PhD Thesis in PDF format


Contraction mechanisms and endothelial interaction in renal afferent arterioles

Torben R. Uhrenholt


The arterial pressure is regulated by a complex interaction between circulating vasoactive substances, regulated salt excretion and the central nervous system. The kidney plays a central role in this regulation through the renin-angiotensin-aldosterone system. The afferent arteriole is central to renal blood flow and the glomerular filtration. Changes in the afferent arterioles structure and contraction mechanisms seem to be important to the pathophysiology of hypertension. The purpose of this study was to (1) elucidate the signaling pathways involved in the non-genomic effect of aldosterone on the afferent arteriole and (2) investigate the calcium signaling in the afferent arteriole during depolarization-induced contraction Aldosterone was suggested to be a vasoactive substance and elicit vessel contraction via a nongenomic mechanism. We tested this proposal in the perfused rabbit renal afferent arterioles.
Aldosterone had no effect on internal diameter in concentrations from 10-10-10-5 M, but aldosterone abolished the ability of K+ to induce vascular contraction. The inhibitory effect of aldosterone was observed from 1 picomol/L. The inhibitory effect was significant after 5 min, maximal after 20 min and was fully reversible. Actinomycin D prolonged the effect of aldosterone. The effect was abolished by the mineralocorticoid receptor (MR) antagonist spironolactone but not by the glucocorticoid receptor antagonist mifepristone. MR was detected by RT-PCR and immunohistochemistry in rat renal vasculature and rabbit endothelial cells. Inhibition of phosphatidylinositol (PI)-3 kinase with LY 294002 restored sensitivity to K+ in the presence of aldosterone and afferent arterioles were immunopositive for PI-3 kinase subunit p110 α. Inhibition of NO-formation by L-NAME or inhibition of soluble guanylyl cyclase with ODQ restored K+-
induced vasoreactivity in the presence of aldosterone. Geldanamycin, an inhibitor of heat shock protein 90, abolished aldosterone-induced asorelaxation. It was concluded that aldosterone inhibits depolarisation-induced vasoconstriction in renal afferent arterioles by a rapid non-genomic mechanism that is initiated by mineralocorticoid receptor activation and involves PI-3 kinase, PKB and hsp90-mediated stimulation of NO-generation.
To explore if depolarization-induced contraction to some part was counteracted by NO production in perfused afferent arterioles, we first tested the effect of L-NAME on isolated microperfused rabbit afferent arterioles. It was demonstrates that L-NAME treatment improved depolarizationinduced contraction by significantly left-shifting the K+ concentration-response curve. Another important observation was that the high concentration of K+ needed for contraction of the perfused afferent arteriole was transient. 40-45 seconds after applying K+. This increase in luminal diameter  during depolarization was fully reversed by pretreatment with L-NAME. The perfused afferent arteriole preparation (one monolayer of smooth muscle) and the specific florescence fura-2 technique used, allowed us to monitor intracellular calcium, [Ca2+]i in smooth muscle and endothelial cells. Ten-15s after the initial [Ca2+]i rise in the smooth muscle cell we observed a calcium increase in the underlying endothelial cell, which could provide the signal for NO release that feeds back to the SMC and curtails contraction. [Ca2+]i reached a maximal plateau within 40s after addition of K+, which was coincident with onset of relaxation. The calcium wave, observed in endothelial cells close to the contracting smooth muscle cell propagated in both upstream and downstream direction. The velocity of the calcium wave was 1.13 – 1.44 µm/s and did not show any regenerative characteristics. Acetylcholine induced a rapid sustained [Ca2+]i increase in the endothelial cells. In contrast to calcium increase in smooth muscle cells, [Ca2+]i increase in endothelial cells did not cause an corresponding calcium increase in the smooth muscle.
To investigate whether perfusion of the arteriole had any impact on calcium signaling during depolarization induced contraction we measured the [Ca2+]i in the smooth muscle cells during K+ induced contraction in perfused and non-perfused arterioles. The basal level of [Ca2+]i in smooth muscle cells was lower in non-perfused compared to the perfused arterioles. Moreover, the nonperfused arterioles also causes a higher [Ca2+]i peak compared to perfused arterioles. Neither the specific L-type voltage-dependent calcium channel blocker calciseptine nor L-NAME had any effect on the basal level of [Ca2+]i. However, the [Ca2+]i peak during depolarization induced contraction was significantly reduced by calciseptine but not L-NAME. It is concluded that endothelial NO production is stimulated during contraction by a local transfer of calcium signaling from SMC to the EC cell layer, where the calcium signal spreads.