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An increase of glomerular filtration rate (GFR) is a common observation in early diabetes and is considered a key risk factor for subsequent kidney injury. However, the mechanisms underlying diabetic hyperfiltration have not been fully clarified. Here, we tested the hypothesis that macula densa neuronal nitric oxide synthase (NOS1) is upregulated via sodium glucose cotransporter type 1 (SGLT1) in diabetes, which then inhibits tubuloglomerular feedback (TGF) promoting glomerular hyperfiltration. Therefore, we examined changes in cortical NOS1 expression and phosphorylation, nitric oxide production in the macula densa, TGF response, and GFR during the early stage of insulin-deficient (Akita) diabetes in wild-type and macula densa-specific NOS1 knockout mice. A set of sophisticated techniques including microperfusion of juxtaglomerular apparatus in vitro, micropuncture of kidney tubules in vivo, and clearance kinetics of plasma fluorescent-sinistrin were employed. Complementary studies tested the role of SGLT1 in SGLT1 knockout mice and explored NOS1 expression and phosphorylation in kidney biopsies of cadaveric donors. Diabetic mice had upregulated macula densa NOS1, inhibited TGF and elevated GFR. Macula densa-selective NOS1 knockout attenuated the diabetes-induced TGF inhibition and GFR elevation. Additionally, deletion of SGLT1 prevented the upregulation of macula densa NOS1 and attenuated inhibition of TGF in diabetic mice. Furthermore, the expression and phosphorylation levels of NOS1 were increased in cadaveric kidneys of diabetics and positively correlated with blood glucose as well as estimated GFR in the donors. Thus, our findings demonstrate that the macula densa SGLT1-NOS1-TGF pathway plays a crucial role in the control of GFR in diabetes.

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.

Macula densa (MD) cells, a chief sensory cell type in the nephron, are endowed with unique microanatomic features including a high density of protein synthetic organelles and secretory vesicles in basal cell processes (“maculapodia”) that suggest a so far unknown high rate of MD protein synthesis. This study aimed to explore the rate and regulation of MD protein synthesis and their effects on glomerular function using novel transgenic mouse models, newly established fluorescence cell biology techniques, and intravital microscopy. Sox2-tdTomato kidney tissue sections and an O-propargyl puromycin incorporation-based fluorescence imaging assay showed that MD cells have the highest level of protein synthesis within the kidney cortex followed by intercalated cells and podocytes. Genetic gain of function of mammalian target of rapamycin (mTOR) signaling specifically in MD cells (in MD-mTOR(gof) mice) or their physiological activation by low-salt diet resulted in further significant increases in the synthesis of MD proteins. Specifically, these included both classic and recently identified MD-specific proteins such as cyclooxygenase 2, microsomal prostaglandin E(2) synthase 1, and pappalysin 2. Intravital imaging of the kidney using multiphoton microscopy showed significant increases in afferent and efferent arteriole and glomerular capillary diameters and blood flow in MD-mTOR(gof) mice coupled with an elevated glomerular filtration rate. The presently identified high rate of MD protein synthesis that is regulated by mTOR signaling is a novel component of the physiological activation and glomerular hemodynamic regulatory functions of MD cells that remains to be fully characterized. NEW & NOTEWORTHY This study discovered the high rate of protein synthesis in macula densa (MD) cells by applying direct imaging techniques with single cell resolution. Physiological activation and mammalian target of rapamycin signaling played important regulatory roles in this process. This new feature is a novel component of the tubuloglomerular cross talk and glomerular hemodynamic regulatory functions of MD cells. Future work is needed to elucidate the nature and (patho)physiological role of the specific proteins synthesized by MD cells.

Background: Regulation of renal hemodynamics and blood pressure (BP) via tubuloglomerular feedback (TGF) may be an important adaptive mechanism during pregnancy. Because the β-splice variant of nitric oxide synthase 1 (NOS1β) in the macula densa is a primary modulator of TGF, we evaluated its role in normal pregnancy and gestational hypertension in a mouse model. We hypothesized that pregnancy upregulates NOS1β in the macula densa, thus blunting TGF, allowing glomerular filtration rate (GFR) to increase and BP to decrease. Methods: We employed sophisticated techniques, including microperfusion of juxtaglomerular apparatus in vitro, micropuncture of renal tubules in vivo, clearance kinetics of plasma FITC-sinistrin, and radio-telemetry BP monitoring, to determine the effects of normal pregnancy or reduced uterine perfusion pressure (RUPP) on macula densa NOS1β/NO levels, TGF responsiveness, GFR, and BP in wild-type and macula densa-specific NOS1 knockout (MD-NOS1KO) mice. Results: Macula densa NOS1β was upregulated during pregnancy, resulting in blunted TGF, increased GFR, and decreased BP. These pregnancy-induced changes in TGF and GFR were largely diminished, with a significant rise in BP, in MD-NOS1KO mice. In addition, RUPP resulted in a downregulation in macula densa NOS1β, enhanced TGF, decreased GFR, and hypertension. The superimposition of RUPP into MD-NOS1KO mice only caused a modest further alteration in TGF and its associated changes in GFR and BP. Finally, in African green monkeys, renal cortical NOS1β expression increased in normotensive pregnancies but decreased in spontaneous gestational hypertensive pregnancies. Conclusions: Macula densa NOS1β plays a critical role in control of renal hemodynamics and BP during pregnancy.

Olfactory receptor 78 (Olfr78) is a G protein‐coupled receptor (GPCR) that is expressed in the juxtaglomerular apparatus (JGA) of the kidney as well as the peripheral vasculature, and is activated by gut microbial metabolites. We previously reported that Olfr78 plays a role in renin secretion in isolated glomeruli, and that Olfr78 knockout (KO) mice have lower plasma renin activity. We also noted that in anesthetized mice, Olfr78KO appeared to be hypotensive. In this study, we used radiotelemetry to determine the role of Olfr78 in chronic blood pressure regulation. We found that the blood pressure of Olfr78KO mice is not significantly different than that of their WT counterparts at baseline, or on high‐ or low‐salt diets. However, Olfr78KO mice have depressed heart rates on high‐salt diets. We also report that Olfr78KO mice have lower renin protein levels associated with glomeruli. Finally, we developed a mouse where Olfr78 was selectively knocked out in the JGA, which phenocopied the lower renin association findings. In sum, these experiments suggest that Olfr78 modulates renin, but does not play an active role in blood pressure regulation at baseline, and is more likely activated by high levels of short chain fatty acids or hypotensive events. This study provides important context to our knowledge of Olfr78 in BP regulation., Olfactory receptor 78 (Olfr78) is a GPCR expressed in the kidney and peripheral vasculature which is activated by gut microbial metabolites. Using radiotelemetry, we determined that Olfr78KO mice do not significantly differ from their WT counterparts with regards to baseline blood pressure. However, we report that Olfr78KO mice have lower renin protein levels associated with glomeruli by immunofluorescence. These experiments suggest that Olfr78 status modulates renin, but does not play an active role in blood pressure regulation at baseline.

Juxtaglomerular (JG) cells, major sources of renin, differentiate from metanephric mesenchymal cells that give rise to JG cells or a subset of smooth muscle cells of the renal afferent arteriole. During periods of dehydration and salt deprivation, renal mesenchymal stromal cells (MSCs) differentiate from JG cells. JG cells undergo expansion and smooth muscle cells redifferentiate to express renin along the afferent arteriole. Gene expression profiling comparing resident renal MSCs with JG cells indicates that the transcription factor Sox6 is highly expressed in JG cells in the adult kidney. In vitro, loss of Sox6 expression reduces differentiation of renal MSCs to renin-producing cells. In vivo, Sox6 expression is upregulated after a low-Na+ diet and furosemide. Importantly, knockout of Sox6 in Ren1d+ cells halts the increase in renin-expressing cells normally seen during a low-Na+ diet and furosemide as well as the typical increase in renin. Furthermore, Sox6 ablation in renin-expressing cells halts the recruitment of smooth muscle cells along the afferent arteriole, which normally express renin under these conditions. These results support a previously undefined role for Sox6 in renin expression.

