Fumihiro Tomoda

Evidence for decreased structurally determined preglomerular resistance in the young spontaneously hypertensive rat after 4 weeks of renal denervation.

Objectives To study the effects of denervation of the kidney on renal vascular resistance at maximal dilatation and renal function during the development of hypertension in the spontaneously hypertensive rat (SHR). Methods SHR aged 6 weeks were subjected to left renal denervation or a sham-operation (n = 18 denervated, n = 13 sham). When they were aged 10 weeks, pairs of denervated and sham-operated left kidneys were perfused with 2% dextran in Tyrodes solution and pressure–flow and pressure–glomerular filtration rate (GFR) relationships at maximal vasodilatation were established. The awake mean arterial blood pressure, in-vivo renal function and renal noradrenaline content were also measured. Results There were no significant differences between the pressure–flow relationships for denervated and sham–operated kidneys. However, there was a marked, parallel, shift leftwards in the pressure–GFR relationship (P < 0.001). Thus, the denervated kidneys commenced filtering at a lower threshold perfusion pressure than did the sham-operated ones. In-vivo renal plasma flow and GFR were significantly greater in the denervated left kidneys of SHR than they were in the contralateral kidneys. The noradrenaline content in denervated kidneys was 5 ± 3% of that in innervated kidneys. The awake mean arterial pressure was 135 ± 1 and 138 ± 2 mmHg in the denervated and sham-operated groups respectively. Conclusion Denervation of the kidney of SHR aged 6 weeks of age altered the pressure–GFR but not the pressure–flow relationship for these rats 4 weeks later. The results are compatible with there having been an increase in average preglomerular and a decrease in post-glomerular vessel lumen diameters. These changes suggest that the renal nerves affect the structural development of the renal vasculature in SHR.

Role of bradykinin receptors in the renal effects of inhibition of angiotensin converting enzyme and endopeptidases 24.11 and 24.15 in conscious rabbits.

1 We tested the effects on systemic haemodynamics and renal function, of inhibition of endopeptidase (EP) 24.15 (E.C. 3.4.24.15), in conscious uninephrectomized rabbits in which the activities of angiotensin converting enzyme (ACE, E.C. 3.4.15.1) and neutral endopeptidase (EP 24.11, E.C. 3.4.24.11) were already inhibited. To test the role of bradykinin B2‐receptors in mediating the effects following inhibition of these enzymes, the antagonist Hoe 140 was used. 2 Hoe 140 (0.1 mg kg−1, i.v.) did not affect resting mean arterial pressure or heart rate, but antagonized the depressor effect of right atrial administration of bradykinin. The dose‐response curve for bradykinin was shifted more than 1000 fold to the right for more than 4 h. Hoe 140 approximately doubled resting urine flow and increased fractional Na+ excretion from 4.2 to 6.0%; consistent with the hypothesis that it exerts a partial agonist effect on the kidney. 3 Combined inhibition of ACE (captopril; 0.25 mg kg−1 plus 0.2 mg kg−1h−1) and EP 24.11 (SCH 39370; 3 mg kg−1 plus 3 mg kg−1h−1) was followed by a sustained reduction in arterial pressure (−6 ± 2 mmHg) and increase in heart rate (35 ± 7 beats min−1). There was a small increase in renal blood flow (by 6.5 ± 3.2% relative to vehicle‐treatment) without a change in glomerular filtration rate, and about a 150% increase in Na+ excretion. Hoe 140 (0.1 mg kg−1, i.v.) pretreatment did not influence the renal effects of captopril and SCH 39370, although it did appear to blunt their hypotensive and tachycardic effects. 4 When EP 24.15 was inhibited with N‐[1‐(R,S)‐carboxy‐3‐phenylpropyl]‐Ala‐Ala‐Tyr‐p‐aminobenzoate (cFP‐AAY‐pAB; 5 mg kg−1 plus 3 mg kg−1h−1, i.v.) in rabbits pretreated with captopril and SCH 39370, no changes in systemic haemodynamics or renal function were observed. 5 We concluded that in conscious uninephrectomized rabbits, EP 24.15 does not play a major role in modulating renal function, at least under conditions where ACE and EP 24.11 are already inhibited. In contrast, ACE and/or EP 24.11 do modulate renal function in this model, but their influences are mediated chiefly through metabolism of peptides other than bradykinin.

