Initiation Condition of Hemodialysis Is Independently Associated with All-Cause Mortality in Maintenance Hemodialysis Patients: A Retrospective Study.

BACKGROUND: Despite the progression of dialysis techniques,  the mortality of hemodialysis (HD) patients is still high in China. Here, a retrospective study was performed to investigate the neglected risk factors of all-cause mortality during maintenance HD (MHD). METHODS: We investigated 117 MHD patients who died between 2011 and 2016 in the Second Xiangya Hospital of Central South University HD center. In order to analyze the risk factors of 48 months all-cause death, the methods of Kaplan-Meier and Cox regression were used. RESULTS: Multivariate analyses of adjusted age and gender showed that MHD patients with estimated glomerular filtration rate <7 or >10 mL/min/1.73 m2 and anemia (hemoglobin <100 g/L) at the initiation of dialysis are independently associated with the higher death risk. Using central venous catheter vascular access, cerebrovascular comorbidities, diabetes, low-flux dialyzer, and dialysis frequency ≤2 times weekly were also the independent risk factors of death within 48 months. CONCLUSIONS: This study indicated that the status of HD initiation is a risk factor of long-term survival in MHD patients, which were usually ignored for lacking of nephrology care prior and could potentially be identified and modified to improve the survival prognosis. Video Journal Club "Cappuccino with Claudio Ronco" at  https://www.karger.com/Journal/ArticleNews/223997?sponsor=52.

[Abnormal percentage of memory B cells in tonsils and peripheral blood in clinical progression of IgA nephropathy].

OBJECTIVE: To observe an abnormal expression of humoral immune response induced by memory B cells in tonsils and peripheral blood of patients with IgA nephropathy (IgAN), the variation of memory B cells after tonsillectomy, and to discover the role of tonsillectomy in IgAN. METHODS: In the study, 28 patients were diagnosed as IgAN via renal biopsy, and 27 patients suffering from chronic tonsillitis without nephritis and 10 normal human beings were selected as controls. The expression of memory B cells in the tonsils and peripheral blood was tested by flow cytometry, and the same method was used to test the variation of the expression of memory B cells in peripheral blood of patients with IgAN after tonsillectomy. RESULTS: In this study, higher percentages of memory B cells were observed in tonsil and peripheral blood of IgAN patients, which were 5.72%±5.26%, 4.92%±5.10%. After tonsillectomy, the percentage of memory B cells was 1.10%±0.65%, lower than that before tonsillectomy (P<0.05). Meanwhile, in tonsils and peripheral blood, the percentage of memory B cells varied with the variation of the urinary findings of the IgAN patients. CONCLUSION: The percentage of memory B cell in tonsils and peripheral blood could predict disease progression of IgAN to a certain extent.

Functional and structural alterations of peritoneum and secretion of fibrotic cytokines in rats caused by high glucose peritoneal dialysis solutions.

OBJECTIVE: To determine functional and structural alterations of peritoneum and fibrotic cytokines expression in peritoneal dialysis (PD) rats. METHODS: 28 Sprague-Dawley (S-D) rats were randomly divided into four groups and dialyzed with various solutions daily for four weeks: (1) no solution (CON group), (2) 0.9% Saline solution (NS group), (3) 1.5% Dianeal (LG group), (4) 4.25% Dianeal (HG group). Peritoneal equilibration tests, ultrafiltration function and effluent protein quantification were measured. Peritoneum morphology was studied and immunohistochemistry were performed for detection of transforming growth factor ß1 (TGF-ß1), connective tissue growth factor (CTGF), and fibronectin (FN) proteins. Reverse transcriptional-polymerase chain reaction was used to analyze the expression of TGF-ß1, CTGF mRNA. RESULTS: Administration of 4.25% Dianeal caused functional and structural changes of peritoneum, including protein loss through the transport process, decrease of peritoneal solute transport rate and ultrafiltration capacity. The collagen of peritoneum in the HG group was thicker than the other groups. The levels of CTGF, TGF-ß1, and FN proteins were significantly the highest in the HG group, followed by the LG group. The liner correlation analysis showed positive correlations between the levels of CTGF, TGF-ß1, and FN proteins and the collagen thickness. The expression of TGF-ß1 and CTGF mRNA in the HG group were significantly higher than those in the other groups and were indicated positive correlation. CONCLUSION: Using high glucose peritoneal dialysis solutions in rats may not only lead to processing of peritoneal fibrosis, which is promoted by ectopic expression of TGF-ß1, but also increase the expression of CTGF. CTGF is an important fibrotic media of peritoneal fibrosis in PD rats.

miRNA589 regulates epithelial-mesenchymal transition in human peritoneal mesothelial cells.

