Agnieszka Walkowska

Early effects of renal denervation in the anaesthetised rat: natriuresis and increased cortical blood flow

1 A novel method of renal denervation was developed based on electro‐coagulation of tissue containing most of the sympathetic fibres travelling towards the kidney. Kidney tissue noradrenaline was decreased to 4.7 % of the content measured in the contralateral innervated kidney when studied 3 days postdenervation. 2 The method was utilised in anaesthetised rats to examine the effects of denervation within the heretofore unexplored first 75 min period postdenervation. Sodium excretion (UNaV) increased significantly (+82 %, P < 0.03) over the 25‐50 min after denervation. In a parallel group, with a lower baseline UNaV, there was also a significant increase in UNaV (+54 %, P < 0.03) within the first 25 min. The renal perfusion pressure was maintained at a constant value and the glomerular filtration rate did not change after denervation. 3 Renal cortical and medullary blood flows (CBF, MBF) were estimated as laser Doppler flux and medullary tissue ion concentration was estimated as electrical admittance (Y). Following denervation, in both groups CBF increased significantly within the first 25 min (+12 %, P < 0.01 and +8 %, P < 0.05, respectively) while MBF did not change or decreased slightly; Y did not change. 4 The data document the development of natriuresis within the first 25‐50 min after denervation. The increase in CBF indicated that, prior to denervation, the cortical, but not medullary, circulation was under a tonic vasoconstrictor influence of the renal nerves. Such a dissociation of neural effects on the renal cortical vs. medullary vasculature has not been previously described.

Combined inhibition of 20-hydroxyeicosatetraenoic acid formation and of epoxyeicosatrienoic acids degradation attenuates hypertension and hypertension-induced end-organ damage in Ren-2 transgenic rats

Recent studies have shown that the renal CYP450 (cytochrome P450) metabolites of AA (arachidonic acid), the vasoconstrictor 20-HETE (20-hydroxyeicosatetraenoic acid) and the vasodilator EETs (epoxyeicosatrienoic acids), play an important role in the pathophysiology of AngII (angiotensin II)-dependent forms of hypertension and the associated target organ damage. The present studies were performed in Ren-2 renin transgenic rats (TGR) to evaluate the effects of chronic selective inhibition of 20-HETE formation or elevation of the level of EETs, alone or in combination, on the course of hypertension and hypertension-associated end-organ damage. Both young (30 days of age) prehypertensive TGR and adult (190 days of age) TGR with established hypertension were examined. Normotensive HanSD (Hannover Sprague-Dawley) rats served as controls. The rats were treated with N-methylsulfonyl-12,12-dibromododec-11-enamide to inhibit 20-HETE formation and/or with N-cyclohexyl-N-dodecyl urea to inhibit soluble epoxide hydrolase and prevent degradation of EETs. Inhibition in TGR of 20-HETE formation combined with enhanced bioavailability of EETs attenuated the development of hypertension, cardiac hypertrophy, proteinuria, glomerular hypertrophy and sclerosis as well as renal tubulointerstitial injury. This was also associated with attenuation of the responsiveness of the systemic and renal vascular beds to AngII without modifying their responses to noradrenaline (norepinephrine). Our findings suggest that altered production and/or action of 20-HETE and EETs plays a permissive role in the development of hypertension and hypertension-associated end-organ damage in this model of AngII-dependent hypertension. This information provides a basis for a search for new therapeutic approaches for the treatment of hypertension.

Similar renoprotection after renin‐angiotensin‐dependent and ‐independent antihypertensive therapy in 5/6‐nephrectomized Ren‐2 transgenic rats: are there blood pressure‐independent effects?

1. Hypertension plays a critical role in the progression of chronic kidney disease (CKD) to end‐stage renal disease (ESRD), but it has also been postulated that antihypertensive drugs that block the renin‐angiotensin system (RAS) show class‐specific renoprotective actions beyond their blood pressure (BP)‐lowering effects.

Effects of ATP on rat renal haemodynamics and excretion: role of sodium intake, nitric oxide and cytochrome P450

Aim:  Adenosine‐5′‐triphosphate (ATP) affects intrarenal vascular tone and tubular transport via P2 receptors; however, the actual role of the system in regulation of renal perfusion and excretion remains unclear and is the subject of this whole‐kidney study.

