Carotid atherosclerosis in virologically suppressed HIV patients: comparison with a healthy sample and prediction by cardiovascular risk equations.

OBJECTIVES: To compare the prevalence of carotid atherosclerosis in virologically suppressed HIV patients with that of a community sample, and to evaluate the capacity of various cardiovascular risk (CVR) equations for predicting carotid atherosclerosis. METHODS: This was a cross-sectional study with two randomly selected groups: HIV patients from an HIV unit and a control group drawn from the community. Participants were matched by age (30-80 years) and sex without history of cardiovascular disease. Carotid plaque, common carotid intima-media thickness (cc-IMT) and subclinical atherosclerosis (carotid plaque and/or cc-IMT > 75th percentile) were assessed by carotid ultrasound. The Systematic Coronary Risk Evaluation (SCORE), Framingham, REGICOR, reduced Data Collection on Adverse Effects of Anti-HIV Drugs (D:A:D), and COMVIH equations were applied, and their abilities to predict carotid plaque were compared using the area under the curve (AUC). RESULTS: Each group included 379 subjects (77.8% men, age 49.7 years). Duration of antiretroviral therapy was 15.5 years. There were no differences between the groups for carotid plaque (HIV, 33.2%; control, 31.3%), mean cc-IMT (HIV, 0.63 mm; control, 0.61 mm) or subclinical atherosclerosis (HIV, 42.9%; control, 47.9%). Thymidine analogues were independently associated with subclinical atherosclerosis in HIV-infected patients. CVR equations revealed AUCs between 0.715 and 0.807 for prediction of carotid plaque; prediction was better in the control group and did not improve when HIV-adapted scales were used. CONCLUSIONS: The features of carotid atherosclerosis did not differ between the HIV-infected and the control group, although CVR equations were more predictive for carotid plaque in controls than in HIV-infected patients. HIV-specific equations did not improve prediction.

Sprouty1 induces a senescence-associated secretory phenotype by regulating NFκB activity: implications for tumorigenesis.

Genes of the Sprouty family (Spry1-4) are feedback inhibitors of receptor tyrosine kinase (RTK) signaling. As such, they restrain proliferation of many cell types and have been proposed as tumor-suppressor genes. Although their most widely accepted target is the Extracellular-regulated kinases (ERK) pathway, the mechanisms by which Spry proteins inhibit RTK signaling are poorly understood. In the present work, we describe a novel mechanism by which Spry1 restricts proliferation, independently of the ERK pathway. In vivo analysis of thyroid glands from Spry1 knockout mice reveals that Spry1 induces a senescence-associated secretory phenotype via activation of the NFκB pathway. Consistently, thyroids from Spry1 knockout mice are bigger and exhibit decreased markers of senescence including Ki67 labeling and senescence-associated ß-galactosidase. Although such 'escape' from senescence is not sufficient to promote thyroid tumorigenesis in adult mice up to 5 months, the onset of Phosphatase and tensin homolog (Pten)-induced tumor formation is accelerated when Spry1 is concomitantly eliminated. Accordingly, we observe a reduction of SPRY1 levels in human thyroid malignancies when compared with non-tumoral tissue. We propose that Spry1 acts as a sensor of mitogenic activity that not only attenuates RTK signaling but also induces a cellular senescence response to avoid uncontrolled proliferation.

[Vascular calcification: types and mechanisms]. / Calcificación vascular: tipos y mecanismos.

Vascular calcification has traditionally been considered to be a passive process that was associated with advanced age, atherosclerosis, uncommon genetic diseases and some metabolic alterations such as diabetes mellitus and end-stage kidney failure. However, in the last years, vascular calcification has been proven to be an active and regulated process, similar to bone mineralisation, in which different bone-related proteins are involved. Recent results question the classic classification of vascular calcification into intimal and medial calcification, at least in capacitance arteries. Pro and anti-calcifying mechanisms play an active role in calcium deposition in vascular cells, making this area an active focus of research. The identification of therapeutic targets which can slow down the progression or even reverse vascular calcification could be an important step forward in the treatment of patients with chronic kidney disease.

