[Renal expression of RANK-RANKL in a rat model of puromycin aminonucleoside nephropathy].

OBJECTIVE: To investigate RANK-RANKL expression in the kidneys of a rat model of puromycin aminonucleoside nephropathy (PAN). METHODS: Thirty-six SD rats were randomly divided into PAN model group and normal control group. PAN was induced by a single intravenous injection of 100 mg/kg puromycin aminonucleoside. Serum creatinine and 24-hour urinary protein were measured on days 3, 7, and 14 after the injection, and renal pathologies were assessed with optical and immune transmission electron microscopy. The expression of RANK and RANKL in the kidneys was examined using reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. RESULTS: The PAN model rats showed massive proteinuria and elevated serum creatinine on day 3, which peaked on day 7. RANK-RANKL protein and mRNA expressions in PAN model group was higher than those in the control group. In the PAN rats, RANK was expressed mainly on the top cell membrane and in the cytoplasm of renal podocytes with a significantly increased expression level compared with that in the control group. CONCLUSION: The PAN rat model shows aberrant RANK and RANKL expressions in the podocytes, indicating their contribution to podocyte injury in PAN.

Probucol combined with valsartan in immunoglobulin A nephropathy: a multi-centre, open labelled, randomized controlled study.

AIM: Angiotensin receptor antagonists (ARBs) and anti-oxidants reduce urinary protein excretion and delay progression of immunoglobulin A (IgA) nephropathy. We investigated the efficacy and safety of probucol (an anti-oxidant) combined with valsartan (an ARB) on the progression of IgA nephropathy. METHODS: Patients with IgA nephropathy (n = 69) were recruited from five centres and randomly assigned to a treatment group (750 mg/day probucol plus 160 mg/day valsartan) or a control group (160 mg/day valsartan) and were followed for 3 years. RESULTS: At baseline, the two groups in any measured clinical information were comparable. The primary endpoint (doubling serum creatinine) showed no significant difference between the two groups during 3-year follow-up. The secondary endpoint (50% reduction in 24-h urinary protein) occurred in 23 patients in the treatment group and 20 patients in the control group. The time to the secondary end-point was shorter in the treatment group than the control group (8.13 months vs 19.63 months, P = 0.019). However, at the 3-year follow-up, the 24-h urinary protein levels were not significantly different from the baseline levels (P = 0.99 and P = 0.66, respectively). At the 1-year follow-up, plasma cholesterol in the treatment group was markedly lower than in the control group (4.12 ± 1.28 vs 5.03 ± 1.01, P = 0.02). CONCLUSION: Kidney function remained stable and there was no significant difference in two group patients. Probucol combined with valsartan led to a more rapid decrease of 24-h urinary protein excretion than valsartan alone. However, the long-term effect needs further investigation.

Receptor activator of NF-kappaB and podocytes: towards a function of a novel receptor-ligand pair in the survival response of podocyte injury.

BACKGROUND: Glomerulosclerosis correlates with reduction in podocyte number that occurs through mechanisms which include apoptosis. Podocyte injury or podocyte loss in the renal glomerulus has been proposed as the crucial mechanism in the development of glomerulosclerosis. However, the mechanism by which podocytes respond to injury is poorly understood. TNF and TNF receptor superfamilies are important in the pathogenesis of podocyte injury and apoptosis. The ligand of receptor activator of NF-kappaB (RANKL) and receptor activator of NF-kappaB (RANK) are members of the TNF and receptor superfamilies. We investigated whether RANK-RANKL is a receptor-ligand complex for podocytes responding to injury. METHODOLOGY/PRINCIPAL FINDINGS: In this study, RANKL and RANK were examined in human podocyte diseases and a rat model of puromycin aminonucleoside nephrosis (PAN). Compared with controls, RANK and RANKL were increased in both human podocyte diseases and the rat PAN model; double immunofluorescence staining revealed that RANK protein expression was mainly attributed to podocytes. Immunoelectron microscopy showed that RANK was localized predominantly at the top of the foot process membrane and the cytoplasm of rat podocyte. In addition, RANK was upregulated in mouse podocytes in vitro after injury induced by puromycin aminonucleoside (PA). Knockdown of RANK expression by small interference RNA (siRNA) exacerbated podocyte apoptosis induced by PA. However, RANKL inhibited significantly the apoptosis of podocytes induced by PA. CONCLUSIONS/SIGNIFICANCE: These findings suggest the increase in RANK-RANKL expression is a response to podocyte injury, and RANK-RANKL may be a novel receptor-ligand complex for the survival response during podocyte injury.

