R. Ariel Gomez

The MicroRNA-Processing Enzyme Dicer Maintains Juxtaglomerular Cells

Juxtaglomerular cells are highly specialized myoepithelioid granulated cells located in the glomerular afferent arterioles. These cells synthesize and release renin, which distinguishes them from other cells. How these cells maintain their identity, restricted localization, and fate is unknown and is fundamental to the control of BP and homeostasis of fluid and electrolytes. Because microRNAs may control cell fate via temporal and spatial gene regulation, we generated mice with a conditional deletion of Dicer, the RNase III endonuclease that produces mature microRNAs in cells of the renin lineage. Deletion of Dicer severely reduced the number of juxtaglomerular cells, decreased expression of the renin genes (Ren1 and Ren2), lowered plasma renin concentration, and decreased BP. As a consequence of the disappearance of renin-producing cells, the kidneys developed striking vascular abnormalities and prominent striped fibrosis. We conclude that microRNAs maintain the renin-producing juxtaglomerular cells and the morphologic integrity and function of the kidney.

Ren1c Homozygous Null Mice Are Hypotensive and Polyuric, but Heterozygotes Are Indistinguishable from Wild-Type

Mice lacking Ren1c were generated using C57BL/6-derived embryonic stem cells. Mice homozygous for Ren1c disruption (Ren1c-/-) are born at the expected ratio, but approximately 80% die of dehydration within a few days. The surviving Ren1c-/- mice have no renin mRNA expression in the kidney, hydronephrosis, thickening of renal arterial walls, and fibrosis in the kidney. Plasma renin and angiotensins I and II are undetectable. Urinary aldosterone is 6% wild-type. They have low tail-cuff BP (84 +/- 4 versus 116 +/- 5 mmHg in +/+) and excrete large amounts of urine (5.2 +/- 0.8 ml/d, 725 +/- 34 mOsm versus 1.1 +/- 0.1 ml/d, 2460 +/- 170 mOsm in +/+). After 5 d of drinking 5% dextrose, desmopressin does not increase the osmolality of the urine in -/- mice (624 +/- 19 to 656 +/- 25 mOsm), whereas in +/+, it increases severalfold (583 +/- 44 to 2630 +/- 174 mOsm). Minipump infusion of angiotensin II to Ren1c-/- mice restores BP to wild-type level, but preexisting damage to the medulla prevents complete restoration of the ability of the kidney to concentrate urine. Heterozygous Ren1c+/- mice, in contrast, are indistinguishable from +/+ in BP, urine volume, and osmolality. Kidney renin mRNA, the number of kidney cells producing renin, and plasma renin concentration in the Ren1c+/- mice are also indistinguishable from +/+. These results demonstrate that renin is the only enzyme capable of maintaining plasma angiotensins and that renin expression in the kidney is very tightly regulated at the mRNA level.

Loss of the VEGF164 and VEGF188 Isoforms Impairs Postnatal Glomerular Angiogenesis and Renal Arteriogenesis in Mice

Vascular endothelial growth factor (VEGF) is transcribed in the VEGF(120), VEGF(164), or VEGF(188) isoforms, which differ in receptor binding, matrix association, and angiogenic activity. This vascular growth factor has been implicated in the development of the renal vasculature, but the role of the distinct VEGF isoforms remains unknown. In the present report, renal angiogenesis and arteriogenesis were studied in VEGF(120/120) mice, expressing only the short VEGF(120) isoform. In VEGF(120/120) mice, ingrowth and survival of capillaries in glomeruli, remodeling of peritubular capillaries, vascular coverage by pericytes, and branching of renal arteries were all severely impaired, causing abnormal glomerular filtration and impairing renal function. The arterial branching defect might be related to a reduced expression of renin, a presumed renal arterial branching factor. Glomerulosclerosis and tubular dilation possibly resulted from renal ischemia caused by vascular defects. Thus, VEGF(164) and VEGF(188) not only mediate angiogenesis, but they also play an essential role in renal branching arteriogenesis.

