Low-protein diet supplemented with ketoacids ameliorates proteinuria in 3/4 nephrectomised rats by directly inhibiting the intrarenal renin-angiotensin system.

Low-protein diet plus ketoacids (LPD+KA) has been reported to decrease proteinuria in patients with chronic kidney diseases (CKD). However, the mechanisms have not been clarified. As over-activation of intrarenal renin-angiotensin system (RAS) has been shown to play a key role in the progression of CKD, the current study was performed to investigate the direct effects of LPD+KA on intrarenal RAS, independently of renal haemodynamics. In this study, 3/4 subtotal renal ablated rats were fed 18 % normal-protein diet (Nx-NPD), 6 % low-protein diet (Nx-LPD) or 5 % low-protein diet plus 1 % ketoacids (Nx-LPD+KA) for 12 weeks. Sham-operated rats fed NPD served as controls. The level of proteinuria and expression of renin, angiotensin II (AngII) and its type 1 receptors (AT1R) in the renal cortex were markedly higher in Nx-NPD group than in the sham group. LPD+KA significantly decreased the proteinuria and inhibited intrarenal RAS activation. To exclude renal haemodynamic impact on intrarenal RAS, the serum samples derived from the different groups were added to the culture medium of mesangial cells. It showed that the serum from Nx-NPD directly induced higher expression of AngII, AT1R, fibronectin and transforming growth factor-ß1 in the mesangial cells than in the control group. Nx-LPD+KA serum significantly inhibited these abnormalities. Then, proteomics and biochemical detection suggested that the mechanisms underlying these beneficial effects of LPD+KA might be amelioration of the nutritional metabolic disorders and oxidative stress. In conclusion, LPD+KA could directly inhibit the intrarenal RAS activation, independently of renal haemodynamics, thus attenuating the proteinuria in CKD rats.

The GSTA1 polymorphism and cyclophosphamide therapy outcomes in lupus nephritis patients.

Pulsed low-dose cyclophosphamide (CTX) therapy has become a very effective approach in improving the clinical outcomes of lupus nephritis (LN) patients. However, variations of CTX therapeutic outcomes in LN patients are incompletely understood. We investigated the contributions of known allelic variants to CTX therapy outcomes in 77 LN patients. Then, 22 out of the 77 patients were randomly enrolled to evaluate the pharmacokinetic profiles. LN patients with a GSTA1*A mutation (CT heterozygous) had more risk of non-remission (44% vs. 20%, P=0.005). Pharmacokinetic data indicated that patients with a GSTA1*A heterozygous variant had a lower exposure to 4-hydroxycyclophosphamide (4OHCTX) compared to wild-type patients (AUC4OHCTX: 12.8 (9.8, 19.5) vs. 27.5 (18.1, 32.8) h mg/l, P=0.023). Clinical remission was significantly related to higher exposure of 4OHCTX (P=0.038). In conclusion, LN patients with GSTA1*A heterozygous genotypes had poor CTX treatment remission due to less exposure to activated metabolites of CTX.

Acid loading stimulates rat glomerular mesangial cells proliferation through Na(+)-H (+) exchanger isoform 1 (NHE1)-dependent pathway.

The role of metabolic acidosis in the progression of chronic kidney disease (CKD) remains unclear. The aim of the present study was to investigate the direct effects of acid loading on the proliferation of rat glomerular mesangial cells (GMCs) in vitro and the possible role of sodium-hydrogen ion exchanger isoform 1 (NHE1). Rat GMCs were treated with acidic medium as acid loading. Growth and proliferation of GMCs was studied by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, thymidine ((3)H-TdR) incorporation, and flow cytometry. NHE1 protein expression and activity were quantified by Western blot and dual wavelength epifluorescent illumination with 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein, respectively. 5-(N,N-dimethyl) amiloride hydrochloride (DMA), a specific inhibitor of NHE1, was used to investigate the possible involvement of NHE1 in the proliferation of GMCs. The MTT assay, (3)H-TdR incorporation, and cell cycle distribution analysis indicated that acid loading stimulated the proliferation of GMCs. Acid loading increased NHE1 activity, but had no effects on NHE1 expression at the protein level. The effects of acid loading on the proliferation of GMCs were inhibited by DMA. Acid loading induced GMC proliferation through NHE1-dependent pathways. Our findings may contribute to the understanding of metabolic acidosis in the progression of CKD.

