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For normal maintenance of blood pressure and blood volume a well-balanced renin-angiotensin-aldosterone system (RAS) is necessary. For this purpose, renin is secreted as the situation demands by the juxtaglomerular cells (also called as granular cells) that are in the walls of the afferent arterioles. Juxtaglomerular cells can sense minute changes in the blood pressure and blood volume and accordingly synthesize, store, and secrete appropriate amounts of renin. Thus, when the blood pressure and blood volume are decreased JGA cells synthesize and secrete higher amounts of renin and when the blood pressure and blood volume is increased the synthesis and secretion of renin is decreased such that homeostasis is restored. To decipher this important function, JGA cells (renin cells) need to sense and transmit the extracellular physical forces to their chromatin to control renin gene expression for appropriate renin synthesis. The changes in perfusion pressure are sensed by Integrin β1 that is transmitted to the renin cell's nucleus via lamin A/C that produces changes in the architecture of the chromatin. This results in an alteration (either increase or decrease) in renin gene expression. Cell membrane is situated in an unique location since all stimuli need to be transmitted to the cell nucleus and messages from the DNA to the cell external environment can be conveyed only through it. This implies that cell membrane structure and integrity is essential for all cellular functions. Cell membrane is composed to proteins and lipids. The lipid components of the cell membrane regulate its (cell membrane) fluidity and the way the messages are transmitted between the cell and its environment. Of all the lipids present in the membrane, arachidonic acid (AA) forms an important constituent. In response to pressure and other stimuli, cellular and nuclear shape changes occur that render nucleus to act as an elastic mechanotransducer that produces not only changes in cell shape but also in its dynamic behavior. Cell shape changes in response to external pressure(s) result(s) in the activation of cPLA2 (cytosolic phospholipase 2)-AA pathway that stretches to recruit myosin II which produces actin-myosin cytoskeleton contractility. Released AA can undergo peroxidation and peroxidized AA binds to DNA to regulate the expression of several genes. Alterations in the perfusion pressure in the afferent arterioles produces parallel changes in the renin cell membrane leading to changes in renin release. AA and its metabolic products regulate not only the release of renin but also changes in the vanilloid type 1 (TRPV1) expression in renal sensory nerves. Thus, AA and its metabolites function as intermediate/mediator molecules in transducing changes in perfusion and mechanical pressures that involves nuclear mechanotransduction mechanism. This mechanotransducer function of AA has relevance to the synthesis and release of insulin, neurotransmitters, and other soluble mediators release by specialized and non-specialized cells. Thus, AA plays a critical role in diseases such as diabetes mellitus, hypertension, atherosclerosis, coronary heart disease, sepsis, lupus, rheumatoid arthritis, and cancer.

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.

As shown in our previous paper (Kriz W, Löwen J, Federico G, van den Born J, Gröne E, Gröne HJ. Am J Physiol Renal Physiol 312: F1101-F1111, 2017), mesangial matrix expansion in diabetic nephropathy (DN) results for a major part from the accumulation of worn-out undegraded glomerular basement membrane material. Here, based on the reevaluation of >900 biopsies of DN, we show that this process continues with the progression of the disease finally leading to the herniation of the matrix-overloaded tuft through the glomerular entrance to the outside. This leads to severe changes in the glomerular surroundings, including a dissociation of the juxtaglomerular apparatus with displacement of the macula densa. The herniation is associated with a prominent outgrowth of glomerular vessels from the tuft. Mostly, these aberrant vessels are an abnormal type of arteriole with frequent intramural insudations of plasma. They spread into glomerular surroundings extending in intertubular and periglomerular spaces. Their formation is associated with elevated mRNA levels of vascular endothelial growth factor-A, angiopoietins 1 and 2, and the corresponding receptors. Functionally, these processes seem to compromise tubuloglomerular feedback-related functions and may be one factor why Na + -glucose cotransporter-2 inhibitors are not effective in advanced stages of DN.

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, N ω -nitro-l-arginine methyl ester (NLA), or the highly selective neuronal NOS inhibitor, N 5 -(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.

Introduction: Embryogenesis of the kidney glomeruli, especially its vascular component, has not been well documented. Glomeruli capillary tuft is surrounded and enveloped by visceral epithelial cells, which is a unique portal system that connects afferent with efferent arteriole without interaction with venular circulation. We hypothesized that the portal system embryologically has developed by extension of the intima of afferent arteriole into the stroma of glomerulus. We also hypothesized that juxtaglomeruli apparatus was developed from remnants of smooth muscle cells of the media of afferent arteriole at the anastomosing site with the Bowman capsule entrance., Materials and Methods: We studied 5 human fetal kidneys by hematoxylin-eosin, periodic acid-Schiff, and immunoperoxidase staining techniques., Results: Hematoxylin-eosin staining of fetal kidney showed presence of erythrocytes in early vesicle form of glomeruli that was confirmed by immunohistochemical staining with CD31, smooth muscle actin, and CD34 markers. These stains showed extension of extraglomerular arterioles to the glomeruli. Periodic acid-Schiff staining showed also the continuity of the basement membrane in extraglomeruli and internal glomerular vascular tufts., Conclusions: This study shows that there is a relationship between the metanephric blast cells and major vessel critical for angiogenesis. When afferent arteriole come in contact with the immature glomeruli, its intima migrates into the glomerular tuft to form intraglomerular capillary system, while its smooth muscle remains at the entrance orifice and develops juxtaglomerular apparatus cells.

