Objective To elucidate the clinical and pathologic characteristics of IgA nephropathy associated with ankylosing spondylitis（AS）. Methods Clinical and pathologic data of 10 patients suffered from IgA nephropathy associated with AS were reviewed. They were admitted to our hospital from 1997 to 2006 and diagnosed by renal biopsy. Results The average age of nine male and one female patients was (28.6±6.8) years old. Four patients presented with asymptomatic hematuria, and six with hematuria and proteinuria. All the patients suffered from microhematuria and two from macrohematuria. The average 24-hour proteinuria was （1.56 ±1.53） g. Two had hypertension. Serum creatinine levels of all the cases were normal. Light microscopy examination showed that eight patients were mild mesangial proliferation with Lee’s classification grade Ⅰ or grade Ⅱ, and the other two were moderate to severe mesangial proliferation with grade Ⅲ or grade Ⅵ. Conclusion Mild mesangioproliferative IgA glomerulonephritis may be the major morphological pattern in AS patients with renal involvement.

Objective To investigate the clinicopathological characteristics and outcome of two subtypes of class Ⅳ lupus nephritis (LN). Methods Seventy-eight patients with class Ⅳ LN proven by renal biopsy were classified into two groups according to the 2003 ISN/RPS modified classification of LN. Class Ⅳ-G (56 patients) referred to involvement in more than 50% of glomerular capillary surface area. Class Ⅳ-S(22 patients) referred to involvement in less than 50% of glomerular capillary surface area. The clinical manifestations, pathology and therapeutic efficacy were compared between these two groups. Results There were no significant differences of age, gender, course of disease, mean arterial pressure, haemoglobin, C4, serum creatinine between two groups. Greater 24 hour urine protein [（4.34±2.67） vs. (3.52±3.46) g, P<0.05）]and lower C3 level [（0.41±0.17） vs. (0.46±0.16) g/L, P<0.05）]were observed in Ⅳ-G group. Systemic lupus erythematosus disease activity index (SLEDAI) was higher in Ⅳ-G group（14.39±4.07 vs. 11.53±4.15, P<0.05）.The immunohistochemical staining revealed that immune complex deposit was higher in Ⅳ-G group than that in Ⅳ-S group (2.11±0.54 vs. 0.35±0.39, P<0.01). The infiltration of mononuclear macrophage was more common in Ⅳ-G group (1.75±0.87 vs. 0.86±0.76, P<0.05), whereas fibrinoid necrosis was more frequent in Ⅳ-S group（11.66±17.26 vs. 13.61±20.01, P<0.05）. The higher active index （9.56±3.17 vs. 6.59±2.12, P<0.01）and lower chronic index ( 4.93±1.59 vs. 5.88±2.15, P<0.05） in Ⅳ-G group were detected. A total of 49 patients (Ⅳ-S 14, Ⅳ-G 35) were followed up for more than 1 year and received immune suppressive agents. There were no significant differences between two groups in the changes of proteinuria, complement, SLEDAI scores and serum creatinine. Conclusions There are some clinicopathological differences between Ⅳ-S and Ⅳ-G patients. The prognostic distinction between them remains to be proven.

Objective To investigate the pathogenesis of lipoprotein glomerulopathy (LPG) by analyzing the apolipoprotein E (apoE) gene of members in a family including four LPG patients and one asymptomatic carrier. Methods Laboratory tests for urinalysis, serum creatinine, plasma lipid and lipoprotein were performed on all the members of above family. Genomic DNA was extracted from peripheral leukocytes. The fragment of the apoE gene covering exon 4 was amplified by PCR. The gel-purified PCR products were directly sequenced and subcloned into the pMD 18-T vector. Recombinant plasmids were sequenced to investigate both alleles of patients. Results A nucleotide G to C point mutation in exon 4 of the apoE gene was confirmed in each patient and one asymptomatic member. This missense mutation encoded a new apoE variant that amino acid substitution of the proline residue for arginine residue at position 150 of apoE. Those carriers were all heterozygous of this novel point mutation apoE(arginine150 proline) with elevated plasma concentrations of apoE. Conclusions A new variant of apoE, apoE(arginine150 proline), has been identified in a family with 3 generations, including four LPG patients and one asymptomatic carrier. Study of this variant characteristics may be important for understanding the pathogenesis of LPG.