Background. microRNA (miRNA, miR) are thought to interact with multiple mRNAs which are involved in the EMT process. But the role of miRNAs in peritoneal fibrosis has remained unknown. Objective. To determine if miRNA589 regulates the EMT induced by TGFß1 in human peritoneal mesothelial cell line (HMrSV5 cells). Methods. 1. Level of miR589 was detected in both human peritoneal mesothelial cells (HPMCs) isolated from continuous ambulatory peritoneal dialysis (CAPD) patients' effluent and HMrSV5 cells treated with or without TGFß1. 2. HMrSV5 cells were divided into three groups: control group, TGFß1 group, and pre-miR-589+TGFß1 group. The level of miRNA589 was determined by realtime PCR. The expressions of ZO-1, vimentin, and E-cadherin in HPMCs were detected, respectively. Results. Decreased level of miRNA589 was obtained in either HPMCs of long-term CAPD patients or HMrSV5 cells treated with TGFß1. In vitro, TGFß1 led to upregulation of vimentin and downregulation of ZO-1 as well as E-cadherin in HMrSV5 cells, which suggested EMT, was induced. The changes were accompanied with notably decreased level of miRNA589 in HMrSV5 cells treated with TGFß1. Overexpression of miRNA589 by transfection with pre-miRNA589 partially reversed these EMT changes. Conclusion. miRNA589 mediates TGFß1 induced EMT in human peritoneal mesothelial cells.

Norcantharidin, a protective therapeutic agent in renal tubulointerstitial fibrosis.

Progressive renal tubulointerstitial fibrosis is a common final pathway of nearly all forms of chronic kidney disease. Many efforts have been done to arrest or prevent renal tubulointerstitial fibrosis but with little progress. Nowadays, few therapeutic agents are available in clinical use. Norcantharidin (NCTD) is of great benefit in anticancer treatment, by inducing cell apoptosis, inhibiting cell proliferation, in addition, blocking tumor metastasis and angiogenesis in cancer, whereas little attention is given to its relationship with other diseases. Our recent studies demonstrated that NCTD was protective against renal tubulointerstitial fibrosis both in vivo and in vitro. The underlying mechanisms may include modulation of TGF-ß1/Smad signal cascade, inhibition of protein serine/threonine phosphatases (PPP) as well as NF-κB. NCTD may be a promising therapeutic agent for renal tubulointerstitial fibrosis. In the present article, we will review the action of NCTD in renal tubulointerstitial fibrosis and discuss its possible mechanisms.

Evaluation of renal clinicopathological changes in IgA nephropathy by urinary podocytes excretion and podocalyxin expression.

OBJECTIVE: To explore the association of urinary podocyte excretion and renal expression of podocyte-specific marker podocalyxin (PCX) with clinicopathological changes in immunoglobulin A nephropathy (IgAN). METHODS: Morning urine samples from IgAN patients and healthy controls were collected. The expression of glomerular PCX was quantified in 50 IgAN patients diagnosed by renal biopsy. IgAN was classified based on the Lee's Grading system and scored according to the Katafuchi semiquantitative criteria. Morphological evaluation of podocyte was determined by electron microscopy. RESULTS: The amount of urinary podocytes in the IgAN patients was significantly higher than that in the healthy controls (p < 0.01). Pairwise comparison among Lee's grades of IgAN showed that the median of urinary podocytes in Lee's I-II group was lower than that in Lee's III, IV, and V groups (p < 0.05); group III lower than group V (p < 0.05). The positive rate of urinary podocytes was the highest in Lee's IV and V groups (100%), and lowest in Lee's I-II group (55%). Multiple comparison among groups of Lee's grades of IgAN showed that the glomerular PCX expression in Lee's I-II group was higher than that in Lee's III, IV, and V groups (p < 0.05); groups III and IV higher than group V (p < 0.05). The amount of urinary podocytes in IgAN patients was negatively correlated with PCX expression (r = -0.702, p < 0.01), but positively correlated with 24-h urinary protein (r = 0.465, p < 0.01) and glomerular (r = 0.233, p < 0.01) and renal tubular pathological scores (r = 0.307, p < 0.05). The glomerular PCX expression was negatively correlated with 24-h urinary protein (r = -0.367, p < 0.05) and glomerular (r = -0.560, p < 0.05) and tubular pathological scores (r = -0.377, p < 0.05). Electron microscopy showed significant changes in podocytes of IgAN, especially in the foot process. CONCLUSION: The amount of urinary podocyte can reflect the loss of podocytes in renal tissue, which may be a marker of IgAN progression.