Synthetic inhibitors of endopeptidase EC 3.4.24.15: potency and stability in vitro and in vivo

1 The role of the metalloendopeptidase EC 3.4.24.15 (EP 24.15) in peptide metabolism in vivo is unknown, in part reflecting the lack of a stable enzyme inhibitor. The most commonly used inhibitor, N‐[1‐(R,S)‐carboxy‐3‐phenylpropyl]‐Ala‐Ala‐Tyr‐p‐aminobenzoate (cFP‐AAY‐pAB, Ki = 16 nM), although selective in vitro, is rapidly degraded in the circulation to cFP‐Ala‐Ala, an angiotensin converting enzyme (ACE) inhibitor. This metabolite is thought to be generated by neutral endopeptidase (NEP; EC 3.4.24.11), as the Ala‐Tyr bond of cFP‐AAY‐pAB is cleaved by NEP in vitro. In the present study, we have examined the role of NEP in the metabolism of cFP‐AAY‐pAB in vivo, and have tested a series of inhibitor analogues, substituted at the second alanine, for both potency and stability relative to the parent compound. 2 Analogues were screened for inhibition of fluorescent substrate cleavage by recombinant rat testes EP 24.15. D‐Ala or Asp substitution abolished inhibitory activity, while Val‐, Ser‐ and Leu‐substituted analogues retained activity, albeit at a reduced potency. A relative potency order of Ala (1) > Val (0.3) > Ser (0.16) > Leu (0.06) was observed. Resistance to cleavage by NEP was assessed by incubation of the analogues with rabbit kidney membranes. The parent compound was readily degraded, but the analogues were twice (Ser) and greater than 10 fold (Leu and Val) more resistant to cleavage. 3 Metabolism of cFP‐AAY‐pAB and the Val‐substituted analogue was also examined in conscious rabbits. A bolus injection of cFP‐AAY‐pAB (5 mg kg−1, i.v.) significantly reduced the blood pressure response to angiotensin I, indicating ACE inhibition. Pretreatment with NEP inhibitors, SCH 39370 or phosphoramidon, slowed the loss of cFP‐AAY‐pAB from the plasma, but did not prevent inhibition of ACE. Injection of 1 mg kg−1 inhibitor resulted in plasma concentrations at 10 s of 23.5 μm (cFP‐AAY‐pAB) and 18.0 μm (cFP‐AVY‐pAB), which fell 100 fold over 5 min. Co‐injection of 125I‐labelled inhibitor revealed that 80–85% of the radioactivity had disappeared from the circulation within 5 min, and h.p.l.c. analysis demonstrated that only 25–30% of the radiolabel remained as intact inhibitor at this time. Both analogues were cleared from the circulation at the same rate, and both inhibitors blunted the pressor response to angiotensin I, indicative of ACE inhibition. 4 These results suggest that both NEP and other clearance/degradation mechanisms severely limit the usefulness of peptide‐based inhibitors such as cFP‐AAY‐pAB. To examine further EP 24.15 function in vivo, more stable inhibitors, preferably non‐peptide, must be developed, for which these peptide‐based inhibitors may serve as useful molecular templates.

Increased levels of small dense low-density lipoprotein cholesterol associated with hemorheological abnormalities in untreated, early-stage essential hypertensives