Renin cells are essential for survival perfected throughout evolution to ensure normal development and defend the organism against a variety of homeostatic threats. During embryonic and early postnatal life, they are progenitors that participate in the morphogenesis of the renal arterial tree. In adult life, they are capable of regenerating injured glomeruli, control blood pressure, fluid-electrolyte balance, tissue perfusion, and in turn, the delivery of oxygen and nutrients to cells. Throughout life, renin cell descendants retain the plasticity or memory to regain the renin phenotype when homeostasis is threatened. To perform all of these functions and maintain well-being, renin cells must regulate their identity and fate. Here, we review the major mechanisms that control the differentiation and fate of renin cells, the chromatin events that control the memory of the renin phenotype, and the major pathways that determine their plasticity. We also examine how chronic stimulation of renin cells alters their fate leading to the development of a severe and concentric hypertrophy of the intrarenal arteries and arterioles. Lastly, we provide examples of additional changes in renin cell fate that contribute to equally severe kidney disorders.

Although macula densa (MD) cells are chief regulatory cells in the nephron with unique microanatomical features, they have been difficult to study in full detail due to their inaccessibility and limitations in earlier microscopy techniques. The present study used a new mouse model with a comprehensive imaging approach to visualize so far unexplored microanatomical features of MD cells, their regulation, and functional relevance. MD-GFP mice with conditional and partial induction of green fluorescent protein (GFP) expression, which specifically and intensely illuminated only single MD cells, were used with fluorescence microscopy of fixed tissue and live MD cells in vitro and in vivo with complementary electron microscopy of the rat, rabbit, and human kidney. An elaborate network of major and minor cell processes, here named maculapodia, were found at the cell base, projecting toward other MD cells and the glomerular vascular pole. The extent of maculapodia showed upregulation by low dietary salt intake and the female sex. Time-lapse imaging of maculapodia revealed highly dynamic features including rapid outgrowth and an extensive vesicular transport system. Electron microscopy of rat, rabbit, and human kidneys and three-dimensional volume reconstruction in optically cleared whole-mount MD-GFP mouse kidneys further confirmed the presence and projections of maculapodia into the extraglomerular mesangium and afferent and efferent arterioles. The newly identified dynamic and secretory features of MD cells suggest the presence of novel functional and molecular pathways of cell-to-cell communication in the juxtaglomerular apparatus between MD cells and between MD and other target cells. NEW & NOTEWORTHY This study illuminated a physiologically regulated dense network of basal cell major and minor processes (maculapodia) in macula densa (MD) cells. The newly identified dynamic and secretory features of these microanatomical structures suggest the presence of novel functional and molecular pathways of cell-to-cell communication in the juxtaglomerular apparatus between MD and other target cells. Detailed characterization of the function and molecular details of MD cell intercellular communications and their role in physiology and disease warrant further studies.

Connexin hemichannels play an important role in the control of cellular signaling and behaviors. Given that lowering extracellular Ca2+, a condition that activates hemichannels, is a well-characterized stimulator of renin in juxtaglomerular cells, we, therefore, tested a potential implication of hemichannels in the regulation of renin in As4.1 renin-secreting cells. Lowering extracellular Ca2+ induced hemichannel opening, which was associated with cAMP signaling pathway activation and increased renin production. Blockade of hemichannels with inhibitors or downregulation of Cxs with siRNAs abrogated the activation of cAMP pathway and the elevation of renin. Further analysis revealed that cAMP pathway activation was blocked by adenylyl cyclase inhibitor SQ 22536, suggesting an implication of adenyl cyclase. Furthermore, the participation of hemichannels in the activation of the cAMP signaling pathway was also observed in a renal tubular epithelial cell line NRK. Collectively, our results characterized the hemichannel opening as a presently unrecognized molecular event involved in low Ca2+-elicited activation of cAMP pathway and renin production. Our findings thus provide novel mechanistic insights into the low Ca2+-initiated cell responses. Given the importance of cAMP signaling pathway in the control of multiple cellular functions, our findings also highlight the importance of Cx-forming channels in various pathophysiological situations.

Supplemental Digital Content is available in the text., We investigated the significance of the primary cilia on the macula densa and thick ascending limb (TAL) in regulation of renal hemodynamics, sodium excretion, and blood pressure in this study. A tissue-specific primary cilia knock-out (KO) mouse line was generated by crossing NKCC2-Cre mice with IFT88-Δ/flox mice (NKCC2CRE; IFT88Δ/flox), in which the primary cilia were deleted from the macula densa and TAL. NO generation was measured with a fluorescent dye (4,5-diaminofluorescein diacetate) in isolated perfused juxtaglomerular apparatus. Deletion of the cilia reduced NO production by 56% and 42% in the macula densa and TAL, respectively. NO generation by the macula densa was inhibited by both a nonselective and a selective nitric oxide synthesis inhibitors, whereas TAL-produced NO was inhibited by a nonselective and not by a selective NO synthesis 1 inhibitor. The tubuloglomerular feedback response was enhanced in the KO mice both in vitro measured with isolated perfused juxtaglomerular apparatuses and in vivo measured with micropuncture. In response to an acute volume expansion, the KO mice exhibited limited glomerular filtration rate elevation and impaired sodium excretion compared with the wild-type mice. The mean arterial pressure measured with telemetry was the same for wild-type and KO mice fed a normal salt diet. After a high salt diet, the mean arterial pressure increased by 17.4±1.6 mm Hg in the KO mice. On the basis of these findings, we concluded that the primary cilia on the macula densa and TAL play an essential role in the control of sodium excretion and blood pressure.

Impaired renal autoregulation permits more transmission of disturbance in systemic blood pressure, which initiates barotrauma in intrarenal microvasculatures such as glomerular and tubulointerstitial capillaries, contributing to the development of kidney damage and deterioration in renal function, especially under the conditions with high blood pressure. Although it has been postulated that autoregulatory efficiency is attenuated in the aging kidney, direct evidence remains lacking. In the present study, we measured the autoregulation of renal blood flow, myogenic response of afferent arteriole (Af-Art), tubuloglomerular feedback in vivo with micropuncture, as well as tubuloglomerular feedback in vitro in isolated perfused juxtaglomerular apparatus in young and aged C57BL/6 mice. We found that renal blood flow was not significantly changed in response to a defined elevation of renal arterial pressure in young mice but significantly increased in aged mice. Additionally, myogenic response of Af-Art measured by microperfusion with a stepwise increase in perfusion pressure was significantly blunted in the aging kidney, which is associated with the attenuation of intraluminal pressure-induced intracellular calcium increases, as well as the reduced expression of integrin α5 (Itga5) in Af-Art. Moreover, both tubuloglomerular feedback in vivo and in vitro were nearly inactive in the aging kidney, which is associated with the significantly reduced expression of adenosine A1 receptor (A1AR) and suppressed vasoconstrictor response to adenosine in Af-Art. In conclusion, this study demonstrates that aging impairs renal autoregulation with blunted myogenic response and inhibited tubuloglomerular feedback response. The underlying mechanisms involve the downregulations of integrin α5 and A1AR in the Af-Art.

Kidney function and blood pressure homeostasis are regulated by purinergic signaling mechanisms. These autocrine/paracrine signaling pathways are initiated by the release of cellular ATP, which influences kidney hemodynamics and steady-state renin secretion from juxtaglomerular cells. However, the mechanism responsible for ATP release that supports tonic inputs to juxtaglomerular cells and regulates renin secretion remains unclear. Pannexin 1 (Panx1) channels localize to both afferent arterioles and juxtaglomerular cells, and provide a transmembrane conduit for ATP release and ion permeability in the kidney and the vasculature. We hypothesized that Panx1 channels in renin-expressing cells regulate renin secretion in vivo. Using a renin cell-specific Panx1 knockout model, we found that male Panx1 deficient mice exhibiting a heightened activation of the renin-angiotensin-aldosterone system have markedly increased plasma renin and aldosterone concentrations, and elevated mean arterial pressure with altered peripheral hemodynamics. Following ovariectomy, female mice mirrored the male phenotype. Furthermore, constitutive Panx1 channel activity was observed in As4.1 renin-secreting cells, whereby Panx1 knockdown reduced extracellular ATP accumulation, lowered basal intracellular calcium concentrations and recapitulated a hyper-secretory renin phenotype. Moreover, in response to stress stimuli that lower blood pressure, Panx1-deficient mice exhibited aberrant “renin recruitment” as evidenced by reactivation of renin expression in pre-glomerular arteriolar smooth muscle cells. Thus, renin-cell Panx1 channels suppress renin secretion and influence adaptive renin responses when blood pressure homeostasis is threatened.