Twelve weeks of pioglitazone therapy significantly attenuates dysmetabolism and reduces inflammation in continuous ambulatory peritoneal dialysis patients--a randomized crossover trial.

BACKGROUND: The aim of the present study was to investigate the effect of oral pioglitazone (PIO) on lipid metabolism, insulin resistance, inflammation, and adipokine metabolism in continuous ambulatory peritoneal dialysis (CAPD) patients. METHODS: In this randomized crossover trial, 36 CAPD patients with serum triglyceride levels above 1.8 mmol/L were randomly assigned to receive either oral PIO 15 mg once daily or no PIO for 12 weeks. Then, after a 4-week washout, the patients were switched to the alternative regimen. The primary endpoint was change in serum triglycerides during the PIO regimen compared with no PIO. Secondary endpoints included changes in other lipid levels, homeostatic model assessment of insulin resistance (HOMA-IR), adipocytokines, and C-reactive protein (CRP). RESULTS: All 36 CAPD patients (age: 64 ± 11 years; 33% men; 27.8% with diabetes mellitus) completed the study. Comparing patients after PIO and no PIO therapy, we found no significant differences in mean serum triglycerides (3.83 ± 1.49 mmol/L vs 3.51 ± 1.98 mmol/L, p = 0.2). However, mean high-density lipoprotein (0.94 ± 0.22 mmol/L vs 1.00 ± 0.21 mmol/L, p = 0.004) and median total adiponectin [10.34 µg/mL (range: 2.59 - 34.48 µg/mL) vs 30.44 µg/mL (3.47 - 93.41 µg/mL), p < 0.001] increased significantly. Median HOMA-IR [7.51 (1.39 - 45.23) vs 5.38 (0.97 - 14.95), p = 0.006], mean fasting blood glucose (7.31 ± 2.57 mmol/L vs 6.60 ± 2.45 mmol/L, p = 0.01), median CRP [8.78 mg/L (0.18 - 53 mg/L) vs 3.50 mg/L (0.17 - 26.30 mg/L), p = 0.005], and mean resistin (32.70 ± 17.17 ng/mL vs 28.79 ± 11.83 ng/mL, p = 0.02) all declined. The PIO was well tolerated, with only one adverse event: lower-extremity edema in a patient with low residual renal function. CONCLUSIONS: Blood triglycerides were not altered after 12 weeks of PIO 15 mg once daily in CAPD patients, but parameters of dysmetabolism were markedly improved, including insulin resistance, inflammation, and adipokine balance, suggesting that PIO could be of value for this high-risk patient group. Larger, more definitive studies are needed to confirm these findings.

[Reasons for not intensifying antihypertensive treatment to diabetes in some regions of China].

OBJECTIVE: To prospectively evaluate the current situation of blood pressure control in the type 2 diabetics in some regions of China. METHODS: Totally 5063 consecutive patients with hypertension were investigated. Among them 1993 with diabetes underwent baseline survey. Individualized target blood pressures were set by physicians. Four follow-up visits, in weeks 2, 4, 8, and 12 were performed to monitor the achievements of these targets and the reasons for not modifying antihypertensive treatments were analyzed when the blood pressure goals were not reached. RESULTS: The prevalence of diabetes among the hypertensive patients was 39.2%. The target blood pressure values defined by the physicians at baseline survey varied with the grade and degree of hypertension. 31.4% of the patients reached the target blood pressure goals (BP < 130/80 mm Hg) at week 12. The reasons for not intensifying antihypertensive treatment when the blood pressure targets were not achieved were manifold. The main reasons were as follows: the physicians considered that the BP value was close to the target, longer treatment and waiting for full drug effect were needed, etc. On average, 2 different antihypertensive agents were used to achieve BP target and some patients used 4-5 different drugs. The most frequent agents used were angiotensin II receptor antagonist and calcium channel blockers. The use of thiazide-type diuretics increased from the first visit to the last visit (weeks 12). CONCLUSION: The co-prevalence of hypertension and diabetes in China is high and the target blood pressure set by physician is almost 130/80 mm Hg as defined in guidelines. Only about 31.4% of patients have reached their goals 12 weeks after treatment. There are many subjective and objective reasons responsible for not intensifying antihypertensive treatment when the blood pressure targets are not achieved. Hypertension in diabetes is poorly controlled compared with that in the whole population.

