Gustavo Parra

Evolution of renal interstitial inflammation and NF-κB activation in spontaneously hypertensive rats

Lymphocytes and macrophages infiltrate the kidney of spontaneously hypertensive rats (SHR) and interventions leading to their reduction are associated with improvement of the hypertension. The present studies examined the evolution of the interstitial inflammation in the natural course of the SHR to gain insight on the potential role of interstitial immune cell accumulation in the development of hypertension. We studied SHR and control WKY rats at 3 weeks (SHR-3wk group, n = 11 and WKY-3wk group, n = 10), 11 weeks (SHR-11wk group, n = 5 and WKY-11wk group, n = 5) and 24 weeks (SHR-24wk group, n = 10 and WKY-24wk group, n = 10). The SHR-3wk group was normotensive and older SHR developed hypertension that was severe in the SHR-24wk group. Tubulointerstitial accumulation of lymphocytes, macrophages, angiotensin II-positive cells, cells expressing the p65 DNA-binding subunit of NF-ĸB and activation of NF-ĸB in the kidney were all significantly increased (p < 0.01) in the prehypertensive SHR-3wk group and augmented progressively, with the highest values in the SHR-24wk group. The SHR-24wk group showed increased (p < 0.001) helper (CD4) T cell infiltration and a high CD4/CD8 ratio. These findings are consistent with the possibility that activation of NF-ĸB and renal interstitial infiltration of immune cells may be part of the pathophysiologic process that drives hypertension in the SHR.

Mycophenolate mofetil treatment reduces cholesterol-induced atherosclerosis in the rabbit

Immunosuppressive therapy has been shown to either improve or, more frequently, enhance the development of atherosclerosis. We tested the effect of mycophenolate mofetil (MMF), an inhibitor of nucleotide synthesis widely used in transplant therapy, in diet-induced atherosclerosis in the rabbit. Two groups (n=10 each) of New Zealand White (NZW) rabbits were fed a 1% cholesterol diet for 12 weeks. One group received MMF (CHOL+MMF group) by gastric gavage (30 mg/kg daily) and the other group (CHOL) received the same volume of saline by the same route. There were no differences in the serum cholesterol (mean values > or =30 mmol/l in both groups after 2 weeks) or in the triglyceride, blood sugar, total protein, and albumin serum levels and weight gain in both groups of animals. The cholesterol-fed untreated rabbits had atherosclerotic plaques covering 43.9.1+/-SD 16.40% of their thoracic aorta and 41.9+/-22. 59% of their abdominal aorta, while the MMF treated group had 18. 5+/-7.17% and 17.7+/-9.71%, respectively (P<0.01). The cholesterol content of the aorta (mg/g) in the cholesterol-fed untreated group was 4.61+/-SD 1.21 in the thoracic aorta and 4.54+/-2.07 in the abdominal aorta, whereas the MMF treated group had and 2.83+/-0.84 and 2.77+/-1.44, respectively (P<0.01). Infiltrating macrophages (RAM 11 positive cells/100 nuclei) in the intimal layer of the aorta were 58.4+/-SD26.16 in the CHOL group and 8.5+/-5.51 in the CHOL+MMF group: (P<0.001). CD18 positive cells/100 nuclei were 27.4+/-17.6 in the CHOL group and 5.3+/-3.82 in the CHOL+MMF group (P<0.01), and the intima/media ratio was 0.66+/-0.11 in the CHOL group and 0. 30+/-0.09 in the MMF treated rabbits (P<0.001). MMF also reduced proliferating smooth muscle cells (HHF35 positive) infiltrating between the macrophages. These results indicate that MMF ameliorates importantly the atherogenic potential of a high cholesterol diet and this effect is associated with a reduction in macrophage and foam cell infiltration and smooth muscle cell proliferation and infiltration. Since chronic treatment with this drug is given routinely in various clinical conditions with relatively minor side effects, consideration may be given to its use as adjuvant therapy in atherosclerotic cardiovascular disease.

Immune reactivity to heat shock protein 70 expressed in the kidney is cause of salt-sensitive hypertension

Hypertension affects one-third of the adult population of the world. The causes of hypertension are incompletely understood, but relative impairment of sodium excretion is central to its pathogenesis. Immune cell infiltration in the kidney is a constant finding in hypertension that in association with local angiotensin and oxidants causes a defect in sodium excretion. However, it is unclear if the T cell influx into the kidney responds to nonspecific chemokine cues or is due to antigen-driven immune attraction. We found that T cells in experimentally induced salt-driven hypertension present a CD4 clonal response to heat shock protein 70 (HSP70) that is overexpressed in the kidney. We used a highly preserved amino acid sequence within the HSP molecule to induce immune tolerance associated with the generation of IL-10 producing regulatory T cells. Immune tolerant rats to HSP70 developed minimal renal inflammation and were protected from the development of salt-sensitive hypertension. Adoptive transfer of T lymphocytes isolated from spleen of tolerized rats also reversed hypertension. HSP70 gene delivery to the renal vein of the kidneys of rats sensitized to HSP70 caused an increment in blood pressure in response to a high-salt diet. The HSP70 peptide used in this work induces a strong proliferative response in peripheral blood lymphocytes of patients with essential hypertension. These studies provide evidence that autoimmunity plays a role in salt-sensitive hypertension and identifies HSP70 expressed in the kidney as one key antigen. These findings raise the possibility of novel approaches to the treatment of this condition.