Synthesis of renin in renal renin-producing cells (RPCs) is controlled via the intracellular messenger cAMP. Interference with cAMP-mediated signaling by inducible knockout of Gs-alpha (Gsα) in RPCs of adult mice resulted in a complex adverse kidney phenotype. Therein, glomerular endothelial damage was most striking. In this study, we investigated whether Gsα knockout leads to a loss of RPCs, which itself may contribute to the endothelial injury. We compared the kidney phenotype of three RPC-specific conditional mouse lines during continuous induction of recombination. Mice expressing red fluorescent reporter protein tdTomato (tdT) in RPCs served as controls. tdT was also expressed in RPCs of the other two strains used, namely with RPC-specific Gsα knockout (Gsα mice) or with RPC-specific diphtheria toxin A expression (DTA mice, in which the RPCs should be diminished). Using immunohistological analysis, we found that RPCs decreased by 82% in the kidneys of Gsα mice as compared with controls. However, the number of tdT-positive cells was similar in the two strains, demonstrating that after Gsα knockout, the RPCs persist as renin-negative descendants. In contrast, both renin-positive and tdT-labeled cells decreased by 80% in DTA mice suggesting effective RPC ablation. Only Gsα mice displayed dysregulated endothelial cell marker expression indicating glomerular endothelial damage. In addition, a robust induction of genes involved in tissue remodelling with microvascular damage was identified in tdT-labeled RPCs isolated from Gsα mice. We concluded that Gsα/renin double-negative RPC progeny essentially contributes for the development of glomerular endothelial damage in our Gsα-deficient mice.

The antidiuretic hormone vasopressin (AVP) regulates renal salt and water reabsorption along the distal nephron and collecting duct system. These effects are mediated by vasopressin 2 receptors (V2R) and release of intracellular Gs-mediated cAMP to activate epithelial transport proteins. Inactivating mutations in the V2R gene lead to the X-linked form of nephrogenic diabetes insipidus (NDI), which has chiefly been related with impaired aquaporin 2-mediated water reabsorption in the collecting ducts. Previous work also suggested the AVP-V2R-mediated activation of Na+-K+-2Cl−-cotransporters (NKCC2) along the thick ascending limb (TAL) in the context of urine concentration, but its individual contribution to NDI or, more generally, to overall renal function was unclear. We hypothesized that V2R-mediated effects in TAL essentially determine its reabsorptive function. To test this, we reevaluated V2R expression. Basolateral membranes of medullary and cortical TAL were clearly stained, whereas cells of the macula densa were unreactive. A dominant-negative, NDI-causing truncated V2R mutant (Ni3-Glu242stop) was then introduced into the rat genome under control of the Tamm-Horsfall protein promoter to cause a tissue-specific AVP-signaling defect exclusively in TAL. Resulting Ni3-V2R transgenic rats revealed decreased basolateral but increased intracellular V2R signal in TAL epithelia, suggesting impaired trafficking of the receptor. Rats displayed significant baseline polyuria, failure to concentrate the urine in response to water deprivation, and hypercalciuria. NKCC2 abundance, phosphorylation, and surface expression were markedly decreased. In summary, these data indicate that suppression of AVP-V2R signaling in TAL causes major impairment in renal fluid and electrolyte handling. Our results may have clinical implications.

The intrarenal renin-angiotensin system (RAS) has an important role in generating and maintaining hypertension in two-kidney, one-clip (2K1C) rats. This study evaluated how various intrarenal RAS components contributed to hypertension not only in the maintenance period (5w; 5 wk after operation) but also earlier (2w; 2 wk after operation). We inserted a 2.5-mm clip into the left renal artery of Sprague-Dawley rats and euthanized them at 2w and 5w following the operation. Systolic blood pressure increased within 1 wk after the operation, and left ventricular hypertrophy occurred in 2K1C rats. At 2w, juxtaglomerular apparatus (JGA) and collecting duct (CD) renin increased in clipped kidney (CK) of 2K1C rats. The tubular angiotensin I-converting enzyme (ACE) was not changed, but peritubular ACE2 decreased in nonclipped kidney (NCK) and CK of 2K1C rats. At 5w, ACE and CD renin were enhanced, and ACE2 was still lessened in both kidneys of 2K1C rats. However, plasma renin activity (PRA) was not different from that in sham rats. In proximal tubules of CK, the ANG II type 1 receptor (AT1R) was not suppressed, but the Mas receptor (MasR) was reduced; thus the AT1R/MasR ratio was elevated. Although hypoxic change in CK could not be excluded, the JGA renin of CK and CD renin in both kidneys was highly expressed independent of time. Peritubular ACE2 changed in the earlier period, and uninhibited AT1R in proximal tubules of CK was presented in the maintenance period. In 2K1C rats, attenuated ACE2 seems to contribute to initiating hypertension while upregulated ACE in combination with unsuppressed AT1R may have a key role in maintaining hypertension.

Renin or a renin-like enzyme evolved in ancestral vertebrates and is conserved along the vertebrate phylogeny. The ontogenic development of renin, however, is not well understood in nonmammalian vertebrates. We aimed to determine the expression patterns and relative abundance of renin mRNA in pre- and postnatal chickens (Gallus gallus, White Leghorn breed). Embryonic day 13 (E13) embryos show renal tubules, undifferentiated mesenchymal structures, and a small number of developing glomeruli. Maturing glomeruli are seen in post-hatch day 4 (D4) and day 30 (D30) kidneys, indicating that nephrogenic activity still exists in kidneys of 4-week-old chickens. In E13 embryos, renin mRNA measured by quantitative polymerase chain reaction in the adrenal glands is equivalent to the expression in the kidneys, whereas in post-hatch D4 and D30 maturing chicks, renal renin expressions increased 2-fold and 11-fold, respectively. In contrast, relative renin expression in the adrenals became lower than in the kidneys. Furthermore, renin expression is clearly visible by in situ hybridization in the juxtaglomerular (JG) area in D4 and D30 chicks, but not in E13 embryos. The results suggest that in chickens, renin evolved in both renal and extrarenal organs at an early stage of ontogeny and, with maturation, became localized to the JG area. Clear JG structures are not morphologically detectable in E13 embryos, but are visible in 30-day-old chicks, supporting this concept.

The aim of the present study was to investigate the renogenic characteristics of amniotic fluid stem cells (AFSCs) and to evaluate their in vitro differentiation potential into renal proximal tubular-like cells and juxtaglomerular-like cells. We culture expanded AFSCs derived from rat amniotic fluid. The AFSCs grew as adherent spindle-shaped cells and expressed mesenchymal markers CD73, CD90, and CD105 as well as renal progenitor markers WT1, PAX2, SIX2, SALL1, and CITED1. AFSCs exhibited an in vitro differentiation potential into renal proximal tubular epithelial-like cells, as shown by the upregulation of expression of proximal tubular cell–specific genes like AQP1, CD13, PEPT1, GLUT5, OAT1, and OCT1. AFSCs could also be differentiated into juxtaglomerular-like cells as demonstrated by the expression of renin and α-SMA. The AFSCs also expressed pluripotency markers OCT4, NANOG, and SOX2 and could be induced into embryoid bodies with differentiation into all the three germ layers, highlighting the pluripotent nature of these cells. Our results show that amniotic fluid contains a population of primitive stem cells that express renal-progenitor markers and also possess the propensity to differentiate into two renal lineage cell types and, thus, may have a therapeutic potential in renal regenerative medicine.