[A survey of factors influencing prognosis and control rate for patients with hypertension in mainland China].

OBJECTIVE: To observe the risk factor stratification and prevalence of target organ damage in hypertensive patients before therapy and blood pressure control rate after 4 or 12 weeks antihypertensive drug therapy. METHODS: In this prospective survey, data on cardiovascular risk factors, target organ damage and concomitant disease were collected in 26 655 hypertensive patients. Among them 26 325 and 3457 patients were recruited for antihypertensive drug therapy for 4 and 12 weeks, respectively and blood pressure control rate was determined. RESULTS: The sedentary lifestyle, smoking, high body mass index, dyslipidemia were found in 52.5%, 34.4%, 31.8%, 24.5%, and microproteinuria, left ventricular hypertrophy, coronary artery disease and diabetes in 21.0%, 23.6%, 20.1%, 26.7% hypertensive patients, respectively. The average systolic and diastolic pressures were 158 +/- 14 mm Hg and 94 +/- 11 mm Hg and 3.2%, 22.2%, 21.1% and 53.3% patients were defined as low, medium, high and very high risk patients in risk stratification to quantify prognosis. There were 77.2%, 20.4% and 2.4% systolic and diastolic, isolated systolic and isolated diastolic hypertensive patients respectively. The goal blood pressure control rate was 50.2% and 56.7% respectively after 4 and 12 weeks antihypertensive drug therapy. The control rate in patients complicated with diabetes and renal disease was significantly lower than patients without them and systolic pressure control rate was remarkably lower than diastolic pressure control rate. Majority patients required 2 or more antihypertensive drugs for effective pressure control (1.5 drug per patients in average in both 4 or 12 weeks groups). CONCLUSION: The prevalence of risk factors, target organ damage and concomitant disease were high in Chinese patients with hypertension and comprehensive interventions were indicated. To reach goal blood pressure control in patients with hypertension, follow up intensifying and drug therapy guidance are required within the context of usual medical care.

Renoprotective effect of breviscapine through suppression of renal macrophage recruitment in streptozotocin-induced diabetic rats.

BACKGROUND/AIMS: Experimental and clinical evidence has consistently demonstrated that renal macrophage infiltration is one of the most important events for the progression of diabetic nephropathy. Breviscapine is a flavonoid extracted from the Chinese herb Erigeron breviscapus. Previously, it was shown that treatment with breviscapine attenuated renal injury in the diabetic rats. The purpose of this study is to investigate whether the renoprotective effect of breviscapine is through suppression of renal macrophage recruitment in diabetic rats. METHODS: Diabetes was induced bystreptozotocin injection, and breviscapine was administered orally at a dose of 20 mg/kg/day for 8 weeks. Control rats received vehicle or breviscapine with the same schedule. RESULTS: Breviscapine treatment markedly inhibited both an increase of albuminuria and glomeruli hypertrophy and tubulointerstitial injury without modifying mean arterial blood pressure and creatinine clearance. Levels of malondialdehyde and protein kinase C activities were markedly higher and antioxidant enzyme activities such as superoxide dismutase, catalase as well as glutathione peroxidase were significantly lower in the kidneys of diabetic rats than of the control group, breviscapine administration markedly remitted these changes. ED-1-positive cells and expression of intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) in glomeruli and tubulointerstitium were all markedly elevated but were significantly reduced by breviscapine. Western blot analysis noted that the expression of transforming growth factor beta1 protein was increased 1.8-fold in the kidney in diabetic rats, breviscapine treatment could reduce increased expression of TGF-beta1 protein by 47%. CONCLUSION: This study describes a novel mechanism by which breviscapine confers a renoprotective effect.