Among subfractions of low-density lipoprotein cholesterol (LDL-C), small dense LDL-C (SdLDL-C) has been highlighted as the most atherogenic lipoprotein cholesterol. The present study aimed to compare the relationship of SdLDL-C with blood viscosity, a surrogate marker for cardiovascular disease, with that of other lipid fractions with blood viscosity in essential hypertensives (EHTs). In 128 untreated, early-stage EHTs, blood viscosity was measured with a falling-ball microviscometer, and serum levels of lipid fractions were determined. Blood and plasma viscosity was significantly higher in 49 patients with dyslipidemia (fasting serum level of LDL-C >140 mg dl−1, triglyceride >150 mg dl−1 or high-density lipoprotein cholesterol (HDL-C)<40 mg dl−1) compared with 79 patients without dyslipidemia, although hematocrit and RBC rigidity index ‘k’ did not differ between the two groups. Together, SdLDL-C, LDL-C, triglyceride and large LDL-C were positively correlated with blood viscosity, but for HDL-C, the correlation was negative. After adjusting for non-lipid variables that correlated with blood viscosity (that is, the age, body mass index, resting diastolic blood pressure, sex, hematocrit, plasma viscosity and homeostasis model of assessment of insulin resistance), SdLDL-C was most strongly associated with blood viscosity among the lipid fractions. These data suggest that SdLDL-C could strongly increase blood viscosity in EHTs.

Blood rheology and platelet function in untreated early-stage essential hypertensives complicated with metabolic syndrome.

We examined whether hemorheology and platelet function are affected in essential hypertensives (EHTs) of the World Health Organization stage I when complicated with metabolic syndrome (Mets). In 156 untreated EHTs, blood viscosity and platelet surface markers were determined. Blood viscosity was significantly elevated in 54 subjects with Mets compared with 102 subjects without Mets. Hematocrit and plasma viscosity increased in the group with Mets, although red blood cell rigidity index “k” did not differ between groups. As a whole group, blood viscosity correlated positively with hematocrit and plasma viscosity. Additionally, plasma viscosity correlated positively with plasma leptin, triglyceride, homeostasis model assessment index, C-reactive protein, and plasma fibrinogen, but negatively with high-density lipoprotein cholesterol. In contrast, no differences were seen in platelet surface markers between groups. In conclusion, EHTs of the early stage complicated with Mets are characterized by increased blood viscosity due to hemoconcentration and increased plasma viscosity.

Effects of uninephrectomy on renal structural properties in spontaneously hypertensive rats

1. To investigate effects of a reduction in nephron numbers on renal structural properties in hypertension, either unilateral nephrectomy (UNX) or sham operation (SO) was performed at 5 weeks of age in spontaneously hypertensive rats (SHR) and Wistar‐Kyoto (WKY) rats (n = 9 for each operation for each strain).

Increased blood viscosity is associated with reduced renal function and elevated urinary albumin excretion in essential hypertensives without chronic kidney disease

Increased blood viscosity reduces blood flow and elevates vascular resistance in the cardiovascular system. The aim of this study was to elucidate how blood viscosity could affect renal function and eventually contribute to renal damage in essential hypertensives (EHT). In 164 untreated EHT without apparent renal damage (96 men, 56±12 years old, creatinine clearance 123±33 ml min−1 per 1.73 m2 and urinary albumin excretion 19±19 mg per day), blood and plasma viscosity was determined using a falling ball microviscometer. Blood viscosity correlated negatively with creatinine clearance (r=−0.185, P=0.018) and positively with urinary albumin excretion (r=0.253, P=0.001). This indicated that increased blood viscosity is associated with reduced renal function and worsening of albuminuria in EHT. Stepwise multiple regression analysis identified blood viscosity as an independent determinant of creatinine clearance (R2=0.058) and urinary albumin excretion (R2=0.216). In conclusion, increased blood viscosity may be a risk for development of renal disease in EHT.

CHRONIC RENAL BLOOD FLOW MEASUREMENT IN DOGS BY TRANSIT-TIME ULTRASOUND FLOWMETRY

To test the validity of transit-time ultrasound flowmetry for chronic measurement of renal blood flow in dogs, we compared this method with the renal clearance of para-aminohippuric acid (CPAH) (corrected for hematocrit), and with direct volumetric measurements. When flow-probes were implanted without silastic sheeting to stabilize the implant, there was significant disparity between the (within-dog) mean levels of renal blood flow estimated by flow-probe and CPAH. In contrast, when the flow-probe implants were stabilized with silicone sheeting, there was close agreement in each dog between the flow rates measured by the two methods. When flow-probes were calibrated volumetrically in situ, there was a close linear relationship between flow derived from the flow-probe and that measured volumetrically (r = 0.98 +/- 0.02). We conclude that valid, chronic measurement of renal blood flow in dogs can be achieved using transit-time ultrasound flowmetry, provided the implant is stabilized with silicone sheeting.