Norcantharidin inhibits the expression of extracellular matrix and TGF-ß1 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-ß1 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-ß1, 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-ß1 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-ß1 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-ß1 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-ß1 was not associated with its inhibition of the CaN/NFAT pathway.

Norcantharidin inhibits proliferation and fibronectin expression of HK-2 cells induced by albumin in vitro.

The present study aims to observe the effects of NCTD (norcantharidin) on proliferation and FN (fibronectin) expression in human renal proximal tubular epithelial cell line (HK-2) induced by albumin in vitro. HK-2 cells were divided into control group, albumin group and different concentration of NCTD intervention groups. Proliferation of HK-2 cells was determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide], FN protein in culture media of HK-2 cells was examined by ELISA, and FN mRNA was analysed by RT-PCR (reverse transcription-PCR). We chose less than 5.0 mg/l of NCTD as the experimental concentration for the cytotoxicity test. MTT score was higher in the albumin group than in the control group (P<0.05). As compared with that of the albumin group, MTT score and FN protein concentration decreased, FN mRNA significantly down-regulated in NCTD intervention groups respectively (P<0.05). Our study showed that NCTD could inhibit the albumin-induced cell proliferation and FN expression in HK-2 cells, which might further prove the anti-fibrotic role of NCTD in proteinuria-associated tubulointerstitial damage.

Expression of CD19(+)CD5(+)B cells and IgA1-positive cells in tonsillar tissues of IgA nephropathy patients.

The hallmark of IgA nephropathy (IgAN) is the mesangial deposits of polymeric IgA. However, the source of IgA1 and the mechanism of deposition of IgA1 in the mesangium remain unknown. To better understand its pathogenesis, we investigated the expression of CD19(+)CD5(+)B cells and IgA1-positive cells in the tonsils of IgAN patients. Immunofluorescence was used to visualize the locations of CD19(+)CD5(+)B cells and IgA1-positive cells in the tonsils. In this study, it was demonstrated that CD19(+)CD5(+)B cells are usually found in germinal centers and in the capsule covering the upper parts of the nodules of lymphoid tissue (cap of the nodule). The expression of IgA1-positive cells in tonsil tissue can be seen in the cap of the nodule and subepithelial tissue. There is a significant relationship between IgA1 and CD19(+)CD5(+)B cells. The level of CD19(+)CD5(+)B cells is positively correlated to the severity of renal pathological changes. These findings suggest that CD19(+)CD5(+)B cells in the tonsils could have an impact on the pathogenesis of IgAN.

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-ß1, 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-ß1 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-ß1 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 NCTD's anti-tubulointerstitial fibrosis and its inhibition to CaN expression remains to be further elucidated.

p66Shc mediates high-glucose and angiotensin II-induced oxidative stress renal tubular injury via mitochondrial-dependent apoptotic pathway.

p66Shc, a promoter of apoptosis, modulates oxidative stress response and cellular survival, but its role in the progression of diabetic nephropathy is relatively unknown. In this study, mechanisms by which p66Shc modulates high-glucose (HG)- or angiotensin (ANG) II-induced mitochondrial dysfunction were investigated in renal proximal tubular cells (HK-2 cells). Expression of p66Shc and its phosphorylated form (p-p66Shc, serine residue 36) and apoptosis were notably increased in renal tubules of diabetic mice, suggesting an increased reactive oxygen species production. In vitro, HG and ANG II led to an increased expression of total and p-p66Shc in HK-2 cells. These changes were accompanied with increased production of mitochondrial H(2)O(2), reduced mitochondrial membrane potential, increased translocation of mitochondrial cytochrome c from mitochondria into cytosol, upregulation of the expression of caspase-9, and ultimately reduced cell survival. Overexpression of a dominant-negative Ser36 mutant p66Shc (p66ShcS36A) or treatment of p66Shc- or PKC-ß-short interfering RNAs partially reversed these changes. Treatment of HK-2 cells with HG and ANG II also increased the protein-protein association between p-p66Shc and Pin1, an isomerase, in the cytosol, and with cytochrome c in the mitochondria. These interactions were partially disrupted with the treatment of PKC-ß inhibitor or Pin1-short interfering RNA. These data suggest that p66Shc mediates HG- and ANG II-induced mitochondrial dysfunctions via PKC-ß and Pin1-dependent pathways in renal tubular cells.

microRNA-630 Regulates Underglycosylated IgA1 Production in the Tonsils by Targeting TLR4 in IgA Nephropathy.

