Objective To study the characteristics of glomerular filtration rate (GFR) and its influential factors in type 2 diabetes mellitus at different stages of albuminuria. Method GFR was measured in 630 cases of type 2 diabetes mellitus between 2002 and 2005 by plasma disappearance of 99m-techmetium-diethylene- triamine- penta-acetic acid (99mTc-DTPA). Body mass index (BMI), blood pressure, plasma glucose, HbA1c, Scr, BUN, uric acid (UA), profile of plasma lipid and 24 h-urinary albumin excretion (24 h-UAE) were also measured. All the patients were divided into 3 groups according to their 24 h-UAE: normoalbuminuric group (group A, 24 h-UAE<30 mg), microalbuminuric group (group B, 24 h-UAE from 30 mg to 300 mg) and macroalbuminuric group (group C, 24 h-UAE>300 mg). Results (1) The mean GFR was (99.8±26.3) ml/min, (96.1±31.2) ml/min and (69.7±29.8) ml/min in A, B and C groups respectively. The GFR in group C was significantly lower than that in group A and group B(P<0.01). (2) Negative correlations were found between GFR and age in all these groups (group A r= -0.533, group B r=-0.612 and group C r=-0.412，respectively, P<0.01）. (3) In each group, GFR of patients with hypertension was significantly lower than that of patients without hypertension（P<0.05）. (4) The Pearson correlation analysis adjusted by age showed that GFR was negatively correlated with 24 h-UAE in group B and group C (r=-0.283 and -0.24 respectively, all P<0.05). The multiple stepwise regression analysis showed that 24 h-UAE was the major influential factor of GFR in these 2 groups. Conclusions Measurement of both GFR performed by non-traumatic plasma disappearance of 99mTc-DTPA method and UAE provides a more precise evaluation on the the development and progression of diabetic nephropathy. Albuminuria should be controlled, especially in microalbuminuric stage.

Objective To report 2 cases of HIV infection complicated with kidney disease for the first time in China. Methods The clinical manifestation of 2 HIV-seropositive male patients and pathological findings by renal biopsy were collected. Results The HIV-1 seropositive male patient was 69 years old. He was diagnosed as nephrotic syndrome （NS） complicated with renal insufficiency. Renal biopsy revealed characteristic focal segmental glomerular sclerosis（FSGS）, non-collapsing, tubulointerstitial prominent lymphocytic infiltration. These characteristics were partially compatible with human immunodeficiency virus-associated nephropathy (HIVAN). According to NS and normal range of CD4, he was recommended with steroid and immunosuppressive therapy while absent of highly active anti-retroviral therapy (HAART). During one-year followed up, he had experienced several episodes of severe infection without remission of NS. After immunosuppressive therapy withdrawal and counterchecked low level of CD4, he received HAART for HIV treatment. The other patient was a hemophile-infected AIDS complicated with chronic hepatitis C， haemophilia A(lack of factor Ⅷ)， and diabetes mellitus（DM）. Proteinuria and chronic renal failure occurred after 1 year of HAART. During the therapy period, the renal function was impaired progressively and irreversibly entered into ESRD at the 3rd year of HAART. Conclusions The clinical and pathological manifestation of HIV infection complicated with kidney disease shonld be known in China. For HIV-infected kidney impaired patients suspected of HIVAN or other pathological pattern, it is important to establish the diagnosis and treatment by renal biopsy.

Objective To examine the effects of connective tissue growth factor (CTGF) on cell proliferation and production of collagen type Ⅰin cultured rat cortical myofibroblasts，and to investigate the role of ERK1/2 siganling pathway. Methods Myofibroblasts were obtained from normal rat renal cortex. 5’-bromodeoxyuridine (BrdU) incorporation assay and cell counting were used to detect cell proliferation. Western blot analysis was used to detect the levels of collagen typeⅠin the supernatant medium and the activation of ERK1/2 signaling pathway in cultured myofibroblasts. Results CTGF could induce the proliferation of myofibroblasts in a dose- and time-dependent manner. Cell number of 100 μg/L CTGF at day 4 was 1.6 folds of control group(P<0.05). Incubation with 100 μg/L CTGF also significantly increased secretion of collagen type I in the supernatant medium compared with control group (2.6±1.2 folds over control, P<0.01). ERK1/2 activation occurred as early as 5 minutes following 100 μg/L CTGF treatment, and persisted till 15 minutes later, and then declined back to the basal level after 30 minutes. Pretreatment with 50 μmol/L PD98059, a specific inhibitor of ERK1/2 pathway, abolished the effects of CTGF-induced cell proliferation (7%±5% vs 85%±7%, P<0.01) and CTGF-increased secretion of collagen type I (1.0±0.1 vs 1.6±0.3, P<0.05). Conclusion CTGF promotes cell proliferation and secretion of collagen type I through ERK1/2 pathway in primary cultured rat myofibroblasts.

