Objective To investigate the effects of nuclear factor κB(NF-κB) antisense oligodeoxynucleotides (AS-ODNs) on transforming growth factor β1 (TGF-β1)-induced epithelial mesenchymal transition (EMT) in human renal tubular epithelial cells. Methods NF-κB AS-ODNs were transferred into the human renal tubular epithelial cells (HK-2), and the cells were stimulated by 10 μg/L TGF-β1 for 24 hours. The expression of NF-κB mRNA and α-SMA mRNA were measured by RT-PCR. α-SMA protein expression was assessed by fluorescence spectrum.Results TGF-β1 significantly up-regulated the expression of NF-κB mRNA, which was 8 folds of blank control (P<0.01). TGF-β1-indueed epithelial mesenchymal transition was inhibited by NF-kB AS-ODN and the NF-KB mRNA expression of AS-ODNs was decreased by 75%(P<0.05).The expression of α-SMA mRNA and protein was also down-regulated obviously (P<0.05).Conclusion NF-κB AS-ODN can inhibit the expression of NF-κB and the epithelial-mesenchymal transition, which may be a new therapeutic strategy against tubulointerstitial fibrosis.

Objective To investigate the correlation of infiltration of mast cells in kidney with renal interstitial fibrosis, expression of TGF-β1 and stem eel] factor (SCF) in rat models withprotein-overload nephropathy. Methods Sixty uninephrectomized SD rats were randomly divided into model group [intraperitoneal injections of bovine serum albumin (BSA)] and control group (intraperitoneal injections of equal volume of saline). Ten rats from both groups were sacrificed respectively at week 3, 7 and 11 after injection. 24 h urinary protein and serum biochemistry of these SD rats at the time of sacrifice were measured. The intensity of mast cell infiltration was examined by toluidine blue (TB) staining and immunohistochemistry using a monoclonal anti-MC chymase antibody. The expression of TGF-β1 and SCF was detected byimmunohistochemistry, using a monoclonal mouse anti-rat TGF-β1 antibody and a polyclonal rabbstanti-rat SCF antibody. Results Severe proteinuria was induced in the rats by BSA injectionpeaked at week 7 [(199.1±98.4) mg/d] after the BSA injection and gradually decreased until week11 [(133.7±67.8) mg/d]. Renal injury was accompanied with chymase-postitive and TB-postitive mast cell infiltration, in close proximity to areas of interstitial fibrosis. With aggravation oflesions degree, the number of mast cells increased,the difference between the modal rats and control rats was significant (P<0.05). Immunostainahle expression of SCIF and TGF-β1 was detected in tubular as well as interstitial cells, and increased with the BSA injection. The difference between the model rats and control rats was significant (P<0.05). Mast cells were positively correlated with interstitial fibrosis (r=0.772, P<0.01), expression of TGF-β1 (r=0.521, P<0.01) and SCF(r=0.916,P<0.01). Conclusions Increased infiltration of mast cells is involved in interstitial fibrosis of rats with protein-overload nephropathy. Proteinuria may attract mast cells to kidney by chemot actions of SCF,and mast cells may contribute to the development of renal fibrosis by secreting chymase and increasing expression of TGF-β1.

Objective To examine the association of urinary neutrophil gelatinase-associated lipocalin(NGAL) and interleukin 18(IL-18) with acute kidney injury (AKI) in patients after cardiac surgery. Methods Thirty-three patients undergone cardiac surgery were divided into AKI group and non-AKI group according to the AKI criteria. The Scr, urinary NGAL and IL-18 were measured at different time points. Results Nine of 33 patients (27.27%)developed postoperative AKI, and Scr concentration in AKI group reached its peak within 12-48 hours after cardiac surgery. Urinary concentrations of NGAL and IL-18 at 2 h and 4 h after cardiac surgery were significantly higher than those before operation in AKI patients (P<0.01). The urinary concentrations of NGAL at each time point and that of IL-18 at 2 h and 4 h after cardiac surgery in AKI patients were significantly higher than those in non-AKI patients. After correction by urinary creatinine, the differences of NGAL/Ucr and IL-18/Ucr ratios were still significant (P< 0.01). For concentrations of urinary NGAL, IL-18 and ratios of NGAL/Ucr, IL-18/Ucr at 2 h after surgery, sensitivities and specificities were good with cutoff values at 250 μg/L, 250 μg/mmol and 1800 ng/L, 1800 ng/mmol, respectively. Urinary concentration of NGAL at 2 h after cardiac surgery was positively correlated with Scr at 12 h postoperation in AKI group (r=0.638, P<0.05).Conclusions The incidence of AKI in patients after cardiac surgery is quite high. Urinary concentrations of NGAL, IL-18 and ratios of NGAL/Ucr, IL-18/Ucr at 2 h after cardiac surgery are the early diagnostic markers for AKI, among which urinary NGAL/Ucr is the most sensitive one.

