Sydney Benchetrit

Calcitriol blunts the deleterious impact of advanced glycation end products on endothelial cells

Advanced glycation end products (AGEs), which are elevated in diabetic and uremic patients, may induce vascular dysfunctions, and calcitriol may improve the cardiovascular complications. Therefore, we examined whether calcitriol may modify the endothelial response to AGEs stimulation. Knowing the importance of nuclear factor-kappaB in endothelial inflammatory responses, the effect of AGEs and calcitriol on this pathway was also studied. Calcitriol was added to endothelial cells previously incubated with AGE-human serum albumin (HSA). AGE-HSA induced a decrease in endothelial nitric oxide synthase (eNOS) mRNA expression and enzyme activity. Addition of calcitriol to AGE-HSA-treated endothelial cells improved the decreased action of AGEs on the eNOS system. AGE-HSA increased the AGEs receptor mRNA and protein, which were both blunted by calcitriol. The parallel elevation of interleukin-6 mRNA in the presence of AGE-HSA was also blunted by calcitriol. The NF-kappaB-p65 DNA binding activity was enhanced and associated with a decrease in inhibitor kappaBalpha (IkappaBalpha) and an increase in phosphorylated (p)-IkappaBalpha levels. Addition of calcitriol blunted the AGEs-induced elevation of NF-kappaB-p65 DNA binding activity, a phenomenon related to an increased expression of IkappaBalpha. This increase was correlated to declined p-IkappaBalpha levels. The present results support the concept that calcitriol may act as a vascular protective agent counteracting the probable deleterious actions of AGEs on endothelial cell activities.

The potential roles of FGF23 and Klotho in the prognosis of renal and cardiovascular diseases

Fibroblast growth factor (FGF) 23 and Klotho are two factors associated with several metabolic disorders. Similar to humans, accelerated aging processes characterized by chronic vascular disease, bone demineralization, skin atrophy and emphysema have been recognized in FGF23-null mice and Klotho-deficient mice. The role of these factors in the control of mineral metabolism homeostasis have been shown recently, particularly at the level of parathyroid cells and also in modulating active vitamin D production, two phenomena which are relevant in the presence of chronic kidney disease. In addition, the hormonal affect of circulating FGF23 and Klotho proteins on vascular reactivity, either directly on endothelial cell functions or indirectly by modulating the brain endothelin-1-dependent sympathetic nervous system activity, has contributed to understanding their role in the pathophysiology of hypertension and atherosclerotic vasculopathies. Consequently, very recent clinical investigations seem to confirm the involvement of Klotho in modulating the severity and prognosis of human cardiovascular (CV) disorders and longevity. The present review reports data related to the possible interactive effects of Klotho and FGF23 on the prognosis of renal and CV diseases.

Parathyroid hormone stimulates the endothelial expression of vascular endothelial growth factor

Background  We showed previously that parathyroid hormone (PTH) may stimulate the endothelial expression of pro‐atherosclerotic and pro‐inflammatory markers. Considering the impact of PTH on vasculature, we decided to evaluate its effect on mRNA and intra‐cellular protein expressions of endothelial vascular endothelial growth factor (VEGF) taking into account that VEGF may play a role in the pathogenesis of endothelial dysfunctions.

Proximal Tubular Hypertrophy and Enlarged Glomerular and Proximal Tubular Urinary Space in Obese Subjects with Proteinuria

Background Obesity is associated with glomerular hyperfiltration, increased proximal tubular sodium reabsorption, glomerular enlargement and renal hypertrophy. A single experimental study reported an increased glomerular urinary space in obese dogs. Whether proximal tubular volume is increased in obese subjects and whether their glomerular and tubular urinary spaces are enlarged is unknown. Objective To determine whether proximal tubules and glomerular and tubular urinary space are enlarged in obese subjects with proteinuria and glomerular hyperfiltration. Methods Kidney biopsies from 11 non-diabetic obese with proteinuria and 14 non-diabetic lean patients with a creatinine clearance above 50 ml/min and with mild or no interstitial fibrosis were retrospectively analyzed using morphometric methods. The cross-sectional area of the proximal tubular epithelium and lumen, the volume of the glomerular tuft and of Bowman’s space and the nuclei number per tubular profile were estimated. Results Creatinine clearance was higher in the obese than in the lean group (P=0.03). Proteinuria was similarly increased in both groups. Compared to the lean group, the obese group displayed a 104% higher glomerular tuft volume (P=0.001), a 94% higher Bowman’s space volume (P=0.003), a 33% higher cross-sectional area of the proximal tubular epithelium (P=0.02) and a 54% higher cross-sectional area of the proximal tubular lumen (P=0.01). The nuclei number per proximal tubular profile was similar in both groups, suggesting that the increase in tubular volume is due to hypertrophy and not to hyperplasia. Conclusions Obesity-related glomerular hyperfiltration is associated with proximal tubular epithelial hypertrophy and increased glomerular and tubular urinary space volume in subjects with proteinuria. The expanded glomerular and urinary space is probably a direct consequence of glomerular hyperfiltration. These effects may be involved in the pathogenesis of obesity-related renal disease.