Inhibition of soluble epoxide hydrolase is renoprotective in 5/6 nephrectomized Ren-2 transgenic hypertensive rats

The aim of the present study was to test the hypothesis that increasing kidney tissue concentrations of epoxyeicosatrienoic acids (EETs) by preventing their degradation to the biologically inactive dihydroxyeicosatrienoic acids (DHETEs) using blockade of soluble epoxide hydrolase (sEH) would attenuate the progression of chronic kidney disease (CKD). Ren‐2 transgenic rats (TGR) after 5/6 renal mass reduction (5/6 NX) served as a model of CKD associated with angiotensin (Ang) II‐dependent hypertension. Soluble epoxide hydrolase was inhibited using cis‐4‐[4‐(3‐adamantan‐1‐yl‐ureido)cyclohexyloxy]benzoic acid (c‐AUCB; 3 mg/L drinking water) for 20 weeks after 5/6 NX. Sham‐operated normotensive transgene‐negative Hannover Sprague‐Dawley (HanSD) rats served as controls. When applied in TGR subjected to 5/6 NX, c‐AUCB treatment improved survival rate, prevented the increase in blood pressure, retarded the progression of cardiac hypertrophy, reduced proteinuria and the degree of glomerular and tubulointerstitial injury and reduced glomerular volume. All these organ‐protective actions were associated with normalization of the intrarenal EETs : DHETEs ratio, an index of the availability of biologically active EETs, to levels observed in sham‐operated HanSD rats. There were no significant concurrent changes of increased intrarenal AngII content. Together, these results show that 5/6 NX TGR exhibit a profound deficiency of intrarenal availability of active epoxygenase metabolites (EETs), which probably contributes to the progression of CKD in this model of AngII‐dependent hypertension, and that restoration of intrarenal availability of EETs using long‐term c‐AUCB treatment exhibits substantial renoprotective actions.

Intrarenal cytochrome P-450 metabolites of arachidonic acid in the regulation of the nonclipped kidney function in two-kidney, one-clip Goldblatt hypertensive rats.

Objective The contribution of epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE) as cytochrome P-450 metabolites of arachidonic acid in the regulation of the nonclipped kidney function in two-kidney, one-clip (2K1C) Goldblatt hypertensive rats was investigated during the phases of initial and stable hypertension, that is, 7 or 27 days after clipping, respectively. Methods Male Hannover Sprague–Dawley rats had the right renal artery clipped or underwent sham operation. Urinary excretion of EETs, their inactive metabolites dihydroxyeicosatrienoic acids and of 20-HETE was measured. Intrarenal cytochrome P-450 protein expression and the activities of epoxygenase, ω-hydroxylase and soluble epoxide hydrolase were also determined. The responses of renal hemodynamics and electrolyte excretion of the nonclipped kidney to left renal artery infusions of inhibitors of EETs or 20-HETE formation (MS-PPOH and DDMS, respectively) were measured. Results In 2K1C rats, urinary excretion of EETs was significantly lower and that of 20-HETE was higher than that in sham-operated animals only on day 27 after clipping. Intrarenal inhibition of EETs significantly decreased renal hemodynamics and sodium excretion in sham-operated but not in 2K1C rats. Intrarenal inhibition of 20-HETE decreased sodium excretion in sham-operated rats but elicited increases in renal hemodynamics and sodium excretion in 2K1C rats. Conclusion Our results indicate that the nonclipped kidney of Goldblatt 2K1C rats in the phase of sustained hypertension exhibits decreased intrarenal EETs and elevated 20-HETE levels as compared with the kidney of sham-operated animals. This suggests that altered production and action of cytochrome P-450-derived metabolites during this stable phase contributes to the mechanism of Goldblatt 2K1C hypertension.

Simultaneous recording of tissue ion content and blood flow in rat renal medulla : evidence on interdependence

The relationship of renal medullary tissue ion concentration and medullary blood flow (MBF) has never been closely evaluated because of limitations of available measuring methods. In an attempt to overcome this difficulty, an integrated probe was developed for simultaneous recording in rat renal medulla of tissue electrical admittance ( Y), an index of interstitial ion concentration, and tissue perfusion with blood (laser-Doppler method). During spontaneous-selective MBF variations tissue Y showed inverse changes ( r = -0.77, P < 0.001). The inverse correlation of the two variables was also seen after MBF has been reduced (-43%) by indomethacin, 5 mg/kg body wt iv ( r = -0.77, P < 0.01). A modest selective MBF reduction (15%) induced by glibenclamide, an inhibitor of ATP-dependent K channels, did not alter medullary tissue admittance. The data support experimentally the concept that the rate of medullary tissue perfusion with blood is one determinant of interstitial solute concentration; however, changes in the latter were demonstrable only with major alterations of the MBF.