[Usefulness of imaging techniques and novel biomarkers in the prediction of cardiovascular risk in patients with chronic kidney disease in Spain: the NEFRONA project]. / Utilidad de las técnicas de imagen y biomarcadores en la predicción del riesgo cardiovascular en pacientes con enfermedad renal crónica en España: Proyecto NEFRONA.

BACKGROUND: Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in patients with chronic kidney disease (CKD). Cardiovascular risk assessment in this population is hampered by the failure of traditional risk factors to fully account for the elevated CVD risk, mainly due to the reverse epidemiology effect, and the presence of risk factors specifically related to uremia. Hereby, we present the protocol of a prospective study aimed to assess the predictive value of imaging techniques and biomarkers for CVD in patients with CKD. METHODS: From November 2009, 2.661 asymptomatic adult patients with stages 3-5D CKD will be recruited from nephrology services and dialysis units throughout Spain. Eight hundred forty-three participants without CKD (control group) will be also recruited. During the follow-up, CVD events and mortality will be recorded from all CKD patients. One trained itinerant team will carry out a carotid ultrasound to assess intima-media thickness and presence of plaques. A composite atherosclerosis score will be constructed based on carotid ultrasound data and ankle-brachial index. Presence and type of calcifications will be assessed in carotid, femoral and brachial arteries, and in cardiac valves, by ultrasound. Finally, blood samples will be collected from all participants to study biomarkers. DISCUSSION: The NEFRONA study will allow us to examine the usefulness of imaging techniques and biomarkers to assess atherosclerosis development and their predictive value in a Spanish population with CKD.

Occult chronic kidney disease: discordance among different methods used to estimate glomerular filtration rate in a healthy population.

BACKGROUND: Chronic kidney disease is a widely recognized cardiovascular risk factor. Its detection within large populations depends upon the method used to estimate glomerular filtration. The Cockcroft and MDRD equations are widely used, although their accuracy is limited in certain cases. METHODS: The present study analyzes glomerular filtration values in 674 young, healthy subjects using five methods: Cockcroft, Cockcroft corrected for body surface, MDRD-4 Lund-Malmö and Sawyer formulas. Glomerular filtration values obtained with the first three methods were compared using ANOVA. The Spearman coefficient was calculated to estimate the correlation between MDRD-4 and Cockcroft values, and between Cockcroft values and body mass index. RESULTS: There was a slight glomerular filtration rate decrease (< 90 ml/min) seen in 394 subjects using the Cockcroft equation, and in 344 subjects using the MDRD-4 formula. The prevalence of chronic kidney disease (glomerular filtration < 60 ml/min) was seen in 3 subjects using the MDRD-4 equation and 161 subjects using the Cockcroft formula. There was significant discordance, by method, between values obtained, with 40% of the population being classified into different stages (> 90 or < 90 ml/min) depending on the formula used. In 8% of the population there was even greater discordance, because they had strictly normal renal function according to MDRD-4 (> 90 ml/min) but fell into chronic kidney disease Stage 3 (< 60 ml/min) according to the Cockcroft formula. There was poor correlation between glomerular filtration rates obtained using the Cockcroft and MDRD-4 equations, suggesting that the subjects with a glomerular filtration rate decrease detected by the two methods were not the same ones. There was correlation between body mass index and glomerular filtration rates obtained with Cockcroft, Cockcroft corrected for body surface and Sawyer formulas and not with MDRD and Lund-Malmö equations. CONCLUSIONS: There are important discrepancies between the methods used to assess renal function in healthy populations. These limitations must be taken into account when deciding on strategies for diagnosis and control of occult chronic kidney disease in the general population.

Mineral metabolism influences pulse pressure increase provoked by chronic kidney disease.