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 α q (Gα q ; released by ANG II) and βγ (Gβγ; released by G i -coupled receptors) to active phospholipase C (PLC). Here we investigated the molecular basis for the enhanced coincident signaling between Gβγ and Gα q in SHR preglomerular vascular smooth muscle cells (PGVSMCs). Because receptor for activated C kinase 1 (RACK1; a scaffolding protein) organizes interactions between Gβγ, Gα q , and PLC, we included RACK1 in this investigation. Cell fractionation studies demonstrated increased levels of membrane (but not cytosolic) Gβ, Gα q , PLCβ 3 , and RACK1 in SHR PGVSMCs compared with Wistar-Kyoto rat PGVSMCs. In SHR PGVSMCs, coimmunoprecipitation demonstrated RACK1 binding to Gβ and PLCβ 3 , 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, Gα q , or PLCβ 3 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 PLCβ 3 recruits RACK1 to membranes and RACK1 then organizes signaling. Consequently, knockdown of RACK1 prevents coincident signaling between ANG II and the G i pathway. This is the first study to implicate RACK1 in vascular smooth muscle cell contraction and suggests that RACK1 inhibitors could be effective cardiovascular drugs., (Copyright © 2017 the American Physiological Society.)

Introduction: Renin synthesis and release is the rate-limiting step in the renin-angiotensin system, because cyclic adenosine monophosphate (cAMP) has been identified as dominant pathway for renin gene expression, and cAMP response element-binding protein (CREB) is found in the human and mouse renin promoter. This study aimed to evaluate the role of CREB in expression of the renin gene., Materials and Methods: We created conditional deletion of CREB in mice with low-sodium diet, specifically in renin cells of the kidney. To assess the effect of CREB on renin expression, immunostaining of renin was used in samples from wild-type mice and mice with gene knock-down of CREB. Cyclic AMP response element-binding-protein-binding protein (CBP) and p300 were measured in cultured renin cells of the mice, and RNA detection was done with real-time polymerase chain reaction., Results: With low-sodium diet, renin was expressed along the whole wall of the afferent glomerular arterioles in wild-type mice, while there was no increase or even decrease in renin expression in CREB-specific deletion mice; RNA level of renin in cultured cells decreased by 50% with single knock-down of CREB, CBP, or p300, and decreased 70% with triple knock-down of CREB, CBP, and p300., Conclusions: This study found that CREB was important for renin synthesis and the role of CREB can be achieved through the recruitment of co-activators CBP and p300.

To observe the formation of blood flow in the juxtaglomerular pathway in comparative-anatomical and age-related aspects, 484 kidneys from the representatives of the five classes of vertebrates, 50 kidneys of human fetuses and 193 kidneys of normal individuals of different ages were studied. Macro-microscopic, histological and morphometric methods were used. In all the mammalian animal species, the significant development of vascular glomeruli and peritubular capillaries was observed. In human kidneys, the relative content of cortical and medullary arterial vessels was maximal in juvenile age. With age, this parameter was found to decrease both in renal cortex and medulla. The differentiation of renal tissue into the cortex and the medulla, that began in birds and was finally formed in mammals, explains the appearance of cortical and juxtamedullary blood flow pathways. During the antenatal period of human development, renal juxtaglomerular pathway of blood flow prevails over the cortical one. The diminished significance of juxtamedullary pathway of blood flow in elderly and senile age determines the decreased adaptive capacities of intraorgan renal arterial bed in the norm.

Renin-expressing cells in the kidney normally appear as mural cells of developing preglomerular vessels and finally impose as granulated juxtaglomerular cells in adult kidneys. The differentiation of renin-expressing cells from the metanephric mesenchyme in general and the potential role of special precursor stages in particular is not well understood. Therefore, it was the aim of this study to search for renin cell precursors in the kidney. As an experimental model, we used kidneys of aldosterone synthase-deficient mice, which display a prominent compensatory overproduction of renin cells that are arranged in multilayered perivascular cell clusters. We found that the perivascular cell clusters contained two apparently distinct cell types, one staining positive for renin and another one staining positive for type I procollagen (PC1). It appeared as if PC1 and renin expression were inversely related at the cellular level. The proportion of renin-positive to PC1-positive cells in the clusters was inversely linked to the rate of salt intake, as was overall renin expression. Our findings suggest that the cells in the perivascular cell clusters can reversibly switch between PC1 and renin expression and that PC1-expressing cells might be precursors of renin cells. A few of those PC1-positive cells were found also in adult wild-type kidneys in the juxtaglomerular lacis cell area, in which renin expression can be induced on demand.

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Lipofuscin-like granules, first described by Biava and West in 1965, are a subcellular, quasi-physiologic finding mainly seen in the smooth muscle cells of renal arterioles, but also in juxtaglomerular cells and the lacis cells of human kidneys. They increase in number in subjects affected by arterial hypertension and diabetes. They do not correlate with a specific primary renal disease. Lipofuscin-like granules are not related to renin granules. The world literature on this subject is almost non-existent, and the awareness of this finding or its clinical significance among either pathologists or nephrologists is very poor. We incidentally observed these lipofuscin-like granules in 8 cases during the routine electron microscope examination of 440 renal biopsies, and report herein on their ultrastructural features. Six of these 8 patients were affected by arterial hypertension, one of whom was also concomitantly affected by diabetes mellitus. These lipofuscin-like granules appear as dense bodies with a lipid component, a coarsely granular matrix, and a crystalloid component which may appear in a band or dot pattern, according to the plane of sectioning. The pathologist has to be aware of these lipofuscin-like granules in order not to confuse them with the semicircularly organized (fingerprint) linear immune deposits associated with some specific glomerulopathies., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

