[The incidence of aspirin resistance and relevant influencing factors in patients on maintenance hemodialysis].

OBJECTIVE: To explore aspirin resistance (AR) and its relevant influencing factors in patients on maintenance hemodialysis (MHD). METHODS: Patients on MHD who visited Beijing Chaoyang Hospital from June 1 to 30, 2011 were enrolled in this study. A total of 150 age and gender matched individuals with normal renal function were taken as control group. Anthropometric data, biochemistry parameters, ultrasonography and thromboelastograph (TEG) were inspected in the both groups. AR was defined as inhibiting rate of acetylsalicylic acid drugs [MA(AA)]>50% by TEG. RESULTS: Among the total 391 patients on MHD, hypercoagulation was found in 18 patients (4.6%), normal coagulation in 288 patients (73.7%) and hypocoagulation in 85 patients (21.7%). Pearson's correlation analysis revealed that the reaction time (R) and the thrombus maxithrombelastic degree (MA) values were not correlated with the levels of hemoglobin and platelet in MHD patients. A total of 306 patients with hypercoagulation and normal coagulation were chosen as the MHD group. Compared with the control group, higher high sensitivity C reactive protein (hsCRP), homocysteine (Hcy) and R value were observed in the MHD group (P < 0.05), while MA was significantly lower in the MHD group. Statistically higher incidence of AR was shown in the MHD group (48.0% vs 20.0%, P = 0.00). Patients in the MHD group were divided into the AR group and the aspirin sensitive (AS) group by the result of TEG. Compared with the AS group, patients in the AR group were found to be older with a higher female/male ratio, longer dialysis sustained time, higher ratio of diabetes history, higher hsCRP, Hcy and fasting blood glucose (FBG) and MA. They also manifested a higher incidence of cardiovascular and cerebrovascular diseases, peripheral vascular disease and arteriovenous fistulas with thrombosis with more spots of carotid artery and higher intima thickness of carotid artery (IMT) (all P values<0.05). Lower R value was shown in the AR group. Binary logistic regressive analysis revealed that the ratio of diabetes history, age and dialysis sustained time. Hcy and hsCRP were the independent risk factors for AR in patients on MHD. A total of 289 patients on MHD with atherosclerosis were followed up for the mean time of 18.0 months with no hemorrhage found in the process. Cox proportional hazards regression modeling demonstrated that AR was associated with the major adverse long-term outcome of the vascular events [HR = 0.40, 95%CI 0.29-0.72, P = 0.00]. CONCLUSIONS: The ratio of platelet activation in patients on MHD is significantly lower than in those with normal renal function. Small dose of aspirin could be prescribed for the patients on MHD with atherosclerosis to prevent vascular events. The incidence of AR is 48.0% in the MHD group and the independent risk factors for AR in MHD patients are the ratio of diabetes history, age, dialysis sustained time, Hcy and hsCRP. AR is associated with the major adverse long-term outcome of acute vascular events.

Is the dialysate calcium concentration of 1.75 mmol/L suitable for Chinese patients on maintenance hemodialysis?

We studied the effects of increasing the dialysate calcium concentration (DCa) to 1.75 mmol/L on controlling chronic kidney disease-mineral and bone disorder in Chinese patients on maintenance hemodialysis (MHD). We reviewed the data of MHD patients in one center (cohort 1) during prior 10 years and analyzed the risk factors of mortality and transference calcification (TC) in120 MHD patients surviving in 2003 (cohort 2). A multicenter, prospective, parallel-group, controlled trial (cohort 3) was also conducted from January 2011 to December 2012. The DCa at one center was increased from 1.5 to 1.75 mmol/L but was not changed at the other two centers. The clinical outcomes, biochemical parameters, medicine treatments, and TC markers [aortic arch calcification score (AoACS)] were compared between groups. In cohort 1, the annual mean serum iPTH increased significantly over 10 years. In cohort 1, 72 patients survived for 10 years, whose doses of calcium salts and active vitamin D3 and AoACs increased progressively. In cohort 2, the main cause of death was cardiocerebrovascular disease (CCVD) (n = 18, 48.6 %). Male sex and lower serum calcium concentrations were independent risk factors for CCVD mortality. In cohort 3, serum phosphorus, iPTH, and 25(OH)D decreased and serum calcium increased significantly; also, the doses of calcium and vitamin D3 decreased from 2011 to 2012 in the DCa 1.75 group. There were no significant differences in clinical outcomes either between groups or between the two calendar years. Our results indicate that increasing DCa to 1.75 mmol/L can decrease the elevated levels of serum iPTH and phosphorus, reduce the doses of calcium and vitamin D3, and be safe for short periods of time.