Effect of Uric Acid on Gentamicin‐Induced Nephrotoxicity in Rats – Role of Matrix Metalloproteinases 2 and 9

In this work, we aimed to study the effect of uric acid on gentamicin-induced nephrotoxicity. Male Sprague-Dawley rats were assigned to one of six groups (six rats each) which received intraperitoneal injections for 9 days: (S) saline; (UA) Uric acid alone; (G) Gentamicin alone; (G + UA) Gentamicin + uric acid; (G rec) Gentamicin recovery and (G + UA rec) Gentamicin + uric acid recovery. In (G rec) and (G + UA rec), rats recovered for 7 days after the last injection. Urine and blood samples were taken on day 0 and at the end of every stage. Kidneys were harvested for histological scoring, determination of renal malondialdehyde (MDA), zymography and western blots for matrix metalloprotease (MMP)-2 and MMP-9. Uric acid alone did not provoke changes in biochemical and histological parameters when compared to controls. Gentamicin alone increased significantly plasma creatinine and blood urea nitrogen and caused a moderate histological damage. When combined with uric acid, these conditions worsened. MMP-9 activity and expression was decreased in rats from group G + UA as compared with rats from group G, while activity of MMP-2 was similarly increased in both groups when compared to controls. The increase in renal MDA induced by gentamicin was not altered when it was combined with uric acid. During the recovery stage, all biochemical parameters returned to normal levels, though a trend for delay of tubular damage recovery was observed in group G + UA rec when compared with group G rec. The results indicate that uric acid worsens gentamicin-induced nephrotoxicity. The mechanism is likely to implicate down-regulation of MMP-9.

Fish oil dietary supplementation reduces Ia expresion in rat and mouse peritoneal macrophages

Preliminary studies suggest that administration of fish oil fatty acids may be beneficial in several immunological diseases; therefore, we studied the effect of fish oil dietary supplementation on the expression of Ia in stimulated murine peritoneal macrophages. Rats (n = 19) and mice (n = 27) on standard rodent feeding were separated in experimental (E) and control (C) groups that received fish oil or saline solution, respectively, daily for 4 weeks by esophageal gavage. Cholesterol serum levels were significantly lowered by fish oil (E vs C, P less than 0.01). E and C groups were injected intraperitoneally with Listeria monocytogenes (LM) and peritoneal cells were harvested 4 and 7 days after infection. Decreased expression of Ia induced by LM was found in rats (C = 49.68 +/- 5.09%, E = 16.95 +/- 4.3%, P less than 0.01) and mice (C = 47.38 +/- 7.63%, E = 26.66 +/- 1.92%, P less than 0.01). Animals with a more pronounced depression of serum cholesterol (reduction of 44.04 +/- 1.52% of baseline levels) had more depression of Ia expression (6.47 +/- 1.22%, P less than 0.001 vs control). Reduction of Ia expression was not related to PGE2 production by peritoneal cells. Reduction of Ia expression by fish oil could induce down-regulation of antigen presentation and alloreactivity.

Experimental induction of salt-sensitive hypertension is associated with lymphocyte proliferative response to HSP70

Renal tubulointerstitial inflammation is a constant feature of experimental models of hypertension and likely plays a role in the pathogenesis of salt-sensitive hypertension. We have previously raised the possibility that the immune cell infiltration is driven by a low grade autoimmune reactivity directed to or facilitated by renal heat shock protein over expression. The present studies were done to gain insight on possible cell-mediated immune mechanisms in experimental hypertension by determining the renal expression of HSP70 and the proliferation index of T lymphocytes cultured with HSP70. We studied male Sprague-Dawley rats with inhibition of nitric oxide (NO) synthase (n=6), protein overload (PO) proteinuria (n=7) and short-term angiotensin II (Ang II) infusion (n=5), and their corresponding control groups. Each model was associated with 2 to 4 fold increase (P<0.05-0.001) in renal HSP70 expression. T cells isolated from the spleens demonstrated a significant two- to nine-fold response compared to controls (P<0.05 or lower for each comparison) when cultured with HSP70. These studies suggest that autoimmunity to stress proteins is involved in the sustained low-grade inflammatory infiltration that occurs in the tubulointerstitial areas of the hypertensive kidney.

Cyclosporin A reduces expression of adhesion molecules in the kidney of rats with chronic serum sickness.