Effect of blood sample type on the measurement of advanced oxidation protein products as a biomarker of inflammation and oxidative stress in hemodialysis patients.

Advanced oxidation protein products (AOPP) is widely used as a uremic biomarker, especially for cardiovascular disease. However, it has not been determined whether it is better to measure AOPP in plasma or serum. In this cross-sectional study, which included 102 patients undergoing maintenance hemodialysis, fibrinogen-free serum and defibrinated plasma samples were prepared. AOPP levels from fibrinogen-free samples displayed a stronger correlation with myeloperoxidase activity and levels of C-reactive protein, interleukin-6 and tumor necrosis factor-alpha, as well as prevalent cardiovascular disease, than AOPP levels obtained from plasma samples. These results demonstrated that fibrinogen interferes with the measurement of AOPP.

[Evaluation of continuous venous-venous hemofiltration combined with coupled plasma filtration adsorption for treatment of systemic inflammation response syndrome with acute renal failure].

OBJECTIVE: To evaluate the clinical efficacy of continuous venous-venous hemofiltration (CVVH) combined with coupled plasma filtration adsorption (CPFA) in the management of systemic inflammation response syndrome (SIRS) complicated by acute renal failure (ARF). METHODS: Thirty patients with SIRS complicated by ARF (including 25 with severe acute pancreatitis, 2 with colonic perforation with infection, and 3 with acute infective endocarditis) were randomly divided into CVVH plus CPFA group (n=14) and CVVH alone group (n=16). The APACHE II score, mean arterial pressure, PaO2/FiO2, TNF-alpha and IL-10 were detected prior to or after the intervention. The feasibility and tolerance of CVVH plus CPFA and the therapy-related adverse reactions were evaluated. RESULTS: The two groups showed no significant differences in the baseline clinical characteristics (P>0.05). The mean arterial pressure and PaO2/FiO2 increased significantly after treatment as compared with the control (P<0.05), with TNF-alpha being reduced and IL-10 elevated. In CVVH plus CPFA group, APACHEII score improved significantly after 10 days (P<0.05). No therapy-related adverse reactions were noted, suggesting good tolerance of CVVH plus CPFA. CONCLUSION: CVVH combined with CPFA is an effective and safe method for improving the clinical outcome of patients with SIRS and ARF.

[Low serum fetuin A is a risk factor of coronary artery calcification in patients starting hemodialysis].

OBJECTIVE: To examine the relationship between reduction of serum fetuin A and coronary artery calcification (CAC) in patients starting hemodialysis. METHODS: Twenty-nine patients on chronic hemodialysis (duration of hemodialysis less than 6 months) were enrolled in this study. Serum fetuin A and such potential CAC-related risk factors as C-reactive protein (CRP), Ca, P, iPTH, body mass index (BMI) were examined. CAC was detected by multislice spiral CT scan (MSCT) and quantified by the modified Agaston's scoring system. All the 29 patients were followed up for 18 months to appraise the cardiovascular events defined as cardiac failure, angina pectoris or myocardial infarction. RESULTS: Eleven patients (78.57%) were found to have CAC as detected by MSCT in low serum fetuin A (below the average serum concentration of 0.71 g/L) group, a rate significantly higher than that in high serum fetuin A group (7 patients, 46.67%, P<0.05). Serum fetuin A in these 29 patients was related with CAC score (Pearson correlation coefficient of -0.734, P=0.001) and stepwise regression analysis indicated that serum fetuin A (standardized beta=-0.568, P=0.003) and age (standardized beta=0.416, P=0.019) were independently correlated to CAC. Such factors as CRP, Ca, P, iPTH, Chol, TG, HDL-C, LDL-C, BMI and blood pressure were excluded from the regression equation. Reduction of serum fetuin A was associated with cardiovascular events (Spearman's rho -0.758, P<0.01). No significant difference was found between low and high serum fetuin A groups by Kaplan-Meier survival analysis (P=0.065). CONCLUSION: Reduced serum fetuin A may be a potential risk factor of coronary artery calcification, and can contribute to cardiovascular events in patients starting hemodialysis.

Combination of urinary kidney injury molecule-1 and interleukin-18 as early biomarker for the diagnosis and progressive assessment of acute kidney injury following cardiopulmonary bypass surgery: a prospective nested case-control study.

