Endothelin ETA receptor blockade with darusentan increases sodium and potassium excretion in aging rats.

This study investigated whether intrarenal endothelin-1(ET-1) contributes to sodium excretion in aged rats. Metabolic function studies were performed in male Wistar rats (3 and 24 months) treated with placebo or the orally active ET(A) receptor antagonist darusentan (20 mg/kg/d) for 4 weeks. Mean arterial pressure was measured using an intra-arterial catheter. Electrolytes, aldosterone levels, renin activity, and angiotensin converting enzyme activity were determined in plasma, and mRNA expression of epithelial sodium channel (ENaC) and Na(+), K(+)-ATPase subunits was measured in the renal cortex and medulla. Aging was associated with a marked decrease in urinary excretion of sodium, chloride, and potassium (all P < 0.001) as well as renin activity (P < 0.05), but had no significant effect on gene expression of ENaC or Na(+), K(+)-ATPase subunits. In aged rats, darusentan treatment increased ion excretion (P < 0.05), reduced cortical gene expression of alphaENaC and alpha(1)-Na(+), K(+)-ATPase (both P < 0.05), and increased plasma aldosterone levels (P < 0.01). These data demonstrate a decrease of sodium and potassium excretion in aged rats, changes that are partly sensitive to ETA receptor blockade. Treatment with darusentan also reduced cortical expression of alphaENaC and alpha(1)-Na(+), K(+)-ATPase and increased plasma aldosterone levels independently of blood pressure, electrolytes, renin activity, or angiotensin converting enzyme activity. These findings may provide new pathogenetic links between aging and sodium sensitivity.

Role of podocytes for reversal of glomerulosclerosis and proteinuria in the aging kidney after endothelin inhibition.

The cause of focal-segmental glomerulosclerosis as a consequence of physiological aging, which is believed to be inexorable, is unknown. This study investigated whether inhibition of endothelin-1, a growth-promoting peptide contributing to renal injury in hypertension and diabetes, affects established glomerulosclerosis and proteinuria in the aged kidney. We also determined the role of endothelin receptors for podocyte injury in vivo and in vitro. Aged Wistar rats, a model of spontaneous age-dependent glomerulosclerosis, were treated with the orally active endothelin subtype A (ET(A)) receptor antagonist darusentan, and evaluation of renal histology, renal function studies, and expression analyses were performed. In vitro experiments using puromycin aminonucleoside to induce podocyte injury investigated the role of ET(A) receptor signaling for apoptosis, cytoskeletal injury, and DNA synthesis. In aged Wistar rats, established glomerulosclerosis and proteinuria were reduced by >50% after 4 weeks of darusentan treatment, whereas blood pressure, glomerular filtration rate, or tubulo-interstitial renal injury remained unaffected. Improvement of structural injury in glomeruli and podocytes was accompanied by a reduction of the expression of matrix metalloproteinase-9 and p21Cip1/WAF1. In vitro experiments blocking ET(A) receptors using specific antagonists or RNA interference prevented apoptosis and structural damage to podocytes induced by puromycin aminonucleoside. In conclusion, these results support the hypothesis that endogenous endothelin contributes to glomerulosclerosis and proteinuria in the aging kidney. The results further suggest that age-dependent glomerulosclerosis is not merely a "degenerative" but a reversible process locally confined to the glomerulus involving recovery of podocytes from previous injury.

Effects of angiotensin II, arginine vasopressin and tromboxane A2 in renal vascular bed: role of rho-kinase.

BACKGROUND: Angiotensin II (Ang II), arginine vasopressin (AVP) and tromboxane A(2) (TxA(2)) are dissimilar vasoconstrictors involved in regulating renal circulation. Whereas Ang II is primarily a physiological modulator, AVP and TxA(2) play important roles under pathological conditions. Previously, we have shown variable importance of intracellular Ca(2+) and protein kinase C for their mode of action (Ang II > AVP >U-46619), but the cell signalling via rho-associated kinase (ROK) is a common pathway. The aim of this study was to determine their sites of action in the renal vascular bed and the corresponding role of ROK at the microvascular level. METHODS: Glomerular blood flow (GBF) and luminal diameter of different vessels (10-70 micro m) were measured in the split hydronephrotic kidney of anaesthetized rats. The tissue bath concentration of Ang II, AVP or the TxA(2) agonist U-46619 was adjusted to reduce GBF by approximately 50%. The measurements were repeated after adding a sub-maximal dose of the ROK inhibitor Y-27632 into the bath. RESULTS: Ang II constricted all vessels significantly, the constriction being least in the proximal segment of the arcuate artery ( approximately 70 micro m). Significant constrictions due to AVP were found only in interlobular and arcuate arteries (20-70 micro m), but not in the afferent and efferent arterioles. U-46619 constricted only the arcuate artery (> or = 50 micro m). Y-27632 (10(-4) M) dilated all vessels significantly and increased GBF by 65%. Thereafter, effects of all agonists were severely attenuated. Control reductions in GBF could be obtained at higher concentrations of AVP (10-fold) and U-46619 (5-fold) and a lesser GBF reduction with Ang II (100-fold) without changes in the respective patterns of vascular constriction. CONCLUSIONS: Our data indicate that the agonists, in the order Ang II, AVP and TxA(2), constrict larger vessels within the renal vascular tree via activation of ROK. Therefore, ROK inhibitors may provide a therapeutic tool to antagonize pathological vasospasm of conduit vessels, which are resistant to other vasodilators.

