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The renin angiotensin system and kidney development: Novel mechanisms of renin release and angiogenic funtion of angiotensin II

Kirsten Madsen


The renin-angiotensin system (RAS) is involved in a variety of functions in the body including blood pressure regulation, sodium and water homeostasis and kidney development.The activity of the system is predominantly controlled by the secretion rate of renin from the renal juxtaglomerular (JG) cells. Renin initiates a sequential proteolytical cascade leading to the production of the biologically active peptide, angiotensin II (ANG II). ANG II mediates its biological effects through binding to two transmembrane receptors, AT1 and AT2.

Pharmacological inhibition or genetic deletion of components of RAS leads to severe, postnatal renal injury. The phenotype is characterized by impaired nephron formation, shortening of the preglomerular arteries, atrophy of the inner medulla and interstitial fibrosis. Similar lesions are seen in human babies exposed to inhibitors of RAS. The mechanisms behind these effects are currently unknown. We tested the hypothesis that ANG II supports capillary angiogenesis during postnatal kidney development. Pharmacological inhibition of AT1 receptors (candesartan 1 mg/kg*day) from postnatal day (P) 1 to P13 led to significantly impaired development of postglomerular microvessels. Unbiased quantitative stereological analysis revealed diminished total capillary length, volume and surface area in both cortex
and medulla. In addition, developmental organization of vasa recta bundles was impaired by AT1 inhibition. Renal expression of Vascular Endothelial Growth Factor (VEGF), angiopoietin-1 and -2 was significantly impaired, and there was a significantly lower level of the mitotic marker proliferating-cell-nuclear-antigen (PCNA) in microdissected vasa recta bundles. The outer medullary interstitial space was expanded by mesenchymal-like cells after AT1 inhibition. After 14 days without candesartan treatment (P30), total renal blood flow was reduced significantly (~20%) in the AT1 inhibited group whereas glomerular filtration rate was unchanged as assessed by magnetic resonance imaging. We conclude that ANG II promotes postnatal expansion of postglomerular capillaries and that this is necessary for development of
normal renal blood flow.

Renin is produced, stored and released by the JG-cells in the distal part of the afferent arterioles. Intracellular signaling transduction pathways controlling renin release is largely unknown. In study II, we tested the hypothesis that the calcium/calmodulin-dependent phosphatase calcineurin (PP2B) regulates renin release directly at the level of JG-cells. Catalytic calcineurin A-β and A-γ but not A-α subunits were expressed in microdissected afferent arterioles and in single collected JG-cells. In rat JG-cells, application of cyclosporine A (CsA) increased membrane capacitance (Cm), an index of cell surface area. This effect on Cm was mimicked by chelating intracellular calcium with EGTA, and when cells were dialyzed with a specific calcineurin inhibitory peptide. The effect of CsA on Cm was independent of protein kinase A. The calcineurin inhibitor tacrolimus did not alter Cm significantly in single JG-cells despite expression of binding proteins for both calcineurin inhibitors. Cyclosporine and a calmodulin antagonist (calmidazolium) but not tacrolimus stimulated renin release concentration-dependently from cultures of JG-cells. Plasma renin concentration, intrarenal renin content and renin mRNA abundance were not different in CnA-α-/- mice compared to wild-type mice. We conclude that calcineurin exerts a tonic suppressor effect on renin release from JG-cells independent of cAMP, and that clinically used calcineurin inhibitors display a differential ability to stimulate renin. In study III, we tested the hypothesis that osmotically-induced renin release depends on water movement through aquaporin-1 (AQP-1) water channels and subsequent prostanoid formation. Slight hypotonic challenges (3.6% and 5.5%) increased Cm significantly and enhanced outward whole-cell current. The Cm-response to hypotonicity was impaired by indomethacin and a prostaglandin transport blocker, bromcresol green. In addition, the response was absent in JG-cells from cyclooxygenase-2 (COX-2)-/- and AQP1 -/- mice. JGcells sampled with patch pipettes expressed COX-2, AQP-1 and prostaglandin transporters PGT and OATP-D mRNA. A reduction in osmolality (5, 10 and 20%) enhanced cAMP accumulation in JG-cells but not in renin-producing As4.1 cells. Forskolin enhanced cAMP in both cell types. AQP-1 expression was not detectable in As4.1 cells. The protein kinase A inhibitor, RpcAMPs blocked the ability of a hypotonic challenge to increase Cm and whole cell current in JG-cells. The data suggest a novel autocrine signaling pathway whereby a modest decrease in extracellular tonicity leads to AQP-1-mediated water influx and subsequent COX-2-mediated prostaglandin efflux, receptor-dependent cAMP formation and activation of PKA which promotes exocytosis of renin.