Objective To explore the mechanism of protecting cells from ischemic reperfusion injury by constructing specific small interference RNA (siRNA) to inhibit Na+-H+exchanger-1 (NHE-1) expression in human renal tubular epithelial cell (HKC). Methods The siRNA was designed and synthesized based on human NHE-1 complete sequence,and was transfected into HKC.The irrespective siRNA transfected group was used as control.The cells were treated with 10 μmol/L antimyein A to induce ischemia and anoxyaemia environment.NHE-1expression was examined by RT-PCR and Western blot.The intraeellular pH (pHi),Ca2+ or Na+ concentrations were detected by BCECF/AM,Fluo-3/AM and SBFI-AM,respectively,combining with laser eonfocal assay system.Nucleic morphology was determined by Hoechst 33342.Cellular apoptosis was examined by Annexin V/PI staining and flow eytometry.Fluorescent probe JC-1 was used to detect the change of mitechondrial transmembrane potential. Results The specific siRNA could efficiently inhibit NHE-1 expression in HKC.Compared with the irrespective siRNA transfected group,the mRNA and protein expression of NHE-1 was significantly down-regulated in NHE-1 siRNA transfeeted group (all P＜0.05).After treatment with antimyein A,the mRNA and protein expression of NHE-1 was significantly up-regulated in both groups,however,it was less than that in irrespective siBNA transfected group.At the same time,the ratio of apoptosis decreased (8.9% +2.9% vs 18.8%±3.2% , 17.4%±3.6% ,P＜0.05) and mitochondrial transmembrane potential rose significantly in NHE-1 siRNA transfected group as compared to irrespective siRNA transfected group and antimycin A group.The intracellular Na+,H+ and Ca2+concentrations increased in NHE-1 siRNA transfected group treated with antimyein A,but their levels were lower than those in irrespective siRNA transfected group with the same treatment(P＜0.05). Conclusions The synthesized siBNA can inhibit the expression of NHE-1 and can protect HKC from isehemia reperfasion injury induced by antimyein A.The mechanism might be via suppressing the expression of NHE-1 to delay intracelluar Na+ accumulation,attenuate intracellular Ca2+ overloading,and inhibit the decrease of mitechondrion transmembrane potential and reduce cellular apoptosis.

Objective To investigate the effects of rapamycin on renal tissue and function of rats with protein overload nephropathy and to explore the protective mechanism of losartan. Methods Experimental rat models with protein overload nephropathy, induced by intraperiotoneal injection of BSA (2. 0 g/d)into female Wistar rats, were divided into three groups: control group, rapamycin group(injected intraperitoneally with rapamycin) and losartan group(injected intraperitoneally with rapamycin and given orally with losartan). At different time points, the quantity of 24-hour urine protein and renal function were measured, and the morphologic changes of renal tissues were evaluated by HE staining and electron microscope. Results Both at day 7 and day 14, rats received BSA developed intense proteinuria. At day 7, compared with control group, 24-hour proteinuria increased markedly in rapamyein group (P<0.05). Nevertheless,proteinufia was notably alleviated in losartan group (P<0.05). At day 14, 24-hour-urine protein of rapamycin group was also significantly higher than that of the losartan group (P<0.05), but therewas no significant difference between control group and losartan group (P>0.05). Proteinuria and intratubular albumin cast formation were alleviated notably in losartan group. The fusion of focal podocytes in rapamycin group was obvious in comparison with control group. Conclusions Rapamycin can agrravate proteinuria in rats with protein overload nephropathy through changing the barrier of glomerular filtration by damaging podocytes. Furthermore, losartan can alleviate severe proteinuria induced by rapamycin.