Vitamin D receptor activation in a diabetic-like environment: potential role in the activity of the endothelial pro-inflammatory and thioredoxin pathways.

High blood and tissue concentrations of glucose and advanced glycation end products (AGEs) are thought to play an important role in the development of diabetic vascular complications. Thioredoxin interacting protein (TXNIP) is up-regulated in response to high levels of glucose and is an endogenous inhibitor of thioredoxin (TRX), and may play a contributory role in the occurrence of diabetic-related vascular diseases. Vitamin D inhibits endothelial proliferation and is a cardiovascular protective agent. The present study evaluated the impact of paricalcitol and calcitriol on the endothelial inflammatory and TXNIP pathways in cultured endothelial cells exposed to a diabetic-like environment. Fresh human umbilical vein cord endothelial cells (HUVEC) were treated for 24h with 200 μg/ml AGE-HSA and 250 mg/dl glucose concentrations, with paricalcitol or calcitriol. IL6, IL8, NFκB (p50/p65), receptor of AGE (RAGE), TXNIP, and TRX expressions were evaluated at the levels of mRNA, protein, and TRX activity. Calcitriol and paricalcitol significantly down-regulated the markers involved in the inflammatory responses. Only paricalcitol induced a significant decrease in TXNIP mRNA and protein expressions. Neither paricalcitol nor calcitriol affected TRX reductase activity or TRX mRNA and protein expressions. Our findings indicate that in an endothelial diabetic-like environment, paricalcitol and calcitriol significantly decreased the expression of genes involved in the inflammatory pathway. In this in vitro study, it seems that the TRX antioxidant system was not involved. The different effects found between paricalcitol and calcitriol might reflect the selectivity of vitamin D receptor (VDR) activation.

Calcitriol counteracts endothelial cell pro-inflammatory processes in a chronic kidney disease-like environment.

In advanced chronic kidney disease (CKD), hypocalcemia, high levels of advanced glycation end products (AGEs), and parathyroid hormone (PTH) coexist and are considered to play a role in the development of chronic vasculopathies. The aim of the present study was to evaluate the impact of a CKD-like environment on cultured endothelial cell (EC) functions and to assess the impact of calcitriol on the expression of parameters such as endothelial nitric oxide synthase (eNOS), receptor of AGEs (RAGE), interleukin 6 (IL-6) and nuclear factor kappa B (NFκB). Human umbilical vein cord endothelial cells (HUVEC) were grown in medium containing low Ca(2+) concentration stimulated with AGE-HSA and PTH and treated with calcitriol for additional incubation. mRNA expression was established by reverse transcriptase-PCR, protein expression by Western blot analysis, IL-6 secretion by ELISA, NOS activity by conversion of [(14)C]arginine to [(14)C]citrulline and DNA-binding activity of NFκB-p65 assayed colorimetrically in nuclear extracts. The CKD-like environment characterized by the association of low Ca(2+) and high levels of AGEs and PTH, depressed eNOS system activity and enhanced RAGE and IL-6 expression/secretion. DNA-binding activity of nuclear NFκB-p65 was increased and the expression of IκBα decreased. Addition of calcitriol normalized the expression, secretion and activity of eNOS, RAGE and IL-6. The enhanced NFκB activity was also counteracted probably due to the increased IκBα expression. The effect of CKD-like environment on EC may partly explain the increased vasculopathies in CKD patients, in contrast to calcitriol, which suggests a vascular protective action.

Parathyroid hormone decreases endothelial osteoprotegerin secretion: role of protein kinase A and C

Parathyroid hormone (PTH), which is elevated in patients with chronic renal failure, has been shown to participate in the development of vascular calcification. Previous studies have demonstrated that PTH may promote endothelial expressions of proinflammatory parameters. On the basis of these data, we evaluated whether PTH may have an impact on endothelial osteoprotegerin (OPG), a vascular-protective factor which may control vascular calcification. Endothelial cells were stimulated with 10(-12) to 10(-10) mol/l PTH. PKC and PKA are the main cellular pathways of PTH. Inhibitors and activators of PKC or PKA were used to determine whether these signaling pathways are involved in the control of endothelial OPG. PTH induced a decrease in OPG secretion and mRNA expression. Treatment of PTH-stimulated cells by calphostin C (PKC inhibitor) induced a further decrease in OPG secretion, while Rp-cAMP (PKA inhibitor) had no additional effect. In nonstimulated cells, a PKC activator significantly stimulated OPG secretion, while a PKA activator was associated with a decline. These effects were blunted in the presence of calphostin C and Rp-cAMP, respectively. An increase in OPG secretion induced by a PKC activator indicates that the basal OPG secretion is mediated through PKC. The decrease induced by a PKA activator, which is similar to the decrease observed with PTH, suggests that the action of PTH on OPG secretion and mRNA expression may be due to the PKA pathway.