Impact of dry weight determined by calf bioimpedance ratio on carotid stiffness and left ventricular hypertrophy in hemodialysis patients.

Our previous study has shown that modification of bioimpedance technique by the measurement of bioimpedance ratio in the calf (calf-BR) was a simple and practical method in assessing fluid status in hemodialysis patients. However, the consequences of periodical dry weight (DW) adjustment under the guidance of calf-BR on target organ damage have not been investigated. One hundred fifteen hemodialysis patients were enrolled in this pilot trial. Patients were divided into bioimpedance group and control group according to their dialysis schedule. In the bioimpedance group, DW was routinely adjusted under the guidance of calf-BR every 3 months. In the control group, the assessment of DW remained a clinical judgment. Carotid stiffness, left ventricular mass index (LVMI), and calf-BR were measured at baseline and at the 12th month in both groups. Home blood pressure (BP) was monitored monthly. Episodes of dialysis-related adverse events were recorded. No significant differences were observed in parameters between the two groups at baseline. Compared with the control group, the bioimpedance group had significantly lower values in terms of the annual averages of systolic home BP (147.4 ± 15.3 mm Hg vs. 152.6 ± 16.9 mm Hg, P = 0.019), carotid stiffness index ß (10.7 ± 3.3 vs. 12.2 ± 3.1, P = 0.003), LVMI (155.21 ± 15.64 g/m(2) vs. 165.17 ± 16.76 g/m(2) , P < 0.001), and the percentage of individuals with calf-BR over target range (P = 0.040) at month 12, with less annual averages of antihypertensive medications used and lower frequency of intradialytic hypotension, muscle cramps, or clotted angioaccess. Continued DW control achieved by periodical calf-BR measurement improved arterial stiffness and left ventricular hypertrophy with good tolerability in hemodialysis patients.

Effects of increasing diffusive sodium removal on blood pressure control in hemodialysis patients with optimal dry weight.

BACKGROUND: Sodium, apart from volume, may have an independent effect on blood pressure (BP) regulation. METHODS: Sixteen hypertensive hemodialysis patients were enrolled, who have achieved their dry weight assessed by bioimpedance methods, with pre-dialysis plasma sodium levels slightly higher than the facility dialysate sodium concentration 138 mmol/l. After a 1-month period of dialysis with standard dialysate sodium concentration of 138 mmol/l, the patients were followed up for a 4-month period with dialysate sodium set at 136 mmol/l. RESULTS: Along with lowering dialysate sodium, there were significant decreases (-10 mm Hg and -6 mm Hg) in 44-hour ambulatory systolic and diastolic BP at 4 months. Interdialytic weight gain adjusted to the estimated dry weight mildly but significantly decreased (4.81 ± 1.51 vs. 4.36 ± 1.37%, p = 0.047). The post-dialysis volume parameters remained constant throughout the study period. CONCLUSION: In selected hypertensive hemodialysis patients with optimal dry weight, increasing diffusive sodium removal resulted in significant BP decrease. It was probably due to a volume-independent effect.

Mortality rates among prevalent hemodialysis patients in Beijing: a comparison with USRDS data.

