson staining. Transforming growth factor-beta1 (TGF-beta1), connective tissue growth factor (CTGF), plasminogen activator inhibitor-1 (PAI-1), tissue inhibitor of metalloproteinase-1 (TIMP-1) and type I collagen (Col I ) in kidney tissue were determined by RT-PCR and immunohistochemical method, respectively. RESULTS: At end of the 1st week, urinary protein excretion, urinary beta2-MG and SCr in the model group were significantly increased to the levels higher than those in the control group (P < 0.01 or 0.05). Relative area of interstitial fibrosis was significantly enlarged in the model group at the end of the 16th week (P<0.01), and at the same time, the mRNA and protein expression of TCF-beta1, CTGF, PAI-1, TIMP-1 and Col I in kidney tissue were significant up-regulated (P<0.01). After intervention with YRP, the above-mentioned up-regulated parameters, except 24-hr urinary protein excretion, were all significantly inhibited (P <0.01 or 0.05). CONCLUSION: YRP could inhibit the accumulation of extracellular matrix in renal interstitial tissue, so as to alleviate the renal interstitial fibrosis and improve the renal function.

[The medicinal serum of yi-shen ruan-jian san antagonized the effect of aristolochic acid on human proximal tubular epithelial cells in vitro].

OBJECTIVE: To evaluate whether the medicinal serum of Yi-shen Ruan-jian san can antagonize the fibrogenic effect of human proximal tubular epithelial cell line (HKC) activated by aristolochic acid (AA) in vitro. METHOD: The HKC was incubated in the media containing 40 mg x L(-1) aristolochic acid sodium salt (AA-Na) with or without 10% concentration of Yi-shen Ruan-jian san medicinal serum. Then the cell proliferation and cytotoxicity of HKC were determined by MTF and lactate dehydrogenase (LDH) release assay respectively, the antigen expression of cytokeratin and alpha-smooth muscle actin on HKC was detected by immunocytochemistry, the mRNA expression of transforming growth factor-beta1 (TGF-beta1), connective tissue growth factor (CTGF), plasminogen activator inhibitor-1 (PAI-1), tissue inhibitor of metalloproteinase-1 (TIMP-1) and type I Collagen (Col I) of HKC was measured by RT-PCR, and their protein expression was measured by ELISA or Western blot. RESULT: No cytotoxic effect was found in HKC after stimulation of AA-Na with or without the medicinal serum of Yi-shen Ruan-jian san (P > 0.05). No epithelial-myofibroblast transdifferentiation was found in HKC after AA-Na stimulation. The mRNA and protein expression of TGF-beta1, CTGF, PAI-1 and TIMP-1 of HKC was significantly upregulated by AA-Na (P < 0.05). The above-mentioned enhanced mRNA and protein expression, except for PAI-1, was significantly downregulated by the medicinal serum of Yi-shen Ruan-jian san, compared with the control (normal rat serum in the same concentration) (P < 0.05). CONCLUSION: The fibrogenic effects of HKC activated by AA are antagonized by Yi-shen Ruan-jian san, through downregulating the expression of promoting excellular matrix (ECM) synthesis factors (TGF-beta1, CTGF) and inhibiting ECM degradation factor (TIMP-1).

Comparisons between oral pulse alfacalcidol therapy and daily therapy in maintenance hemodialysis patients with secondary hyperparathyroidism: a randomized, controlled, and multicenter study.