Effect of Methyldopa on Renal Function in Rats with L-NAME-Induced Hypertension in Pregnancy

Background: Pregnancy-induced hypertension is characterized by an increased sympathetic activity and probably by a decreased synthesis/activity of nitric oxide. The aim of the present study is to evaluate whether the beneficial action of the sympathetic antagonist methyldopa (a first-choice hypotensive agent in the treatment of PIH) may be associated to changes in nitric-oxide synthesis. Methods: Forty pregnant Wistar rats received L-NAME (NO synthase inhibitor, 9–10 mg/kg/day) from mid-pregnancy (day 11) through to term. Some rats were treated with daltroban (TxA receptor antagonist, 60 mg/kg/day), diltiazem (calcium channel blocker, 30 mg/kg/day), methyldopa (central adrenergic antagonist, 400 mg/kg/day) or L-arginine (260 mg/kg/day) from mid-pregnancy. The effect of the different treatments on systolic blood pressure (SBP), creatinine clearance (CCR), urine protein excretion (UP) and urinary nitrate excretion (UNO3, representing urine NO metabolite) were evaluated and the results compared with those found in normal pregnancy. Normal pregnant rats receiving similar treatment were used as controls. Results: In normal pregnant (P) rats, SBP values decreased from 94 ± 2 to 83 ± 3 mm Hg at the end of pregnancy (p < 0.01) and CCR augmented significantly. Drug treatment had no significant effect. In NAME-treated rats, at the same period, the SBP augmented from 92 ± 1 to 129 ± 1.8 mm Hg (p < 0.01). At the end of pregnancy, NAME rats had significantly lower CCR values and higher UP excretion when compared with P rats. UNO3 increased significantly in P and in P rats treated with methyldopa. As expected, in NAME rats UNO3 excretion was significantly reduced. Treatment with methyldopa normalized SBP, improved CCR and proteinuria and was associated with an increase in UNO3. Similar results were obtained with L-arginine treatment. Diltiazem lowered SBP significantly but had no effect on renal function or UNO3 and daltroban had no effect. Conclusion: The increased UNO3 found in NAME rats treated with methyldopa suggests that the vasoconstriction secondary to chronic NO inhibition may be partially related to an increased sympathetic activity. The efficient action of the sympathetic antagonist methyldopa may be due not only to its antihypertensive effects but also by its stimulating effect on NO synthesis leading also to an improvement of renal function.

Early endothelial dysfunction following renal mass reduction in rats

Background Endothelial dysfunction has been previously described in severely hypertensive rats with renal mass reduction (RMR) receiving large dietary Na loads. Because hypertension and Na loading reduce endothelium‐dependent vasodilation, the effect of renal failure per se is unclear.

Low Molecular Weight Heparin Reduces Proteinuria and Modulates Glomerular TNF-α Production in the Early Phase of Adriamycin Nephropathy

Background/Aim: Heparin has been shown to be renoprotective in a number of experimental nephropathies. The inflammatory component in the early phase of Adriamycin (ADR) induced nephropathy has been established. A microdose of low molecular weight heparin (Fragmin; F) has been noted to exert immunomodulatory effects independent of its anticoagulant activity. We assessed the effects of microdoses of F on daily proteinuria and glomerular production of tumor necrosis factor alpha (TNF-α) and prostaglandins 8 and 15 days after induction of ADR nephropathy. Methods: Following intravenous injection of ADR (7 mg/kg) to Wistar rats weighing 200 ± 20 g, F 20 µg/day/rat s.c. was administered for 8 and 15 days (groups F8 and F15). The respective control groups (C8 and C15) received normal saline subcutaneously. Proteinuria, serum albumin, and creatinine clearance were evaluated on days 8 and 15. The production of TNF-α and prostaglandins from glomerular supernatants was measured by radioimmunoassay on days 8 and 15. Results: F significantly reduced proteinuria (mg/day) on day 8: 13.6 ± 1.2 in F8 versus 40.3 ± 2.7 in C8 (p = 0.008). The glomerular production of TNF-α (pi/ml) was significantly lower on day 8 in rats treated with F: 356 ± 33 in F8 versus 764 ± 81 in C8 (p = 0.006). A decrease in the prostaglandin E2/thromboxane B2 ratio was noted in the F group between 8 and 15 days (1.1 in F8 vs. 0.9 in F15, p = 0.005) which principally reflects an increase of thromboxane B2. The antiproteinuric effect of F shown after 8 days was no longer present after 15 days (354 ± 91 mg/day in F15 vs. 499 ± 69 mg/day in C15, p = 0.33). The same trend was seen for the glomerular production of TNF-α. Light microscopy and immunohistochemistry for interstitial and glomerular macrophages were negative. Conclusion: The lowering effect of microdoses of F on the proteinuria seen during the early phase of ADR nephropathy may be mediated by a decreased production of glomerular TNF-α, supporting the anti-inflammatory action of low molecular weight heparin.