Guanghui Ling

Low-dose paclitaxel ameliorates renal fibrosis in rat UUO model by inhibition of TGF- β /Smad activity

Transforming growth factor-β (TGF-β) has a pivotal function in the progression of renal fibrosis in a wide variety of renal diseases. Smad proteins have been identified to have an important function in regulating the expression of extracellular matrix (ECM) proteins through TGF-β signaling pathway. Aberrant TGF-β/Smad signaling can be modulated by stabilization of microtubules with paclitaxel. In this study, we investigated if paclitaxel can attenuate tubulointerstitial fibrosis in a rat model of unilateral ureteral obstruction (UUO). Rats in groups of six were subjected to UUO and received low-dose intraperitoneal injection of paclitaxel (0.3 mg/kg) twice a week. They were killed at day 7 and 14 after UUO or Sham operation. TGF-β signaling cascade and status of various ECM proteins were evaluated by RT–PCR, western blotting and immunohistochemical or immunofluorescence staining. The paclitaxel treatment markedly suppressed Smad2 and Smad3 phosphorylation. This was associated with attenuated expression of integrin-linked kinase, collagens I and III, fibronectin (FN) and α-smooth muscle actin, and a substantial decrease in renal fibrosis in animals that underwent UUO and received paclitaxel. These data indicate that the low-dose paclitaxel ameliorates renal tubulointerstitial fibrosis by modulating TGF-β signaling, and thus, the paclitaxel may have some therapeutic value in humans.

Low-dose paclitaxel ameliorates fibrosis in the remnant kidney model by down-regulating miR-192

Transforming growth factor (TGF)‐β has been shown to play a central role in the development of tubulointerstitial fibrosis, which can be corrected via treatment with paclitaxel. The biology of microRNA (miR) can be modulated by paclitaxel. We hypothesized that paclitaxel may attenuate renal fibrosis in a rat model of remnant kidney disease by inhibiting TGF‐β induced‐miRs. Rats in groups of 12 were subjected to 5/6 nephrectomy and received low‐dose intraperitoneal injection of paclitaxel. Renal functions were assessed at 8 weeks. The TGF‐β signalling cascade and ECM proteins were evaluated by real‐time polymerase chain reaction (TRT–PCR) and immunofluorescence microscopy. Animals with remnant kidneys developed hypertension, which was not relieved with paclitaxel treatment. However, paclitaxel treatment resulted in dampening the proteinuric response, reduction in serum BUN, creatinine levels and urine protein : creatinine ratio and normalization of creatinine clearance. These effects were accompanied by the inhibition of Smad2/3 activation, attenuation of renal fibrosis and normalization of integrin‐linked kinase (ILK), COL(I)A1, COL(IV)A2 and α‐SMA expression. Also, paclitaxel down‐regulated the expression of miR‐192, miR‐217 and miR ‐377, while miR‐15 was up‐regulated in the remnant kidney. In vitro, in tubular epithelial cells (NRK‐52E), paclitaxel also inhibited TGF‐β1‐induced Smad2/3 activation and normalized ILK, COL(I)A1, COL(IV)A2 and α‐SMA expression. Furthermore, ChIP analyses indicated that Taxol suppressed Smad3‐mediated miR‐192 transcriptional activity. Over‐expression of miR‐192 in NRK‐52E mimicked the changes seen in the remnant kidney, while inclusion of miR‐192 inhibitor in the culture medium blocked TGF‐β1‐induced COL(I)A1 and COL(IV)A2 expression, while ILK and α‐SMA were unaffected. These data suggest that low‐dose paclitaxel ameliorates renal fibrosis via modulating miR‐192 pathobiology and TGF‐β/Smad signalling. Copyright

Nondiabetic Renal Disease in Type 2 Diabetic Patients: A Review of our Experience in 220 Cases