Objective To investigate the effects of dietary salt intake on the expression of cyclooxygenase 2(COX2) in renal medulla of spontaneously hypertensive rats (SHR) and normaltensive Wistar-Kyoto rats (WKY). Methods Rats at age of 6 weeks were randomly assigned to the following groups (n=6): WKY rats were fed with low salt diet containing 0.04% NaCl (WKY-LS), WKY with high salt diet containing 8% NaCl (WKY-HS), and SHR with low salt diet (SHR-LS), SHR with high salt diet (SHR-HS). The blood pressure (BP) was determined by automatic sphygmomanometer at the tail artery. The urine volume（UV） and urinary sodium excretion（UNa） were also measured. Urinary level of 6-keto prostaglandin F1α（6k-PGF1α） was detected by radioimmunity. Protein expressive level of COX2 in the renal medulla was assessed by immunohistochemistry and Western blot. Results The average baseline BP of SHR groups, measured 1 day before the start of the salt diet, was significantly higher than that of WKY groups（P<0.05）. After 3 days of different salt diet, BP of WKY-LS, WKY-HS and SHR-LS did not change obviously (P>0.05), while BP of SHR-HS group was significantly enhanced compared to basal level (P<0.05）. Both UV and UNa of SHR groups were significantly lower compared with those of WKY groups regardless of normal, low or high salt diet provided（P<0.05）. Levels of UV and UNa significantly increased in all the groups treated with high salt diet and decreased in those allotted low salt food, compared to baseline levels（P<0.05）. Urinary levels of COX2 metabolite 6k-PGF1α in both SHR and WKY groups responded similarly to dietary salt intake. Groups of high salt diet exerting an obvious increase and groups of low salt diet exhibiting a drop（P<0.05） with no variance were found between SHR and WKY having the same salt-loading（P>0.05）. Renal COX2 staining was mainly localized in medullary interstitial cells by immunohistochemistry. High salt diet for 1 week increased enormously renal medulla COX2 expression equally in both SHR and WKY groups, while low salt diet had no effects. The same tendency was further confirmed by Western blot. Conclusions High salt dietary induces further elevation of blood pressure and sodium retention in SHR group compared with WKY group. The expression of COX2 in the renal medulla is significantly enhanced in all rats fed with high salt diet and there is no significant difference between SHR and WKY. Renal medullary COX2 may perform normal expression and be involved in the regulation of water and sodium excretion in SHR, but take no part in the occurrence of hypertension.

Objective To study the regulation of renin in juxtaglomerular granular cells (JG cells) cultured from β1/β2 adrenergic receptor-deficient mice. Methods Real-time PCR was used to demonstrate the expression of β1-adrenergic receptors in primary cultures of JG cells. JG cells were stained with quinacrine and Lyso Tracker-Red and observed by confocal microscopy in order to observe the effect of isoproterenol on renin release in real time. Blood was taken from conscious wild type (WT) and β1/β2 adrenergic receptor knockout mice (β1/β2 ADR-/-) by tail vein puncture. Plasma renin concentration was measured by radioimmunoassay. Forskolin, isoproterenol and PGE2 were used to stimulate the renin activity in supernatant and cell lysate of primary cultures of JG cells from WT and β1/β2 ADR-/- mice. The renin mRNA expression of kidney cortex and JG cells was measured by real-time PCR. Results Real-time PCR confirmed the expression of β-adrenergic receptor mRNA in JG cells of WT mice, but not in β1/β2 ADR-/- mice. Isoproterenol stimulated granule exocytosis and the emptying of granule content. Basal plasma renin concentration and renin mRNA expression was low in β1/β2 ADR-/- mice. PGE2, isoproterenol and forskolin increased the renin activity in the JG cell lysate and supernatant in cultures of WT mice, but isoproterenol had no effect on renin expression in JG cells from β1/β2 ADR-/- mice while higher doses of forskolin were needed to stimulate renin secretion. This effect was accompanied by increased renin mRNA expression of JG cells. Forskolin stimulated the cAMP content of JG cells. Conclusions β-adrenergic receptors play an important role in regulating renin secretion in primary cultures of JG cells. This effect is associated with the activation of the cAMP pathway.