The aim of this nested case-control study was to assess the combined use of urinary kidney injury molecule (KIM)-1 and interleukin (IL)-18 for acute kidney injury (AKI) after cardiopulmonary bypass surgery (CPB). From a cohort of 122 subjects who underwent CPB, serial urinary KIM-1 and IL-18 concentrations were determined in 30 AKI and 92 non-AKI patients. An increased level of urinary KIM-1 was associated with the occurrence of AKI, whereas an increased level of IL-18 was related to progressive AKI. The combination of these two biomarkers facilitates the early diagnosis and assessment of the likely progression of AKI after CPB.

Podocyte injury is suppressed by 1,25-dihydroxyvitamin D via modulation of transforming growth factor-beta 1/bone morphogenetic protein-7 signalling in puromycin aminonucleoside nephropathy rats.

1. Accumulating evidence suggests that vitamin D and its analogues are renoprotective. However, the precise mechanisms and the molecular targets by which active vitamin D exerts its beneficial effects remain obscure. The objective of the present study was to evaluate the effect of active vitamin D on rats with puromycin aminonucleoside (PAN) nephropathy, a model that is characterized by predominant podocyte injury. 2. The PAN nephropathy rats were created by a single intravenous injection of 100 mg/kg PAN. Changes in renal pathology and podocyte numbers were observed. Real-time polymerase chain reaction (PCR) was performed to examine mRNA expression of nephrin, transforming growth factor (TGF)-beta1 and bone morphogenetic protein (BMP)-7. Protein expression of nephrin, TGF-beta1, BMP-7 and p-Smad2/3 and p-Smad1/5/8 was examined by immunofluorescence, immunohistochemistry and western blotting, respectively. Rats were treated with 1,25(OH)(2)D(3) by gastric gavage at a dose of 2.5 microg/kg per day, starting 2 days before PAN injection and continuing throughout the experiment. 3. A single injection of PAN induced massive proteinuria and elevated serum creatinine on Day 7, both of which were significantly suppressed by 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). Immunofluorescence and real-time PCR of the podocyte-associated protein nephrin revealed reduced and discontinuous staining and this change was reversed by 1,25(OH)(2)D(3). In PAN nephropathy rats, TGF-beta1 and p-Smad2/3 expression was upregulated, whereas that of BMP-7 and p-Smad1/5/8 was downregulated. Treatment with 1,25(OH)(2)D(3) significantly restored BMP-7/Smad signalling while suppressing TGF-beta1/Smad signalling. 4. In conclusion, 1,25(OH)(2)D(3) can ameliorate podocyte damage and proteinuria induced by PAN. The beneficial effects of 1,25(OH)(2)D(3) on podocytes may be attributable, in part, to direct modulation of TGF-beta1/BMP-7 signalling.

[Effect of bone morphogenic protein 7 on nephrin expression and distribution in diabetic rat kidneys].

OBJECTIVE: To evaluate the effect of bone morphogenic protein 7 (BMP-7) on nephrin expression and distribution in diabetic rat kidneys. METHODS: Twenty rats with diabetes mellitus (DM) induced by streptozotocin (STZ) injection were randomly divided into DM model group and BMP-7 treatment group, with another 10 normal rats serving as the normal control group. The rats in BMP-7 group received intraperitoneal human recombinant BMP-7 injections at 30 microg/kg twice a week for 24 consecutive weeks, while normal saline was administered in rats of the other two groups. Blood glucose and 24 hour urinary protein and creatinine (Ccr) were measured at 8, 16 and 24 weeks, and the rats were sacrificed at 24 weeks to obtain the renal tissues for detecting the expression and distribution of nephrin using immunofluorescence assay and RT-PCR and for examining the expressions of transforming growth factor-beta1 (TGF-beta1) and WT1 using immunohistochemistry. RESULTS: Compared with the normal control group, the DM model group showed significantly increased 24 hour urinary protein, kidney to body weight ratio and TGF-beta1 expression, but had lowered Ccr, glomerular podocyte number and nephrin expression. The linear distribution of nephrin along the capillary loops as found in the normal control group became granular in the kidney of diabetic rats. The rats in BMP-7 group showed less urinary protein excretion, lower TGF-beta1 expression and greater glomerular podocyte number than those in the DM group, and the expression and distribution of nephrin remained normal in the kidney. CONCLUSION: Administration of BMP-7 can significantly suppress the down-regulation of nephrin expression and maintain its normal distribution in the podocytes in diabetic rats possibly in association with a direct suppression of TGF-betasignaling.