The prorenin receptor (PRR) was originally proposed to be a member of the renin-angiotensin system (RAS); however, recent work questioned their association. The present paper describes a functional link between the PRR and RAS in the renal juxtaglomerular apparatus (JGA), a classic anatomical site of the RAS. PRR expression was found in the sensory cells of the JGA, the macula densa (MD), and immunohistochemistry-localized PRR to the MD basolateral cell membrane in mouse, rat, and human kidneys. MD cell PRR activation led to MAP kinase ERK1/2 signaling and stimulation of PGE2 release, the classic pathway of MD-mediated renin release. Exogenous renin or prorenin added to the in vitro microperfused JGA-induced acute renin release, which was inhibited by removing the MD or by the administration of a PRR decoy peptide. To test the function of MD PRR in vivo, we established a new mouse model with inducible conditional knockout (cKO) of the PRR in MD cells based on neural nitric oxide synthase-driven Cre-lox recombination. Deletion of the MD PRR significantly reduced blood pressure and plasma renin. Challenging the RAS by low-salt diet + captopril treatment caused further significant reductions in blood pressure, renal renin, cyclooxygenase-2, and microsomal PGE synthase expression in cKO vs. wild-type mice. These results suggest that the MD PRR is essential in a novel JGA short-loop feedback mechanism, which is integrated within the classic MD mechanism to control renin synthesis and release and to maintain blood pressure.

Renin immunoreactivity was localized at the light and electron microscopic level in two elasmobranch fish species, the Atlantic stingray, Dasyatis sabina, and river ray, Potamotrygon humerosa. At the light microscopic level, the peroxidase-anti-peroxidase method showed a positive immunoreactivity in modified smooth muscle cells in kidney afferent arterioles as well as in arterioles of several organs: rectal gland, inter-renal gland, conus arteriosus, and gill. Electron microscopic renin-positive immunogold localization was confined to the contents of membrane bound granules in the modified smooth muscle cells of these arterioles. The presence of renin-containing granules in the modified smooth muscle, "granular cells," of the renal glomerular afferent arteriole of these two stingray species adds support to earlier studies which showed the structural components of a complete juxtaglomerular apparatus and some of the biochemical and molecular components of a renin-angiotensin system (RAS) as found in teleost fish, reptiles, birds, and mammals. A notable result, however, was the renin-positive immunoreaction in the arteriolar wall of all other organs studied here. The presence of this "diffuse renin system" in the connective tissue of various organs suggests that in these two stingray species in addition to local organ-specific functions, the RAS may act as a systemic mechanism to regulate blood pressure and blood flow in the body.

Nitric oxide (NO) is an important negative modulator of tubuloglomerular feedback responsiveness. We recently found that macula densa expresses a-, b-, and g-splice variants of neuronal nitric oxide synthase 1 (NOS1), and NOS1b expression in the macula densa increases on a high-salt diet. This study tested whether upregulation of NOS1b expression in the macula densa affects sodium excretion and saltsensitive hypertension by decreasing tubuloglomerular feedback responsiveness. Expression levels of NOS1b mRNA and protein were 30-and five-fold higher, respectively, than those of NOS1a in the renal cortex of C57BL/6 mice. Furthermore, macula densa NO production was similar in the isolated perfused juxtaglomerular apparatus of wild-type (WT) and nitric oxide synthase 1a-knockout (NOS1aKO) mice. Compared with control mice, mice with macula densa-specific knockout of all nitric oxide synthase 1 isoforms (MD-NOS1KO) had a significantly enhanced tubuloglomerular feedback response and after acute volume expansion, significantly reduced GFR, urine flow, and sodium excretion. Mean arterial pressure increased significantly in MD-NOS1KO mice (P,0.01) but not NOS1flox/flox mice fed a high-salt diet. After infusion of angiotensin II, mean arterial pressure increased by 61.6 mmHg in MD-NOS1KO mice versus 32.0mmHg in WT mice (P,0.01) fed a high-salt diet. These results indicate that NOS1b is a primary NOS1 isoformexpressed in themacula densa and regulates the tubuloglomerular feedback response, the natriuretic response to acute volume expansion, and the development of salt-sensitive hypertension. These findings show a novel mechanism for salt sensitivity of BP and the significance of tubuloglomerular feedback response in long-term control of sodium excretion and BP.

On the basis of previous observations that deletion of the von Hippel–Lindau protein (pVHL) in juxtaglomerular (JG) cells of the kidney suppresses renin and induces erythropoietin expression, this study aimed to characterize the events underlying this striking change of hormone expression. We found that renin cell-specific deletion of pVHL in mice leads to a phenotype switch in JG cells, from a cuboid and multiple vesicle-containing form into a flat and elongated form without vesicles. This shift of cell phenotype was accompanied by the disappearance of marker proteins for renin cells (e.g., aldo-keto reductase family 1, member 7 and connexin 40) and by the appearance of markers of fibroblast-like cells (e.g., collagen I, ecto-5′-nucleotidase, and PDGF receptor-β). Furthermore, hypoxia-inducible transcription factor-2α (HIF-2α) protein constitutively accumulated in these transformed cells. Codeletion of pVHL and HIF-2α in JG cells completely prevented the phenotypic changes. Similar to renin expression in normal JG cells, angiotensin II negatively regulated erythropoietin expression in the transformed cells. In summary, chronic activation of HIF-2 in renal JG cells leads to a reprogramming of the cells into fibroblast-like cells resembling native erythropoietin-producing cells located in the tubulointerstitium.

We have previously reported significant increases in neuronal nitric oxide synthase (NOS) immunostaining in renal arterioles of angiotensin type 1A receptor (AT1A) knockout mice, and in arterioles and macula densa cells of AT1A/AT1B knockout mice. The contribution of nitric oxide derived from endothelial and macula densa cells in the maintenance of afferent arteriolar tone and acetylcholine-induced vasodilation was functionally determined in kidneys of wild-type, AT1A, and AT1A/AT1B knockout mice. Acetylcholine-induced changes in arteriolar diameters of in vitro blood-perfused juxtamedullary nephrons were measured during control conditions, in the presence of the nonspecific NOS inhibitor, Nω-nitro-l-arginine methyl ester (NLA), or the highly selective neuronal NOS inhibitor, N5-(1-imino-3-butenyl)-l-ornithine (VNIO). Acetylcholine (0.1 mM) produced a significant vasoconstriction in afferent arterioles of AT1A/AT1B mice (−10.9 ± 5.1%) and no changes in afferent arteriolar diameters of AT1A knockout mice. NLA (0.01–1 mM) or VNIO (0.01–1 μM) induced significant dose-dependent vasoconstrictions (−19.8 ± 4.0% 1 mM NLA; −7.8 ± 3.5% 1 μM VNIO) in afferent arterioles of kidneys of wild-type mice. VNIO had no effect on afferent arteriole diameters of AT1A knockout or AT1A/AT1B knockout mice, suggesting nonfunctional neuronal nitric oxide synthase. These data indicate that acetylcholine produces a significant renal afferent arteriole vasodilation independently of nitric oxide synthases in wild-type mice. AT1A receptors are essential for the manifestation of renal afferent arteriole responses to neuronal nitric oxide synthase-mediated nitric oxide release.

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.

The so-called calcium paradoxon of renin describes the phenomenon that exocytosis of renin from juxtaglomerular cells of the kidney is stimulated by lowering of the extracellular calcium concentration. The yet poorly understood effect of extracellular calcium on renin secretion appears to depend on the function of the gap junction protein connexin 40 (Cx40) in renin-producing cells. This study aimed to elucidate the role of Cx40 for the calcium dependency of renin secretion in more detail by investigating if Cx40 function is really essential for the influence of extracellular calcium on renin secretion, if and how Cx40 affects intracellular calcium dynamics in renin-secreting cells and if Cx40-mediated gap junctional coupling of renin-secreting cells with the mesangial cell area is relevant for the influence of extracellular calcium on renin secretion. Renin secretion was studied in isolated perfused mouse kidneys. Calcium measurements were performed in renin-producing cells of microdissected glomeruli. The ultrastructure of renin-secreting cells was examined by electron microscopy. We found that Cx40 was not essential for stimulation of renin secretion by lowering of the extracellular calcium concentration. Instead, Cx40 increased the sensitivity of renin secretion response towards lowering of the extracellular calcium concentration. In line, the sensitivity and dynamics of intracellular calcium in response to lowering of extracellular calcium were dampened when renin-secreting cells lacked Cx40. Disruption of gap junctional coupling of renin-secreting cells by selective deletion of Cx40 from mesangial cells, however, did not change the stimulation of renin secretion by lowering of the extracellular calcium concentration. Deletion of Cx40 from renin cells but not from mesangial cells was associated with a shift of renin expression from perivascular cells of afferent arterioles to extraglomerular mesangial cells. Our findings suggest that Cx40 is not directly involved in the regulation of renin secretion by extracellular calcium. Instead, it appears that in renin-secreting cells of the kidney lacking Cx40, intracellular calcium dynamics and therefore also renin secretion are desensitized towards changes of extracellular calcium. Whether the dampened calcium response of renin-secreting cells lacking Cx40 function results from a direct involvement of Cx40 in intracellular calcium regulation or from the cell type shift of renin expression from perivascular to mesangial cells remains to be clarified. In any case, Cx40-mediated gap junctional coupling between renin and mesangial cells is not relevant for the calcium paradoxon of renin secretion.