BACKGROUND: The raw annual mortality rate reported in Chinese patients on maintenance hemodialysis (MHD) was around 10% between 2005 to 2010, and it was around 20% in the US as reported by the United States Renal Data System (USRDS). Our hypothesis was that the large survival difference was caused by differences in race and practice pattern between nations in addition to differences in patient characteristics. METHODS: Annual mortality in Beijing prevalent MHD patients per year in 2007, 2008, 2009 and 2010 was reported and relative risks of death were compared with the corresponding mortality of USRDS prevalent MHD patients (in whites, African-Americans and Asian-Americans) after age, gender and primary cause of end-stage renal disease (ESRD) were adjusted. A total of 11 675 MHD patients from 104 dialysis facilities under control of Beijing Blood Purification Quality Control and Improvement Center (BJBPQCIC) from 31 December 2006 to 31 December 2010 were included. A total of 1 937 819 MHD patients (only white, African-American and Asian-American were eligible for inclusion) were subtracted from the USRDS No-60-Day prevalent dataset from the year 2004 to 2009, using the RenDER system. Raw annual mortality for each race was reported as a number per 1000 MHD patients at risk for each year. Age, gender and primary cause of ESRD, adjusted annual mortality and relative risk race of death were reported comparing the Beijing patients and each race of the USRDS. RESULTS: The raw annual mortality for the Beijing cohort increased gradually from 47.8 per 1000 patient-years in 2007 to 76.8 in 2010. The raw annual mortality for the white cohort in 2007 was 250.7 per 1000 patient-years, and gradually decreased to 236.3 in 2009. The raw annual mortality for African-Americans (167.8 and 156.7 per 1000 patient-years in 2007 and 2009, respectively) was much lower than that for whites. The annual mortality for Asian-Americans was slightly lower than that for African-Americans. After adjustment, Beijing MHD still had a survival benefit compared with each of the examined USRDS race. The annual mortality rates were 99.4, 80.6 and 94.3 per 1000 patient-years when adjusted to whites, African-Americans and Asian-Americans, respectively, in cohort 2009. CONCLUSIONS: The annual mortality for the Beijing MHD patients was lower than that for their USRDS counterparts, and this difference existed after baseline demographics were adjusted. This survival difference between the Beijing and the USRDS MHD cohorts could be attributed to differences in race or practice pattern. More studies are needed to validate our hypothesis.

Estimating the hydration status in nephrotic patients by leg electrical resistivity measuring method.

AIM: For years, bioelectrical impedance analysis has been widely used to evaluate the hydration status in dialysis patients. However, its value in assessment and controlling the hydration status in non-dialysis patients with kidney disease, such as nephrotic syndrome, is little mentioned. Because a simple and accurate method to evaluate the hydration status of nephrotic patients is not available, the aim of the present study was to assess the value of leg electrical resistivity measurement in controlling the hydration status of nephrotic patients. METHODS: The study investigated 46 nephrotic patients with a mean age of 41.65 +/- 17.15 years, 47.8% of whom were female. The patients were divided into remission and relapse groups according to their serum albumin concentration and oedema. Four hundred and twenty-seven healthy persons were studied as normal control. Their hydration status estimated by leg electrical resistivity was studied. RESULTS: There was significant negative correlation between leg electrical resistivity and percentage of extracellular fluid (ECF) measured by the bromide dilution method. The percentage of ECF estimated by the leg electrical resistivity in the relapse group was significantly larger than that of the remission group, but it was approximately the same in the remission group as in the normal control. For nephrotic patients in the relapse group, after they ahcieved remission, their percentage of ECF estimated by the leg electrical resistivity was significantly less than that before treatment, and was close to that of the normal control. CONCLUSION: Leg electrical resistivity measurement is a simple, non-invasive and valuable method for controlling the hydration status in patients with nephrotic syndrome.

Calf bioimpedance ratio improves dry weight assessment and blood pressure control in hemodialysis patients.

