Age-dependent ultrastructural changes of coronary artery in spontaneously hypertensive rats

The age-dependent differences in basic cardiovascular parameters, geometry and structure of coronary arteries between Wistar and spontaneously hypertensive rats (SHR) were evaluated. SHR of the age 3-, 9-, 17-, and 52-week and age-matched Wistar rats were used. Blood pressure (BP) was measured by the plethysmographic method. Animals were perfused with a glutaraldehyde fixative under pressure of 90 mmHg (3-week-old) and 120 mmHg (9-, 17-, 52-week-old). Coronary arteries were processed for electron microscopy. The proportions and cross sectional areas (CSA) of extracellular matrix in intima and media, endothelial and muscle cells were determined by point counting method. Cardiac hypertrophy and except of 3-week-old rats also BP increase and coronary wall hypertrophy was found in all ontogenic periods in SHR compared to Wistar rats. Arterial wall hypertrophy was evoked by increase of CSA of medial extracellular matrix and smooth muscle cells. In 52-week-old SHR, CSA of muscle cells did not differ from that in 17-week-old SHR but the CSA of intimal and medial extracellular matrix significantly increased. The CSA of endothelial cells and CSA of intimal extracellular matrix were increased only in 52-week-old SHR. The independency between BP and trophicity of individual components of the coronary wall during ontogeny of SHR was documented.     

Angiotensinase activities in the kidney of renovascular hypertensive rats

In spite of the well-known contribution of angiotensin II (Ang II) in the pathogenesis of Goldblatt two-kidney one clip (G2K1C) hypertension, the importance of other Ang peptides, such as Ang III, Ang IV or Ang 2-10, is scarcely understood. The functional status of these peptides depends on the action of several aminopeptidases called angiotensinases. The metabolism of Ang III to Ang IV by aminopeptidase M (AlaAP) and of Ang I to Ang 2-10 by aspartyl aminopeptidase (AspAP) was evaluated in the renal cortex and medulla of normotensive (Sham-operated) and hypertensive (G2K1C) rats, treated or not with the AT(1) receptor antagonist valsartan. The results demonstrated a highly significant increase of membrane-bound (MEMB) AlaAP in the cortex of the non-ischemic kidney of G2K1C rats compared with the kidney of normal rats and with the clipped kidney of G2K1C rats. This suggests an increased formation of Ang IV in the non-clipped kidney of G2R1C rats. Valsartan reduced MEMB AlaAP and AspAP activities in the renal cortex of normotensive and in the clipped kidney of hypertensive rats. The reduced metabolism of Ang III may prolong its half-life in valsartan-treated animals. These results suggest a role for AlaAP in renovascular hypertension. In addition, the higher AspAP activity of the renal cortex compared to medulla reflects its relative functional difference between both locations.     

Beta-adrenoceptors in kidney tubules of spontaneously hypertensive and normotensive rats

Beta-adrenoceptor binding characteristics were determined in different fractions of rat kidney tubules using a [125Iodo]-(-)-cyanopindolol (ICYP) binding assay. The highest amount of binding sites was found in a fraction containing predominantly distal tubular fragments. In a separate series of experiments the ICYP binding characteristics were compared in whole tubular fractions from spontaneously hypertensive (SHR) and normotensive Wistar Kyoto rats (WKY) of different ages. The maximum number of binding sites was significantly higher both in young (3 weeks) and adult (14 weeks) SHR when compared to age-matched WKY. These studies showed the presence of beta-adrenoceptor binding sites in rat kidney tubules and support the potential importance of tubular beta-adrenoceptors in the development of spontaneous hypertension and in the mechanism of antihypertensive action of beta-blockers.     

Endothelium-derived relaxing factors in the kidney of spontaneously hypertensive rats

Acetylcholine (ACh)-induced vasodilation is mainly due to endothelium-derived nitric oxide (EDNO) and hyperpolarizing factor (EDHF). To explore the mechanisms underlying attenuated endothelium-dependent vasodilation in hypertensive arteries, we measured the EDNO released from isolated kidneys of spontaneously hypertensive rats (SHR) using a sensitive chemiluminescence assay system of NO. ACh-induced renal vasodilation was significantly smaller in SHR than in the normotensive control, Wistar-Kyoto rats (WKY). However, ACh-induced NO release did not differ between SHR and WKY (10⁻⁷ M: SHR +37 ± 2 [SE] vs. WKY +32 ± 4 fmol/min/g kidney). Perfusion with a 20 mEq/L high-K⁺ buffer, which is reported to inhibit action of EDHF, significantly reduced ACh-induced vasorelaxation in WKY but not in SHR, resulting in identical renal perfusion pressure in SHR and wKY under these conditions. These results indicate that attenuated ACh-induced vasorelaxation in the SHR kidney may be attributed to a decrease in EDHF rather than that in EDNO.     