[The establishment of plasmid over-expressing mouse peroxisome proliferator-activated receptor gamma1 and functional identification in mesangial cells].

OBJECTIVE: To construct a plasmid expressing peroxisome proliferator-activated receptor gamma1 (PPARgamma1) and to study its antifibrotic effects on transfected mesangial cells under the condition of high glucose. METHODS: Wild type full length of mouse PPARgamma1 (mPPARgamma1/WT) cDNA was ligated into an expressing vector pIRES-EGFP. This constructed pIRES-EGFP-mPPARgamma1/WT was then transfected into cultured glomerular mesangial cells facilitated by lipofectin. The transfection efficiency was determined by RT-PCR, Western bloting for the expression level of PPARgamma1, as well as by specific PPRE binding activity. The effects of over-expressed PPARgamma1 on the expressions of TGF-beta1, PAI-1 and FN in the mesangial cells stimulated by high glucose (30 mmol/L) was examined by RT-PCR and ELISA. A vector expressing dominant negative type of mPPARgamma1, pIRES-EGFP-mPPARgamma1/DN, lacking the biological activity of PPARgamma1, was also constructed and transfected by using the same technology to serve as a control throughout the study. RESULTS: The accuracy of constructed and selected plasmids was confirmed by restriction enzymatic analyses and DNA sequencing. The expression level and activity of PPARgamma1 in the mesangial cells reached the peaks 48 hours after transfection. Over-expressed PPARgamma1 significantly inhibited the high glucose-induced increases in TGF-beta1, PAI-1 and fibronectin syntheses (all P < 0.05). CONCLUSION: The constructed expressing vector pIRES2-EGFP-mPPARgamma1/WT provides a useful tool for further study on the effects and mechanisms of PPARgamma1. Over-expressed PPARgamma1 may protect the mesangial cells from fibrosis caused by high glucose.

[Protection of rosiglitazone against renal interstitial lesion and its mechanism].

OBJECTIVE: To investigate the protection of rosiglitazone (RSG) against renal interstitial lesion and its mechanism. METHODS: Male adult SD rats were randomly divided into 5 groups: Sham group, undergoing sham operation; Sham + RSG group, undergoing sham operation and treated with RSG (30 mg.kg(-1).d(-1)); UUO group, undergoing unilateral left ureter obstruction; UUO + RSG5 group, undergoing UUO and treated with RSG (5 mg.kg(-1).d(-1)); and UUO + RSG30 group, undergoing UUO and treated with RSG (30 mg.kg(-1).d(-1)). Except the Sham group, the other groups were divided into 3 subgroups of 6 approximately 8 rats to be killed 3, 7, and 14 days after the intervention and their left kidneys were taken out to undergo light microscopy by Masson staining to observe the renal interstitial fibrosis index and to undergo immunohistochemistry with mice anti-rat ED-1 antibody to calculate the ED-1 (specific marker antigen on the surface of monocyte/macrophage) positive cells. Homogenate of renal cortex was made. ELISA was used to detect the protein expression of transforming growth factor beta(1) (TGF-beta(1)), bone morphogenetic protein (BMP)-7, and PAI-1 gene. RT-PCR was used to detect the mRNA expression of BMP-7 and TGF-beta(1) downstream effector genes: CTGF and Smad6. RESULTS: Fourteen days after the intervention, the fibrosis index of the UUO group were significantly increased and the fibrosis index values of both UUO + RSG5 and UUO + RSG30 groups were significantly lower than that of the UUO group (both P = 0.000) with a significant difference between the 2 UUO + RSG groups. Three days after the intervention, the number of ED-1 positive cell began to increase in the UUO group and peaked 7 days after, however, the number of ED-1 positive cells of the 2 UUO + RSG groups were significantly lower than that of the UUO group. TGF-beta(1) mRNA and protein were highly expressed in the UUO group, however, the expression of TGF-beta(1) mRNA in the 2 UUO + RSG groups was significantly lower, especially in the UUO + RSG30 group, in comparison with the UUO group (both P < 0.05). The CTGF mRNA expression was increased time-dependently in the UUO group, however the CTGF mRNA expression of the 2 UUO + RGS groups was significantly inhibited dose- and time-dependently (all P < 0.05). In comparison with that in the Sham group, the Smad6 mRNA expression was significantly increased in the Sham + RSG group (P < 0.05), and was decreased time-dependently in the UUO group; however, the decrease of Smad6 mRNA expression was significantly reversed in the 2 UUO + RSG groups, especially in the UUO + RSG30 group (P < 0.05). The expression of BMP-7 protein and mRNA was significantly lower in the UUO group time-dependently. However, the BMP-7 mRNA expression of the 2 UUO + RSG group, especially of the UUO + RSG30 group, was significantly higher than that of the UUO group (both P < 0.05). The PAI-1 protein expression of the UUO group was significantly higher 7 and 14 days after (both P < 0.05), however, the PAI-1 protein expression of the 2 UUO + RSG groups was significantly lower than that of the UUO group (both P < 0.05). CONCLUSION: RSG inhibits the renal interstitial macrophage infiltration, downregulates the expression of the down-stream target genes, and up-regulate the BMP-7 expression, thus blocking the renal fibrosis.