BACKGROUND: Pulse pressure (PP) increase has been associated with hypertension, ageing and chronic kidney disease. Although hyperparathyroidism and phosphate imbalance have been suspect in PP increase in hemodialysis patients, the link between these parameters and pulse pressure, in renal disease before dialysis, has not been established. METHODS AND PATIENTS: 1966 chronic kidney disease (CKD) patients. STATISTICS: ANOVA, Student's t-and Chi-square, rank correlations (Spearman) and multivariate analysis, with PP as the dependent variable, while adjusting for other covariables. RESULTS: There was an increase of pulse pressure parallel to renal function deterioration, and a significant influence of age, diabetes, hypertension, phosphate and PTH on pulse pressure in the whole population, as well as in patients with glomerular filtration rate < 60 ml/min. The impact of phosphate was particularly high after the age of 50. CONCLUSION: PP increase present in renal disease patients might be primarily due to the underlying mineral metabolism disturbances.

Influence of mineral metabolism parameters on pulse pressure in healthy subjects.

BACKGROUND: The role of mineral metabolism in cardiovascular pathologies has been studied almost exclusively in chronic kidney disease patients. There are no studies that relate mineral metabolism to pulse pressure in healthy populations. METHODS: 692 subjects were initially selected. After applying clinical exclusion criteria, 659 subjects were recruited. Creatinine clearance was then calculated to detect subjects with occult chronic kidney disease. Statistical analysis was applied to the remaining population after excluding subjects with occult chronic kidney disease (n = 466). Pulse pressure, creatinine clearance, calcium, phosphorus, intact parathormone, 25-hydroxivitamin D3 and Bsm I genotype of the vitamin D receptor were determined. Means and frequencies were compared by ANOVA and Chi-square, respectively. Multivariate analysis was applied to the whole population and then to Caucasians, Sub-Saharans, Caucasian men and Caucasian women separately. Pulse pressure (PP) was the dependent variable, and adjustments were made for clinical and laboratory data. RESULTS: The prevalence of occult chronic kidney disease was 32%. In subjects without kidney disease, phosphorus and vitamin D were independent predictors of elevated PP in Caucasian males whereas Bsm I genotype of the vitamin D was an independent predictor of elevated PP in the Caucasian population in both genders. No covariable showed relationship with PP in Sub-Saharan subjects. CONCLUSION: Mineral metabolism influences pulse pressure in Caucasian men.

1,25-Dihydroxyvitamin D3 stimulates vascular smooth muscle cell proliferation through a VEGF-mediated pathway.

Atherosclerosis is a complex process characterized by an increase in the wall thickness owing to the accumulation of cells and extracellular matrix between the endothelium and the smooth muscle cell wall. This process is associated with different pathologies and it is accelerated in patients with chronic renal failure. In these patients, decreased synthesis of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) leads to secondary complications, like hyperparathyroidism, and treatment with 1,25(OH)(2)D(3) is a common practice. The effect of 1,25(OH)(2)D(3) on vascular smooth muscle cells (VSMCs) calcification has been widely studied, but the role of 1,25(OH)(2)D(3) on VSMC proliferation remains obscure. We have analyzed the effects of 1,25(OH)(2)D(3) in the proliferation of VSMC. We found that 1,25(OH)(2)D(3) (5-100 nM) induces a dose-dependent increase in VSMC proliferation in quiescent cells and in cells stimulated to grow. This increase in proliferation is achieved by shortening the G1 phase. The effect of 1,25(OH)(2)D(3) on VSMC proliferation is mediated by an increase of the expression of vascular endothelial growth factor A (VEGF), as the inhibition of VEGF activity totally blunted the 1,25(OH)(2) D(3)-induced VSMC proliferation. We found this increase in proliferation in vitro, ex vivo in aortic rings incubated with 1,25(OH)(2)D(3), and in vivo in animals with a model of chronic renal failure (5/6 nephrectomy) treated with 1,25(OH)(2)D(3) (1 mug/kg three times a week for 8 weeks). Thus, we conclude that 1,25(OH)(2)D(3) induces increases in VSMC proliferation through an increase on VEGF expression.

Renal ischemia in the rat stimulates glomerular nitric oxide synthesis.

