FREQUENCE OF JUXTAGLOMERULAR GRANULATED CELIS IN DEHYDRATED AND SODIUM-LOADED MICE

Fifteen albino mice were subjected to three days dehydration and fifteen albino mice were given 1% NaCl in their drinking water. Control animals were given drinking water All animals were sacrificed and kidneys were fixed in different fixatives and processed for light microscopy Section were stained for juxtaglomerular cells demonstration and the juxtaglomerular index (GI) and percentage of granulated nephron(%GN) were calculated. Kidneys from dehydrated and sodium-loaded animals showed significant decrease in both JGI and % GN and was discussed in relation to renin secretion and release.

Deficiency of MicroRNA-181a Results in Transcriptome-Wide Cell-Specific Changes in the Kidney and Increases Blood Pressure

MicroRNA miR-181a is downregulated in the kidneys of hypertensive patients and hypertensive mice. In vitro, miR-181a is a posttranslational inhibitor of renin expression, but pleiotropic mechanisms by which miR-181a may influence blood pressure (BP) are unknown. Here, we determined whether deletion of miR-181a/b-1 in vivo changes BP and the molecular mechanisms involved at the single-cell level. We developed a KO (knockout) mouse model lacking miR-181a/b-1 genes using CRISPR/Cas9 technology. Radiotelemetry probes were implanted in 12-week-old C57BL/6J WT (wild type) and miR-181a/b-1 KO mice. Systolic and diastolic BP were 4- to 5-mm Hg higher in KO compared with WT mice over 24 hours ( P <0.01). Compared with WT mice, renal renin was higher in the juxtaglomerular cells of KO mice. BP was similar in WT mice on a high- (3.1%) versus low- (0.3%) sodium diet (+0.4±0.8 mm Hg), but KO mice showed salt sensitivity (+3.3±0.8 mm Hg; P <0.001). Since microRNAs can target several mRNAs simultaneously, we performed single-nuclei RNA sequencing in 6699 renal cells. We identified 12 distinct types of renal cells, all of which had genes that were dysregulated. This included genes involved in renal fibrosis and inflammation such as Stat4 , Col4a1 , Cd81 , Flt3l , Cxcl16 , and Smad4 . We observed upregulation of pathways related to the immune system, inflammatory response, reactive oxygen species, and nerve development, consistent with higher tyrosine hydroxylase in the kidney. In conclusion, downregulation of the miR-181a gene led to increased BP and salt sensitivity in mice. This is likely due to an increase in renin expression in juxtaglomerular cells, as well as microRNA-driven pleiotropic effects impacting renal pathways associated with hypertension.

Deficiency of MicroRNA-181a Results in Transcriptome-Wide Cell Specific Changes in the Kidney that Lead to Elevated Blood Pressure and Salt Sensitivity

MicroRNA miR-181a is down-regulated in the kidneys of hypertensive patients and hypertensive mice. In vitro, miR-181a is a posttranslational inhibitor of renin expression, but pleiotropic mechanisms by which miR-181a may influence blood pressure (BP) are unknown. Here we determined whether deletion of miR-181a/b-1 in vivo changes BP and the molecular mechanisms involved at the single-cell level. We developed a knockout mouse model lacking miR-181a/b-1 genes using CRISPR/Cas9 technology. Radio-telemetry probes were implanted in twelve-week-old C57BL/6J wild-type and miR-181a/b-1 knockout mice. Systolic and diastolic BP were 4-5mmHg higher in knockout compared with wild-type mice over 24-hours (P<0.01). Compared with wild-type mice, renal renin was higher in the juxtaglomerular cells of knockout mice. BP was similar in wild-type mice on a high (3.1%) versus low (0.3%) sodium diet (+0.4±0.8mmHg) but knockout mice showed salt sensitivity (+3.3±0.8mmHg, P<0.001). Since microRNAs can target several mRNAs simultaneously, we performed single-nuclei RNA-sequencing in 6,699 renal cells. We identified 12 distinct types of renal cells, all of which had genes that were dysregulated. This included genes involved in renal fibrosis and inflammation such as Stat4, Col4a1, Cd81, Flt3l, Cxcl16, Smad4. We observed up-regulation of pathways related to the immune system, inflammatory response, reactive oxygen species and nerve development, consistent with higher tyrosine hydroxylase. In conclusion, downregulation of the miR-181a gene led to increased BP and salt sensitivity in mice. This is likely due to an increase in renin expression in juxtaglomerular cells, as well as microRNA-driven pleiotropic effects impacting renal pathways associated with hypertension.