BACKGROUND: Chronic fluid overload due to overestimation of dry weight (DW) is the major factor in the development of hypertension in hemodialysis (HD) patients. The present study was undertaken to investigate whether bioimpedance ratio in the calf (Calf-BR = impedance at 200 kHz/impedance at 5 kHz) could be a useful hydration marker for estimation of DW and facilitate better control of blood pressure (BP) in HD patients. METHODS: Target range of Calf-BR was derived from 157 healthy Chinese subjects. Post-dialysis Calf-BR was measured in 117 stable, non-edematous HD patients. Those with Calf-BR(s) above target level had their DW(s) gradually reduced under the guidance of Calf-BR. RESULTS: The Calf-BR was normally distributed and increased with age, but was independent of BMI and gender in both healthy subjects and dialysis patients. HD patients with Calf-BR above age-stratified target range had significantly higher home BP, in spite of more antihypertensive treatments (p = 0.058). The patients who reached the target range of Calf-BR by decreasing DW, had their home BP significantly decreased, along with reduction in antihypertensive medications (p = 0.012). CONCLUSION: Recognition and correction of chronic fluid overload based on age-stratified Calf-BR is helpful in hypertension control in Chinese HD patients.

Association of serum pentosidine with arterial stiffness in hemodialysis patients.

Pentosidine is an advanced glycation end product (AGE). The present study was undertaken to investigate the association of serum pentosidine with carotid distensibility as a measure of arterial stiffness in hemodialysis patients. One hundred and three patients on maintenance hemodialysis were recruited. The distensibility coefficient of the common carotid artery was evaluated by an ultrasonic phase-locked echo-tracking system. Serum pentosidine was measured by competitive enzyme-linked immunosorbent assay. Serum albumin, lipid profile, calcium, phosphorus, intact parathyroid hormone (iPTH), high-sensitivity C-reactive protein (hs-CRP), and oxidized low-density lipoprotein (ox-LDL) levels were also measured. Correlation was determined by linear and multiple stepwise regression analysis. Serum pentosidine level studied in hemodialysis patients was 0.54 +/- 0.13 microg/mL. No significant difference in serum pentosidine level was noted between patients with and without diabetes (0.59 +/- 0.10 microg/mL vs. 0.53 +/- 0.13 microg/mL, P = 0.062) as well as between patients with and without prior cardiovascular disease (CVD) history (0.56 +/- 0.14 microg/mL vs. 0.53 +/- 0.12 microg/mL, P = 0.206). In multivariate regression analysis, only age (beta = 0.363, P < 0.001) and ox-LDL (beta = 0.262, P = 0.004) were identified as independent determinants for serum pentosidine. Serum pentosidine was significantly correlated with carotid distensibility (r = -0.387, P < 0.001), as well as age, ox-LDL, and hs-CRP. After adjustment for age, blood pressure, history of diabetes, prior CVD history, lipid profile, calcium, phosphorus, iPTH, hs-CRP, and ox-LDL, serum pentosidine was still negatively correlated with distensibility (beta = -0.175, P = 0.044). Serum pentosidine was independently associated with carotid distensibility in hemodialysis patients. This finding suggested that the accumulation of AGE might be an important pathway in the development of arterial stiffness in end-stage renal disease.

Tumor suppressor cylindromatosis: expressed in IgA nephropathy and negatively associated with renal tubulo-interstitial lesion.

BACKGROUND: IgA nephropathy is the major cause of end-stage renal failure in patients with primary glomerular diseases. Tumor suppressor cylindromatosis (CYLD), the recently identified member of the deubiquitinating enzymes, has been actively involved in regulation of inflammation. This study was undertaken to investigate the CYLD expression profile in IgA nephropathy and identify factors associated with CYLD expression. METHODS: Forty-one cases of IgA nephropathy were selected. CYLD expression in the kidney biopsy tissue was measured by immunohistochemical staining. Relevant clinical and pathological data were analyzed, and Logistic regression analysis was carried out to identify factors associated with CYLD expression. RESULTS: CYLD was specifically expressed in renal tubular epithelial cells in 70% of the studied patients with IgA nephropathy. All patients with positive CYLD staining had proteinuria, while only 72.7% of patients with negative CYLD had proteinuria (P = 0.003). Among studied proteinuric patients, those with positive CYLD had significantly less tubulo-interstitial lesions and higher estimated glomerular filtration rate (eGFR) levels when compared with those patients showed negative CYLD results. Logistic regression analysis indicated that the urinary protein excretion and eGFR were identified as predictors for the CYLD expression. CONCLUSION: CYLD is expressed in renal tubular epithelial cells and appears to be associated negatively with tubulointerstitial lesions, however, its exact functional role remains to be clarified in further experiments.