Normal renin synthesis and secretion is important for the maintenance of juxtaglomerular apparatus architecture. Mice lacking a functional Ren-1(d) gene are devoid of renal juxtaglomerular cell granules and exhibit an altered macula densa morphology. Due to the species-specificity of renin activity, transgenic mice are ideal models for experimentally investigating and manipulating expression patterns of the human renin gene in a native cellular environment without confounding Renin-angiotensin-system interactions. A 55 kb transgene encompassing the human renin locus was crossed onto the mouse Ren-1(d)-null background, restoring granulation in juxtaglomerular cells. Correct processing of human renin in dense core granules was confirmed by immunogold labelling. After stimulation of the renin-angiotensin system, juxtaglomerular cells contained rhomboid protogranules with paracrystalline contents, dilated rough endoplasmic reticulum and electron-lucent granular structures. However, complementation of Ren-1(d-/-) mice with human renin was unable to rescue the abnormality seen in macula densa structure. The juxtaglomerular apparatus was still able to respond to tubuloglomerular feedback in isolated perfused juxtaglomerular apparatus preparations, although minor differences in glomerular tuft contractility and macula densa cell calcium handling were observed. This study reveals that the human renin protein is able to complement the mouse Ren-1(d-/-) non-granulated defect and suggests that granulopoiesis requires a structural motif that is conserved between the mouse Ren-1(d) and human renin proteins. It also suggests that the altered macula densa phenotype is related to the activity of the renin-1d enzyme in a local juxtaglomerular renin-angiotensin system.

See related article, pp 119–128 Organisms tend to compensate for the need of a cellular function by increasing the amount or the size of the responsible cell type. Hypertrophy of all muscle types under increased workload is a prominent example. Also several hormone systems react by increasing the amount of hormone-producing cells, when the main effector of the system is missing. This has been shown for thyroid hormones, leptin, glucagon, and corticosteroids only to name a few. In most cases, negative feedback mechanisms controlling hormone synthesis and release are inactivated by the depletion of the effector hormone. An article in this issue of Hypertension 1 confirms that the renin–angiotensin system is no exception. In adult animals and humans, the first and rate-limiting enzyme in the synthesis of its effector peptide angiotensin II (Ang II), renin, is only expressed in a small amount of kidney cells (0.01%–0.02% of all cells) in the juxtaglomerular apparatus of afferent arterioles, the renin-producing cells (RPCs). Already since 1939 it is known that a fall in renal perfusion pressure2 or salt depletion induces the recruitment of additional RPCs along the afferent arteriole and larger renal vessels upstream. In this way, and not by augmenting renin mRNA in already synthesizing cells, the organism increases its capacity to synthesize renin and respond to a physiological need of more Ang II. This recruitment of RPCs reverses the process of their development in embryogenesis, when first most renal vascular wall cells express renin, which is successively reduced to the few juxtaglomerular cells after birth. Oka et al1 use lineage tracing methods to show that the recruited RPCs are the same which produced renin in embryogenesis and gave it up after birth. Although these cells are not able to produce renin in this study using renin-deficient mice, they still …

We reported earlier that PPAR-gamma regulates renin transcription through a human-specific atypical binding sequence termed hRen-Pal3. Here we developed a mouse model to investigate the functional relevance of the hRen-Pal3 sequence in vivo since it might be responsible for the increased renin production in obesity and thus for the development of accompanying arterial hypertension. We used bacterial artificial chromosome construct and co-placement strategy to generate two transgenic mouse lines expressing the human renin gene from identical genomic locus without affecting the intrinsic mouse renin expression. One line carried a wild-type hRen-Pal3 in the transgene (Pal3wt strain) and the other a mutated non-functional Pal3 (Pal3mut strain). Human renin expression was correctly targeted to the renin-producing juxtaglomerular (JG) cells of kidney in both lines. However, Pal3mut mice had lower basal human renin expression. Since human renin does not recognize mouse angiotensinogen as substrate, the blood pressure was not different between the strains. Stimulation of renin production with the angiotensin-converting enzyme inhibitor enalapril equipotentially stimulated the human renin expression in Pal3wt and Pal3mut mice. High-fat diet for 10 weeks which is known to activate PPAR-gamma failed to increase human renin mRNA in kidneys of either strain. These findings showed that the human renin PPAR-gamma-binding sequence hRen-Pal3 is essential for basal renin expression but dispensable for the cell-specific and high-fat diet regulated renin expression in the kidney.

Aim Tubuloglomerular feedback (TGF) is an important mechanism in control of signal nephron glomerular filtration rate. The oxidative stress in the macula densa, primarily determined by the interactions between nitric oxide (NO) and superoxide (O2−), is essential in maintaining the TGF responsiveness. However, few studies examining the interactions between and amount of NO and O2− generated by the macula densa during normal and hypertensive states. Methods In this study, we used isolated perfused juxtaglomerular apparatus to directly measure the amount and also studied the interactions between NO and O2− in macula densa in both physiological and slow pressor Angiotensin II (Ang II)-induced hypertensive mice. Results We found that slow pressor Ang II at a dose of 600 ng kg−1 min−1 for two weeks increased mean arterial pressure by 26.1 ± 5.7 mmHg. TGF response increased from 3.4 ± 0.2 μm in control to 5.2 ± 0.2 μm in hypertensive mice. We first measured O2− generation by the macula densa and found it was undetectable in control mice. However, O2− generation by the macula densa increased to 21.4 ± 2.5 unit min−1 in Ang II-induced hypertensive mice. We then measured NO generation and found that NO generation by the macula densa was 138.5 ± 9.3 unit min−1 in control mice. The NO was undetectable in the macula densa in hypertensive mice infused with Ang II. Conclusions Under physiological conditions, TGF response is mainly controlled by the NO generated in the macula densa; in Ang II induced hypertension, the TGF response is mainly controlled by the O2− generated by the macula densa.

Iodinated contrast media (CM) have adverse effects that may result in contrast-induced acute kidney injury. Oxidative stress is believed to play a role in CM-induced kidney injury. We test the hypothesis that oxidative stress and reduced nitric oxide in tubules are consequences of CM-induced direct cell damage and that increased local oxidative stress may increase tubuloglomerular feedback. Rat thick ascending limbs (TAL) were isolated and perfused. Superoxide and nitric oxide were quantified using fluorescence techniques. Cell death rate was estimated using propidium iodide and trypan blue. The function of macula densa and tubuloglomerular feedback responsiveness were measured in isolated, perfused juxtaglomerular apparatuses (JGA) of rabbits. The expression of genes related to oxidative stress and the activity of superoxide dismutase (SOD) were investigated in the renal medulla of rats that received CM. CM increased superoxide concentration and reduced nitric oxide bioavailability in TAL. Propidium iodide fluorescence and trypan blue uptake increased more in CM-perfused TAL than in controls, indicating increased rate of cell death. There were no marked acute changes in the expression of genes related to oxidative stress in medullary segments of Henle's loop. SOD activity did not differ between CM and control groups. The tubuloglomerular feedback in isolated JGA was increased by CM. Tubular cell damage and accompanying oxidative stress in our model are consequences of CM-induced direct cell damage, which also modifies the tubulovascular interaction at the macula densa, and may therefore contribute to disturbances of renal perfusion and filtration.