Background: The purpose of this retrospective study was to evaluate the renal biopsies performed on type 2 diabetic patients for suspicion of nondiabetic renal disease (NDRD) and to correlate the pathological finding with the clinical and laboratory findings. Methods: From January 1999 to December 2009, 220 people with type 2 diabetes for clinically suspected NDRD underwent renal biopsy. The case records of these patients were retrospectively analyzed. Based on the biopsy findings, patients were divided into two groups: Group I, isolated diabetic glomerulosclerosis (DGS), and Group II, NDRD with underlying DGS. Clinical and laboratory data were analyzed in relation to the histopathological findings. Results: Of the 220 patients studied, 153 were males and 67 were females. The average age was 51.35 years (30–79). Renal biopsy showed that 100 patients (45.5%) had NDRD with underlying DGS. Group II had a significantly higher level of proteinuria and hematuria but less frequent diabetic retinopathy. There was no significant difference between the two groups in age, duration of diabetes, presence of hypertension, serum creatinine, and glomerular filtration rate. IgA nephropathy was the most common, accounting for 34% of Group II, membranous nephropathy ranked second accounting for 22.0%, followed by mesangial proliferative nephritis for 14%. Conclusion: This study showed that IgA nephropathy is the commonest NDRD among diabetics. The absence of retinopathy, especially when associated with nephritic proteinuria and hematuria, strongly predicts NDRD superimposed on DGS. Renal biopsy should be performed in diabetics when the clinical scenario is atypical.

Norcantharidin Ameliorates Proteinuria, Associated Tubulointerstitial Inflammation and Fibrosis in Protein Overload Nephropathy

Norcantharidin (NCTD), the demethylated analog of cantharidin isolated from Mylabris, is an anticancer drug routinely used against various human cancers in China. The aims of this study are to learn if NCTD has a protective action against severe proteinuria and consequent interstitial inflammation and fibrosis, and if the inhibition of nuclear factor-ĸB (NF-ĸB) and connective tissue growth factor (CTGF) by NCTD might be involved. Male Sprague-Dawley rats with protein overload nephropathy induced by intraperitoneally injected bovine serum albumin were used as a model. The histopathological examination of kidney tissue in the 9th week by light microscopy and scanning electron microscopy revealed that inflammatory cells had extensively infiltrated into the tubulointerstitial areas with interstitial fibrosis. The administration of NCTD at 0.1 mg/kg/day to the bovine-serum-albumin-injected animal models effectively reduced the proteinuria, and prevented the proteinuria-induced interstitial inflammation and fibrosis. Expressions of the NF-ĸB p65 subunit and CTGF, detected by immunohistochemistry, Western blotting and reverse-transcription polymerase chain reaction, were upregulated in protein overload nephropathy and were attenuated by NCTD. Inhibition of the expressions of the NF-ĸB p65 subunit and CTGF was one beneficial effect of NCTD. These results suggest that in addition to the antiproteinuric action of NCTD, due to its anti-inflammatory and antifibrotic effects as shown in the present study, it may become a therapeutic agent for proteinuria and its associated chronic inflammatory and fibrotic nephropathy.

Mast cell infiltration is involved in renal interstitial fibrosis in a rat model of protein-overload nephropathy.

Aims: The present study investigated the relationship between mast cells (MCs) and the protein expression of stem cell factor (SCF) and transforming growth factor-β1 (TGF-β1) in the regions of renal interstitial fibrosis with protein-overload nephropathy, in order to provide a good animal model to study the mechanism of renal fibrosis induced by proteinuria. Methods: 60 male Sprague-Dawley rats were divided into a bovine serum albumin (BSA) group and a control group. The intensity of MCs infiltration was examined by toluidine blue and chymase and tryptase staining. The protein expression of SCF and TGF-β1 was respectively examined by immunohistochemical and immunofluorescence staining. Results: Severe proteinuria was induced in rats of the BSA group. Expression of SCF and TGF-β1 was detected in the tubular and the interstitial cells. The number of MCs positively correlated with the severity of interstitial lesions and the expression of SCF and TGF-β1. Conclusion: Our results demonstrated that in protein-overload nephropathy, MCs infiltrated into the kidney, and the expression of SCF and TGF-β1 gradually increased. They might play important roles in the development of renal interstitial fibrosis, but the underlying mechanism needs to be further studied.