An emerging role of deubiquitinating enzyme cylindromatosis (CYLD) in the tubulointerstitial inflammation of IgA nephropathy.

Immunoglobulin A (IgA) nephropathy is an important cause of end-stage kidney disease (ESKD). Tubulointerstitial inflammation and subsequent fibrosis appear to be a major contributor of the disease progression to ESKD; however, the underlying mechanism is poorly understood. Herein, we report that a unique feature of CYLD expression in kidneys of patients with IgA nephropathy and a CYLD-mediated negative regulation of inflammatory responses in human tubular epithelial cells. Immunochemical staining revealed that CYLD was predominantly expressed in renal tubular epithelial cells in 81% of the patients (37 cases) with proteinuric IgA nephropathy. Patients with positive CYLD had significantly less tubulointerstitial lesions and higher estimated glomerular filtration rate (eGFR) levels when compared with those negative. Logistic regression analysis indicated that eGFR was a predictor for the CYLD expression. In cultured human tubular epithelial HK-2 cells, tumor necrosis factor-alpha (TNFalpha) up-regulated CYLD expression. Adenoviral knockdown of CYLD did not affect albumin-, hydrogen peroxide (H(2)O(2))-, tunicamycin- or thapsigargin-induced cell death; however, it enhanced TNFalpha-induced expression of intracellular adhesion molecule (ICAM)-1 as well as activation of c-Jun N-terminal kinase (JNK). Moreover, monocyte adhesion to the TNFalpha-inflamed HK-2 cells was significantly increased by the CYLD shRNA approach. Taken together, our results suggest that CYLD negatively regulates tubulointertitial inflammatory responses via suppressing activation of JNK in tubular epithelial cells, putatively attenuating the progressive tubulointerstitial lesions in IgA nephropathy.

Association between insulin resistance and carotid arterial stiffness in nondiabetic hemodialysis patients.

BACKGROUND: Patients with end-stage renal disease are known to have insulin resistance and advanced arterial stiffness. The present study was undertaken to investigate whether insulin resistance was correlated with carotid arterial stiffness in hemodialysis patients. METHODS: Eighty nondiabetic hemodialysis patients were recruited. Stiffness index beta of the common carotid artery was assessed by ultrasound using echo-tracking system. Insulin resistance was evaluated by the homeostasis model assessment method (HOMA-IR). Serum albumin, lipid profile, calcium, phosphorus, intact parathyroid hormone, high-sensitivity C-reactive protein and oxidized low-density lipoprotein (ox-LDL) were also measured. RESULTS: Patients with higher HOMA-IR levels had higher BMI, ox-LDL, and stiffness index beta than patients with lower HOMA-IR levels. HOMA-IR was independently correlated with stiffness index beta (beta = 0.260, p = 0.016). BMI and ox-LDL were independent contributors to HOMA-IR. CONCLUSION: Insulin resistance assessed by HOMA-IR is closely associated with carotid arterial stiffness in nondiabetic hemodialysis patients.

A randomized trial comparing conventional swan-neck straight-tip catheters to straight-tip catheters with an artificial subcutaneous swan neck.