We used the whole cell patch-clamp technique to investigate the regulation of descending vasa recta (DVR) pericyte Ca(2+)-dependent Cl(-) currents (CaCC) by cytoplasmic Ca(2+) concentration ([Ca](CYT)), voltage, and kinase activity. Murine CaCC increased with voltage and electrode Ca(2+) concentration. The current saturated at [Ca](CYT) of ∼1,000 nM and exhibited an EC(50) for Ca(2+) of ∼500 nM, independent of depolarization potential. Activation time constants were between 100 and 200 ms, independent of electrode Ca(2+). Repolarization-related tail currents elicited by stepping from +100 mV to varying test potentials exhibited deactivation time constants of 50-200 ms that increased with voltage when electrode [Ca](CYT) was 1,000 nM. The calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7, 30 μM) blocked CaCC. The myosin light chain kinase blockers 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-7, 1-50 μM) and 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-9, 10 μM) were similarly effective. Resting pericytes were hyperpolarized by ML-7. Pericytes exposed to ANG II (10 nM) depolarized from a baseline of -50 ± 6 to -29 ± 3 mV and were repolarized to -63 ± 7 mV by exposure to 50 μM ML-7. The Ca(2+)/calmodulin-dependent kinase inhibitor KN-93 reduced pericyte CaCC only when it was present in the electrode and extracellular buffer from the time of membrane break-in. We conclude that murine DVR pericytes are modulated by [Ca](CYT), membrane potential, and phosphorylation events, suggesting that Ca(2+)-dependent Cl(-) conductance may be a target for regulation of vasoactivity and medullary blood flow in vivo.

Macula densa cells in the distal nephron, according to the classic paradigm, are salt sensors that generate paracrine chemical signals in the juxtaglomerular apparatus to control vital kidney functions, including renal blood flow, glomerular filtration, and renin release. Renin is the rate-limiting step in the activation of the renin-angiotensin system, a key modulator of body fluid homeostasis. Here, we discuss recent advances in understanding macula densa sensing and suggest these cells, in addition to salt, also sense various chemical and metabolic signals in the tubular environment that directly trigger renin release.

The mechanotransduction mechanism underlying the myogenic response is poorly understood, but evidence implicates participation of epithelial sodium channel (ENaC)-like proteins. Therefore, the role of ENaC on the afferent arteriolar myogenic response was investigated in vitro using the blood-perfused juxtamedullary nephron technique. Papillectomy was used to isolate myogenic influences by eliminating tubuloglomerular feedback signals. Autoregulatory responses were assessed by manipulating perfusion pressure in 30-mm Hg steps. Under control conditions, arteriolar diameter increased by 15% from 13.0+/-1.3 to 14.7+/-1.2 microm (P<0.05) after reducing perfusion pressure from 100 to 70 mm Hg. Diameter decreased to 11.3+/-1.1 and 10.6+/-1.0 microm after increasing pressure to 130 and 160 mm Hg (88+/-1 and 81+/-2% of control diameter, P<0.05), respectively. Pressure-mediated autoregulatory responses were significantly inhibited by superfusion of 10 micromol/L amiloride (102+/-2, 97+/-4, and 94+/-3% of control diameter), or 10 micromol/L benzamil (106+/-5, 100+/-3, and 103+/-3% of control diameter), and when perfusing with blood containing 5 micromol/L amiloride (106+/-2, 97+/-4, and 97+/-4% of control diameter). Vasoconstrictor responses to 55 mmol/L KCl were preserved as diameters decreased by 67+/-4, 55+/-8, and 60+/-4% in afferent arterioles superfused with amiloride or benzamil, and perfused with amiloride, respectively. These responses were similar to responses obtained from control afferent arterioles (64+/-6%, P>0.05). Immunofluorescence revealed expression of the alpha, beta, and gamma subunits of ENaC in freshly isolated preglomerular microvascular smooth muscle cells. These results demonstrate that selective ENaC inhibitors attenuate afferent arteriolar myogenic responses and suggest that ENaC may function as mechanosensitive ion channels initiating pressure-dependent myogenic responses in rat juxtamedullary afferent arterioles.

Tubuloglomerular feedback (TGF) and the myogenic mechanism control afferent arteriolar diameter in each nephron and regulate blood flow. Both mechanisms generate self-sustained oscillations, the oscillations interact, TGF modulates the frequency and amplitude of the myogenic oscillation, and the oscillations synchronize; a 5:1 frequency ratio is the most frequent. TGF oscillations synchronize in nephron pairs supplied from a common cortical radial artery, as do myogenic oscillations. We propose that electrotonic vascular signal propagation from one juxtaglomerular apparatus interacts with similar signals from other nephrons to produce synchronization. We tested this idea in tubular-vascular preparations from mice. Vascular smooth muscle cells were loaded with a fluorescent voltage-sensitive dye; fluorescence intensity was measured with confocal microscopy. Perfusion of the thick ascending limb activated TGF and depolarized afferent arteriolar smooth muscle cells. The depolarization spread to the cortical radial artery and other afferent arterioles and declined with distance from the perfused juxtaglomerular apparatus, consistent with electrotonic vascular signal propagation. With a mathematical model of two coupled nephrons, we estimated the conductance of nephron coupling by fitting simulated vessel diameters to experimental data. With this value, we simulated nephron pairs to test for synchronization. In single-nephron simulations, the frequency of the TGF oscillation varied with nephron length. Coupling nephrons of different lengths forced TGF frequencies of both pair members to converge to a common value. The myogenic oscillations also synchronized, and the synchronization between the TGF and the myogenic oscillations showed an increased stability against parameter perturbations. Electronic vascular signal propagation is a plausible mechanism for nephron synchronization. Coupling increased the stability of the various oscillations.