Objective To investigate the changes of protein expression in rat kidney proximal tubular epithelial NRK52E cells during the epithelial to mesenchymal transitions (EMT) process. Methods Comparative proteomics was applied. Proteins extracted from NRK52E cells(three plates treated with TGF-β1 and three plates without treatment) were separated by two-dimensional gel electrophoresis(2-DE). Comparative analysis of 2-DE protein patterns between the two groups were carried out using computerized image analysis. Selected proteins exhibiting significant alternations were identified by mass spectrometry. Western blotting and RT-PCR were performed to examine the expression of the candidated proteins. Results The expression of 22 proteins including transgelin, ARP2/3 complex 21 000 subunit, destrin, ATP synthase α, ubiquitin-conjugating enzyme 9 (Ubc9), and malate dehydrogenase involved in the organization of the cytoskeleton, energy metabolism and post-translational modification were altered during the EMT process. Western blotting and RT-PCR confirmed the differences of several altered proteins, which was consistent with the findings of 2-DE. Conclusion TGF-β1 induces specific changes in the expression of structural, regulatory and metabolic proteins relevant to the complex process of EMT in NRK52E cells. This findings are helpful to understand the molecular mechanism of EMT and expound the chronic progress mechanism of the kidney disease.

Objective To investigate whether JAK/STAT signaling pathway is associated with the tissue inhibitor of metallproteinase-1(TIMP-1)-induced apoptosis in rat mesangial cells(RMC). Methods The human sense and antisense TIMP-1 recombinant plasmids were transfected into RMC through liposome. After RMC was stimulated under the condition of serum deprivation and AG490(JAK2 specific inhibitor) for 24 hours in vitro, the apoptosis rate was measured by flow cytometry. The expression of TIMP-1, bcl-xl, cyclin D1, p27kip1 and JAK2 mRNA was assayed by RT-PCR. The expression of JAK2, STAT3, STAT5, p-JAK2, p-STAT3 and p-STAT5 protein was analyzed by Western blot. Results The apoptosis rates of the non-transfected group, sense TIMP-1 group and antisense TIMP-1 group in the serum-deprived culture medium without serum and AG490 were （10.59±0.96）％, （7.08±0.43）％ and （21.91±0.25）％,respectively. There were statistical differences among those apoptosis rates. The expression of bcl-xl and cyclin D1 mRNA was the highest in sense group, while it was the lowest in antisense one. The expression of p27kip1 mRNA was the highest in antisense group. AG490 treatment enhanced the apoptosis rates of RMC significantly(P<0.01). AG490 decreased the expression of TIMP-1 mRNA and bcl-xl, as well as cyclin D1 mRNA, and increased the expression of p27kip1 mRNA. Before RMC was stimulated with AG490, the expression of p-JAK2, p-STAT3 and p-STAT5 was the highest in sense TIMP-1 group, and it was the lowest in antisense one. AG490 treatment significantly suppressed the expression of the phosphorylation proteins in all groups. Conclusions The expression of TIMP-1 can be regulated by JAK/STAT signaling pathway. The JAK/STAT can inhibit RMC apoptosis through up-regulating the expression of the TIMP-1. TIMP-1 inhibits the apoptosis of RMC induced by serum deprivation through JAK/STAT signal pathway. Bcl-xl, cyclin D1 and p27kip1 are involved in the above-mentioned courses

Objective To investigate the effect of TNF-α on the expression of bone alkaline phosphatase (BAP), osteopontin (OPN) and bone morphogenetic protein 2 (BMP-2), and the deposit of extracellular calcium in human umbilical artery smooth muscle cells (hUASMC). Method hUASMC were cultured primarily with tissue explants-attached method. The mRNA expression of BAP and OPN was determined by real time-PCR. The protein expression of BAP, OPN and BMP-2 was determined by Western blotting. The calcium deposit was detected by O-cresolphthalein complexone method. Results TNF-α promoted the proliferation of hUASMC and increased the calcium deposit in a time- and dose-dependent manner. The mRNA expression of BAP and OPN in hUASMC was up-regulated by TNF-α（50 μg/L） treatment at day 3(BAP mRNA 1.908±0.034 vs 1.000±0.033, OPN mRNA 3.600±0.073 vs 1.000±0.079, all P<0.05). The protein expression of BAP, OPN and BMP-2 in hUASMC was up-regulated by TNF-α（50 μg/L） treatment at day 5 (BAP protein 3.394±0.083 vs 1.000±0.030, OPN protein 1.967±0.134 vs 1.000±0.070, BMP-2 protein 2.745±0.289 vs 1.000±0.208, all P<0.05）. Conclusion TNF-α of certain concentrations can promote the proliferation of hUASMC, induce the ossific calcification of hUASMC, increase the calcium deposit, and participate in the development of vascular calcium