OBJECTIVE: To evaluate the safety and efficacy of inserting a straight-tip Tenckhoff catheter configured with a subcutaneous artificial swan neck. DESIGN: Clinical outcomes of conventional swan-neck straight-tip catheters and Tenckhoff straight-tip catheters implanted with an artificial subcutaneous swan neck were compared in a prospective randomized controlled trial in a single-center setting. PATIENTS AND METHODS: Patients undergoing peritoneal dialysis catheter insertion were randomized to receive either a double-cuff straight-tip Tenckhoff catheter with an artificial subcutaneous swan-neck (TC) or a conventional double-cuff straight-tip swan-neck catheter (SN). The primary outcome was catheter exit-site infection rate; the secondary outcomes were catheter-related mechanical events and surgery-related bleeding. RESULTS: A total of 39 consecutive patients were enrolled: 20 into the TC group and 19 into the SN group. More exit-site infections were observed in the SN group than in the TC group, although the difference was not statistically significant (0.97 vs 0.51 episodes per patient-year, p = 0.0657). However, there were more peritonitis episodes in the TC group than in the SN group (0.35 vs 0.15 episodes per patient-year, p = 0.0256). Exit-site and main wound bleeding post surgery were generally mild and similar in the 2 groups. No events of dialysate leakage, catheter tip migration, or subcutaneous cuff protrusion were observed in patients of either group. Outflow failure due to mechanical causes occurred in 2 patients in the TC group and in 1 patient in the SN group during the intermittent peritoneal dialysis period; all were corrected successfully by laparoscopic omentectomy. CONCLUSIONS: Placement of the double-cuff straight-tip Tenckhoff catheter configured with an artificial subcutaneous swan neck appears to be an effective and safe procedure. It may be a good alternative to the conventional swan-neck catheter.

[Treatment of proliferative lupus nephritis with leflunomide and steroid: a prospective multi-center controlled clinical trial].

OBJECTIVE: Leflunomide (LEF) is a selective inhibitor of de novo pyrimidine synthesis, currently used in the treatment of rheumatoid arthritis. To evaluate the efficacy and safety of LEF in the treatment of proliferative lupus nephritis, a prospective multi-center controlled clinical trial was conducted. METHODS: Patients with biopsy-confirmed proliferative lupus nephritis were recruited. Patients of recent onset who had not used any immunosuppressive drug were given either oral LEF (group A) or IV cyclophosphamide (group B); relapsed patients who had received immunosuppressive therapy 3 months before were given LEF (group C). Efficacy and safety were evaluated at 6 months after treatment. RESULTS: Total 51 patients were enrolled, 4 patients withdrew due to adverse events. For those initial treated patients, total response rate were 80% in group A and 75% in group B, complete remission rate were 40% and 25% respectively, not statistically different. Renal parameters (proteinuria, serum albumin and serum creatinine) and systemic lupus erythematosus disease activity index (SLEDAI) improved similarly in both groups. For 14 relapsed patients, total response rate was 60% and complete remission rate was 6.7%. Major adverse events reported in LEF treated patients were infection and alopecia. Herpes zoster was the most often type among infectious events, and one case of severe lung infection was reported. CONCLUSION: LEF combined with steroid was effective in the induction therapy of proliferative lupus nephritis. LEF was generally well-tolerated, its efficacy in maintenance therapy and long-term safety remains to be clarified.

VEGF165b, an inhibitory vascular endothelial growth factor splice variant: mechanism of action, in vivo effect on angiogenesis and endogenous protein expression.

Growth of new blood vessels (angiogenesis), required for all tumor growth, is stimulated by the expression of vascular endothelial growth factor (VEGF). VEGF is up-regulated in all known solid tumors but also in atherosclerosis, diabetic retinopathy, arthritis, and many other conditions. Conventional VEGF isoforms have been universally described as proangiogenic cytokines. Here, we show that an endogenous splice variant, VEGF(165)b, is expressed as protein in normal cells and tissues and is circulating in human plasma. We also present evidence for a sister family of presumably inhibitory splice variants. Moreover, these isoforms are down-regulated in prostate cancer. We also show that VEGF(165)b binds VEGF receptor 2 with the same affinity as VEGF(165) but does not activate it or stimulate downstream signaling pathways. Moreover, it prevents VEGF(165)-mediated VEGF receptor 2 phosphorylation and signaling in cultured cells. Furthermore, we show, with two different in vivo angiogenesis models, that VEGF(165)b is not angiogenic and that it inhibits VEGF(165)-mediated angiogenesis in rabbit cornea and rat mesentery. Finally, we show that VEGF(165)b expressing tumors grow significantly more slowly than VEGF(165)-expressing tumors, indicating that a switch in splicing from VEGF(165) to VEGF(165)b can inhibit tumor growth. These results suggest that regulation of VEGF splicing may be a critical switch from an antiangiogenic to a proangiogenic phenotype.