Sodium homeostasis in transplanted rats with a spontaneously hypertensive rat kidney

Recipients of a kidney from spontaneously hypertensive rats (SHR) but not from normotensive Wistar-Kyoto rats (WKY) develop posttransplantation hypertension. To investigate whether renal sodium retention precedes the development of posttransplantation hypertension in recipients of an SHR kidney on a standard sodium diet (0.6% NaCl), we transplanted SHR and WKY kidneys to SHR x WKY F1 hybrids, measured daily sodium balances during the first 12 days after removal of both native kidneys, and recorded mean arterial pressure (MAP) after 8 wk. Recipients of an SHR kidney (n = 12) retained more sodium than recipients of a WKY kidney (n = 12) (7.3 +/- 10 vs. 4.0 +/- 0.7 mmol, P < 0.05). MAP was 144 +/- 6 mmHg in recipients of an SHR kidney and 106 +/- 5 mmHg in recipients of a WKY kidney (P < 0.01). Modest sodium restriction (0.2% NaCl) in a further group of recipients of an SHR kidney (n = 10) did not prevent posttransplantation hypertension (MAP, 142 +/- 4 mmHg). Urinary endothelin and urodilatin excretion rates were similar in recipients of an SHR and a WKY kidney. Transient excess sodium retention after renal transplantation may contribute to posttransplantation hypertension in recipients of an SHR kidney.     

ETA receptor-mediated role of endothelin in the kidney of doca-salt hypertensive rats

Renal effects of FR139317, an endothelin ET_A receptor antagonist, were examined using anesthetized normotensive and deoxycorticosterone acetate (DOCA)-salt hypertensive rats. The intravenous bolus injection of FR139317 (10 mg/kg) produced a slight decrease in mean blood pressure (MAP; −13%) in the control rats and this hypotension was accompanied by a moderate renal vasodilation (renal vascular resistance: RVR; −12%). In the DOCA-salt hypertensive rat, FR139317 had a more pronounced hypotensive effect (MAP; −26%) accompanied by a potent renal vasodilation (RVR; −33%). FR 139317 significantly increased renal blood flow only in the DOCAsalt rats. In contrast, FR139317 produced a significant decrease in urine flow and urinary sodium excretion only in control rats. Northern blot analysis revealed that the renal prepro endothelin-1 (ET-1) mRNA level was significantly increased in DOCA-salt hypertensive rats. Thus, it seems likely that endogenous ET-1 is responsible for the maintenance of DOCA-salt-induced hypertension. We also suggest that at least in part, ET-1 and £ta receptors are involved in renal hemodynamic abnormalities in DOCA-salt-induced hypertension. The augmentation of renal ET-1 production may possibly have a function in the development and maintenance of DOCA-salt-induced hypertension.     

Proteomic identification of tyrosine nitration targets in kidney of spontaneously hypertensive rats

Nitrosative and oxidative stress are implicated in the development of hypertension. Events in the renal medulla may play a key role in the development and progression of hypertension. This may arise through disruption of nitric oxide signalling in the medulla and be accompanied by enhanced nitrosative and oxidative stress as indicated by the presence of proteins containing 3-nitrotyrosine. Here we demonstrate enhanced protein nitration in the medulla of spontaneously hypertensive rats. We have identified several nitrated proteins with both varied subcellular location and functional roles. These proteins are involved in nitric oxide signalling, antioxidant defense and energy metabolism. Moreover, increased nitration was observed in conjunction with enhanced oxidative damage as evidenced by the presence of protein carbonyl oxidative stress biomarkers. Our results suggest that kidney medulla is subject to enhanced nitrosative and oxidative stress, and that resulting protein modifications may contribute to the progression of hypertension.     

Increased phospholipase A2 activity in the kidney of spontaneously hypertensive rats

Phospholipase A2 activity was studied in the renal cortex and medulla of stroke-prone spontaneously hypertensive rat (SHRSP) and normotensive rat (WKY), and the subcellular localization of its activity was determined. Enhanced activity was found in both the cortical and medullary microsomes in SHRSP kidneys. In SHRSP, but not in WKY, phospholipase A2 activity progressively increased with age. This phospholipase A2 had substrate specificity toward phosphatidylethanolamine. There were no differences in optimal pH, substrate specificity, heat lability, and responses to Triton X-100 and deoxycholate between SHRSP and WKY. Ca2+ stimulated phospholipase A2 activity in both animals. The maximal activation was achieved at 5 mM Ca2+, and EDTA strongly inhibited the activity. But the response to Ca2+ was different in each. Ca2+ enhanced this activity in SHRSP markedly compared with WKY. It seems that Ca2+ is specifically required for phospholipase A2 activity in SHRSP. Though the influx of Ca2+ into microsomal membranes was not enhanced, the Ca2+ efflux of microsomal membranes decreased in SHRSP. This results in increases of intramicrosomal Ca2+, which may cause the subsequent activation of phospholipase A2. The Ca2+ permeability may be one of the factors in the increased phospholipase A2 activity in SHRSP.     