1093 THE RELATION OF DIETARY SALT TO INSULIN SENSITIVITY IN PRIMARY ALDOSTERONISM

Objectives: Whether or not the influence of aldosterone on insulin sensitivity is modified by dietary salt is unknown. In this study, the relationship between dietary salt and insulin sensitivity was investigated in primary aldosteronism (PA). Design and Methods: After the measurements of plasma renin activity (PRA), plasma aldosterone concentration (PAC) and urinary sodium excretion (UNaV) during rest, blood sugar (BS) and insulin (IRI) were evaluated before and after glucose loading of 75 g in 20 PA patients. According to dietary salt estimated by UNaV, subjects were divided into patients with 10.3±2.2 (High salt) and 6.2±2.1 g/day (Low salt) (n=10 of each group). Thereafter, in ten patients, the same study was repeated following low-salt diet instruction (6 g/day) for three months. Results: There was no difference in age, blood pressure, PAC and PAC/PRA between the two groups. Before glucose loading, IRI and HOMA-IR index (i.e., index for insulin sensitivity) was higher in High (12.8±6.8 &mgr;U/ml, 3.13±1.86) than in Low group (5.6±2.7, 1.31±0.72) despite no differences in BS among the two groups (96.8±13.1vs 94.0±7.2 mg/dl). After glucose loading, the increase of IRI estimated by AUC of IRI was greater in High (456.5±267.8 U/ml) than in Low group (254.9±177.1), although BS elevated to the similar extent in both groups. Following low-salt diet instruction, both IRI and HOMA-IR decreased (9.87±7.63 to 7.54±5.03 &mgr;U/ml, 2.54±2.08 to 1.88±1.39) without changes in BS. Thus, in PA, salt restriction enhanced insulin sensitivity and reduced blood level of insulin. Conclusions: These results suggest that insulin sensitivity could be salt-sensitive in PA.

294 RELATION OF SMALL DENSE LOW-DENSITY LIPOPROTEIN CHOLESTEROL TO CAROTID ATHEROSCLEROSIS IN ESSENTIAL HYPERTENSIVES WITH NORMAL RENAL FUNCTION

Objectives: Recently, small dense low-density lipoprotein cholesterol (sdLDL-C) has been highlighted as the most atherogenic lipoprotein in cardiovascular disease. In this study, the relations of sdLDL-C and other lipid parameters to surrogate markers of atherosclerosis were investigated in essential hypertensives (EHT) with normal renal function. Design and Methods: In 137 untreated EHT with GFR≥60 mL/min, plasma levels of sdLDL-C (using assay kit supplied by Denka Seiken Co., Ltd., Niigata, Japan), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), adiponectin and norepinephrine, and blood sugar were measured. Intima-media thickness (IMT) and stiffness index &bgr; (&bgr;) of carotid artery (i.e., index for wall thickness and arterial stiffness of large artery, respectively) was also evaluated by B-mode ultrasonography and ultrasonic phase-locked echo-tracking system, respectively. Results: Although LDL, TG and HDL-C did not associate with IMT, sdLDL-C and ratio of sdLDL-C to LDL significantly correlated with IMT (r = 0.226, 0.181, p < 0.05 of each). Multiple regression analysis demonstrated that sdLDL-C was an independent determinant for IMT together with age, blood sugar and plasma levels of adiponectin and norepinephrine (r2 = 0.323, P < 0.05). In contrast, sdLDL-C and the other lipoproteins did not relate to &bgr;. In addition, either blood pressure or pulse rate did not associate with IMT and &bgr;. Conclusions: These results indicate that in EHT with normal renal function, sdLDL was the best marker for large arterial wall thickness among lipid parameters, and suggest that quantitative measurement of sdLDL could give the useful information for atherosclerosis compared with systemic hemodynamics.