Smad Anchor for Receptor Activation Regulates High Glucose-Induced EMT via Modulation of Smad2 and Smad3 Activities in Renal Tubular Epithelial Cells.

Background: SARA is an essential adaptor protein in TGF-β1 signaling and it is also involved in the process of Epithelial-mesenchymal transition (EMT) and fibrosis. Our aim was to investigate the effect of SARA on high glucose (HG)-induced EMT and extracellular matrix (ECM) synthesis in renal tubular epithelial cells. Methods: The cells were transfected with the following plasmids: wild-type SARA (SARA-WT), SARA-dSBD (SARA with Smad binding domain deletion) and then subjected to HG ambience (30 mM). The expression levels were assessed by Western-blot, real-time PCR and immunofluorescence. Results: HG-induced EMT phenotype with increased expression of ECM protein in HK-2 cells. This was associated with the decreased expression of SARA and Smad2. In comparison with the HG group, overexpression of SARA in HK-2 cells, a relatively high upregulation of ZO-1 expression was seen, while that of Vimentin, fibronectin and collagen I was decreased. The Smad2 protein expression was increased in HK-2 cells after transfection with SARA (WT) plasmid. Interestingly, the overexpression of SARA prolonged the activity period of Smad2 and shortened that of Smad3, which seemed consistent with the change of EMT phenotype and ECM changes in HK-2 cell induced by HG. Conclusions: SARA regulates HG-induced EMT and ECM excretion via modulation of the activation of Smad2 and Smad3 in renal tubular epithelial cells. In view of these findings, it is conceivable that SARA may serve as a potential novel target in pre-EMT states for the amelioration renal fibrosis seen in chronic kidney diseases.

Norcantharidin Attenuates Tubulointerstitial Fibrosis in Rat Models with Diabetic Nephropathy

Objective: To investigate the effects of norcantharidin (NCTD) on tubulointerstitial fibrosis of diabetic nephropathy (DN) in streptozotocin-induced rat model. Methods: Sprague–Dawley rats were randomly divided into control group, model group, low-dose NCTD (0.05 mg/kg/day) group, and high-dose NCTD (0.1 mg/kg/day) group. The model group was induced by injection intraperitoneally with 30 mg/kg streptozotocin in 0.1 mol/L sodium citrate solution (pH 4.5), after high-calorie foods were given for 2 months. NCTD was administered daily after the DN rat model was built. Rats were sacrificed at the end of the third and the eighth week; renal fibrosis and the expression of FN, collagen IV, TGF-β1, and calcineurin (CaN) were detected by Masson and immunohistochemistry staining, respectively. Results: Tubulointerstitial fibrosis was observed in DN rats, this kind of pathological changes was ameliorated in NCTD treatment group (p < 0.05). The expressions of FN, collagen IV, and TGF-β1 protein increased in the tubulointerstitial field of DN rats compared with the rats in control group. NCTD treatment could dose-dependently decrease their expression and reverse the fibrotic degree (p < 0.05). Meanwhile, the expression of CaN was detected in tubular fields of normal kidney and increased in the tubulointerstitial field in DN rats. However, NCTD downregulated its expression in a dose-dependent manner (p < 0.05). Conclusions: NCTD could downregulate FN, collagen IV, and TGF-β1 expression in tubulointerstitial fields and attenuate tubulointerstitial fibrosis in the early stage of DN rats. NCTD also alleviated the expression of CaN in tubules in DN. The relationship between the role of NCTDs anti-tubulointerstitial fibrosis and its inhibition to CaN expression remains to be further elucidated.

Norcantharidin inhibits the expression of extracellular matrix and TGF-β1 in HK-2 cells induced by high glucose independent of calcineurin signal pathway.