IgA nephropathy (IgAN) is the most common primary glomerular disease. The characteristic pathology involves immune complexes formed by the deposition of IgA1 and underglycosylated IgA1 aggregates in the mesangial area, which may be accompanied by the deposition of IgG and/or IgM and complement components. However, the molecular mechanisms of IgAN remain unclear. In the present study, microarray analysis showed that the expression of microRNA-630 (miR-630) was significantly reduced in palatal tonsils from IgAN patients compared with chronic tonsillitis. Additionally, bioinformatic analysis showed that Toll-like receptor 4 (TLR4) was the predicted target gene of miR-630 and was regulated by miR-630. When miR-630 was overexpressed in palatal tonsil mononuclear cells from IgAN patients, the expression of TLR4 was reduced and the content of IgA1 in the cell culture supernatant was decreased, and the level of galactosylation in the IgA1 hinge region was increased. Moreover, immunohistochemical analysis showed that the expression of TLR4 in IgAN patients was significantly increased. After knocking down the expression of TLR4, both the concentration of IgA1 and the binding force of IgA1 with broad bean lectin were significantly reduced in IgAN. Furthermore, the mechanism study demonstrated that TLR4 might regulate the expression of IL-1ß and IL-8 through NF-κB signaling pathway to modulate the concentration of IgA1 and the glycosylation level of IgA1. This interesting finding may offer new insight into the molecular mechanism of IgAN.

The protective role of telmisartan against nephrotoxicity induced by X-ray contrast media in rat model.

BACKGROUND: Contrast-induced nephropathy is a serious complication of diagnostic and interventional procedures. PURPOSE: To evaluate the nephrotoxicity of high- and low-osmolar contrast media (HOCM, LOCM) on kidneys in Sprague-Dawley rats. Telmisartan was administered to confirm its protective role against nephrotoxicity induced by contrast media. MATERIAL AND METHODS: Sixty male rats were randomly divided into six groups (n=10/group). Glycerin was given to all rats except controls to induce renal injury. HOCM (diatrizoate) or LOCM (iohexol) (10 ml/kg b.w., 300 mg I/ml) was given through a caudal vein. Serum creatinine level was measured by an automatical biochemical analyzer. Caspase-3 activity and Angiotensin II (Ang II) level of renal tissue were detected by fluorometric method and radioimmunoassay, respectively. The renal injury was also assessed by hematoxylin and eosin and TdT-mediated deoxyuridine nick end-labeling staining. RESULTS: In diatrizoate-injected rats, serum creatinine level was increased (P<0.001). There was no significant difference between iohexol animals and glycerol controls in the level of serum creatinine. The renal caspase-3 activity and Ang II levels in HOCM and LOCM groups were higher than those in glycerol control group (P<0.001). The percentage of apoptotic tubular cells and pathological scores were lower in the iohexol animals than that in the diatrizoate animals (P<0.001). In the groups pretreated with telmisartan, no increase in the levels of serum creatinine, renal Ang II, and caspase-3 activity was observed (P>0.05). The renal injuries induced by contrast media were alleviated. CONCLUSION: Both HOCM (diatrizoate) and LOCM (iohexol) could cause renal tubular cell apoptosis in the kidneys damaged by glycerin. LOCM was less toxic to rat kidneys than HOCM. Caspase-3 and Ang II might play a role in renal tubular cell apoptosis induced by contrast media. Telmisartan protected the renal tissue from nephrotoxicity induced by contrast media.

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-kappaB (NF-kappaB) 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-kappaB 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-kappaB 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.

Arkadia-Smad7-mediated positive regulation of TGF-beta signaling in a rat model of tubulointerstitial fibrosis.