Decreased level of calpain inhibitor activity in kidney from Milan hypertensive rats

Rat kidney contains two different calpain isozymes distinguishable on the basis of their Ca2+ requirement and of their activation mechanisms. The two calpain isozymes are present in comparable amounts in kidney of normotensive and hypertensive rats of the Milan strain. Conversely, the level of the natural inhibitor of calpain is significantly decreased in kidney of hypertensive rats as compared to control normotensive rats. This deficiency is more pronounced in the cortical region than in other kidney fractions. These results taken together with previous observations indicating the existence of an identical defect in red cells from the same hypertensive rat strain, (Pontremoli, S., Melloni, E., Salamino, F., Sparatore, B., Viotti, P., Michetti, M., Duzzi, L., and Bianchi, G. (1986) Biochem. Biophys. Res. Commun. 138, 1370-1375) emphasize the possible role of an unbalanced intracellular proteolytic system in the development of genetically determined hypertension.     

The activity of kidney lysosomal hydrolases and renin in hypertensive rats.

In renal cortices of hypertensive rats the activity of renin and β-glucuronidase is significantly enhanced, while the activity of acid phosphatase remains pratically unchanged. A significant decrease in protein content accompanies the rise in enzyme activity. The distribution pattern of renin, β-glucuronidase and acid phosphatase after isopycnic centrifugation of cortical homogenates from hypertensive animals is different from that of controls.     

Peritubular membrane potential in kidney proximal tubular cells of spontaneously hypertensive rats

Peritubular membrane potential in kidney proximal tubular cells of spontaneously hypertensive rats (SHR-Okamoto strain adult rats) was measured with conventional 3 mol KCl microelectrodes, in vivo. Peritubular cell membrane potential was not different in SHR (-66.5 ± 0.7 mV) as compared with normotensive control Wistar rats (-67.5 ± 1.2 mV). To test the effects of possible altered sodium membrane transport in SHR on proximal tubule peritubular membrane potential, we allowed SHR and control rats to drink 1% NaCl for two weeks. Again, proximal tubule peritubular membrane potential was not different in SHR on 1% NaCl (-67.0 ± 1.0 mV) as compared with control rats on 1% NaCl (-64.7 ± 1.3 mV). From these results we concluded that peritubular membrane potential in kidney proximal tubular cells of SHR was not different from normotensive Wistar control rats, and if some alteration of sodium transport in kidney proximal tubular cells of SHR could exist, that was not possible to evaluate from the measurements of peritubular membrane potential in kidney proximal tubular cells.     

Age-dependent alterations of linoleic, arachidonic and eicosapentaenoic acids in renal cortex and medulla of spontaneously hypertensive rats

The lipid content as well as the fatty acid pattern of triglycerides, free fatty acids (FFA), phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were estimated in renal cortex and medulla of spontaneously hypertensive rats (SHR) and normotensive Wistar rats (WR) at 4,8,26 and 52 weeks of age. In general, the level of triglycerides in renal medulla appeared higher when compared with the cortex. On the other hand, PC and PE, increasing with age, were usually higher in the cortex. A decreased percentage of linoleic acid (LA) in triglycerides, of arachidonic acid (AA) in PC and of eicosapentaenoic acid (EPA) in triglycerides, FFA, PC and PE could be found in the kidneys of SHR at 8 weeks of age, i.e. during the development of hypertension. This was accompanied with a rise of AA in FFA of SHR at 8 weeks of age, which occurred with delay in WR (at 26 weeks of age). From the data presented it can be concluded that systematic alterations in the availability of individual polyunsaturated fatty acids (PUFA) in various renal lipids might be related to the onset of hypertension in SHR which should be elucidated in more detail.     