OSMOTIC HYPERTONICITY OF THE RENAL MEDULLA DURING CHANGES IN RENAL PERFUSION PRESSURE IN THE RAT

1 The relationship between renal perfusion pressure (RPP) and ion concentration in renal medulla was studied in anaesthetized rats. RPP was changed in steps within the pressure range 130‐80 mmHg, while tissue electrical admittance (Y, index of interstitial ion concentration) and medullary and cortical blood flow (MBF and CBF; laser Doppler flowmetry) were measured, along with glomerular filtration rate (Cin) and renal excretion. 2 With a RPP reduction from 130 to 120 mmHg, tissue Y remained stable; at 100 and 80 mmHg, Y was 5 and 17 % lower, respectively, than at 120 mmHg. 3 CBF fell less than RPP (partial autoregulation) in the range 130‐100 mmHg only. MBF was autoregulated within 120‐100 mmHg, but not above or below this range. 4 Each step of RPP reduction was followed by a decrease in sodium and water excretion (UNaV and V). The osmolality of excised inner medulla fragments was similar at 120 and 105 mmHg (586 ± 45 and 618 ± 35 mosmol (kg H2O)−1, respectively) but lower at 80 mmHg (434 ± 31 mosmol (kg H2O)−1, P < 0.01); the ion concentration changed in parallel. 5 The data show that medullary hypertonicity was well preserved during RPP fluctuations within 130‐100 mmHg, but not below this range. RPP‐dependent changes of UNaV and V were not clearly associated with changes in solute concentration in medullary tissue.

Renal nerves and nNOS: roles in natriuresis of acute isovolumetric sodium loading in conscious rats

It was hypothesized that renal sympathetic nerve activity (RSNA) and neuronal nitric oxide synthase (nNOS) are involved in the acute inhibition of renin secretion and the natriuresis following slow NaCl loading (NaLoad) and that RSNA participates in the regulation of arterial blood pressure (MABP). This was tested by NaLoad after chronic renal denervation with and without inhibition of nNOS by S-methyl-thiocitrulline (SMTC). In addition, the acute effects of renal denervation on MABP and sodium balance were assessed. Rats were investigated in the conscious, catheterized state, in metabolic cages, and acutely during anesthesia. NaLoad was performed over 2 h by intravenous infusion of hypertonic solution (50 micromol.min(-1).kg body mass(-1)) at constant body volume conditions. SMTC was coinfused in amounts (20 microg.min(-1).kg(-1)) reported to selectively inhibit nNOS. Directly measured MABPs of acutely and chronically denervated rats were less than control (15% and 9%, respectively, P < 0.005). Plasma renin concentration (PRC) was reduced by renal denervation (14.5 +/- 0.2 vs. 19.3 +/- 1.3 mIU/l, P < 0.005) and by nNOS inhibition (12.4 +/- 2.3 vs. 19.6 +/- 1.6 mlU/l, P < 0.005). NaLoad reduced PRC (P < 0.05) and elevated MABP modestly (P < 0.05) and increased sodium excretion six-fold, irrespective of renal denervation and SMTC. The metabolic data demonstrated that renal denervation lowered sodium balance during the first days after denervation (P < 0.001). These data show that renal denervation decreases MABP and renin secretion. However, neither renal denervation nor nNOS inhibition affects either the renin down-regulation or the natriuretic response to acute sodium loading. Acute sodium-driven renin regulation seems independent of RSNA and nNOS under the present conditions.

Nitric oxide inhibition and the impact on renal nerve‐mediated antinatriuresis and antidiuresis in the anaesthetized rat

The contribution of nitric oxide (NO) to the antinatriuresis and antidiuresis caused by low‐level electrical stimulation of the renal sympathetic nerves (RNS) was investigated in rats anaesthetized with chloralose–urethane. Groups of rats, n= 6, were given i.v. infusions of vehicle, l‐NAME (10 μg kg−1 min−1), 1400W (20 μg kg−1 min−1), or S‐methyl‐thiocitrulline (SMTC) (20 μg kg−1 min−1) to inhibit NO synthesis non‐selectively or selectively to block the inducible or neuronal NOS isoforms (iNOS and nNOS, respectively). Following baseline measurements of blood pressure (BP), renal blood flow (RBF), glomerular filtration rate (GFR), urine flow (UV) and sodium excretion (UNaV), RNS was performed at 15 V, 2 ms duration with a frequency between 0.5 and 1.0 Hz. RNS did not cause measurable changes in BP, RBF or GFR in any of the groups. In untreated rats, RNS decreased UV and UNaV by 40–50% (both P < 0.01), but these excretory responses were prevented in l‐NAME‐treated rats. In the presence of 1400W i.v., RNS caused reversible reductions in both UV and UNaV of 40–50% (both P < 0.01), while in SMTC‐treated rats, RNS caused an inconsistent fall in UV, but a significant reduction (P < 0.05) in UNaV of 21%. These data demonstrated that the renal nerve‐mediated antinatriuresis and antidiuresis was dependent on the presence of NO, generated in part by nNOS. The findings suggest that NO importantly modulates the neural control of fluid reabsorption; the control may be facilitatory at a presynaptic level but inhibitory on tubular reabsorptive processes.