BACKGROUND/AIMS: Upregulation of transforming growth factor beta (TGF-beta)/Smad signaling has been implicated in the primary pathogenesis of renal fibrosis. The ubiquitin-proteasome pathway has an important influence on TGF-beta signaling through regulating Smad degradation. As E3 ubiquitin ligases, both Arkadia and Smurf2 are involved in this prosess. In this study, we focused on Arkadia, Smurf2, Smad7, and TGF-beta type I receptor (TbetaRI), principal molecules in the regulation of TGF-beta signaling, to understand the regulatory mechanism of ubiquitin-proteasomal degradation of TGF-beta signaling in the pathogenesis of renal fibrosis. METHODS: A unilateral ureteral obstruction (UUO) model was employed, and sham-operated rats were used as controls. Renal lesions and the expression of Arkadia, Smurf2, Smad7, TbetaRI, TGF-beta1, and type 1 collagen (COL-1) were detected by Western blot, immunoprecipitation, immunohistochemistry, and/or reverse transcription-polymerase chain reaction. RESULTS: The results indicated progressive tubulointerstitial fibrosis, high expression levels of Arkadia, Smurf2, TbetaRI, TGF-beta1 mRNA, type 1 collagen mRNA, and Smad7 mRNA, and low levels of Smad7 protein in the kidneys of rats with unilateral ureteral obstruction, in which Smurf2 interacted with both Smad7 and TbetaRI, and Arkadia only interacted with Samd7 but not with TbetaRI. CONCLUSION: Reduction of Smad7 resulting from ubiquitin-dependent degradation may be mainly attributed to Arkadia, and Arkadia-Smad7-mediated positive regulation of TGF-beta signaling may play a promoting role in the progression of tubulointerstitial fibrosis.

Mast cell, a promising therapeutic target in tubulointerstitial fibrosis.

Tubulointerstitial fibrosis is a final common pathway to the eventual structural desolation of kidneys. However, the mechanism involved in this phenomenon is still poorly understood, and current therapies are ineffective or only marginally effective. Mast cell has a variety of physiological and pathological functions through the production of heparin, histamine, neutrophil chemoattractants, immunoregulatory cytokines, and mast cell-specific serine proteases tryptase and chymase. The survival and proliferation of mast cell are dependent upon stem cell factor. Presently, mast cells are known to participate in the pathogenesis of tubulointerstitial fibrosis in many kidney diseases. Several therapeutic approaches to inhibit mast cell activation have already demonstrated some clinical utility in tissue fibrosis or inflammatory diseases such as the use of mast cell stabilizers, inhibitors of tryptase or chymase, blockade of stem cell factor and anti-IgE therapy. We hypothesize that mast cell has a significant role in the progression of tubulointerstitial fibrosis, thus the treatment strategies based on mast cell appear to be promising in these conditions. Development of these novel therapeutic approaches will enable us to target any types of renal disease.

Inhibition effect of small interfering RNA of connective tissue growth factor on the expression of vascular endothelial growth factor and connective tissue growth factor in cultured human peritoneal mesothelial cells.

BACKGROUND: The peritoneum response to peritoneal dialysis can lead to fibrosis. The transforming growth factor beta1 (TGF-beta1) plays a key role in regulating tissue repair and remodelling after injury. Connective tissue growth factor (CTGF), a downstream mediator of TGF-beta1 inducing fibrosis, has been implicated in peritoneal fibrosis. Vascular endothelial growth factor (VEGF) plays a key role in angiogenesis that can hasten peritoneal fibrosis. In this study, we investigated the effect of small interfering RNA (siRNA) of CTGF by pRETRO-SUPER (PRS) retrovirus vector on the expression of CTGF and VEGF in human peritoneal mesothelial cells. METHODS: Retrovirus producing CTGF siRNA were constructed from the inverted oligonucleotides and transferred into packaging cell line PT67 with lipofectamine, and the virus supernatant was used to infect human peritoneal mesothelial cell (HPMC). The cells were divided into seven groups: low glucose DMEM, low glucose DMEM + TGF-beta1 5 ng/ml, low glucose DMEM + TGF-beta1 5 ng/ml + PRS-CTGF-siRNA(1-4) and low glucose DMEM + TGF-beta1 5 ng/ml + PRS. The expression of CTGF and VEGF were measured by semiquantitative RT-PCR and Western blot. RESULTS: Low levels of CTGF and VEGF were detected in confluent HPMCs. Following stimulation with TGF-beta1, the levels of CTGF and VEGF were significantly upregulated (P < 0.01). Introduction of PRS-CTGF-siRNA(1-4) resulted in the significant reduction of CTGF mRNA and protein, and VEGF mRNA (P < 0.01), especially in groups PRS-CTGF-siRNA1 and PRS-CTGF-siRNA4. The introduction of PRS void vector did not have these effects (P > 0.05). CONCLUSIONS: The expression of CTGF siRNA mediated by PRS retrovirus vector can effectively reduce the level of CTGF and VEGF induced by TGF-beta1 in cultured HPMCs. This study may provide potential therapeutic strategies to prevent the peritoneal fibrosis.