Pathways of bradykinin degradation in blood and plasma of normotensive and hypertensive rats

Kinins are vasoactive peptide hormones that can confer protection against the development of hypertension. Because their efficacy is greatly influenced by the rate of enzymatic degradation, the activities of various kininases in plasma and blood of spontaneously hypertensive rats (SHR) were compared with those in normotensive Wistar-Kyoto rats (WKY) to identify pathogenic alterations. Either plasma or whole blood was incubated with bradykinin (10 microM). Bradykinin and kinin metabolites were measured by high-performance liquid chromatography. Kininase activities were determined by cumulative inhibition of angiotensin I-converting enzyme (ACE), carboxypeptidase N (CPN), and aminopeptidase P (APP), using selective inhibitors. Plasma of WKY rats degraded bradykinin at a rate of 13.3 +/- 0.94 micromol x min(-1) x l(-1). The enzymes ACE, APP, and CPN represented 92% of this kininase activity, with relative contributions of 52, 25, and 16%, respectively. Inclusion of blood cells at physiological concentrations did not extend the activities of these plasma kininases further. No differences of kinin degradation were found between WKY and SHR. The identical conditions of kinin degradation in WKY and SHR suggest no pathogenic role of kininases in the SHR model of genetic hypertension.     

Accumulation of copper in kidney of spontaneously hypertensive rats, a preliminary study

Mineral levels such as Cu, Fe, Zn, Ca and Mg in organs of spontaneously hypertensive osteogenic disorder Shionogi rat (SHR-od) were compared with those of normotensive osteogenic disorder Shionogi rat (ODS). The effect of vitamin C deficiency was also examined for both strains. Cu levels in kidney of SHR-od increased to 2.2 times and 5.0 times that of ODS at 6-week old and 30-week old, respectively, although the effect of vitamin C deficiency was not observed in Cu levels of five organs examined. The effect of vitamin C deficiency appeared in the change in the other mineral levels almost similarly for both SHR-od and ODS rats at 30-week old; the decrease of Fe in liver, spleen and lung, whereas the increase of Zn in liver, Ca in heart and Mg in spleen, respectively.     

Calcium transport in basolateral plasma membranes from kidney cortex of Milan hypertensive rats

Ca2+ transport was investigated in basolateral plasma membranes (BLM) isolated from kidney cortex of the Milan strain of genetically hypertensive rats (MHS) and their normotensive controls (MNS) during a pre-hypertensive stage (age 3-4 weeks). It was found that the Vmax of ATP-dependent Ca2+ transport (in the presence of calmodulin) was about 16% lower in MHS than in control rats. In membranes from MNS rats which had been isolated in the presence of EGTA, the ATP-dependent Ca2+ transport showed a hyperbolic Ca2+ concentration dependence, a high Km (Ca2+) and a low Vmax; upon addition of exogenous calmodulin, the kinetics became sigmoidal, the Km (Ca2+) was decreased and the Vmax was increased. In membranes from MHS rats, the Ca2+ concentration dependence of ATP-driven Ca2+ transport was sigmoidal and the Ca2+ affinity was high in the absence of added calmodulin. Addition of exogenous calmodulin to these membranes resulted in an increase in Vmax, but no change in other kinetic parameters. Low-affinity hyperbolic kinetics of Ca2+ transport could only be obtained in MHS rats if the membranes were extracted with hypotonic EDTA and hypertonic KCl. These data suggest that the plasma membrane Ca2+-ATPase, which catalyses the ATP-dependent Ca2+ transport, exists in BLM of pre-hypertensive MHS rats predominantly in an activated, high-affinity form.     

Increased reactive oxygen species contributes to kidney injury in mineralocorticoid hypertensive rats.

Hypertension is associated with increased reactive oxygen species (ROS). Renal ROS production and their effects on renal function have never been investigated in mineralocorticoid hypertensive rats. In this study we hypothesized that increased ROS production in kidneys from deoxycorticosterone (DOCA)-salt rats contributes to adverse renal morphological changes and impaired renal function in DOCA-salt hypertensive rats. We also determined whether ROS-induced renal injury was dependent on blood pressure. DOCA-salt hypertensive rats exhibited a marked increase in blood pressure, renal ROS production, glomerular and tubular lesions, and microalbuminuria compared to sham rats. Treatment of DOCA-salt hypertensive rats with apocynin for 28 days resulted in attenuation of systolic blood pressure and improvement of renal morphology. Renal superoxide level in DOCA-salt rats was 215% of sham-operated rats and it was significantly decreased to 140% with apocynin treatment. Urinary protein level was decreased from 27 +/- 3 mg/day in DOCA-salt hypertensive rats to 9 +/- 2 mg/day. 28 days of Vitamin E treatment also reduced renal injury in regard to urinary protein level and renal morphology but had no effect on blood pressure in DOCA-salt rats. Increased urinary 8-isoprostane, a marker for oxidative stress, in DOCA-salt hypertensive rats (55 +/- 8 ng/day) was diminished by vitamin E treatment (24 +/- 6 ng/day). These data suggest that renal injury characteristic of mineralocorticoid hypertension is associated with oxidative stress and is partly independent of blood pressure.