In the renal circulation, the afferent arterioles leading to juxtamedullary glomeruli are anatomically under high pressure load in order to reduce the high arterial pressure and keep the pressure of the following glomeruli at about 50 mmHg. Because of the pressure overload, it is possible that vascular injury begins in the afferent arterioles of juxtamedullary glomeruli. We call such high pressure-loaded vessels branching directly from relatively large vessels 'strain vessels'. Coronary arteries and penetrating cerebral arteries are also strain vessels. They are also susceptible to pressure-induced injury. This review presents a hypothesis that microalbuminuria reflects the early stage of vascular injury, especially strain vessels' injury, and that strain vessels' injury connects chronic kidney disease with the risk of cardiovascular disease.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a systemic arterial disease characterized by impairment of vascular smooth muscle cell structure and function related to NOTCH3 mutations. Pathological findings include pathognomonic granular osmiophilic material (GOM) deposition with nonspecific hyalinization within the artery wall in a variety of tissues. The main clinical presentation is iterative strokes in young adults despite the lack of cardiovascular risk factors, leading to early dementia. Although arteriosclerosis and GOM have been found in kidneys from patients with CADASIL, kidney disease has been described only once up to now, in association with immunoglobulin A nephropathy. We report the case of a 61-year-old patient with a medical history of CADASIL and recent mild hypertension. His mother also showed neuropsychiatric symptoms and end-stage renal disease of unknown cause. The patient had a chronic kidney disease defined by means of estimated glomerular filtration rate using the 4-variable Modification of Diet in Renal Disease Study equation of 58 mL/min/1.73 m(2) associated with mild proteinuria and intermittent microscopic hematuria. Renal histological analysis showed severe arteriosclerosis and mild interstitial fibrosis. Glomeruli did not show mesangial immunoglobulin A deposition or focal segmental proliferation. Electron microscopic analysis showed typical GOM deposition in the vicinity of altered vascular smooth muscle cells in interlobular and juxtaglomerular arteries. The nephroangiosclerosis-like lesions were unusually severe in contrast to the recent mild hypertension. The presence of GOM strongly suggests that renal lesions were related to the NOTCH3 mutation. Here, we describe the first case of familial occurrence of kidney disease with decreased kidney function in the absence of coexisting nephropathy in patients with CADASIL. We discuss the role of NOTCH3 mutation in the pathogenesis of nephroangiosclerosis through functional impairment of renal microcirculation or primary Notch3-related vascular disease.

Connexin (Cx) proteins are known to play a role in cell-to-cell communication via intercellular gap junction channels or transiently open hemichannels. Previous studies have identified several connexin isoforms in the juxtaglomerular apparatus (JGA), but the vascular connexin isoform Cx45 has not yet been studied in this region. The present work aimed to identify in detail the localization of Cx45 in the JGA and to suggest a functional role for Cx45 in the kidney using conditions where Cx45 expression or function was altered. Using mice that express lacZ coding DNA under the control of the Cx45 promoter, we observed beta-galactosidase staining in cortical vasculature and glomeruli, with specific localization to the JGA region. Renal vascular localization of Cx45 was further confirmed with the use of conditional Cx45-deficient (Cx45fl/fl:Nestin-Cre) mice, which express enhanced green fluorescence protein (EGFP) instead of Cx45 only in cells that, during development, expressed the intermediate filament nestin. EGFP fluorescence was found in the afferent and efferent arteriole smooth muscle cells, in the renin-producing juxtaglomerular cells, and in the extra- and intraglomerular mesangium. Cx45fl/fl:Nestin-Cre mice exhibited increased renin expression and activity, as well as higher systemic blood pressure. The propagation of mechanically induced calcium waves was slower in cultured vascular smooth muscle cells (VSMCs) from Cx45fl/fl:Nestin-Cre mice and in control VSMC treated with a Cx45 gap mimetic peptide that inhibits Cx45 gap junctional communication. VSMCs allowed the cell-to-cell passage of the gap junction permeable dye Lucifer yellow, and calcium wave propagation was not altered by addition of the ATP receptor blocker suramin, suggesting that Cx45 regulates calcium wave propagation via direct gap junction coupling. In conclusion, the localization of Cx45 to the JGA and functional data from Cx45fl/fl:Nestin-Cre mice suggest that Cx45 is involved in the propagation of JGA vascular signals and in the regulation of renin release and blood pressure.

The aim of the study is to evaluate the impact of nitric oxide (NO) produced by endothelial NO synthase (eNOS) and neuronal NOS (nNOS) on the angiotensin II response in afferent arterioles (Af). Dose responses were assessed for angiotensin II in microperfused Af of mice homozygous for disruption of the eNOS gene [eNOS(-/-)], or nNOS gene [nNOS(-/-)], and their wild-type controls, eNOS(+/+) and nNOS(+/+). Angiotensin II at 10(-8) and 10(-6) mol/l reduced the lumen to 69% and 68% in eNOS(+/+), and to 59% and 50% in nNOS(+/+). N(G)-nitro-L-arginine methyl ester (L-NAME) did not change basal arteriolar diameters, but augmented angiotensin II contraction, reducing diameters to 23% and 13% in eNOS(+/+), and 7% and 10% in nNOS(+/+) at 10(-8) and 10(-6) mol/l. The response to angiotensin II was enhanced in nNOS(-/-) mice (41% and 25% at 10(-8) and 10(-6) mol/l) and even more enhanced in eNOS(-/-) mice (12% and 9%) compared with nNOS(+/+) and eNOS(+/+). L-NAME led to complete constriction of Af in these groups. Media-to-lumen ratios of Af did not differ between controls and gene-deficient mice. mRNA expression of angiotensin II receptor types 1A and 1B and type 2 also did not differ. The results reveal that angiotensin II-induced release of NO from both eNOS and nNOS significantly contributes to the control of Af. Results also suggest that eNOS-derived NO is of greater importance than nNOS-derived NO in this isolated arteriolar preparation.