OBJECTIVE: To investigate the efficacy and safety of 1alpha-(OH)-D3 high-dose pulse therapy or daily low-dose therapy in secondary hyperthyroidism in maintenance hemodialysis patients in China. METHODS: Maintenance hemodialysis patients of both gender with intact parathyroid hormone (iPTH) level above 200 pg/mL were randomly divided into a pulse group and a daily group. They were treated for 20 weeks, with 2 microg oral Alfacalcidol twice weekly or thrice weekly in the pulse group, and 0.5 microg oral Alfacalcidol per day in the daily group. The therapeutic end point was parathyroid hormone level < 200 pg/ mL. The iPTH levels during the study were monitored, and parameters representative of calcium and phosphate metabolism and side effects were also observed. RESULTS: One hundred and fifty-eight patients were initially enrolled, 91 in the pulse therapy group and 67 in the daily therapy group. There was no significant difference in age, hemodialysis duration, proportion of diabetic nephropathy and systemic diseases, proportion of patients who had received active vitamin D therapy previously, mean initial iPTH level (pulse group 570.47 +/- 295.86 pg/mL; daily group 498.33 +/- 207.84 pg/mL), serum calcium, serum phosphate, alkaline phosphatase (AKP), and albumin between two groups. In the pulse therapy group there were more patients with iPTH levels of 500 to approximately 1,000 pg/mL and > 1,000 pg/mL, so stratified analysis according to iPTH level was used. In therapeutic end point, iPTH levels in both groups were significantly lower compared with those before therapy (pulse group 261.29 +/- 234.97 pg/mL, P < .01; daily group 262.17 +/- 274.82 pg/mL, P < .01). After 4 weeks, the ratio of reaching end point in the pulse group was 35.2%, which was significantly higher than that (19.4%) in the daily group (P < .05). More obvious change was seen in the 200 to approximately 500 pg/mL subgroup by stratified analysis (P < .05), whereas there was no significant difference between the 500 to approximately 1,000 pg/mL and > 1,000 pg/mL subgroup (P > .05). At therapeutic end point, the total ratio of reaching end point did not differ between the two groups, and there were no obvious differences between each subgroup. In the iPTH 200 to approximately 500 pg/mL subgroup, mean iPTH%/week in the pulse group was significantly higher than that in the daily group, and no obvious difference was seen in other subgroups. AKP levels decreased significantly in both groups at therapeutic end point (pulse group 98.42 +/- 54.52 vs. 74.21 +/- 30.68 IU/L, P < .01; daily group 103.3 +/- 68.04 vs. 75.40 +/- 34.12 IU/L, P < .01). On the 4th week, AKP level in pulse group (82.39 +/- 35.23 IU/L) was significantly lower than the initial level (98.42 +/- 54.52 IU/L, P < .05), whereas in the daily group there was no difference between each week. The mean serum calcium, phosphate, and [Ca2+] x [P3+] levels in both groups did not change greatly. Nine patients in the pulse group (9.9%) and 8 patients in the daily group (11.9%) suffered hypercalcemia at least once. Persistent hypercalcemia occurred in 8 patients in the pulse group (8.8%) and 9 patients in the daily group (13.4%), but the difference in proportion did not show statistical significance. The serum phosphate in the daily group was higher after the therapy (1.74 +/- 0.36 vs. 1.89 +/- 0.36 mmol/L, P < .05), whereas that in the pulse group remained unchanged. At therapeutic end point, [Ca2+] x [P3+] level in the daily group was higher than that before the therapy (48.04 +/- 11.71 vs. 55.46 +/- 12.66, P < .05), whereas in the pulse group there was no significant difference. Side effects for both groups were minimal and well tolerated. CONCLUSIONS: Alfacalcidol [1alpha-(OH)-D3] has good and safe effects on secondary hyperparathyroidism in maintenance hemodialysis patients. The efficacy and early effects of pulse therapy are superior to those of daily therapy in moderate hyperparathyroidism patients.

[Antagonistic effect of yishen ruanjian san contained serum against aristolochic acid in antagonizing human renal interstitial fibroblasts].

OBJECTIVE: To study whether yishen ruanjian san contained serum (S-YRS) could intervene the action of aristolochic acid (AA) in antagonizing human renal interstitial fibroblasts (hRIFs) to induce extracellular matrix (ECM) accumulation. METHODS: AA-Na 40 microg/ml, with or without 10% S-YRS, was co-cultured with hRIFs, then the hRIFs mRNA of transforming growth factor-beta1 (TGF-beta1), connective tissue growth factor (CTGF), plasminogen activator inhibitor-1 (PAI-1), tissue inhibitor of metalloproteinase-1 (TIMP-1) and type I collagen (Col I) in the cultured cells were detected by RT-PCR, and their protein expression monitored with ELISA and Western blot respectively. RESULTS: The mRNA and protein expression of all the above-mentioned factors were significantly up-regulated by AA-Na (P < 0.05). Excepting PAI-1, the enhanced mRNA and protein expression were significantly down-regulated by S-YRS (P < 0.05). CONCLUSION: S-YRS could down-regulate the hRIF to promote the expression of ECM synthesis factors and inhibit the ECM degradation factors in hRIFs, so as to antagonize the AA stimulated accumulation of ECM such as Col I.

[Multicenter prospective study on relationship of TCM syndrome type and renal pathology in 286 patients with IgA nephropathy].

OBJECTIVE: To study the distribution pattern of TCM Syndrome type and its relationship with renal pathology in patients with IgA nephropathy. METHODS: Apopting multicenter coordinated method, patients' TCM Syndrome type was differentiated according to their clinical manifestations, data concerning laboratory examination and renal pathology were collected to establish a database for analyzing the relationship between TCM Syndrome type and renal pathology in 286 patients. RESULTS: Patients of Pi-Fei Qi-deficiency type (type 1) and both Qi-Yin deficiency type (type 2) showed rather milder pathological changes, by Lee classification, most of them belonged to grade I-III (72.3%, 70.2%); patients of Gan-Shen Yin deficiency type (type 3) had severe pathological change and majority of them belonged to grade III-IV (84.6%); and the most severe pathological change was shown in patients of Pi-Shen Yang-deficiency type (type 4), and the Lee's grade IV-V was dominant (88.0%) in them. Syndrome type of patients was significantly correlated with their Lee's grade (r = 0.26, P < 0.01). Percentage of glomerular sclerosis in patients of type 4 was higher than that in patients of the other three types. Semi-quantitative scoring upon pathological changes showed that the total, glomerular, tubulo-interstitial and vascular scores were significantly higher in patients of type 4 than in those of the other three types; those scores were higher in patients of type 3 than in type 1; the total, glomerular and vascular scores were higher in patients of type 3 than in type 2; and scores in patients of type 1 and type 2 showed insignificant difference. CONCLUSION: Multicentric prospective study proves that the TCM Syndrome typeof patients with IgA nephropathy is significantly correlated with the grade and severity of their renal pathological changes, thus, the TCM Syndrome typing shows definite referential importance to conclude the severity of renal pathological change in patients with IgA nephropathy.