Edu Suarez

Effects of Paricalcitol and Enalapril on Atherosclerotic Injury in Mouse Aortas

Aims: This study investigated the protective effect of vitamin D analog paricalcitol combined with angiotensin-converting enzyme inhibitor (enalapril) on aortic oxidative injury in atherosclerotic mice. Methods: Female mice were treated for 16 weeks as follows: (1) ApoE deficient + vehicle, (2) ApoE deficient + paricalcitol (200 ng 3 times a week), (3) ApoE deficient + enalapril (30 mg/l in drinking water), (4) ApoE deficient + paricalcitol + enalapril, and (5) wild-type controls. Results: ApoE-deficient mice developed hypertension which was prevented by enalapril or enalapril + paricalcitol treatment but not by paricalcitol treatment. Histology showed atherosclerotic plaque in the aorta of ApoE-deficient mice which was prevented by paricalcitol, enalapril, and paricalcitol + enalapril treatments. Aortic malondialdehyde levels, NADPH oxidase subunit p22phox, manganese-superoxide dismutase (Mn-SOD), inducible nitric oxide synthase, monocyte chemoattaractant protein-1, tumor necrosis factor (TNF)-α, and cyclooxygenase-2 protein expressions increased, whereas glutathione levels, CuZn-SOD, and endothelial protein expressions decreased in ApoE-deficient mice compared to controls. Treatment with paricalcitol and enalapril alone or in combination protected the inflammatory and oxidative endothelial injury of the aorta in atherosclerotic mice. Conclusion: Combination therapy affords greater protection against aortic inflammatory and oxidative injury in atherosclerosis than monotherapy.

Effect of combining an ACE inhibitor and a VDR activator on glomerulosclerosis, proteinuria, and renal oxidative stress in uremic rats.

Angiotensin-converting enzyme (ACE) inhibitors ameliorate the progression of renal disease. In combination with vitamin D receptor activators, they provide additional benefits. In the present study, uremic (U) rats were treated as follows: U+vehicle (UC), U+enalapril (UE; 25 mg/l in drinking water), U+paricalcitol (UP; 0.8 μg/kg ip, 3 × wk), or U+enalapril+paricalcitol (UEP). Despite hypertension in UP rats, proteinuria decreased by 32% vs. UC rats. Enalapril alone, or in combination with paricalcitol, further decreased proteinuria (≈70%). Glomerulosclerosis and interstitial infiltration increased in UC rats. Paricalcitol and enalapril inhibited this. The increase in cardiac atrial natriuretic peptide (ANP) seen in UC rats was significantly decreased by paricalcitol. Enalapril produced a more dramatic reduction in ANP. Renal oxidative stress plays a critical role in inflammation and progression of sclerosis. The marked increase in p22(phox), a subunit of NADPH oxidase, and decrease in endothelial nitric oxide synthase were inhibited in all treated groups. Cotreatment with both compounds inhibited the uremia-induced increase in proinflammatory inducible nitric oxide synthase (iNOS) and glutathione peroxidase activity better than either compound alone. Glutathione reductase was also increased in UE and UP rats vs. UC. Kidney 4-hydroxynonenal was significantly increased in the UC group compared with the normal group. Combined treatment with both compounds significantly blunted this increase, P < 0.05, while either compound alone had no effect. Additionally, the expression of Mn-SOD was increased and CuZn-SOD decreased by uremia. This was ameliorated in all treatment groups. Cotreatment with enalapril and paricalcitol had an additive effect in increasing CuZn-SOD expression. In conclusion, like enalapril, paricalcitol alone can improve proteinuria, glomerulosclerosis, and interstitial infiltration and reduce renal oxidative stress. The effects of paricalcitol may be amplified when an ACE inhibitor is added since cotreatment with both compounds seems to have an additive effect on ameliorating uremia-induced changes in iNOS and CuZn-SOD expression, peroxidase activity, and renal histomorphometry.

Aging increases mitochondrial DNA damage and oxidative stress in liver of rhesus monkeys.

While the mechanisms of cellular aging remain controversial, a leading hypothesis is that mitochondrial oxidative stress and mitochondrial dysfunction play a critical role in this process. Here, we provide data in aging rhesus macaques supporting the hypothesis that increased oxidative stress is a major characteristic of aging and may be responsible for the age-associated increase in mitochondrial dysfunction. We measured mitochondrial DNA (mtDNA) damage by quantitative PCR in liver and peripheral blood mononuclear cells of young, middle age, and old monkeys and show that older monkeys have increases in the number of mtDNA lesions. There was a direct correlation between the amount of mtDNA lesions and age, supporting the role of mtDNA damage in the process of aging. Liver from older monkeys showed significant increases in lipid peroxidation, protein carbonylations and reduced antioxidant enzyme activity. Similarly, peripheral blood mononuclear cells from the middle age group showed increased levels in carbonylated proteins, indicative of high levels of oxidative stress. Together, these results suggest that the aging process is associated with defective mitochondria, where increased production of reactive oxygen species results in extensive damage at the mtDNA and protein levels. This study provides valuable data based on the rhesus macaque model further validating age-related mitochondrial functional decline with increasing age and suggesting that mtDNA damage might be a good biomarker of aging.