Objective To investigate the signal transduction events that regulate CTGF synthesis and secretion in response to TGF-β1, especially the role of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) in TGF-β1-induced CTGF expression. Methods PKC was inhibited with Go6850 and ERK, JNK, p38 MAPK was blocked by PD98059, U0126, SP600125 and SB203580 respectively, and their effects on CTGF expression and Smad2/Smad3 phosphorylation wereanalyzed. Results TGF-β1 （5 μg/L） induced Smad2/Smad3 phosphorylation in a time-dependent manner from baseline 0.87±0.09 to 2.35±0.11 for 2 hours. The PKC inhibitor Go6850（5 μmol/L） and the ERK inhibitor PD98059（10 μmol/L） and U0126 （10 μmol/L）partially attenuated TGF-β1-stimulated expression of CTGF, while the p38 MAPK inhibitor SB203580（20 μmol/L） or the JNK inhibitor SP600125（10 μmol/L） had no such effect. In addition, phosphorylation of Smad2/Smad3 was not blocked by U0126（10 μmol/L） or PD98059 （10 μmol/L）, but by Go6850. Conclusions Ras/MEK/ERK and PKC activity are required for the TGF-β1 induction of CTGF in tubular epithelial cells. Furthermore, PKC activity is involved in the TGF-β1-induced CTGF expression in a smad-dependent manner, while Ras/MEK/ERK modulates CTGF expression independently on Smads.

Objective To explore the role of TNF-related apoptosis inducing ligand （TRAIL） system in the pathogenesis of diabetic nephropathy. Methods The expression of TRAIL, death receptor 4(DR4), decoy receptor 2 (DcR2) and nuclear factor-κB （NF-κB） in kidney tissue of diabetic rats in different periods was examined and their relationships with kidney function deterioration were evaluated. Eighty rats were randomly divided as follows: control group (NC), diabetic group (DM). Uninephrectomized male Wistar rats were induced by single intraperitoneal injection of streptozotocin (STZ, 55 mg/kg) in diabetic group. Eight rats were selected randomly from each group at the end of the 4th, 8th, 12th, 16th week of diabetic groups. Serum biochemistry, 24 h urinary protein and the ratio of kidney weight/body weight were determined. Quantitive real time RT-PCR and immunohistochemistry were performed to detect the expression of TRAIL, DR4, DcR2 and NF-κB mRNA and protein. Results DM group showed a higher 24 h urinary protein and hypertrophy index compared with NC group (P<0.05). Blood albumin(Alb) decreased from the 8th week （P<0.01）. BUN and Scr did not change obviously until the 16th week. Compared with NC group, the expression of TRAIL and DR4 decreased significantly at the end of 4th， 8th， 12th week, and increased significantly at the end of 16th week（P<0.01）. DcR2 increased significantly at the end of 4th， 8th， 12th week（P<0.01）. Compared with NC group, the expression of NF-κB increased significantly in diabetic group in every time point（P<0.05）. The protein expressions of TRAIL and its receptors were mainly located in tubuli contorti, and no expression was detectable in the glomeruli or renal vasculature. The expression of NF-κB protein was seen in glomeruli and tubuli contorti. Above expression was significantly related with mononuclear macrophage infiltration, kidney function and structural lesion. The protein expression was consistent with the results of PCR. Conclusion TRAIL system, as important monitoring factors in the regulation of apoptosis, participates in the pathogenesis of diabetic nephropathy.

Objective To establish a method for DNA extraction and PCR amplification from renal biopsy paraffin sections. Methods Six paraffin embedded renal biopsy tissue blocks, which pathological diagnoses were hepatitis B associated glomerulonephritis(2 case), primary IgA nephropathy(2 case) and membrane nephropathy(2 case), were employed. Two or three sections at 2 μm thickness were cleanly cut and placed on microscope slides. Sections were incubated at 70℃ for 30 min to melt down paraffin and deparaffinized with three changes of xylene. Digestion buffer was freshly made by mixing 150 μl of protein K solution(10 mg/ml) with 850 μl of a mixture of the followings: 0.5 ml Tween 20, 0.2 ml 0.5 mol/L EDTA(pH 8.0), 5.0 ml 1 mol/L Tris(pH 8.5), 94.3 ml of distilled water. Tissues on the slides were removed by 10 μl Eppendoff pipette tip and each sample was placed in a separate tube. Samples were digested for 48 h and DNA concentration was measured. Angiotensin converting enzyme gene and HBcAg gene were amplified by PCR. Results DNA extraction of samples ranged from 10 μg to 30 μg. ACE gene PCR amplification results showed all samples displayed 490 bp and/or 190 bp lanes. HBcAg gene PCR amplification results showed the two hepatitis B associated glomerulonephritis samples displayed 132 bp lane while the primary IgA nephropathy and membrane nephropathy samples were negative. Conclusion This method is able to obtain comparable quality and quantity of DNA extracts which yields uniform PCR products from renal biopsy paraffin sections and provides the possibility for large genetics analysis of renal biopsy tissues.