Earlier electron microscopy studies demonstrated morphological signs of fluid flow in the juxtaglomerular apparatus (JGA), including fenestrations of the afferent arteriole (AA) endothelium facing renin granular cells. We aimed to directly visualize fluid flow in the JGA, the putative function of the fenestrated endothelium, using intravital multiphoton microscopy of Munich-Wistar rats and C57BL6 mice. Renin content of the AA correlated strongly with the length of the fenestrated, filtering AA segment. Fluorescence of the extracellular fluid marker lucifer yellow (LY) injected into the cannulated femoral vein in bolus was followed in the renal cortex by real-time imaging. LY was detected in the interstitium around the JG AA before the plasma LY filtered into Bowman's capsule and early proximal tubule. The fluorescence intensity of LY in the JGA interstitium was 17.9 +/- 3.5% of that in the AA plasma (n = 6). The JGA fluid flow was oscillatory, consisting of two components: a fast (one every 5-10 s) and a slow (one every 45-50 s) oscillation, most likely due to the rapid transmission of both the myogenic and tubuloglomerular feedback (TGF)-mediated hemodynamic changes. LY was also detected in the distal tubular lumen about 2-5 s later than in the AA, indicating the flow of JGA interstitial fluid through the macula densa. In the isolated microperfused JGA, blocking the early proximal tubule with a micropipette caused significant increases in MD cell volume by 62 +/- 4% (n = 4) and induced dilation of the intercellular lateral spaces. In summary, significant and dynamic fluid flow exists in the JGA which may help filter the released renin into the renal interstitium (endocrine function). It may also modulate TGF and renin signals in the JGA (hemodynamic function).

ATP release from macula densa (MD) cells into the interstitium of the juxtaglomerular (JG) apparatus (JGA) is an integral component of the tubuloglomerular feedback (TGF) mechanism that controls the glomerular filtration rate. Because the cells of the JGA express a number of calcium-coupled purinergic receptors, these studies tested the hypothesis that TGF activation triggers a calcium wave that spreads from the MD toward distant cells of the JGA and glomerulus. Ratiometric calcium imaging of in vitro microperfused isolated JGA-glomerulus complex dissected from rabbits was performed with fluo-4/fura red and confocal fluorescence microscopy. Activation of TGF by increasing tubular flow rate at the MD rapidly produced a significant elevation in intracellular Ca(2+) concentration ([Ca(2+)](i)) in extraglomerular mesangial cells (by 187.6 +/- 45.1 nM) and JG renin granular cells (by 281.4 +/- 66.6 nM). Subsequently, cell-to-cell propagation of the calcium signal at a rate of 12.6 +/- 1.1 microm/s was observed upstream toward proximal segments of the afferent arteriole and adjacent glomeruli, as well as toward intraglomerular elements including the most distant podocytes (5.9 +/- 0.4 microm/s). The same calcium wave was observed in nonperfusing glomeruli, causing vasoconstriction and contractions of the glomerular tuft. Gap junction uncoupling, an ATP scavenger enzyme cocktail, and pharmacological inhibition of P(2) purinergic receptors, but not adenosine A(1) receptor blockade, abolished the changes in [Ca(2+)](i) and propagation of the calcium wave. These studies provided evidence that both gap junctional communication and extracellular ATP are integral components of the TGF calcium wave.

Background: Gap junctions may play an important role in regulating renal blood flow and glomerular responses. We have therefore made a comprehensive analysis of connexin expression in the renal vasculature of control and diabetic mice since elevated glucose has been reported to down-regulate connexin 43 in vascular cells in vitro., Methods: Connexin distribution was determined with immunohistochemistry using subtype-specific and cell type-specific antibodies. Diabetes was induced with streptozotocin (120/80 mg/kg, intraperitoneally) in C57BL/6 mice., Results: Connexins 37, 40, and 43 were expressed in endothelial cells of the renal, lobar, arcuate, and interlobular arteries and afferent arterioles, although connexin 43 was weak in the renal and arcuate arteries. Connexin 37 was detected in the media of arcuate, interlobular arteries and afferent arterioles and connexins 37 and 40 were found in renin-secreting cells. Both connexins 37 and 40 were expressed in extraglomerular mesangial cells, connexin 40 was abundantly expressed in intraglomerular mesangial cells, but connexin 37 was limited to mesangial cells at the vascular pole. In contrast, only connexin 43 was detected in endothelial cells of efferent arterioles and there was no connexin staining in the media. In diabetes, connexin 40 was expressed in smooth muscle cells along afferent arterioles, glomerular connexin staining was more extensive and connexin 43 was detected in renin-secreting cells. In contrast connexin 43 expression in endothelial cells of efferent arterioles was markedly reduced., Conclusion: The renal vasculature and mesangial cells are well coupled on the preglomerular side but there is little evidence that the coupling extends into the efferent arteriole. This pattern of cell coupling is accentuated during diabetes.

Experiments were performed to investigate the potential role of Src family kinase(s) in the rat afferent arteriolar contractile response to ANG II. The in vitro blood-perfused juxtamedullary nephron technique was employed to monitor afferent arteriolar lumen diameter responses to 1-100 nM ANG II before and during Src family kinase inhibition (10 microM PP2). PP2 did not alter baseline diameter but attenuated ANG II-induced contractile responses by 33 +/- 6%. An inactive analog of PP2 (PP3) had no effect on ANG II-induced afferent arteriolar contraction. The effect of Src kinase inhibition on ANG II-induced intracellular free Ca(2+) concentration ([Ca(2+)](i)) responses was probed in fura 2-loaded preglomerular microvascular smooth muscle cells (PVSMCs) obtained from explants and studied after 3-5 days in culture. In untreated PVSMCs, ANG II evoked peak (Delta = 293 +/- 66 nM) and plateau (Delta = 23 +/- 8 nM) increases in [Ca(2+)](i). In PVSMCs pretreated with PP2, baseline [Ca(2+)](i) was unaltered, but both the peak (Delta = 140 +/- 22 nM) and plateau (Delta = 3 +/- 2 nM) phases of the ANG II response were significantly reduced compared with untreated cells. PP3 did not alter [Ca(2+)](i) responses to ANG II. Immunoprecipitation and Western blot analysis confirmed that 100 nM ANG II increased phosphorylation of c-Src (at Y(416)) in PVSMCs. The phosphorylation response was maximal 1 min after ANG II exposure and was prevented by PP2. We conclude that the preglomerular vasoconstriction evoked by ANG II involves rapid c-Src activation with subsequent effects that contribute to the [Ca(2+)](i) response to the peptide.