Guyton suggested over 40 years ago that the kidney, by regulating volume homeostasis and sodium reabsorption, is critically involved in maintaining normal blood pressure through the pressure natriuresis response and that alterations in these processes lead to hypertension.1 The importance of the kidney in the pathophysiology of hypertension is also evidenced by kidney transplantation studies in rodents, where grafting of kidneys from normotensive rats normalized blood pressure in bilaterally nephrectomized hypertensive rats, and vice versa.2 These data suggest that the blood pressure trait follows the kidney phenotype and may relate to the nephron number, where fewer nephrons are associated with impaired pressure natriuresis and predisposition to hypertension.3 To further support a role for the kidney in hypertension are human genetic studies, which demonstrated that the majority of inherited monogenetic forms of hypertension affect the distal nephron and volume and sodium homeostasis.4 Moreover, increased renal sympathetic nerve activity causes an increase in blood pressure, whereas renal sympathectomy lowers blood pressure in experimental models of hypertension5 and in human hypertensive patients, indicating the importance of the sympathetic nervous system in renal function in hypertension.6 Article, see p 547 The functional significance of the renal sympathetic nerves in the kidney became more apparent when it was shown anatomically that nerve fibers are in direct contact with tubular segments and that they directly influence renal tubular function through norepinephrine-containing renal sympathetic nerve terminals.7 Activation of the renal sympathetic system increases norepinephrine release with increased sodium and water reabsorption by renal tubular epithelial cells, smooth muscle contraction, and renin release from granular cells of the juxtaglomerular apparatus,1 processes that contribute to blood pressure elevation. In addition, growing evidence indicates that activation of renal sympathetic nerves promotes renal inflammation through adaptive and innate immune mechanisms. In hypertension, T …

Cyclic guanosine monophosphate (cGMP) is synthesized by nitric oxide or natriuretic peptide-stimulated guanylyl cyclases and exhibits pleiotropic regulatory functions in the kidney. Hence, integration of cGMP signaling by cGMP-dependent protein kinases (cGKs) might play a critical role in renal physiology; however, detailed renal localization of cGKs is still lacking. Here, we performed an immunohistochemical analysis of cGKIα and cGKIβ isozymes in the mouse kidney and found both in arterioles, the mesangium, and within the cortical interstitium. In contrast to cGKIα, the β-isoform was not detected in the juxtaglomerular apparatus or medullary fibroblasts. Since interstitial fibroblasts play a prominent role in interstitial fibrosis, we focused our study on cGKI function in the interstitium, emphasizing a functional differentiation of both isoforms, and determined whether cGKIs influence renal fibrosis induced by unilateral ureter obstruction. Treatment with the guanylyl cyclase activators YC1 or isosorbide dinitrate showed stronger antifibrotic effects in wild-type than in cGKI-knockout or in smooth muscle-cGKIα-rescue mice, which are cGKI deficient in the kidney except in the renal vasculature. Moreover, fibrosis influenced the mRNA and protein expression levels of cGKIα more strongly than cGKIβ. Thus, our results indicate that cGMP, acting primarily through cGKIα, is an important suppressor of kidney fibrosis.

Renin is synthesized and released from juxtaglomerular (JG) cells. Adenosine inhibits renin release via an adenosine A1 receptor (A1R) calcium-mediated pathway. How this occurs is unknown. In cardiomyocytes, adenosine increases intracellular calcium via transient receptor potential canonical (TRPC) channels. We hypothesized that adenosine inhibits renin release via A1R activation, opening TRPC channels. However, higher concentrations of adenosine may stimulate renin release through A2R activation. Using primary cultures of isolated mouse JG cells, immunolabeling demonstrated renin and A1R in JG cells, but not A2R subtypes, although RT-PCR indicated the presence of mRNA of both A2AR and A2BR. Incubating JG cells with increasing concentrations of adenosine decreased renin release. Different concentrations of the adenosine receptor agonist N-ethylcarboxamide adenosine (NECA) did not change renin. Activating A1R with 0.5 μM N6-cyclohexyladenosine (CHA) decreased basal renin release from 0.22 ± 0.05 to 0.14 ± 0.03 μg of angiotensin I generated per milliliter of sample per hour of incubation (AngI/ml/mg prot) ( P < 0.03), and higher concentrations also inhibited renin. Reducing extracellular calcium with EGTA increased renin release (0.35 ± 0.08 μg AngI/ml/mg prot; P < 0.01), and blocked renin inhibition by CHA (0.28 ± 0.06 μg AngI/ml/mg prot; P < 0. 005 vs. CHA alone). The intracellular calcium chelator BAPTA-AM increased renin release by 55%, and blocked the inhibitory effect of CHA. Repeating these experiments in JG cells from A1R knockout mice using CHA or NECA demonstrated no effect on renin release. However, RT-PCR showed mRNA from TRPC isoforms 3 and 6 in isolated JG cells. Adding the TRPC blocker SKF-96365 reversed CHA-mediated inhibition of renin release. Thus A1R activation results in a calcium-dependent inhibition of renin release via TRPC-mediated calcium entry, but A2 receptors do not regulate renin release.

The renin-angiotensin-aldosterone system (RAAS) regulates BP and salt-volume homeostasis. Juxtaglomerular (JG) cells synthesize and release renin, which is the first and rate-limiting step in the RAAS. Intense pathologic stresses cause a dramatic increase in the number of renin-producing cells in the kidney, termed JG cell recruitment, but how this occurs is not fully understood. Here, we isolated renal CD44(+) mesenchymal stem cell (MSC)-like cells and found that they differentiated into JG-like renin-expressing cells both in vitro and in vivo. Sodium depletion and captopril led to activation and differentiation of these cells into renin-expressing cells in the adult kidney. In summary, CD44(+) MSC-like cells exist in the adult kidney and can differentiate into JG-like renin-producing cells under conditions that promote JG cell recruitment.

Despite the well documented and very effective non pharmacologic and pharmacologic therapies, hypertension remains often poorly controlled. There is still room for improvements in blood pressure control and recent technological advances have generated a regained interest in the physiopathology of renal sympathetic innervation in hypertension. In this article we review the evidence that renal sympathetic activity is increased in essential hypertension. The postganglionic sympathetic fibers are directed to the afferent and efferent renal arterioles, the juxtaglomerular apparatus, the proximal renal tubule, the loop of Henle, as well as the distal renal tubule and are under the control of many reflex loops, which are summarized in a most comprehensive manner for an unfamiliar reader within this field of research. Studies on renal denervation have provided further insights on the role of the sympathetic system in the kidneys, however their proper interpretation requires a special attention to the experimental protocols, as is explained in the text. Last, the possibility of kidney reinnervation is discussed, as well as the emerging evidence that the kidney is also a sensory organ. In summary, this review article provides a strong scientific background to understand not only the mechanisms of the hypotensive effects, but also those of possible pitfalls, of renal denervation.

Background Renin is synthesized in the collecting duct and is regulated differently than renin in the juxtaglomerular apparatus. However, the physiological relevance of collecting duct renin remains unknown, particularly with regard to its ability to regulate blood pressure. Methods We used gene targeting to generate mice with overexpression of renin in the collecting duct. A conditional mutant mouse line was created with the mouse renin transcript distal to a "transcriptional stop sequence" such that gene expression only occurred when the stop sequence was excised. These mice were bred with mice transgenic for the aquaporin-2 promoter driving Cre recombinase in order to achieve collecting duct-specific overexpression of renin. Results RNA analysis confirmed kidney-specific recombination, and medullary renin mRNA levels were increased 5-fold in collecting duct renin mice. Blood pressure was recorded by telemetry and plasma and urine was collected in 24-hour metabolic cages on normal, high-, and low-Na+ diets. Although no significant differences in 24-hour urinary Na+ excretion between targeted and control mice were detected, renin overexpresser mice had elevated blood pressure compared with controls on a high-Na+ diet. Urinary renin excretion was 2-fold higher in targeted mice as compared with controls on normal and low-Na+ diets. Plasma renin concentration was significantly suppressed in targeted mice as compared with controls on normal and high-Na+ diets. Conclusion Taken together, these results suggest that collecting duct-derived renin has the potential to modulate blood pressure independent of the systemic renin-angiotensin system.