A report with consensus statements of the International Society of Nephrology 2004 Consensus Workshop on Prevention of Progression of Renal Disease, Hong Kong, June 29, 2004.

This report summarizes the discussions of the International Society of Nephrology (ISN) 2004 Consensus Workshop on Prevention of Progression of Renal Disease, which was held in Hong Kong on June 29, 2004. Three key areas were discussed during the workshop: (1) screening for chronic kidney disease; (2) evaluation and estimating progression of chronic kidney disease; and (3) measures to prevent the progression of chronic kidney disease. Fifteen consensus statements were made in these three areas, as endorsed by the participants of the workshop. The ISN can make use of and take reference to these statements in formulating its policy for tackling chronic kidney disease, a disease with significant global impact.

[Production of aldosterone by rat mesangial cell and the accumulation of extracellular matrix induced by aldosterone].

OBJECTIVE: To investigate whether aldosterone (Aldo) may be synthesized by glomerular mesangial cells and whether Aldo promotes the synthesis of fibronectin (FN) and type IV collagen in mesangial cells. METHODS: Rat mesangial cells (RMC) were cultured and then divided into 4 groups: control group, AngII group (AngII of the concentrations of 10(-10), 10(-9), 10(-8), 10(-7), and 10(-6) mol/L was added), losartan group (AngII 1 type inhibitor losartan and AngII were added), and KCL group (KCL of the concentrations of 7 and 9 mmol/L was added). 48 hours after the RNAs of the cells in different groups were collected to detect the Aldo synthesized by the RMCs themselves. Another RMCs were cultured and divided into 4 groups: control group, Aldo group (Aldo of the concentration of 10(-7) mol/L was added), spironolactone + Aldo group (spironolactone 10(-9) mol/L and Aldo 10(-7) mol/L were added), and spironolactone group (spironolactone 10(-9) mol/L was added). RT-PCR was used to detect the expression of the Aldo synthase CYP11B2, mineralocorticoid receptor (MR), and 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD2 (an enzyme required for MR ligand specificity) mRNAs and proteins. The level of Aldo in the incubation medium was detected by radioimmunoassay. After being exposed to 10(-7) mol/L Aldo for 24 h, the level of FN and type IV collagen in the incubation medium were examined by ELISA and Western blotting respectively. RESULTS: CYP11B2, MR, and 11 beta-HSD2 mRNA expressions were detectable in RMCs by RT-PCR and confirmed by sequencing. The concentration of Aldo in the incubation medium was 1.605 pg/10(6) cells. The mRNA expression of CYP11B2 was significantly up-regulated by both AngII and potassium. Immunocytochemistry showed that MR and 11 beta-HSD2 were distributed in both the cytoplasm and the nucleolus. After treatment of 10(-7) mol/L Aldo for 24 hours, the concentration of FN was 74 +/- 16 ng/ml, significantly higher than the baseline value (12.4 +/- 1.9 ng/ml, P < 0.05) and those of spironolactone group and spironolactone + Aldo groups (17.8 +/- 3.6 ng/ml and 25.9 +/- 5.3 ng/ml respectively); and the level of type IV collagen was 136% +/- 14%, significantly higher than the baseline level (100%, P < 0.05), and those of spironolactone group and spironolactone + Aldo groups (112% +/- 33% and 102% +/- 10% respectively). CONCLUSION: RMCs synthesize Aldo. Aldosterone also acts on RMCs, leading to extracellular matrix accumulation AngII and potassium are involved in the regulation of local production of aldosterone.