The signaling pathways of endothelin (ET)-1-mediated vasoconstriction in the renal circulation have not been elucidated but appear to be distinct between ET(A) and ET(B) receptors. The purpose of this study was to determine the role of L-type Ca(2+) channels in the vasoconstrictor response to ET-1 and the ET(B) receptor agonist sarafotoxin 6c (S6c) in the rat kidney. Renal blood flow (RBF) was measured with an ultrasonic flow probe in anesthetized rats, and a microcatheter was inserted into the renal artery for drug infusion. All rats were given vehicle (0.9% NaCl) or three successive bolus injections (1, 10, and 100 pmol) of ET-1 or S6c at 30-min intervals (n = 6 in each group). ET-1 and S6c produced dose-dependent decreases in RBF. The Ca(2+) channel blocker nifedipine (1.5 mug) significantly attenuated the RBF response only at the highest doses of ET-1 and S6c. In the isolated blood-perfused juxtamedullary nephron preparation, Ca(2+) channel blockade with diltiazem had a very small inhibitory effect on ET-1-induced decreases in afferent arteriolar diameter only at the lowest concentrations of ET-1. In vascular smooth muscle cells isolated from preglomerular vessels, ET-1 produced a typical biphasic Ca(2+) response, whereas S6c had no effect on cytosolic Ca(2+). Furthermore, Ca(2+) channel blockade (diltiazem or Ni(2+)) had no effect on the peak or sustained increase in cytosolic Ca(2+) produced by ET-1. These results support the hypothesis that L-type Ca(2+) channels play only a minor role in the constrictor responses to ET-1 in the renal microcirculation.

Background: Recent studies have shown that adenosine triphosphate (ATP) is liberated from macula densa cells in response to increased tubular NaCl in vitro. We tested the hypothesis that increased NaCl in the macula densa stimulates the release of ATP, resulting in extracellular formation of adenosine which is involved in signal transmission of the tubuloglomerular feedback response., Methods: Rabbit afferent arterioles and attached macula densas were simultaneously microperfused in vitro. Tubuloglomerular feedback was induced by increasing macula densa Na/Cl from 11/10 to 81/80 mmol/L and was measured before and after treatment., Results: We first tested whether hydrolysis of ATP is required for tubuloglomerular feedback. When we enhanced conversion of ATP to adenosine by adding hexokinase or apyrase to the bath and arteriole lumen, the tubuloglomerular feedback response was augmented. During the control period, tubuloglomerular feedback decreased arteriole diameter by 2.2 +/- 0.2 microm. In the presence of hexokinase, tubuloglomerular feedback decreased diameter by 3.4 +/- 0.3 microm (N= 8) (P < 0.05, with vs. without hexokinase). In the apyrase group, tubuloglomerular feedback decreased diameter by 2.7 +/- 0.4 microm during the control period. When apyrase was added, tubuloglomerular feedback decreased diameter by 4.7 +/- 0.4 microm (N= 8) (P < 0.05, with vs. without apyrase). When hydrolysis of adenosine monophosphate (AMP) to adenosine was blocked by supplementing the bath with 100 micromol/L alpha,beta-methylene adenosine 5'-diphosphate (MADP), an inhibitor of 5'-nucleotidase, tubuloglomerular feedback response was blocked and diameter remained unchanged. We next studied whether ATP released from the macula densa binds to P(2) receptors and activates the tubuloglomerular feedback response. The P(2) purinergic receptor inhibitor suramin was added to both arteriole lumen and bath. During the control period, tubuloglomerular feedback decreased diameter by 3.7 +/- 0.5 microm. Suramin (100 micromol/L) did not significantly inhibit tubuloglomerular feedback, since in the presence of suramin diameter decreased by 3.8 +/- 0.3 microm (N= 7). Finally, we added the adenosine A(1) receptor inhibitor FK838 to both bath and lumen and found that it completely blocked high NaCl-induced tubuloglomerular feedback., Conclusion: We concluded that ATP released from the macula densa is broken down to form AMP in the extracellular space. AMP in turn is degraded by ecto-5'-nucleotidases to adenosine, which mediates signal transmission of the tubuloglomerular feedback response.