Previous studies have indicated that 20-hydroxyeicosatetraeonic acid (20-HETE) modulates vascular tone in large cerebral and renal arteries through inhibition of the large conductance, calcium sensitive potassium (BK) channel activity. However, the role of 20-HETE in modulating tubuloglomerular feedback (TGF) and the myogenic response in the afferent arteriole (Af-Art) is unknown. The present study examined the effects of inhibitors of the synthesis and action of 20-HETE on the myogenic and TGF responses of isolated rabbit and mouse Af-Arts. Luminal diameter decreased by 9.2 ± 0.5% in mice and 8.9 ± 1.3% in rabbit Af-Art when the perfusion pressure was increased from 60 to 120 mmHg. Administration of a 20-HETE synthesis inhibitor, HET0016 (1 μM), or a selective 20-HETE antagonist, 6, 15-20-hydroxyeicosadienoic acid (6, 15-20-HEDE, 10 μM) completely blocked the myogenic response of both rabbit and mouse Af-Art, while addition of 5, 14-20-HEDE (10 μM), a 20-HETE agonist, restored the myogenic response in vessels treated with HET0016. Increases in NaCl concentration from 10 to 80 mM of the solution perfusing the macula densa constricted the Af-Art of rabbits by 6.0 ± 1.4 μm (n = 5). Addition of a 20-HETE agonist to the tubular perfusate potentiated the TGF-mediated vasoconstrictor response. This response was blocked by addition of a 20-HETE antagonist (6, 15-20-HEDE, 10 μM) to the vascular perfusate. These studies indicate that locally produced 20-HETE plays an important role in modulating the myogenic and TGF responsiveness of the Af-Art and may help explain how deficiencies in the renal formation of 20-HETE could promote the development of hypertension induced glomerular injury.

During development, renin cells are precursors for arteriolar smooth muscle, mesangial cells, and interstitial pericytes. Those seemingly differentiated descendants retain the memory to re-express renin when there is a threat to homeostasis. Understanding how such molecular memory is constructed and regulated would be crucial to comprehend cell identity which is, in turn, intimately linked to homeostasis.

Intracellular cAMP, the production of which is catalyzed by the

Although renin cells are crucial for blood pressure homeostasis, little is known about their nature. We now know that renin cells are precursors that appear early and in multiple tissues during embryonic development. They participate in morphogenetic events, vascular development and injury, tissue repair, and regeneration. When confronted to a homeostatic threat, renin cell descendants have the capability to switch the renin gene on or off. This poorly understood switch or molecular memory enables the organism to maintain constancy of the internal milieu and tissue perfusion. Here, we discuss briefly the major events that govern the acquisition and maintenance of renin cell identity and how manipulations that alter the fate of renin cells can lead to serious disease. We also advance the concept that renin cells are at the center of an ancestral system of defense linking the endocrine, the immune, and the repair responses of the organism. The renin–angiotensin system (RAS) is crucial in the regulation of blood pressure and fluid–electrolyte homeostasis.1,2 In the traditional view of the RAS, renin is released by the kidney juxtaglomerular cells, and on reaching the circulation, it acts on its only known substrate, angiotensinogen, produced mainly in the liver to yield angiotensin I (Ang I), a decapeptide and Des-Ang I-angiotensinogen, a large molecule of unclear function. Thereafter, Ang I is hydrolyzed by angiotensin-converting enzyme to yield the octapeptide Ang II, a fast acting and powerful vasoconstrictor that regulates peripheral vascular resistance, renal hemodynamics, and sodium reabsorption via several mechanisms, including the stimulation of aldosterone secretion by the adrenal glands. Most of the known cardiovascular and renal actions of the RAS are achieved by the actions of Ang II on its receptors, mainly AT1 receptors. It should be noted that for the system to operate properly, it needs to respond accurately …

The preglomerular microcirculation of spontaneously hypertensive rats (SHR) is hypersensitive to angiotensin (ANG) II, and studies have shown that this is likely due to enhanced coincident signaling between G protein subunits αq(Gαq; released by ANG II) and βγ (Gβγ; released by Gi-coupled receptors) to active phospholipase C (PLC). Here we investigated the molecular basis for the enhanced coincident signaling between Gβγ and Gαqin SHR preglomerular vascular smooth muscle cells (PGVSMCs). Because receptor for activated C kinase 1 (RACK1; a scaffolding protein) organizes interactions between Gβγ, Gαq, and PLC, we included RACK1 in this investigation. Cell fractionation studies demonstrated increased levels of membrane (but not cytosolic) Gβ, Gαq, PLCβ3, and RACK1 in SHR PGVSMCs compared with Wistar-Kyoto rat PGVSMCs. In SHR PGVSMCs, coimmunoprecipitation demonstrated RACK1 binding to Gβ and PLCβ3, but only at cell membranes. Pertussis toxin (which blocks Gβγ) and U73122 (which blocks PLC) reduced membrane RACK1; however, RACK1 knockdown (shRNA) did not affect membrane levels of Gβ, Gαq, or PLCβ3. In a novel gel contraction assay, RACK1 knockdown in SHR PGVSMCs attenuated contractions to ANG II and abrogated the ability of neuropeptide Y (which signals via Gβγ) to enhance ANG II-induced contractions. We conclude that in SHR PGVSMCs the enlarged pool of Gβγ and PLCβ3recruits RACK1 to membranes and RACK1 then organizes signaling. Consequently, knockdown of RACK1 prevents coincident signaling between ANG II and the Gipathway. This is the first study to implicate RACK1 in vascular smooth muscle cell contraction and suggests that RACK1 inhibitors could be effective cardiovascular drugs.

Renin is the initiator and rate-limiting factor in the renin-angiotensin blood pressure regulation system. Although renin is not exclusively produced in the kidney, in nonmurine species the synthesis and secretion of the active circulatory enzyme is confined almost exclusively to the dense core granules of juxtaglomerular (JG) cells, where prorenin is processed and stored for release via a regulated pathway. Despite its importance, the structural organization and regulation of granules within these cells is not well understood, in part due to the difficulty in culturing primary JG cells in vitro and the lack of appropriate cell lines. We have streamlined the isolation and culture of primary renin-expressing cells suitable for high-speed, high-resolution live imaging using a Percoll gradient-based procedure to purify cells from RenGFP+ transgenic mice. Fibronectin-coated glass coverslips proved optimal for the adhesion of renin-expressing cells and facilitated live cell imaging at the plasma membrane of primary renin cells using total internal reflection fluorescence microscopy (TIRFM). To obtain quantitative data on intracellular function, we stained mixed granule and lysosome populations with Lysotracker Red and stimulated cells using 100 nM isoproterenol. Analysis of membrane-proximal acidic granular organelle dynamics and behavior within renin-expressing cells revealed the existence of two populations of granular organelles with distinct functional responses following isoproterenol stimulation. The application of high-resolution techniques for imaging JG and other specialized kidney cells provides new opportunities for investigating renal cell biology.

Renin, a key component in the regulation of blood pressure in mammals, is produced by the rare and highly specialized juxtaglomerular cells of the kidney. Chronic stimulation of renin release results in a recruitment of new juxtaglomerular cells by the apparent conversion of adjacent smooth muscle cells along the afferent arterioles. Because juxtaglomerular cells rapidly dedifferentiate when removed from the kidney, their developmental origin and the mechanism that explains their phenotypic plasticity remain unclear. To overcome this limitation, we have performed RNA expression analysis on 4 human renin-producing tumors. The most highly expressed genes that were common between the reninomas were subsequently used for in situ hybridization in kidneys of 5-day-old mice, adult mice, and adult mice treated with captopril. From the top 100 genes, 10 encoding for ligands were selected for further analysis. Medium of human embryonic kidney 293 cells transfected with the mouse cDNA encoding these ligands was applied to (pro)renin-synthesizing As4.1 cells. Among the ligands, only platelet-derived growth factor B reduced the medium and cellular (pro)renin levels, as well as As4.1 renin gene expression. In addition, platelet-derived growth factor B–exposed As4.1 cells displayed a more elongated and aligned shape with no alteration in viability. This was accompanied by a downregulated expression of α-smooth muscle actin and an upregulated expression of interleukin-6, suggesting a phenotypic shift from myoendocrine to inflammatory. Our results add 36 new genes to the list that characterize renin-producing cells and reveal a novel role for platelet-derived growth factor B as a regulator of renin-synthesizing cells.