Renal ischemia in humans and in experimental animals is associated with a complex and possibly interrelated series of events. In this study, we have investigated the glomerular nitric oxide (NO) production after renal ischemia. Unilateral or bilateral renal ischemia was induced in Wistar rats by clamping one or both renal arteries. NO production was assessed by measuring glomerular production of nitrite, a stable end product of NO catabolism, and NO-dependent glomerular cGMP production and by assessing the glomerular NADPH diaphorase (ND) activity, an enzymatic activity that colocalizes with NO-synthesis activity. Furthermore, we determined the isoform of NO synthase (NOS) implicated in NO synthesis by Western blot and immunohistochemistry. Glomeruli from rats with bilateral ischemia showed elevated glomerular nitrite and cGMP production. Besides, glomeruli from this group of rats showed an increased ND activity, whereas glomeruli from the ischemic and nonischemic rats with unilateral ischemia did not show this increase in nitrite, cGMP, and ND activity. In addition, glomeruli from ischemic kidneys showed an increased expression of endothelial NOS without changes in the inducible isoform. Addition of L-NAME in the drinking water induced a higher increase in the severity of the functional and structural damage in rats with bilateral ischemia than in rats with unilateral ischemia and in sham-operated animals. We can conclude that after renal ischemia, there is an increased glomerular NO synthesis subsequent to an activation of endothelial NOS that plays a protective role in the renal damage induced by ischemia and reperfusion.

F(2)-isoprostanes mediate high glucose-induced TGF-beta synthesis and glomerular proteinuria in experimental type I diabetes.

BACKGROUND: The recently discovered arachidonic acid derivatives, isoprostanes, are increased in pathological conditions associated with oxidative stress, such as diabetes. No role has yet been described for isoprostanes during the development of diabetic nephropathy. Cell culture in high ambient glucose has been used as a model in elucidating cellular mechanisms underlying diabetic nephropathy. Among the growth factors involved in the effect of high glucose, transforming growth factor-beta (TGF-beta) has been described as playing a key role in the development of nephropathy. METHODS: Streptozotocin-induced diabetic rats were supplemented in their diet with the antioxidant vitamin E (1000 U/kg diet). Blood and urine samples were taken to determine renal function and isoprostane concentration, as determined by gas chromatography/mass spectrometry. Glomerular mesangial and endothelial cells were cultured in high ambient glucose to determine the synthesis of isoprostanes and the role of isoprostanes in high glucose-induced synthesis of TGF-beta. RESULTS: Streptozotocin-induced diabetic rats had marked increases in plasma levels and urinary excretion rates of F(2)-isoprostanes. Dietary supplementation with vitamin E normalized (plasma) and reduced (urine) isoprostane levels and, surprisingly, improved proteinuria and blood urea nitrogen (BUN) levels. High ambient glucose increased F(2)-isoprostane synthesis in glomerular endothelial and mesangial cells in culture. Incubation of glomerular cells with F(2)-isoprostanes stimulated the production of TGF-beta. CONCLUSIONS: Increased F(2)-isoprostane synthesis during diabetes appears to be responsible in part for the increase in renal TGF-beta, a well-known mediator of diabetic nephropathy.

Role of glomerular nitric oxide in glycerol-induced acute renal failure.

Myoglobinuric acute renal failure remains one of the least understood clinical syndromes and the mediators involved remain obscure. The aim of the present study was to assess the role of nitric oxide in glycerol-induced acute renal failure under normal conditions and after uninephrectomy. Acute renal failure was induced in rats by injection of 50% glycerol (10 mL x kg(-1) body weight). Half of the animals were subjected to uninephrectomy two days before glycerol injection. Two days after the induction of acute renal failure, glomeruli from some animals were isolated and glomerular nitrite production was measured. Another group of animals was used for acute clearance studies. In this case, the effect of infusing either L-NAME or L-arginine was assayed. Glomerular nitrite production was significantly decreased in glycerol-induced acute renal failure. Glomeruli from uninephrectomized animals showed an increase in nitrite production, both in normal conditions and after glycerol injection, as compared with glomeruli from non-nephrectomized animals. L-NAME infusion worsened renal function in all the study groups, but more slowly in animals with glycerol-induced acute renal failure than in control rats. In uninephrectomized animals L-NAME reduced renal function more than in animals with two kidneys. In conclusion, in this model of acute renal failure the decrease in glomerular nitric oxide production plays an important role in the decrease in renal function. After uninephrectomy, an increase in glomerular nitric oxide synthesis plays a protective role against glycerol-induced acute renal failure.