Introduction: Earlier publications show the endothelial fenestration at the juxtaglomerular part of the afferent arteriole facing mesangial and granular cells and finger-like protrusions of urinary space into the region of the lacis in experimental animals such as the rat, the mouse, and Tupaia belangery. In human kidney both the endothelial fenestration in the wall of the afferent arteriole and protrusions with filtration slits of urinary space into the lacis, were observed with remarkable heterogeneity. There was found the fenestrated entdothelium facing the reningranulated cells of Amphiuma kidney too. Arteriolosclerosis frequently occurs in IgA nephritis, and is the hallmark of benign nephrosclerosis. The quantitative ultrastructure of juxtaglomerular arterioles is not known in these disorders., Aims and Methods: The aim of both studies was to characterize the arteriolar parameters using stereology. In normal kidney was the permeability studied along the full length of afferent arteriole using ferritin particles as indicator of permeability/fenestration. In renal biopsies from patients with IgA nephritis and patients with benign nephrosclerosis the afferent and efferent arterioles were examined. Age-matched living renal transplant donors were used as controls. The thickness of the media (myomedial cells plus the mátrix) and the thickness of the medial matrix were estimated. From these estimates, the mátrix/myomedia ratio was calculated., Results: In the normal kidney the density of ferritin particles was high close to the glomerulus and decreased continuously similarly to the profile of renin distribution. There was a correlation between the length of ferritin-positive and renin-positive portions. Based on these results, the afferent arteriole, according to its endothelial permeability, can be divided into two distinct portions i.e. the permeable and the non-permeable, the length and ratio of which may be related to the actual renin formation. In IgA nephritis with normotension or hypertension, the afferent media and its components did not exhibit significant thickening compared with the controls, whereas in benign nephrosclerosis the afferent media and its layers were markedly and significantly thickened. The efferent media in IgA nephritis and benign nephrosclerosis displayed mild and significant thickening, with normotension., Conclusion: The results indicate that the afferent arterioles are not the main sites of IgA nephritis-related arteriolosclerosis, and that benign nephrosclerosis represents different lesions. The stereological method was successfully used and provided essential information about the arterioles in both study.

ANG II controls the vascular tone of pre- and postglomerular arterioles, and thereby glomerular filtration, through binding to either AT1A, AT1B, or AT2 receptors. AT1 receptors, which are coupled to intracellular Ca2+ signaling, have vasoconstricting effects, whereas AT2 receptors, whose signaling mechanism is unknown, induce vasodilatation. The angiotensin receptors have been characterized in afferent arterioles, which express the three types of receptors, but not in efferent arterioles. Two subpopulations of juxtamedullary efferent arterioles, muscular ones which terminate as vasa rectae and thin ones which terminate as peritubular capillaries, have been described. They display functional heterogeneity with regard to the ANG II response. To evaluate whether these differences are associated with differential expression of ANG II receptors, we examined the expression pattern of AT1A, AT1B, and AT2 receptor mRNAs by RT-PCR in these arterioles and studied the effect of valsartan, a specific AT1-receptor antagonist. Results indicate that muscular arterioles express AT1A, AT1B, and AT2 receptors, whereas thin arterioles only express the AT1A and AT2 types, and at a much lower level. Valsartan fully inhibited ANG II-induced increases in intracellular Ca2+ in both arteriolar types, but with different kinetics. In muscular arterioles, inhibition was monoexponential, whereas it displayed a marked positive cooperativity in thin arterioles. Finally, the apparent affinity for valsartan was higher in muscular than in thin arterioles. In conclusion, this study further documents the differences between muscular and thin efferent arterioles with regard to ANG II signalization in the rat kidney.

The molecular mechanisms with which the juxtaglomerular apparatus accomplishes its twin functions, acute regulation of glomerular blood flow and secretion of renin, are still not clearly understood. Least understood is the role of the extraglomerular mesangial (EM) cells, also known as lacis or Goormaghtigh cells, which lie sandwiched between the macula densa and the afferent and efferent arterioles. Here, we report that immunoreactivity for phospholemman (FXYD1), a single-span membrane protein homologous to the gamma (gamma) sub-unit of the Na,K-ATPase, is found in the kidney in EM cells with the Na,K-ATPase beta2-subunit and in cortical blood vessels and the afferent arteriole with Na,K-ATPase alpha2 and beta2. Phospholemman's distribution in EM cells is distinct from that of the Na,K-ATPase gamma-subunit, which is found on the basolateral surface of macula densa cells with Na,K-ATPase alpha1 and beta1. Phospholemman is a major kinase target, and its location in the juxtaglomerular apparatus suggests that it is involved in tubuloglomerular feedback.

Because renin and angiotensin I (ANG I) level are high in the renal circulation, the conversion of ANG I is a critical step in the regulation of glomerular hemodynamics. We studied this conversion by investigating the effect of ANG I on intracellular Ca(2+) concentration ([Ca(2+)](i)) in rat juxtamedullary glomerular afferent and efferent arterioles (AA and EA, respectively). Two types of EA were considered, thin EA and muscular EA, terminating as peritubular capillaries and vasa rectae, respectively. In all arterioles, ANG I elicited [Ca(2+)](i) elevations. Maximal responses of 171 +/- 28 (AA), 183 +/- 7 (muscular EA), and 78 +/- 11 nM (thin EA) (n = 6), similar to those obtained with ANG II, were observed with 100 nM ANG I. The EC(50) values were 20 times higher for ANG I than for ANG II in AA (10.2 vs. 0.5) and muscular EA (6.8 vs. 0.4 nM) and 150 times higher in thin EA (15.2 vs. 0.1 nM). ANG I effect was blocked by losartan, indicating that AT(1) receptors were involved. The ANG-converting enyzme (ACE) inhibitor lisinopril inhibited the maximal response to ANG I in AA and muscular EA by 75 +/- 9% (n = 13) and 70 +/- 7% (n = 13), respectively, but had no effect in thin EA (n = 14). The serine protease inhibitor aprotinin, the chymase inhibitor chymostatin, and the cysteine protease inhibitors E64 and leupeptin had no effect on ANG I action. These data show that ANG I effects are mainly mediated by ACE in AA and muscular EA but not in thin EA. The lisinopril-insensitive response may be related to conversion by unknown enzyme(s) and/or to activation of AT(1) receptors by ANG I.