Objective To investigate the effects of TNF-α on the expression of matrix metalloproteinases (MMP) and their inhibitors (TIMP) in human peritoneal mesothelial cells,meanwhile, to investgate the effects of treatment of TNF-α+TGF-β1, TNF-α+TGF-β1+IL-1 or TNF-α alone on the MMP-9 activity of the HPMC. Methods After 24-hour stimulation by different concerntrations of cytokines（TNF-α，TGF-β， IL-1）, the serum-free conditioned HPMC culture supernatants and the HPMC were collected. The mRNA expression of MMP-2, MMP-9, TIMP-2 and TIMP-1 was detected by semi-quantitative RT-PCR. The activity of active and pro MMP-9 in the conditioned media was quantitatively measured by Biotrak MMP-9 activity assay system. Collagen Ⅰ protein level was examined by ELISA. Results In vitro by the treatment of TNF-α, the expression of MMP-9 mRNA of HPMC was significantly up-regulated in a dose- and time-dependent manner（2.3~4.9 folds of base value, P＜0.05）; the expression of TIMP-1 mRNA and TIMP-2 mRNA was slightly down-regulated（77.2%, 61.3% of base value, P＜0.05）, however the expression of MMP-2 mRNA did not change obviously. Activity and secretion of MMP-9 in the conditioned cell supernatant increased significantly by the treatment of TNF-α+TGF-β１, TNF-α+TGF-β１+IL-1 or TNF-α alone. Collagen Ⅰ protein expression of HPMC increased significantly as well by 24-hour stimulation of TNF-α. Conclusion The increase of MMP-9 activity and expression induced by TNF-α alone and TNF-α with other cytokines may play a role in the peritoneal fibrogenesis process.

Objective To investigate the effect of large dosage of spironolactone on renal fibrosis in spontaneously hypertensive rats. Methods Twenty-four 8-week aged spontaneously hypertensive rats were divided into low and large dosage of spironolactone group and control group. Eight normal Wistar-Kyoto rats were as normal group. Low and large dosage groups were given spironolactone 20 mg&#8226;kg-1&#8226;d-1 and 100 mg&#8226;kg-1&#8226;d-1 for 8 weeks. Then systolic blood pressure, proteiuria, albumin, K＋,Na＋, Scr, the aldosterone level of kidney tissue and plasma were measured. Renal tissue sections were stained by HE and Masson for evaluating glomerular injury and collagen deposition. The protein expression of TGF-β1 and aldosterone receptor in renal tissue were detected by immunohistochemical SABC method, and the mRNA levels were detected by RT-PCR. Results Compared to the controls, in low dosage of spironolactone group, urinary protein decreased (P<0.05), serum albumin increased (P<0.05), plasma and renal tissue aldosterone levels decreased, without significant diffeence. In large dosage of spironolactone group, blood pressure had no significant change, whereas the levels of urinary protein[(27.3±4.5) vs (24.5±3.2) mg/d] as well as plasma and renal tissue aldosterone [(28.3±1.5) vs (22.2±0.6) ng/g] increased significantly (P<0.05), and serum albumin [(20.2±4.2) vs (22.7±3.5) g/L] decreased significantly (P<0.05). Compared to the controls, in low dosage of spironolactone group, protein cast and inflammatory cells infiltration around tubules reduced (P<0.05); the renal tissue aldosterone receptor mRNA and protein expression did not change significantly, TGF-β1 mRNA and protein expression reduced significantly (P<0.05). In high-dose spironolactone group, protein cast and inflammatory cells infiltration around tubules increased significantly (P <0.05), and glomerular collagen formation also increased significantly (P<0.05); the renal tissue aldosterone receptor and TGF-β1 mRNA and protein expression were significantly increased (P<0.05). Conclusions Large dosage of spironolacton can aggravate renal fibrosis maybe through up-regulating level of renal aldosterone and its receptor.

Objective To promote the proliferation of seeding cells, obtain plenty of engineering cells for bioartificial kidney(BAK) in relatively short time and facilitate the construction of BAK system. Methods Recombinant adenovirus-associated virus II type harboring human Nanog gene (rAAV2- hNanog) was prepared, then rAAV2-hNanog was transfected into seeding cell HKC. The expression of human Nanog in seeding cells was detected by RT-PCR after transfection. The effects of human Nanog on HKC cell line were examined by tetrazolium salt colorimetry (MTT), immunofluorescence and confocal laser scanning microscope（CLSM）. Results The human Nanog could be expressed in engineering cells. The proliferation activity of transfected seeding cell was significantly improved（P<0.05）. Meanwhile, no obvious morphology changes could be found under inverted microscope in the seeding cells transfected by rAAV2-hNanog compared with control cells. Conclusions The proliferation of seeding cells modified by human Nanog gene is strikingly accelerated. This new approach will overcome the insufficiency of seeding cells for BAK system.