Norcantharidin (NCTD) was shown in our previous studies to attenuate renal tubulointerstitial fibrosis in rat models with diabetic nephropathy (DN). The aim of this study was to determine the effects of NCTD on the expression of extracellular matrix (ECM) and TGF-β1 in HK-2 cells stimulated by high glucose and on calcineurin (CaN)/NFAT pathway. Whether or not the antifibrotic effect of NCTD on renal interstitium was dependent on its inhibition of CaN pathway was also investigated. Experimental concentrations of NCTD were verified by cytotoxic test and MTT assay. HK-2 cells were transfected with CaN small interference RNA (siRNA). The mRNA and protein expressions of FN, ColIV, TGF-β1, and CaN in HK-2 cells were detected by real-time PCR and western blot. The CaN/NFAT pathway was examined by indirect immunofluorescence and western blot. Our study revealed that NCTD concentrations over 5 mg/l had overt cytotoxicity on HK-2 cells. Meanwhile, both 2.5 and 5 mg/l NCTD inhibited HK-2 cell proliferation (P<0.05). NCTD inhibited the upregulation of FN, ColIV, and TGF-β1 of HK-2 cells stimulated by high glucose (P<0.05), and also significantly downregulated the expression of CaN mRNA and protein in HK-2 cells (P<0.05). In addition, not only was the nuclear translocation of NFATc inhibited, but its protein level in the nucleus was also reduced. Following CaN siRNA transfection, CaN mRNA and protein expression were significantly decreased. In contrast, the protein levels of FN, ColIV, and TGF-β1 increased in HK-2 cells stimulated by high glucose (P<0.05). However, NCTD treatment downregulated their expression. These results indicated that NCTD could decrease the expression of ECM and TGF-β1 in HK-2 cells stimulated by high glucose, downregulate CaN expression, and block the CaN/NFAT signaling pathway. However, the effect of NCTD on inhibition of the expression of ECM and TGF-β1 was not associated with its inhibition of the CaN/NFAT pathway.

The relationship between the TGF-beta1 gene -509C/T polymorphism and tubulointerstitial damage resulting from primary nephrotic syndrome.

Background: The aim of this study was to investigate the correlation between the transforming growth factor (TGF)-β1 gene −509C/T polymorphism and the susceptibility to primary nephrotic syndrome (PNS), and in particular to the severe degree of tubulointerstitial damage (TID) seen in Chinese. Methods: Ninety-eight PNS patients and 128 normal controls were studied. The extent of tubulointerstitial changes was evaluated and patients were divided into two groups according to the severe or mild degree of TID. The TGF-β1gene −509C/T polymorphism was detected with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique, and the serum level of TGF-β1 was determined by enzyme-linked immunosorbent assay (ELISA). Results: No statistical differences in genotype or allele frequency of the TGF-β1 gene −509C/T were found between PNS and normal subjects. However, T allele and CT + T T genotype frequency were higher in the PNS with severe TID than the mild TID and controls. Additionally, the serum concentration of TGF-β1 was significantly higher in the PNS with severe TID group than the other two groups and in the T T genotype individuals than the CC and CT genotype individuals. A logistic regression analysis demonstrated that TGF-β1 gene −509C/T genotype was the risk factor of TID in PNS patients [OR (odd ratio) 2.34, confidence interval (CI) 0.98–3.46, p = 0.012]. Conclusion. TGF-β1 gene −509C/T polymorphism was associated with severe TID. The higher value in serum concentration of TGF-β1 was also associated with severe TID and the T T genotype/T allele. T allele gene might be the important risk factor for susceptibility.

Maltose, a promising osmotic agent in peritoneal dialysis solution.

Peritoneal dialysis has undergone considerable development from a technological point of view, and osmotic agent has played the essential role in peritoneal dialysis fluid. Because the most commonly used osmotic agent is glucose and icodextrin, there are some disadvantages related to the use of glucose-based solutions and icodextrin. So it is urgent to develop a new peritoneal dialysis osmotic agent. According to these characteristics of glucose and icodextrin, it is promising to explore a better osmotic agent of peritoneal dialysis solution which is able to allow maintenance of the maximum ultrafiltration gradient, and prevent toxicity or accumulation of unwanted substances in the blood, being non-toxic or less-toxic, furthermore the metabolite should not cause significant metabolic disturbance. Maltose may be one of promising osmotic agent and may put an important influence on development of peritoneal dialysis.