Objective To investigate the role of C-Jun N-terminal kinase (JNK) in epithelial mesenchymal transition (EMT) induced by transforming growth factor β1 (TGF-β1) in rat peritoneal mesothelial cells(RPMCs). Methods RPMCs were harvested from the peritoneum of male Sprague-Dawley rats, then cultured in DMEM/F12 medium with 15% (V/V) FBS. After stimulation with TGF-β1, the expression of a-smooth muscle actin (α-SMA), E-cadherin and collagen I were detected in RPMCs. In some groups, the ceils were pretreated with SP600125, a specific inhibitor of JNK, for 4 hours before incubation with TGF-β1. The protein expression of phosphorylated JNK was detected by Western blotting. The mRNA and protein expression ofα-SMA, E-cadherin and collagen I were examined with RT-PCR and Western blotting, respectively.The intracellular distribution and expression of α-SMA was determined by indirect immunofluorescence. Results TGF-β1 could significantly increase the expression of α-SMA and collagen I, and decrease the expression of E-cadherin in RPMCs. TGF-α1 could stimulate the expression of phosphorylated JNK at 5 minutes with the peak at 10 minutes (P<0.01). The addition of SP600125 effectively inhibited TGF-β1-induced high expression of α-SMA and collagen I (P<0.05), and prevented TGF-β1-induced down-regulation of E-cadherin expression in RPMCs (P<0.05). The indirect immunofluorescence showed that the expression of intracellular α-SMA in RPMCs stimulated by TGF-β1 for 48 h increased significantly, which could be inhibited by SP600125. Conclusions JNK regulates epithelial mesenchymal transition induced by TGF-β1 in rat peritoneal mesothelial cells. JNK inhibitor may be used as a novel therapeutic agent for peritoneal fibrosis.

Objective To investigate the protective effects of atorvastatin on hyperphosphate-induced rat vascular smooth muscle ceils (RVSMCs) calcification and to discuss the mechanism. Methods RVSMCs were placed in various culture media, including normal phosphate medium, high phosphate medium, ZVAD-FMK medium and atorvastatin medium.Calcium content and cell protein content were quantified by the o-cresolphthalein complexone method and BCA protein assay respectively. Calcification was visualized by yon Kossa staining. And cell apoptosis was quantified by ELISA. Results (1)At day 3, 6, 9, RVSMCs calcification occurred more frequently in high phosphate medium than that in normal phosphate medium (P<0.05). (2)At day 6, RVSMCs calcification was significantly inhibited in 1.0 μmol/L and 2.0 μmol/LZVAD-FMK medium (P<0.05). And in 10 nmol/L and I00 nmol/L statin medium, RVSMCscalcium deposition significantly decreased (P<0.05). (3)RVSMCs apoptosis and calcification occurredfrequently in high phosphate medium. And atorvastatin significantly inhibited RVSMCs apoptosisboth in long-term and short-term (P<0.05). Conclusions Hyperphosphate can induce the calcium deposition of RVSMCs in vitro. Atorvastatin protects RVSMCs from phosphate-induced calcification by inhibiting apoptosis.

Objective To explore the effects of peroxisome proliferator-activated receptorγ (PPARγ) agonist rosiglitazone and 15-deoxy-delta-12,14-prostaglandin J2 (15d-PGJ2) on the expression of PPARγ, toll-like receptor 4 (TLR4) and the activation of STAT1 as well as the local inflammation reaction of abdominal cavity in sprague dawley (SD) rats with peritoneal dialysis- related acute peritonitis induced by lipopolysaccharide (LPS). Methods Twenty-four male SD rats were equally randomized to four groups(n=6 each): control group, injected with 4.25% dextrose peritoneal dialysate (PDF) via abdominal cavity(90 ml/kg); LPS group, injected with LPS(1 mg/kg) via abdominal cavity 4 hours later follewed by PDF injection; rosiglitazone plus LPS group (Rosi group), preconditioned with rosiglitazone (20 mg·kg-1·d-1) by intragastric way for 3 days, then injected with LPS and PDF via abdominal cavity; 15d-PGJ2 plus LPS group (15d-PGJ2 group), preconditioned with 15d-PGJ2 (0.3 mg·kg-1·d-1)via abdominal cavity injection for 3 days, then injected with LPS and PDF via abdominal cavity. The rats were killed 4 hours after PDF injection, IL-6 level in abdominal dropsy was determined by ELISA. Peritoneum tissue was stained by Masson. Leucocyte count in abdominal dropsy was performed. The mRNA expression of PPARγ and TLR4 in peritoneum tissue was determined by RT-PCR; the protein expression of PPARγ, TLR4, p-STAT1 and STAT1 in peritoneum tissue was analyzed by Western blot. Results IL-6 level of abdominal dropsy in LPS group [median 268.53 (range 201.87-335.19) ng/L] was significantly higher than that of control group [median 147.62 (range 130.60-164.64) ng/L] (P<0.01). The IL-6 level of abdominal dropsy in Rosi group [median 110.20 (range 77.60-142.80) ng/L] was significantly lower than that of LPS group (P<0.05). Compared to that of control group, the edematous degree of peritoneum in LPS group was significantly severer, meanwhile, mRNA and proteins expression of PPARγ and TLR4 in rat peritoneum were also significantly higher (P<0.05, P<0.01). Compared to that of LPS group, the edematous degree of peritoneum in Rosi group was lighter, the expression of PPARγ and TLR4 mRNA was significantly up-regulated (P<0.05), meanwhile their proteins expression was down-regulated (P<0.05); and in 15d-PGJ2 group, the edematous degree of peritoneum, the expression of PPARγ mRNA and protein was also decreased (P<0.05), but TLR4 mRNA expression was up-regulated (P<0.01), however, its protein expression was down-regulated (P<0.05). There were no significant differences in leucocyte count of abdominal dropsy among the four groups. The p-STAT1 expression in the rats peritoneum induced by LPS was markedly increased by both rosiglitazone and 15d-PGJ2 (P<0.01). Conclusions Both rosiglitazone and 15d-PGJ2 can down-regnlate the inflammatory reaction in rat peritonitis induced by LIPS, which may be involved in modulating the expression of associated functional protein during LPS signal pathway.