Increased renal glomerular endothelin-1 release in gentamicin-induced nephrotoxicity.

Gentamicin-induced acute renal failure is characterized by a decrease in renal plasma flow and creatinine clearance. Endothelins (ET) are potent renal vasoconstrictors. The aim of this work is to assess the role of ET-1 in gentamicin-induced renal failure. Renal glomerular release of ET-1 was measured in rats with gentamicin-induced nephrotoxicity (100 mg/kg/day, s.c. for 2, 4 or 6 days). Glomeruli were isolated and incubated for 24 h in RPMI-1640. Glomerular supernatant and plasma concentration of ET-1 were measured by RIA. Renal failure was assessed by insulin, para-aminohippuric and creatinine clearance and histological studies. Gentamicin induced a dose number-dependent increase in plasma creatinine and a decrease in creatinine clearance. This was accompanied by a marked decrease in inulin and para-aminohippuric acid clearance, as well as by a marked tubular necrosis, without alterations in glomerular structures. Plasma ET-1 concentration and glomerular ET-1 release were also increased in gentamicin-treated rats. When 10-5 M gentamicin was added to control glomeruli, ET-1 production was not modified (36.4 +/- 2.2 vs. 35.2 +/- 1.7 pg/ml/24 h). All these results suggest that elevated ET-1 plasma levels and increased glomerular release of ET-1 could mediate, at least in part, the decrease in glomerular filtration rate observed in gentamicin-induced ARF.

Transfection of rat kidney with human 15-lipoxygenase suppresses inflammation and preserves function in experimental glomerulonephritis.

The human 15-lipoxygenase (15-LO) gene was transfected into rat kidneys in vivo via intra-renal arterial injection. Three days later, acute (passive) or accelerated forms of antiglomerular basement membrane antibody-mediated glomerulonephritis were induced in transfected and nontransfected or sham-transfected controls. Studies of glomerular functions (filtration and protein excretion) and ex vivo glomerular leukotriene B(4) biosynthesis at 3 hr, and up to 4 days, after induction of nephritis revealed preservation or normalization of these parameters in transfected kidneys that expressed human 15-LO mRNA and mature protein, but not in contralateral control kidneys or sham-transfected animals. The results provide in vivo-derived data supporting a direct anti-inflammatory role for 15-LO during immune-mediated tissue injury.

Role of nitric oxide in the early renal hemodynamic response after unilateral nephrectomy.

We evaluated the involvement of nitric oxide (NO) in the early hemodynamic response to uninephrectomy (UNX) in rats. Animals were uninephrectomized, and 48 h after removal of the kidney, the effect of infusing NG-nitro-L-arginine methyl ester (L-NAME) on renal function was studied. Glomeruli were isolated, and glomerular nitrite and cGMP productions were measured. In addition, endothelial constitutive NO synthase (NOS III) and inducible NO synthase (iNOS) were assessed by Western blot and by measuring the conversion of arginine to citrulline. UNX animals showed an increase in renal plasma flow that was inhibited by L-NAME in a higher proportion than in sham-operated (SO) animals. No differences were observed in systemic NO-dependent vascular tone, since mean arterial pressure showed similar increments in SO and UNX rats. Glomeruli from UNX animals showed an increase in glomerular nitrite production that was blunted by L-NAME addition. Also, cGMP levels were increased in glomeruli from UNX animals, and this increase was inhibited by L-NAME. Western blot analysis showed no differences in NOS III but a higher iNOS amount in glomeruli from UNX than in those from SO rats. No significant differences between UNX and SO rats were found in calcium-dependent NOS enzymatic activity in the renal cortex. However, calcium-independent enzymatic activity was markedly higher in the renal cortex of UNX than in those from SO animals. In conclusion, glomeruli from rats 48 h after UNX had a greater production of NO than those from SO animals. This increased glomerular NO production is based on an increase in the iNOS isoform. Increased glomerular NO synthesis seems to play a role in the decreased renal vascular resistance observed after unilateral nephrectomy in rats.

