Chronic Stimulation of Renin Cells Leads to Vascular Pathology

Abstract

Experimental or spontaneous genomic mutations of the renin-angiotensin system or its pharmacological inhibition in early life leads to renal abnormalities, including poorly developed renal medulla, papillary atrophy, hydronephrosis, inability to concentrate the urine, polyuria, polydipsia, renal failure, and anemia. At the core of such complex phenotype is the presence of unique vascular abnormalities: the renal arterioles do not branch or elongate properly and they have disorganized, concentric hypertrophy. This lesion has been puzzling because it is often found in hypertensive individuals whereas mutant or pharmacologically inhibited animals are hypotensive. Remarkably, when renin cells are ablated with diphtheria toxin, the vascular hypertrophy does not occur, suggesting that renin cells per se may contribute to the vascular disease. To test this hypothesis, on a Ren1^c-/- background, we generated mutant mice with reporter expression (Ren1^c-/-;Ren1^c-Cre;R26R.mTmG and Ren1^c-/-;Ren1^c-Cre;R26R.LacZ) to trace the fate of renin^null cells. To assess whether renin^null cells maintain their renin promoter active, we used Ren1^c-/-;Ren1^c-YFP mice that transcribe YFP (yellow fluorescent protein) directed by the renin promoter. We also followed the expression of Akr1b7 and miR-330-5p, markers of cells programmed for the renin phenotype. Contrary to what we expected, renin^null cells did not die or disappear. Instead, they survived, increased in number along the renal arterial tree, and maintained an active molecular memory of the myoepitheliod renin phenotype. Furthermore, null cells of the renin lineage occupied the walls of the arteries and arterioles in a chaotic, directionless pattern directly contributing to the concentric arterial hypertrophy.

Keywords: kidney; mice; mutation; phenotype; renin-angiotensin system.

Figure 1

Distribution of YFP + renin null cells in the kidneys of Ren1^c−/− mice. A, B. Low magnification view of kidneys from Ren1^c+/− (A) and Ren1^c−/− (B) mice immunostained for renin. Whereas renin expression is detected in the cortex of the Ren1^c+/− kidney (A), as expected, no renin is observed in the Ren1^c−/− kidney (B). Scale bars: 750 μm. C-E. YFP expression in kidneys from Ren1^c+/− and Ren1^c−/− mice. In control animals, YFP+ cells are restricted to the juxtaglomerular areas (C). In Ren1^c−/− mice, YFP+ cells are observed throughout afferent arterioles, interlobular and arcuate arteries and glomeruli (D). Scale bar: 100 μm (*), glomeruli. E. Quantification of YFP+ cells in kidneys. The number of YFP+ cells is increased threefold in Ren1^c−/− mice when compared to heterozygous controls (***P<0.0001).

Figure 2

Histology of kidneys from Ren1^c+/− and Ren1^c−/− mouse. A, B. αSMA immunostaining shows that Ren1^c−/− kidneys exhibit granular surface (arrows) correlated with marked interstitial and peri-glomerular fibrosis, tubular dilatation and atrophy. Scale bars: 750 μm. C, D. Higher magnificacion of kidney sections from control and Ren1^c−/− mice immunostained for αSMA. Ren1^c−/− kidneys show thicker intrarenal arterial walls than Ren1^c+/− kidneys and and display interstitial fibrosis. Moreover, Ren1^c−/− kidneys show αSMA expression in glomeruli and tubular expansions. Scale bars: 75 μm. E, F. Quantification of the wall thickness shows that Ren1^c−/− mice have thicker arterioles than Ren1^c+/− controls (E, ***P<0.0001). F. Relative frequency distribution histograms show that the distribution curve corresponding to the Ren1^c−/− animals is displaced to the right indicating that a higher proportion of vessels possess thicker walls than in control mice.

Figure 3

Expression of renin cell markers in Ren1^c−/− mice. A, B. αSMA and AKR1B7 immunostaining of consecutive sections of Ren1^c−/− kidneys. AKR1B7 is expressed even when the cells are unable to synthesize renin (B). AKR1B7 positive cells coincide with cells expressing αSMA in the outer layers of the arteriole (A, B, top). In some vessels, AKR1B7+ cells are also distributed deeper inside the arteriolar wall (A, B, bottom). Few AKR1B7+ cells are observed in areas of peri glomerular fibrosis. As expected in those areas, αSMA expression is prominent (A, B, bottom). Scale bars: 75 μm (top panels); 50 μm (bottom panels). C, D. In situ hybridization staining for miR-330-5p, a renin cell marker expressed under conditions known to induce renin expression along the kidney vasculature. In contrast to Ren1^c+/− controls (C), miR-330-5p is highly expressed in the vessel walls and inside glomeruli in Ren1^c−/− kidneys, consistent with its presence when the renin program is activated (D). Scale bars: 100 μm.

Figure 4

Distribution of cells from the renin lineage in Ren1^c−/− mice. In control Ren1^c+/− kidneys, cells from the renin lineage (GFP+) are found in the afferent arterioles including its juxtaglomerular area (A, top). In Ren1^c−/− mice, renin lineage cells are found surrounding arterioles and around glomeruli (arrows) (B, top). In larger vessels, cells from the renin lineage are found throughout the arterial walls both in Ren1^c+/−control and Ren1^c−/− animals but the intramural arrangement of the cells is markedly different (A, B, bottom). In control animals, cells from the renin lineage arranged themselves in an organized circular pattern (A, bottom) and cells from knock out animals were disorganized without a clear directional pattern (B, bottom, D). Labeled in green (GFP+) are cells from the renin lineage. Labeled in red (RFP+) are non-renin lineage cells. C. A similar disorganized pattern of renin null cells is also observed when examining kidneys using the Lac-Z reporter. Stained in blue are cells from the renin lineage. Scale bars: 75 μm (A, B, top); 100 μm (A, B, bottom; C); 50 μm (D).

Figure 5

Schematic of Renin^null cells contribution to vascular pathology. During normal development, renin precursors and/or their descendants are present along large intra-renal arteries, afferent arterioles and inside glomeruli. In the normal adult mouse, renin cells become confined to the juxtaglomerular area. When the renin gene is knocked out, mice exhibit concentric hypertrophy of the kidney arterioles leading to advanced kidney disease. The cells programmed for the renin phenotype survive and increase in numbers along the renal arterial. Those renin^null cells maintain the molecular program of the renin phenotype, i.e. expression of renin cell markers AKR1B7, miR-330-5p and YFP, and integrate in a disorderly manner inside the vessel wall, thus contributing to the vascular disease. Thus, renin null cells not only persist, their distribution and number along the renal arterial tree is expanded in a pattern that replicates the distribution of renin cells during fetal development and in response to physiological stress in adult animals. RPC: renin progenitor cell; SMC: smooth muscle cell; YFP: yellow fluorescent protein, GFP: green fluorescent protein; EC: endothelial cell; JG cell: juxtaglomerular cell.