Objective To observe the changes of CD4+CD25+ regulatory T cells, Foxp3 mRNA and soluble interlukin 2 receptor (sIL-2R) in the peripheral blood of kidney transplantation recipients and to evaluate their effect on the diagnosis of acute rejection. Methods Forty-two renal transplant recipients and 30 healthy controls were enrolled in this study. CD4+CD25+ regulatory T cells proportion, Foxp3 mRNA and sIL-2R of pre-transplantation and those of day 7,14, 28, 56 of post-transplantation were measured by flow cytometer, fluorescent quantization PCR and enzyme-linked immunosorbent assay (ELISA), respectively. Biochemistry appliance was used to detect serum creatinine. The diagnosis of acute rejection in transplanted kidney was based on the clinical symptoms, the laboratory examinations, Doppler ultrasound and biopsy. Results (1)At day 7, 14, 28, 56 of post-transplantation, CD4+CD25+ regulatory T ceils proportion, Foxp3 mRNA level in acute rejection group were significantly decreased compared with those in non-acute rejection group. (2) There were significant differences of peripheral blood CD4+CD25+ regulatory Tcells[(9.22±3.53)% vs (6.09±1.99)%, P<0.01], Foxp3 mRNA[(0.82±0.36)×10-3 vs (0.50±0.28)×10-3, P<0.01] and sIL-2R levels [(856.30±108,24) U/ml vs (247.35±11.24) U/ml, P<0.01]between patients of pre-transplantation and healthy control group. (3)Plasma CD4+CD25+ regulatory T cells [(16.53±4.14)%] and the expression of Foxp3 mRNA [(4.97±1.94)×10-3] was significantly increased, but sIL-2R level [(463.72±31.23) U/ml] was significantly decreased as the transplanted renal function was restored (all P<0.01). (4) Plasma CD4+CD25+regulatory T cells [(12.18~2.86)%] and the expression of Foxp3 mRNA [(3.15±1.22)×10-3] was significantly decreased (P<0.01), and sIL-2R level [(748.36±115.41) U/ml] was significantly increased (P<0.01) when acute rejection occurred. The above changes had an earlier onset than the change of Scr. (5)The percentage of CD4+CD25+ regulatory T cells was positively correlated with the Foxp3 mRNA level (P<0.01), but was not correlated with sIL-2R level in all the patients. Conclusion The measurement of these markers in peripheral blood may be an important guideline to the diagnosis and prognosis of acute rejection in renal transplant recipients.

Objective To report the clinicopathological features of 2 cases of nephronophthisis-medullary cystic kidney disease (NPH-MCKD). Methods The clinical data and pathological changes of renal biopsy in two patients of NPH-MCKD from our hospital were analyzed, and associated literatures were reviewed simultanously. The clinicopathological featuresand diagnosis of NPH-MCKD were discussed. Results Two adolescent patients were admitted to our hospital for indolent renal insufficiency, polyuria accompanied by polydipsia as first signs.Urine analysis showed low specific density urine, mild proteinuria, and few formed elements in urinary sediments. The ability of urine concentration and acidification was decreased. Familial history of renal disease and extra-renal lesions were not found. Renal ultrasound presented an increased echogenicity with diminished cortico-meduUary differentiation, and multiple small cysts in renal corticomedullary border were identified in one case by computed tomography. Pathological examination of renal biopsy revealed diffuse tubular interstitial lesion which was characterized by the triad of tubular basement disintegration, tubular atrophy with cyst development, and interstitial fibrosis. Some of glomerular sclerosis occurred. Cyst development at the corticomedullary border of the kidneys was the specific feature of NPH-MCKD. Conclusions Young patients with impaired tubular function should be suspected of NPH-MCKD. Renal ultrasound or computed tomography can provide an important clue. Multiple renal cysts at the corticomedullary border identified by renal biopsy can be a diagnostic indication for NPH-MCKD.