Effect of cicletanine on renal cGMP production.

The aim of the present study was to assess the effect of cicletanine on renal cGMP production. To do so we measured mean arterial pressure (MAP), creatinine clearance (CC), and urinary excretion of electrolytes and cGMP under basal conditions and after 6 h of cicletanine administration (10 and 15 mg/kg body weight by oral gavage) in conscious Wistar rats. Also, the in vitro effect of cicletanine was assessed by incubating renal slices and isolated rat glomeruli with two concentrations of cicletanine (0.1 and 1 mM) for different times (1, 2, 5, and 30 min) in the presence of 3-isobutyl-1-methylxanthine. Oral administration of cicletanine induced an increase in urinary flow (V) and the urinary excretion of electrolytes and cGMP, with no changes in CC. In addition, a significant decrease in MAP was observed, but only with the lower dose. Incubation with cicletanine did not induce significant changes in cGMP production in glomeruli or renal slices. These results show that cicletanine, administered in vivo at diuretic and antihypertensive doses, induces an increase in urinary cGMP excretion.

Cardiovascular effects of elgodipine and nifedipine compared in anaesthetized rats.

The cardiovascular effects of elgodipine were studied and compared with those of nifedipine in the presence or absence of ganglion blockade. A bolus of elgodipine (5-25 microg/kg) or nifedipine (60-120 microg/kg) was given and sequential cardiovascular effects in rats were recorded. Both dihydropyridines induced a dose-dependent decrease in mean arterial pressure but, whereas nifedipine induced reflex tachycardia, elgodipine induced a dose-dependent bradycardia. Both substances induced decreases in left ventricular d P/dt(max) without significant changes in central venous pressure. Good linear correlation was observed between the elgodipine-induced decrease in mean arterial pressure and those of heart rate and left ventricular dP/dt(max). The profile of the decrease in mean arterial pressure in animals pretreated with hexametonium chloride (20 mg/kg) was the same but the nifedipine-induced tachycardia was abolished without changes in elgodipine-induced bradycardia. These characteristics of elgodipine makes this dihydropyridine a potentially beneficial therapeutic agent in the case of severe hypertension accompanied by obstructive coronopathy.

Effect of nitric oxide synthesis modification on renal function in gentamicin-induced nephrotoxicity.

We evaluated the effect of acute or chronic nitric oxide (NO) synthesis activation or inhibition in rats with gentamicin-induced acute renal failure. Rats received gentamicin 100 mg/kg per day for 6 days, or isotonic saline. Some animals of each group also received N(G)-monomethyl-l-arginine (l-NAME, 4 mg/kg per day) or l-arginine (1%) in the drinking water for 6 days (chronic NO synthesis modification). In another experimental set, animals were treated with gentamicin or saline for 6 days and glomerular filtration rate (GFR) and renal plasma flow (RPF) were measured before and after the infusion of l-NAME (50 mg/h per kg) or l-arginine (60 mg/h per kg) (acute NO synthesis modification). Acute l-NAME administration induced a decrease in GFR and RPF both in control and gentamicin treated animals. Chronic l-NAME treatment induced an impairment in GFR only in gentamicin-treated animals. Acute l-arginine administration did not modify renal function in any experimental group whereas chronic l-arginine administration improved renal function only in gentamicin-treated animals. Urinary excretion of N-acetyl-ß-d-glucosaminidase and alkaline phosphatase was increased by chronic treatment with l-NAME in both groups, whereas l-arginine had no effect. In conclusion, NO synthesis inhibition aggravates gentamicin-induced renal damage. However, chronic NO synthesis stimulation partially prevents against gentamicin nephrotoxicity, thus suggesting that increased renal NO synthesis during gentamicin-induced nephrotoxicity plays a protector role on renal function.