[Effect of mannitol on vasoactive substances].

OBJECTIVE: To observe the changes of vasoactive substances in rabbits administered with mannitol at different dosages and to investigate the mechanism of acute renal failure (ARF) induced by massive mannitol administration. METHODS: Eighteen healthy male New Zealand rabbits were randomly divided into 3 groups: a minor mannitol group (n=6, mannitol 8 g/kg within 2 hours), a control group (n=6, saline of the same volume), and a massive mannitol group with free water taking (n=6, mannitol 40~60 g/kg within 3 days). The changes of renin, angiotensin-I (ang-I), angiotensin-II (ang-II), endothelin (ET), and atrial natriuretic factor(ANF) in the serum were observed. RESULTS: No significant changes in the renin, ang-I, ang-II, ET, and ANF in the serum were found between the minor mannitol group and the saline control group (P> 0.05). In the massive mannitol group with free water taking, renin, ang-I, and ang-II in the serum increased significantly compared with the other 2 groups; ET in the serum decreased significantly compared with the saline control group (P< 0.05); no significant changes in the ANF in the serum were found compared with the other 2 groups(P> 0.05). CONCLUSION: ARF induced by massive mannitol administration is associated with a significant change of vasoactive substances.

[Inhibitive effect of troglitazone on TGF-beta(1) and fibronectin expression in human peritoneal mesothelial cells].

OBJECTIVE: To investigate the effect of the peroxisome proliferator activated receptor-gamma (PPAR-gamma) agonist troglitazone on TGF-beta(1) and fibronectin (Fn) expression in human peritoneal mesothelial cells (HPMCs). METHODS: HPMCs were cultured from human omentum by an enzyme digestion method, growing in medium containing 30 mmol/L D-glucose. TGF-beta(1) and Fn expression were measured in HPMCs in the presence and absence of 15 micromol/L troglitazone. The mRNA expressions of PPAR-gamma,TGF-beta(1) and Fn were determined by semi-quantification reverse transcriptive PCR (RT-PCR). The protein of TGF-beta(1) was determined by enzyme-linked immunosorbent assay (ELISA) and proteins of PPAR-gamma and Fn were determined by Western blot. RESULTS: The mRNA and protein expression of TGF-beta(1) and Fn were significantly increased in HPMCs stimulated with 30 mmol/L D-glucose compared with the control group with F12 media (P<0.01). Obvious decrease of TGF-beta(1) was found in troglitazone(15 micromol/L) treated group compared with group stimulated with 30 mmol/L D-glucose (P<0.05). Exposure of HPMCs to troglitazone reduced the Fn secretion (P<0.05). CONCLUSION: Troglitazone reduced the expression of TGF-beta(1) in HPMCs stimulated by 30mmol/L D-glucose, and reduced Fn production. PPAR-gamma agonists may have a specific role in ameliorating the course of progressive peritoneal fibrosis under long-term peritoneal dialysis states.

[Construction and activity evaluation of pSUPER RNAi system that inhibits Smad3].

OBJECTIVE: To construct the expressing vector of siRNA which can inhibit the Smad3 activity. METHODS: Sixty-four bases of 2 pair oligos for hairp in RNA expression which targeted Smad3 gene were chemically synthesized and annealed. pSUPER vector was linearized with BgL II and Hin d III treated with alkaline phosphatase (CIP). Anneled oligos were inserted into the downstream of the treated pSUPER's pol III H1 promoter to construct RNAi plasmid (pSUPER Smad3). Oligos with a scrambled sequence were used as a negative control. pSUPER Smad3 was transfected into human renal tubular epithelial cells (HKC). RESULTS: Recombinant pSUPER Smad3 vector was identified by the digestion with Eco R I and Hin d III, and confirmed by the sequencing analysis with T3 primer. Sixty-four bases had been inserted into the expected site. Furthermore, the insertion sequence was exactly corrected. The activity evaluation indicated that mRNA and protein of Smad3 but not Smad2 were inhibited by pSUPER Smad3 in HKC. CONCLUSION: The pSUPER Smad3 system has been constructed successfully, and has high inhibition and specificity in vitro.