Developmental consequences of the renin-angiotensin system

Molecular, cellular, and physiological studies indicate that the renin-angiotensin system (RAS) is highly expressed during early kidney development. We propose that a major function of the RAS during early embryonic development is the modulation of growth processes that lead the primitive kidney into a properly differentiated and architecturally organized organ suited for independent extrauterine life. As development progresses, the RAS acquires new and overlapping functions such as the endocrine and paracrine regulation of blood pressure and renal hemodynamics. Disease states in adult mammals often result in expression of RAS genes and phenotypic changes resembling the embryonic pattern, emphasizing the importance of undertaking developmental studies. Because of their importance in health and disease, the immediate challenge is to identify the mechanisms that regulate the unique development of the RAS and its role(s) in normal and abnormal growth processes.

Aberrant Renal Vascular Morphology and Renin Expression in Mutant Mice Lacking Angiotensin-Converting Enzyme

To determine whether angiotensin-converting enzyme plays a role in the development and maintenance of normal renal architecture, the renal morphology of 10- to 12-month-old female mice homozygous for a disruption of the converting enzyme gene was compared with that of age-matched wild-type mice. Tubular obstruction, dilatation, and atrophy were present in all kidneys from the homozygous mutant mice but absent in wild types; two kidneys from 4 mutant mice but none from the wild types were hydronephrotic. The entire arterial vascular tree, microdissected from mice with no converting enzyme, was grossly distorted in comparison to the vasculature of wild-type mice; all intrarenal arterial vessels were widened and thickened, including the terminal (afferent) arterioles. In wild-type mice kidneys, renin-positive cells were detected exclusively in a juxtaglomerular localization. In contrast, abnormal distribution of renin immunostaining was observed in mice without converting enzyme; scattered renin-positive cells were seen along the arterial vessels, often in a perivascular localization, and interstitial renin-positive cells surrounded glomeruli. Kidney renin mRNA was increased more than 32-fold in the mutant mice compared with wild types. Northern blot analysis revealed that this increase included the accumulation of large amounts of smaller renin RNA transcripts. In summary, mice lacking the converting enzyme exhibit abnormal renal vessels and tubules. Renin synthesis is increased, accompanied by the presence of small renin mRNA species, and renin is present mainly in interstitial and perivascular cells. We conclude that angiotensin-converting enzyme is necessary to preserve normal kidney architecture and the normal pattern of renin expression.

Development of the Renal Arterioles

The kidney is a highly vascularized organ that normally receives a fifth of the cardiac output. The unique spatial arrangement of the kidney vasculature with each nephron is crucial for the regulation of renal blood flow, GFR, urine concentration, and other specialized kidney functions. Thus, the proper and timely assembly of kidney vessels with their respective nephrons is a crucial morphogenetic event leading to the formation of a functioning kidney necessary for independent extrauterine life. Mechanisms that govern the development of the kidney vasculature are poorly understood. In this review, we discuss the anatomical development, embryological origin, lineage relationships, and key regulators of the kidney arterioles and postglomerular circulation. Because renal disease is associated with deterioration of the kidney microvasculature and/or the reenactment of embryonic pathways, understanding the morphogenetic events and processes that maintain the renal vasculature may open new avenues for the preservation of renal structure and function and prevent the progression of renal disease.