Phosphate restriction significantly reduces mortality in uremic rats with established vascular calcification

The role of hyperphosphatemia in the pathogenesis of secondary hyperparathyroidism, cardiovascular disease, and progression of renal failure is widely known. Here we studied effects of dietary phosphate restriction on mortality and vascular calcification in uremic rats. Control and uremic rats were fed a high-phosphate diet and at 3 months a portion of rats of each group were killed. Serum phosphate and the calcium phosphate product increased in uremic rats, as did aortic calcium. Of the rats, 56% had positive aortic staining for calcium (von Kossa), RUNX2, and osteopontin. The remaining uremic rats were continued on diets containing high phosphate without and with sevelamer, or low phosphate, and after 3 more months they were killed. Serum phosphate was highest in uremic rats on high phosphate. Serum PTH and FGF-23 were markedly lower in rats on low phosphate. Mortality on high phosphate was 71.4%, with sevelamer reducing this to 37.5% and phosphate restriction to 5.9%. Positive aortic staining for von Kossa, RUNX2, and osteopontin was increased, but phosphate restriction inhibited this. Kidneys from low-phosphate and sevelamer-treated uremic rats had less interstitial fibrosis, glomerulosclerosis, and inflammation than those of uremic rats on high phosphate. Importantly, kidneys from rats on low phosphate showed improvement over kidneys from high-phosphate rats at 3 months. Left ventricles from rats on low phosphate had less perivascular fibrosis and smaller cardiomyocyte size compared to rats on high phosphate. Thus, intensive phosphate restriction significantly reduces mortality in uremic rats with severe vascular calcification.

Cardiac and Renal Effects of Atrasentan in Combination with Enalapril and Paricalcitol in Uremic Rats

Background/Aims: The search for new therapies providing cardiorenal protection in chronic kidney disease (CKD) has led to treatments that combine conventional renin-angiotensin-aldosterone-system inhibitors with other drugs that exhibit potential in disease management. Methods: In rats made uremic by renal ablation, we examined the effects of addition of the endothelin-A receptor antagonist atrasentan to a previously examined combination of enalapril (angiotensin converting enzyme inhibitor) and paricalcitol (vitamin D receptor activator) on cardiac and renal parameters. The effects of the individual and combined drugs were examined after a 3-month treatment. Results: A decrease in systolic blood pressure, serum creatinine and proteinuria, and improvement of renal histology in uremic rats were attributed to enalapril and/or paricalcitol treatment; atrasentan alone had no effect. In heart tissue, individual treatment with the drugs blunted the increase in cardiomyocyte size, and combined treatment additively decreased cardiomyocyte size to normal levels. Perivascular fibrosis was blunted in uremic control rats with atrasentan or enalapril treatment. Conclusions: We found distinct cardiac and renal effects of atrasentan. Combination treatment with atrasentan, enalapril and paricalcitol provided positive effects on cardiac remodeling in uremic rats, whereas combination treatment did not offer further protective effects on blood pressure, proteinuria or renal histology.

Effect of paricalcitol and enalapril on renal inflammation/oxidative stress in atherosclerosis.

AIM To investigate the protective effect of paricalcitol and enalapril on renal inflammation and oxidative stress in ApoE-knock out mice. METHODS Animals treated for 4 mo as group (1) ApoE-knock out plus vehicle, group (2) ApoE-knock out plus paricalcitol (200 ng thrice a week), (3) ApoE-knock out plus enalapril (30 mg/L), (4) ApoE-knock out plus paricalcitol plus enalapril and (5) normal. Blood pressure (BP) was recorded using tail cuff method. The kidneys were isolated for biochemical assays using spectrophotometer and Western blot analyses. RESULTS ApoE-deficient mice developed high BP (127 ± 3 mmHg) and it was ameliorated by enalapril and enalapril plus paricalcitol treatments but not with paricalcitol alone. Renal malondialdehyde concentrations, p22(phox), manganese-superoxide dismutase, inducible nitric oxide synthase (NOS), monocyte chemoattractant protein-1, tumor necrosis factor-alpha and transforming growth factor-β1 levels significantly elevated but reduced glutathione, CuZn-SOD and eNOS levels significantly depleted in ApoE-knock out animals compared to normal. Administration of paricalcitol, enalapril and combined together ameliorated the renal inflammation and oxidative stress in ApoE-knock out animals. CONCLUSION Paricalcitol and enalapril combo treatment ameliorates renal inflammation as well as oxidative stress in atherosclerotic animals.