Variations in cell signaling pathways for different vasoconstrictor agonists in renal circulation of the rat.

BACKGROUND: Major cell signaling pathways involved in agonist-induced vasoconstriction are recognized to be Ca2+ mobilization via inositol-1,4,5 triphosphate (IP3), Ca2+ influx through l-type channels, activation of protein kinase C (PKC), and of Rho-associated kinase (ROK). However, their contribution for renal vasoconstriction induced by different agonists is not well characterized. METHODS: Increasing doses of angiotensin II (Ang II), norepinephrine, and arginine vasopressin (AVP) were infused into the left renal artery of anesthetized rats to reduce renal blood flow from a threshold value to about 50%. Rightward shift of the dose-response curves due to coinfusion of inhibitors served to assess contribution of different pathways: trimethoxybenzoate (TMB-8) against Ca2+ mobilization, nifedipine against Ca2+ influx, staurosporine and Ro-318220 against PKC, and Y-27632 and HA-1077 against ROK. Effects of inhibitors were also determined for renal response to a single dose of U-46619, a thromboxane A2 agonist. Composite response to U-46619 consisting of a fast and slow component did not permit determination of dose-response curves. RESULTS: Inhibition of ROK by Y-27632 or HA-1077 had the largest effect on renal responses to agonists. They shifted dose-response curves of Ang II, norepinephrine, and AVP to sevenfold and higher values. Staurosporine, nifedipine, and TMB-8 had variable effect on agonist responses. They attenuated effects of Ang II and norepinephrine in an additive manner, and each of them increased effective dose values about fourfold. TMB-8 did not attenuate response to AVP and U-46619. Staurosporine and nifedipine diminished effects of AVP in a nonadditive manner, and attenuated additively the fast component of U-46619 response. CONCLUSION: In contrast to other cell signaling pathways, ROK plays a common role for all vasoconstrictor agonistsis in renal circulation.

Rho-kinase inhibition blunts renal vasoconstriction induced by distinct signaling pathways in vivo.

In addition to intracellular calcium, which activates myosin light chain (MLC) kinase, MLC phosphorylation and hence contraction is importantly regulated by MLC phosphatase (MLCP). Recent evidence suggests that distinct signaling cascades of vasoactive hormones interact with the Rho/Rho kinase (ROK) pathway, affecting the activity of MLCP. The present study measured the impact of ROK inhibition on vascular F-actin distribution and on vasoconstriction induced by activation/inhibition of distinct signaling pathways in vivo in the microcirculation of the split hydronephrotic rat kidney. Local application of the ROK inhibitors Y-27632 or HA-1077 induced marked dilation of pre- and postglomerular vessels. Activation of phospholipase C with the endothelin ET B agonist IRL 1620, inhibition of soluble guanylyl cyclase with 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), or inhibition of adenylyl cyclase with the adenosine A1 agonist N6-cyclopentyladenosine (CPA) reduced glomerular blood flow (GBF) by about 50% through vasoconstriction at different vascular levels. ROK inhibition with Y-27632 or HA-1077, but not protein kinase C inhibition with Ro 31-8220, blunted ET B-induced vasoconstriction. Furthermore, the reduction of GBF and of vascular diameters in response to ODQ or CPA were abolished by pretreatment with Y-27632. ROK inhibitors prevented constriction of preglomerular vessels and of efferent arterioles with equal effectiveness. Confocal microscopy demonstrated that Y-27632 did not change F-actin content and distribution in renal vessels. The results suggest that ROK inhibition might be considered as a potent treatment of renal vasoconstriction, because it interferes with constriction induced by distinct signaling pathways in renal vessels without affecting F-actin structure.