1,25-Dihydroxyvitamin D(3) prevents puromycin aminonucleoside-induced apoptosis of glomerular podocytes by activating the phosphatidylinositol 3-kinase/Akt-signaling pathway.

BACKGROUND: Accumulating evidence suggests that vitamin D and its analogs reduce proteinuria and slow the decline in kidney function in chronic kidney disease. Given a rich literature identifying podocyte apoptosis as an early step in the pathophysiological progression to proteinuria and glomerulosclerosis, we hypothesized that vitamin D protects podocytes from undergoing apoptosis. METHODS: A rat model of podocyte apoptosis was created by a single intravenous injection of 100 mg x kg(-1) puromycin aminonucleoside (PAN) and received either solvent or 1,25(OH)(2)D(3) treatment. Proteinuria, podocyte apoptosis, the expression of nephrin protein and mRNA, TGF-beta/Smad and phosphatidylinositol 3-kinase (PI3K)/Akt-signaling pathway were evaluated, respectively. RESULTS: PAN induced massive proteinuria, serum creatinine elevation and podocyte apoptosis in PAN nephropathy rats, which was associated with the loss of nephrin, an adhesion molecule specific for the glomerular slit and the reduced of p-Akt/Akt ratio. Moreover, PAN induced foot process retraction, redistribution of nephrin and the activation of TGF-beta/Smad-signaling pathway. Compared with PAN nephropathy rats, 1,25(OH)(2)D(3) significantly prevented loss of nephrin, foot process retraction and podocyte apoptosis by stimulating Akt phosphorylation and suppressing TGF-beta/Smad-signaling pathway. CONCLUSION: 1,25(OH)(2)D(3) reduced the PAN-induced podocyte apoptosis and loss of nephrin in PAN nephropathy rat. The anti-apoptotic effects of 1,25(OH)(2)D(3 )on podocytes may be partly attributable to activation of a PI3K/Akt survival pathway.

[Prospective study of cystatin C for diagnosis of acute kidney injury after cardiac surgery].

OBJECTIVE: To prospectively study the value of cystatin C in diagnosis of acute kidney injury (AKI) in patients after cardiac surgery. METHODS: A total of 132 patients undergoing cardiopulmonary bypass were enrolled in this prospectively study. From each patient, blood samples were collected everyday before and after operation to detect the serum creatinine (Scr) and cystatin C levels by enzymatic method and particle-enhanced turbidimetric immunoassay (PETIA), respectively, and the glomerular filtration rate (eGFR) was estimated using MDRD equation. AKI diagnosis was made according to the RIFLE criteria of the Acute Dialysis Quality Initiative (ADQI) (R: Scr increased by > or =50%; I: Scr increased by > or =100%; F: Scr increased by > or =200%; L: Loss of kidney function; E: End-stage renal disease). Another AKI diagnostic criterion was also adopted according to the levels of cystatin C increment, namely an increase by > or =50%, > or =100%, and > or =200%. RESULTS: Twenty-nine patients (21.9%) developed AKI of varied severities, including 10 meeting the R-criteria, 12 the I-criteria, 7 the F-criteria, with the other 103 patients without AKI serving as the control group. Cystatin C of the 29 AKI patients was drastically increased in comparison with that of the control group (P<0.001). Significant linear correlation was found between cystatin C and Scr (r=0.732, P<0.001) and between [cystatin C]-1 and estimated GFR (R=0.803, P<0.001). By the two diagnostic criteria based on cystatin C and Scr levels, respectively, the median diagnostic time of AKI was 2 days (range 1-4 days) and 3 days (range 2-5 days) for R criteria (10 patients, P=0.014), 3.5 days (range 1-6 days) and 5 days (range 2-8 days) for I criteria (12 patients, P=0.008), and 5 days (range 3-7 days) and 6.5 days (range 4-9 days) for F criteria (7 patients, P=0.02), respectively. ROC analysis confirmed excellent accuracy of cystatin C in AKI diagnosis (AUC=0.992). With the cut-off value of cystatin C increment by > or =50%, the diagnostic sensitivity and specificity of AKI was 92% and 95%, respectively. CONCLUSION: Cystatin C can serve as a good indicator for AKI diagnosis to allow earlier detection of AKI than Scr-based diagnosis in patients after cardiac surgery.