[Renal protective effects of specific cyclooxygenase-2 inhibitor in rats with subtotal renal ablation].

OBJECTIVE: To investigate the renoprotective effect of specific cyclooxygenase-2 (COX-2) inhibitor rofecoxib and its possible mechanism of retarding progressive renal injury in rats with subtotal renal ablation. METHODS: Rats were randomly divided into six groups: sham, subtotal renal ablation (SNX). SNX treated with rofecoxib (10 mg.kg(-1).d(-1)). SNX treated with indomethacin (2 mg.kg(-1).d(-1)). SNX treated with losartan (100 mg.kg(-1).d(-1)). SNX treated with rofecoxib and losartan. Blood pressure, urinary protein and thromboxane B(2) (TXB(2)) were measured at the 6th week after operation and morphological changes were examined with light microscopy. The mRNA expression of transforming growth factor-beta type I and type II receptors (TbetaRI, TbetaRII) was detected by way of reverse transcription polymerase chain reaction. The expression of plasminogen activator inhibitor type1 (PAI-1), fibronectin (FN) and angiotensin II type 1 receptor was examined utilizing Western blotting or immunohistochemistry. RESULTS: The levels of urinary protein and TXB(2) as well as cortical COX-2 expression in SNX group were significantly increased while COX-1 expression remained undisturbed in comparison with those in sham group. The levels of systolic blood pressure and angiotensin II in renal cortex significantly increased. The expression of TbetaRI and TbetaRII mRNA, PAI-1 and AT1 protein was up-regulated. The glomerulosclerosis index (GSI) and tubular injury index were increased in SNX group. Rofecoxib significantly inhibited the increase in proteinuria and reduced GSI and tubular injury index. The expression of TbetaRI, TbetaRII and PAI-1 was down-regulated by 36.44%, 45.02% and 31.16% respectively, similar to the effect of losartan treatment. Indomethacin significantly decreased proteinuria and slightly reduced GSI. However the tubular injury index was exacerbated. Systolic blood pressure was not significantly blunted in the groups of rofecoxib and indomethacin. There was no significant additive effect of combined therapy with losartan and rofecoxib, though proteinuria was reduced to a lower level. CONCLUSION: Rofecoxib attenuates proteinuria and retards the progressive renal injury in rats with subtotal renal ablation partly by inhibition of COX-2 activity and modulation of activation of renal renin-angiotensin system as well as the down-regulation of transforming growth factor-beta type I and type II receptors and PAI-1.

Determination of vitamin C in plasma and dialysate from uremia patients by high performance liquid chromatography with electrochemical detection.

A convenient and valid method for the determination of ascorbic acid(AA) and dehydroascorbic acid(DHAA) in plasma and dialysate from patients with uremia by high performance liquid chromatography with electrochemical detection is described. A mixture of 0.8 g/L metaphosphoric acid and 18% (volume fraction) perchloric acid was used as a protein precipitant and the extractant for AA from biosamples. It was also a good stabilizer for AA in samples. The proposed method is satisfied for routine screening of vitamin C in clinical applications with a correlation coefficient of more than 0.99 in the range of 2 mumol/L-40 mumol/L AA. The within-day precision was less than 8.9% and 10.55% for AA and DHAA, respectively. The recoveries of AA in plasma and dialysate were over 95% and 78%, respectively.