Selective deletion of Connexin 40 in renin-producing cells impairs renal baroreceptor function and is associated with arterial hypertension

Renin-producing juxtaglomerular cells are connected to each other and to endothelial cells of afferent arterioles by gap junctions containing Connexin 40 (Cx40), abundantly expressed by these two cell types. Here, we generated mice with cell-specific deletion of Cx40 in endothelial and in renin-producing cells, as its global deletion caused local dissociation of renin-producing cells from endothelial cells, renin hypersecretion, and hypertension. In mice lacking endothelial Cx40, the blood pressure, renin-producing cell distribution, and the control of renin secretion were similar to wild-type mice. In contrast, mice deficient for Cx40 in renin-producing cells were hypertensive and these cells were ectopically localized. Although plasma renin activity and kidney renin mRNA levels of these mice were not different from controls, the negative regulation of renin secretion by pressure was inverted to a positive feedback in kidneys lacking Cx40 in renin-producing cells. Thus, our findings show that endothelial Cx40 is not essential for the control of renin expression and/or release. Cx40 in renin-producing cells is required for their correct positioning in the juxtaglomerular area and the control of renin secretion by pressure.

Homeostasis in mice with genetically decreased angiotensinogen is primarily by an increased number of renin-producing cells.

Here we investigate the biochemical, molecular, and cellular changes directed toward blood pressure homeostasis that occur in the endocrine branch of the renin-angiotensin system of mice having one angiotensinogen gene inactivated. No compensatory up-regulation of the remaining normal allele occurs in the liver, the main tissue of angiotensinogen synthesis. No significant changes occur in expression of the genes coding for the angiotensin converting enzyme or the major pressor-mediating receptor for angiotensin, but plasma renin concentration in the mice having only one copy of the angiotensinogen gene is greater than twice wild-type. This increase is mediated primarily by a modest increase in the proportion of renal glomeruli producing renin in their juxtaglomerular apparatus and by four times wild-type numbers of renin-producing cells along afferent arterioles of the glomeruli rather than by up-regulating renin production in cells already committed to its synthesis.

CBP and p300 are essential for renin cell identity and morphological integrity of the kidney

The mechanisms that govern the identity of renin cells are not well understood. We and others have identified cAMP as an important pathway in the regulation of renin synthesis and release. Recently, experiments in cells from the renin lineage led us to propose that acquisition and maintenance of renin cell identity are mediated by cAMP and histone acetylation at the cAMP responsive element (CRE) of the renin gene. Ultimately, the transcriptional effects of cAMP depend on binding of the appropriate transcription factors to CRE. It has been suggested that access of transcription factors to this region of the promoter is facilitated by the coactivators CREB-binding protein (CBP) and p300, which possess histone acetyltransferase activity and may be, in turn, responsible for the remodeling of chromatin underlying expression of the renin gene. We hypothesized that CBP and p300 are therefore required for expression of the renin gene and maintenance of the renin cell. Because mice homozygous for the deletion of CBP or p300 die before kidney organogenesis begins, no data on kidney or juxtaglomerular cell development in these mice are available. Therefore, to define the role of these histone acetyltransferases in renin cell identity in vivo, we used a conditional deletion approach, in which floxed CBP and p300 mice were crossed with mice expressing cre recombinase in renin cells. Results show that the histone acetyltransferases CBP and p300 are necessary for maintenance of renin cell identity and structural integrity of the kidney.

Molecular and cellular aspects of renin during kidney ontogeny

The activity of the renin-angiotensin system is subjected to remarkable developmental changes. Circulating as well as renal concentrations of renin are high in early life, decreasing progressively as maturation evolves. This review summarizes the current molecular framework underlying those changes during kidney development. Evidence is presented demonstrating that expression of the renin gene is developmentally regulated. Renin messenger ribonucleic acid (mRNA) levels are higher in fetuses and newborns than in adult mammals. As maturation progresses, the distribution of renin and its mRNA shifts from large intrarenal arteries in the fetus to the classic juxtaglomerular localization in the adult. Potential explanations for these changes as well as the cytosolic events mediating renin release and gene expression are discussed. Evidence is also presented demonstrating that under diverse physiologic and pathologic conditions the adult kidney vasculature has the capability to recruit renin gene expressing and/or containing cells. Throughout, an effort has been made to identify gaps in our knowledge. Not without bias, we hope that future research in this area will lead to a better understanding of the biology of renin in the developing as well as the adult individual.