Renin is the first and rate-limiting step of the renin-angiotensin system. The exclusive source of renin in the circulation are the juxtaglomerular cells of the kidney, which line the afferent arterioles at the entrance of the glomeruli. Normally, renin production by these cells suffices to maintain homeostasis. However, under chronic stimulation of renin release, for instance during a low-salt diet or antihypertensive therapy, cells that previously expressed renin during congenital life re-convert to a renin-producing cell phenotype, a phenomenon which is known as "recruitment". How exactly such differentiation occurs remains to be clarified. This review critically discusses the phenotypic plasticity of renin cells, connecting them not only to the classical concept of blood pressure regulation, but also to more complex contexts such as development and growth processes, cell repair mechanisms and tissue regeneration.

Evidence for coexpression of two or more classic neurotransmitters in neurons has increased, but less is known about cotransmission. Ventral tegmental area (VTA) neurons corelease dopamine (DA), the excitatory transmitter glutamate, and the inhibitory transmitter GABA onto target cells in the striatum. Olfactory bulb (OB) short axon cells (SACs) form interglomerular connections and coexpress markers for DA and GABA. Using an optogenetic approach, we provide evidence that mouse OB SACs release both GABA and DA onto external tufted cells (ETCs) in other glomeruli. Optical activation of channelrhodopsin specifically expressed in DAergic SACs produced a GABAAreceptor-mediated monosynaptic inhibitory response, followed by DA–D1-like receptor-mediated excitatory response in ETCs. The GABAAreceptor-mediated hyperpolarization activatesIhcurrent in ETCs; synaptically released DA increasesIh, which enhances postinhibitory rebound spiking. Thus, the opposing actions of synaptically released GABA and DA are functionally integrated byIhto generate an inhibition-to-excitation “switch” in ETCs. Consistent with the established role ofIhin ETC burst firing, we show that endogenous DA release increases ETC spontaneous bursting frequency. ETCs transmit sensory signals to mitral/tufted output neurons and drive intraglomerular inhibition to shape glomerulus output to downstream olfactory networks. GABA and DA cotransmission from SACs to ETCs may play a key role in regulating output coding across the glomerular array.

The mode of renin release from renal juxtaglomerular cells into circulation is still unsolved in several aspects. Here we studied the intracellular organization of renin-storage vesicles and their changes during controlled stimulation of renin release. This was accomplished using isolated perfused mouse kidneys with 3-dimensional electron microscopic analyses of renin-producing cells. Renin was found to be stored in a network of single granules and cavern-like structures, and dependent on the synthesis of glycosylated prorenin. Acute stimulation of renin release led to increased exocytosis in combination with intracellular fusion of vesicles to larger caverns and their subsequent emptying. Renin release from the kidneys of SCID-beige mice, which contain few but gigantic renin-storage vesicles, was no different from that of kidneys from wild-type mice. Thus, our findings suggest that renin is released by mechanisms similar to compound exocytosis.

Background/Aims: Despite extensive studies, the intracellular regulatory mechanism of renin production and release is still poorly understood. The present study was designed to test whether CD38-ADP-ribosylcyclase signaling pathway contributes to the regulation of renin production and release, and to examine whether CD38 gene knockout (CD38 -/- ) can change this important renal endocrinal function. Methods: ADP–ribosylcyclase activity was estimated utilizing HPLC, cADPR levels from western blot, plasma renin activity from RIA kit, urinary sodium and potassium excretion from flame photometry. Results: The expression of CD38 and the activity of ADP-ribosylcyclase to produce cyclic ADP-ribose (cADPR) were nearly abolished in the kidney from CD38 -/- mice, indicating that CD38 gene is a major enzyme responsible for the generation of cADPR in vivo. Mice lacking CD38 gene showed increased plasma renin activity (PRA) in either conscious or anesthetized status (P

Increase in blood pressure in the renal afferent arteriole is known to induce an increase in cytosolic calcium concentration ([Ca2+]i) of juxtaglomerular (JG) cells and to result in a decreased secretion of renin.Mechanical stimulation of As4.1 JG cells induces an increase in [Ca2+]i that is inhibited by HC067047 and RN1734, two inhibitors of TRPV4, or by siRNA‐mediated repression of TRPV4.Inhibition of TRPV4 impairs pressure‐induced decrease in renin secretion.Compared to wild‐type mice, Trpv4 −/− mice present increased resting plasma levels of renin and aldosterone and present a significantly altered pressure–renin relationship.We suggest that TRPV4 channel participates in mechanosensation at the juxtaglomerular apparatus.

Albuminuria is an indicator of renal injury and is closely linked with cardiovascular disease (CVD). However, the mechanism by which albumin is excreted in the urine remains unclear. As the juxtamedullary region of the kidney is highly susceptible to pressure increase, juxtamedullary injury is observed from an early phase in hypertensive rat models. Anatomical similarities are observed between the pre-glomerular vessels of the juxtamedullary nephron and the cerebral vasculature. We previously named these 'strain vessels' for their high vascular tone and exposure to higher pressures. The current studies were designed to determine whether albuminuria is the result of juxtamedullary nephron injury, indicating the presence of pressure injury to the strain vessels in spontaneously hypertensive stroke-prone rats (SHR-SP) fed a high-salt diet. Albuminuria was associated with juxtamedullary nephron injury, and the enhanced expression of monocyte chemotactic protein-1 (MCP-1) and tumor growth factor-beta (TGF-β) in 12-week-old SHR-SP rats fed a 4% high-salt diet from the age of 6 weeks. The wall thickness of the pre-glomerular vessels of the juxtamedullary nephron was also associated with that of the perforating artery of the middle cerebral artery. Reducing the blood pressure with nifedipine reduced the degree of albuminuria and juxtamedullary nephron injury as well as MCP-1 and TGF-β expression in the SHR-SP rats fed an 8% high-salt diet from the age of 9 weeks. Nifedipine inhibited stroke events in these animals until they were 14 weeks old. These results indicate that albuminuria is a result of juxtamedullary nephron injury and a marker of pressure-induced injury of the strain vessels.

A major rate-limiting step in the renin-angiotensin-aldosterone system is the release of active renin from endocrine cells (juxtaglomerular (JG) cells) in the media layer of the afferent glomerular arterioles. The number and distribution of JG cells vary with age and the physiological level of stimulation; fetal life and chronic stimulation by extracellular volume contraction is associated with recruitment of renin-producing cells. Upon stimulation of renin release, labeled renin granules "disappear;" the number of granules decrease; cell membrane surface area increases in single cells, and release is quantal. Together, this indicates exocytosis as the predominant mode of release. JG cells release few percent of total renin content by physiological stimulation, and recruitment of renin cells is preferred to recruitment of granules during prolonged stimulation. Several endocrine and paracrine agonists, neurotransmitters, and cell swelling converge on the stimulatory cyclic AMP (cAMP) pathway. Renin secretion is attenuated in mice deficient in beta-adrenoceptors, prostaglandin E(2)-EP4 receptors, Gsα protein, and adenylyl cyclases 5 and 6. Phosphodiesterases (PDE) 3 and 4 degrade cAMP in JG cells, and PDE3 is inhibited by cyclic GMP (cGMP) and couples the cGMP pathway to the cAMP pathway. Cyclic AMP enhances K(+)-current in JG cells and is permissive for secretion by stabilizing membrane potential far from threshold that activates L-type voltage-gated calcium channels. Intracellular calcium paradoxically inhibits renin secretion likely through attenuated formation and enhanced degradation of cAMP; by activation of chloride currents and interaction with calcineurin. Connexin 40 is necessary for localization of JG cells in the vascular wall and for pressure- and macula densa-dependent suppression of renin release.