Differentiated human podocytes endogenously express an inhibitory isoform of vascular endothelial growth factor (VEGF165b) mRNA and protein.

Despite production by podocytes of the proangiogenic molecule vascular endothelial growth factor-A (VEGF), the glomeruli are not sites of angiogenesis. We recently described mRNA expression of an inhibitory splice variant of VEGF (VEGF165b) in normal kidney (Bates DO, Cui TG, Doughty JM, Winkler M, Sugiono M, Shields JD, Peat D, Gillatt D, and Harper SJ. Cancer Res 62: 4123-4131, 2002). Available anti-VEGF antibodies do not distinguish stimulatory from inhibitory VEGF families. To assess the production of VEGF165 (stimulatory) and VEGF165b (inhibitory) isoforms by human podocytes, we examined both primary cultured and conditionally immortalized human podocytes using family- and isoform-specific RT-PCR. In addition, VEGF protein production was analyzed in podocytes, using isoform-specific double-strand small-interference RNAs (siRNA). RT-PCR demonstrated the production of VEGF189 mRNA by podocytes of both phenotypes. In contrast, on differentiation there was a splicing change from VEGF165 to VEGF165b mRNA. In addition, VEGF protein in the supernatant of conditionally immortalized, differentiated podocytes was reduced by VEGF165b siRNA to 20+/-11% of the level of mock-transfected cells (P < 0.01). No reduction was seen with mismatch siRNA. Moreover, there was no reduction in VEGF protein concentration in the supernatant of primary cultured, dedifferentiated human podocytes (109+/-8% of mismatch siRNA, P > 0.1). In conclusion, differentiated but not dedifferentiated human podocytes secrete significant amounts of VEGF165b protein. It is possible that this may explain the paradox of high VEGF production in the glomerulus but no angiogenesis. Furthermore, the existence of this splicing switch in relation to podocyte phenotype suggests that alternative splicing of the VEGF pre-RNA is a regulated process that is open to manipulation and therefore could be a target for novel cancer therapies.

VEGF165b, an inhibitory splice variant of vascular endothelial growth factor, is down-regulated in renal cell carcinoma.

Angiogenesis is essential for tumor growth. Vascular endothelial growth factor (VEGF) is the most potent growth factor of tumor neovasculature, has been shown to be up-regulated in every tumor studied thus far, and is correlated with tumor stage and progression. To determine whether specific VEGF splice variants were differentially expressed in renal cell carcinomas, 18 polar tumor samples were analyzed by reverse transcription-PCR using primers designed to differentiate between VEGF splice variants. Control tissue was derived from the opposite normal pole. An amplicon of length consistent with the previously described variant VEGF(148) was found in normal kidney tissue. Subsequent sequencing revealed a new VEGF isoform formed by differential splicing from the end of exon 7 into the 3' untranslated region of the mRNA. Cloning of this transcript showed that translation would result in a 165-amino acid peptide with an alternative terminal 6 amino acids, followed by a stop codon. We have termed this new isoform VEGF165b. This isoform was present in 17 of 18 normal kidney samples but only 4 of 18 cases from matched malignant tissue. VEGF165b was therefore expressed in a significantly higher proportion of normal tissue than malignant tissue from the same patients (P < 0.001). To determine the functional significance of this new isoform, we expressed the full-length protein in a heterologous expression system. Conditioned medium containing this isoform significantly and dose dependently inhibited VEGF165-mediated proliferation, migration of endothelial cells, and vasodilatation of mesenteric arteries. This novel isoform VEGF165b is therefore an endogenous inhibitory form of VEGF that is down-regulated in renal tumors and, therefore, may be anti-angiogenesis.