Treatment with cyclosporin A (CsA) improves proteinuria and reduces renal cellular infiltration in chronic serum sickness (CSS). We examined if these effects were associated with a reduced renal expression of CD54 and its ligands, interferon‐gamma (IFN‐γ), tumour necrosis factor‐alpha (TNF‐α) and MHC class II molecules. We studied two groups of rats in which CSS was induced by daily injections of ovalbumin (OVA): a group treated with CsA (OVA.CsA group, n = 11) and a group that received no treatment (OVA.CSS group, n = 11). An additional group of five rats (control group) received only phosphate buffer. Immunostaining techniques were used to follow CSS and to study the expression of CD54, CD18, CD11b/c, IFN‐γ, TNF‐α and MHC class molecules. Proteinuria (mg/24 h) was reduced from 248·2 ± 73·1 (OVA.CCS group) to 14·5 ± 13·1 with CsA treatment (P < 0·0001). The renal expression of CD54 and its ligands (CD18 and CD11b/c) was reduced by 50% to 75%. Correspondingly, there was a 60% to 85% reduction in the number of infiltrating leucocytes. The number of cells expressing TNF‐α, IFN‐γ and MHC II molecules was also reduced. CsA reduces expression of CD54 and its ligands. This effect is associated with a reduction of cellular infiltration, IFN‐γ, TNF‐α‐producing cells and with MHC II expression in the kidney. These findings suggest that expression of adhesion molecules plays a critical role in CSS and underline the importance of cellular immunity in this experimental model.

Mycophenolate mofetil aggravates postischemic acute renal failure in rats.

MYCOPHENOLATE MOFETIL (MMF) is an inhibitor of purine synthesis commonly used as an immunosupressant in clinical transplantation. Since transplanted kidneys are subject to ischemia-reperfusion injury resulting from variable periods of ischemia prior to grafting, the potential effects of MMF in affecting the process of regeneration and repair after ARF are of considerable interest; yet, there is scarce information on this subject. Only recently, Jones and Shoskes have reported that the combination of MMF and bioflavonoids reduces injury and facilitates repair after renal ischemia. The present studies were designed to establish the effects of MMF on the clinical and pathologic course of postischemic ARF of variable severity in rats.

Effect of uric acid on nephrotoxicity induced by mercuric chloride in rats

Oxidative stress is an important mechanism in mercury poisoning. We studied the effect of uric acid, a natural and potent reactive oxygen species and peroxynitrite scavenger, in HgCl 2-induced nephrotoxicity. Rats were injected with a unique dose of HgCl2 (2.5 mg/kg body weight, subcutaneously) and then vehicle (for 3 days, twice daily) or HgCl2 (unique dose) and intraperitoneal uric acid suspension (250 mg/kg body weight, twice daily, for 3 days), and then killed at 24, 48 and 72 hours after HgCl2 administration (n = 5 for each group). At the end of the experimental study, kidneys and blood samples were taken. Tissues were prepared and examined under light microscopy. Uric acid significantly prevented the increase in plasma levels of creatinine and blood urea nitrogen (BUN); it helped maintain systemic nitrate/nitrite concentration and total antioxidant capacity. Uric acid attenuated the increase of renal lipid peroxidation and it markedly diminished nitrotyrosine signal and histopathological changes as early as 24 hours after HgCl2 administration. Uric acid did not prevent a decrease in β-actin signal caused by mercuric chloride, but it promoted a faster recovery when compared to the HgCl2 alone group. Our results indicate that UA could play a beneficial role against HgCl2 toxicity by preventing systemic and renal oxidative stress and tissue damage.

Atrial natriuretic peptide levels in brain venous outflow during cardiopulmonary bypass in humans: Evidence for extracardiac hormonal production☆

Atrial natriuretic peptide (ANP) is a hormone with an important role in the regulation of extracellular fluid volume. The ANP gene is not only expressed in the heart, but the high concentration of ANP in cardiac blood makes it difficult to demonstrate extraatrial hormonal secretion in vivo. This issue was addressed during complete cardiopulmonary bypass (CPB) in 13 patients undergoing elective cardiac surgery in whom ANP concentrations were followed in the internal jugular vein, representing largely brain venous outflow, as well as a peripheral vein and radial artery, after the heart and lungs were excluded from circulation. Plasma ANP levels in the peripheral venous circulation showed no significant changes during extracorporeal circulation, although they tended to decrease (from 6.75 +/- 2.16 fmol/mL to 4.76 +/- 0.69 fmol/mL; P greater than 0.05). ANP levels in the radial artery decreased significantly after the exclusion of the heart (from 16.84 +/- 3.51 fmol/mL to 6.83 +/- 0.97 fmol/mL; P less than 0.01). In contrast, ANP concentration in the internal jugular vein increased in 12 of 13 patients during the first 15 minutes of CPB (from 9.49 +/- 1.96 fmol/mL to 15.96 +/- 2.8 fmol/mL; P less than 0.01) and remained above the levels found simultaneously in other sampling sites during CPB. High-performance liquid chromatography analysis of plasma extracts showed multiple peaks of ANP, but the elution patterns of peripheral venous blood and brain outflow were similar. One of the immunoreactive peaks was located at the position of standard human ANP (Ser99-Met110-Tyr126).(ABSTRACT TRUNCATED AT 250 WORDS)