Phosphorylation of β1-integrin in juxtaglomerular cells helps control blood pressure during the progression of diabetic nephropathy

The induction of a high blood pressure due to diabetic nephropathy depends on the increase in renin secretion from juxtaglomerular cells, but many aspects of how juxtaglomerular cells sense blood pressure changes in the afferent arteriole and consequently react remain unclear. In this study, we detected the juxtaglomerular cell-specific phosphorylation of the threonine-788/789 site of β1-integrin, and its expression was negatively correlated with renin production. This relationship was also observed in a culture system of a juxtaglomerular cell line, suggesting that β1-integrin is deeply involved in the regulation of renin production. The knockdown of β1-integrin in the culture system increased renin production, but the degree of the increase was comparable to the increase in renin production by knockdown of connexin-40, which is considered to be an important molecule that plays a role in the pressure sensing mechanism of juxtaglomerular cells. This suggests that the mechanism underlying the regulation of renin production by β1-integrin in juxtaglomerular cells may contribute to the pressure-sensing function of juxtaglomerular cells themselves. Threonine-788/789 phosphorylation of β1-integrin may be involved in the regulation of this pressoreceptor function. (176 words)

Renin-Expressing Cells Require β1-Integrin for Survival and for Development and Maintenance of the Renal Vasculature

Juxtaglomerular cells are crucial for blood pressure and fluid-electrolyte homeostasis. The factors that maintain the life of renin cells are unknown. In vivo, renin cells receive constant cell-to-cell, mechanical, and neurohumoral stimulation that maintain their identity and function. Whether the presence of this niche is crucial for the vitality of the juxtaglomerular cells is unknown. Integrins are the largest family of cell adhesion molecules that mediate cell-to-cell and cell-to-matrix interactions. Of those, β1-integrin is the most abundant in juxtaglomerular cells. However, its role in renin cell identity and function has not been ascertained. To test the hypothesis that cell-matrix interactions are fundamental not only to maintain the identity and function of juxtaglomerular cells but also to keep them alive, we deleted β1-integrin in vivo in cells of the renin lineage. In mutant mice, renin cells died by apoptosis, resulting in decreased circulating renin, hypotension, severe renal-vascular abnormalities, and renal failure. Results indicate that cell-to-cell and cell-to-matrix interactions via β1-integrin is essential for juxtaglomerular cells survival, suggesting that the juxtaglomerular niche is crucial not only for the tight regulation of renin release but also for juxtaglomerular cell survival—a sine qua non condition to maintain homeostasis.

Analysis of the calcium paradox of renin secretion

The secretion of the protease renin from renal juxtaglomerular cells is enhanced by subnormal extracellular calcium concentrations. The mechanisms underlying this atypical effect of calcium have not yet been unraveled. We therefore aimed to characterize the effect of extracellular calcium concentration on calcium handling of juxtaglomerular cells and on renin secretion in more detail. For this purpose, we used a combination of experiments with isolated perfused mouse kidneys and direct calcium measurements in renin-secreting cells in situ. We found that lowering of the extracellular calcium concentration led to a sustained elevation of renin secretion. Electron-microscopical analysis of renin-secreting cells exposed to subnormal extracellular calcium concentrations revealed big omega-shaped structures resulting from the intracellular fusion and subsequent emptying of renin storage vesicles. The calcium concentration dependencies as well as the kinetics of changes were rather similar for renin secretion and for renovascular resistance. Since vascular resistance is fundamentally influenced by myosin light chain kinase (MLCK), myosin light chain phosphatase (MLCP), and Rho-associated protein kinase (Rho-K) activities, we examined the effects of MLCK-, MLCP-, and Rho-K inhibitors on renin secretion. Only MLCK inhibition stimulated renin secretion. Conversely, inhibition of MCLP activity lowered perfusate flow and strongly inhibited renin secretion, which could not be reversed by lowering of the extracellular calcium concentration. Renin-secreting cells and smooth muscle cells of afferent arterioles showed immunoreactivity of MLCK. These findings suggest that the inhibitory effect of calcium on renin secretion could be explained by phosphorylation-dependent processes under control of the MLCK.