Recently, multiphoton excitation fluorescence microscopy has been developed that offers important advantages over confocal imaging, particularly for in vivo visualization of thick tissue samples. We used this state-of-the-art technique to capture high-quality images and study the function of otherwise inaccessible cell types and complex cell structures of the juxtaglomerular apparatus (JGA) in living preparations of the kidney. This structure has multiple cell types that exhibit a complex array of functions, which regulate the process of filtrate formation and renal hemodynamics. We report, for the first time, on high-resolution three-dimensional morphology and Z-sectioning through isolated, perfused kidney glomeruli, tubules, and JGA. Time-series images show how alterations in tubular fluid composition cause striking changes in single-cell volume of the unique macula densa tubular epithelium in situ and how they also affect glomerular filtration through alterations in associated structures within the JGA. In addition, calcium imaging of the glomerulus and JGA demonstrates the utility of this system in capturing the complexity of events and effects that are exerted by the specific hypertensive autacoid angiotensin II. This imaging approach to the study of isolated, perfused live tissue with multiphoton microscopy may be applied to other biological systems in which multiple cell types form a functionally integrated syncytium.

Experiments were performed in mice to investigate whether cyclooxygenase-2 (COX-2) in epithelial cells near the tubulovascular contact point (macula densa and TAL cells) may regulate renin gene expression in juxtaglomerular granular cells. Renin activity, afferent arteriolar granularity, and renin mRNA were determined in wild-type mice and in COX-2-knockout mice on control and low-NaCl diets. Renin activity in microdissected glomeruli assessed as angiotensin I formation in the presence of excess substrate and afferent arteriolar granularity determined by direct visualization and immunostaining were significantly reduced in COX-2 -/- compared with wild-type animals. Similarly, renal cortical mRNA levels were lower in COX-2 -/- than in wild-type mice. Maintaining mice on a low-salt diet for 14 days induced an increase in renin mRNA, afferent arteriolar granularity, and renin activity in wild-type mice. In contrast, renin mRNA and renin granularity did not significantly increase in low-salt-treated COX-2 -/- mice, whereas the increase in juxtaglomerular renin enzyme activity was markedly attenuated, but not fully blocked. In additional experiments we found that COX-2 mRNA was increased in angiotensin type 1A receptor-knockout mice compared with wild-type mice. We conclude that COX-2 in the tubulovascular contact region is a critical determinant of renin synthesis in granular cells under resting conditions and that it participates in the stimulation of renin expression caused by a low-NaCl intake.

The presence and distribution of S100-like protein in the goldfish (Carassius auratus L.) kidney has been studied by the use of immunohistochemical and histochemical methods. Simple immunohistochemistry (peroxidase anti-peroxidase method) was carried out with a polyclonal antibody against a mixture of both S100alpha and S100beta proteins. In order to confirm the cell-type containing S-100-like immunoreactivity, the colocalization of S-100-like protein immunoreactivity with periodic acid-Schiff (PAS) reaction was investigated by using double staining with indirect immunofluorescence and PAS histochemistry. S100-like immunoreactivity was detected only in juxtaglomerular cells located in the renal arterial branch and never on afferent arterioles. No immunoreactivity was observed in other tracts of the nephron or in the interstitial cells. Double staining confirmed that S-100-like immunoreactivity and PAS reactivity were colocalized in juxtaglomerular cells. These findings are the first regarding the presence and distribution of S100-like protein in the teleost kidney; they add a new member to the list of extra-neural S100-like-containing cell types and confirm that the antigen cannot be regarded as nervous-system-specific. In addition, a concentration of S100-like immunoreactivity in juxtaglomerular cells suggests the presence of S100-like calcium-binding protein-mediated activities in these cell types.

The balance of the vascular tone between afferent and efferent arterioles (AAs and EAs, respectively) is a crucial determinant of glomerular hemodynamics. Thus, it is important to study the mechanisms that control their vascular tone to understand renal physiology and pathophysiology. In order to directly study the mechanisms that regulate their vascular tone, we have developed several in vitro microperfusion preparations of these arterioles, which have the advantage of allowing us to observe the arteriolar diameter directly in the absence of systemic hemodynamic and hormonal influences. In the AA but not EA, we have directly demonstrated the presence of two intrinsic mechanisms, namely the myogenic response and macula densa-mediated tubuloglomerular feedback, that play an important role in the control of vascular tone. We also found that both mechanism-induced constrictions of AAs can be modulated by endogenous nitric oxide. In addition, several humoral factors (such as angiotensin II or prostaglandins) play an important role in the control of AA tone. On the other hand, angiotensin II is one major factor that controls the vascular tone of the EA. We have found that the vasoconstrictor effect of angiotensin II on EAs is modulated by prostaglandins produced by the upstream glomerulus. Thus, this may be a mechanism whereby the glomerulus controls its own capillary pressure by releasing prostaglandins and thereby adjusting the resistance of the downstream EA. These varying mechanisms regulating AA and EA tone play an important role in the precise control of glomerular hemodynamics., (Copyright 2000 S. Karger AG, Basel)

Through the use of microanatomic techniques, we investigated the effects of treatment with some dihydropyridine-type calcium antagonists (CAs) (ie, lercanidipine, manidipine, and nicardipine) and with the nondihydropyridine-type vasodilator hydralazine on hypertension-dependent glomerular injury and on the morphology of afferent and efferent arterioles in spontaneously hypertensive rats (SHR). Fourteen-week-old male SHR and age-matched normotensive Wistar-Kyoto rats were left untreated (control groups). Four additional groups of 14-week-old SHR were treated for 12 weeks with daily oral doses of 2.5 mg/kg lercanidipine, 5 mg/kg manidipine, 3 mg/kg nicardipine, or 10 mg/kg hydralazine. These treatments decreased systolic blood pressure values to a similar extent in SHR. Signs of glomerular injury, as characterized by glomerulosclerosis, hypertrophy, and an increased number of mesangial cells, were observed in control SHR. The treatment with CAs improved glomerular morphology and decreased the number of mesangial cells. Lercanidipine and manidipine were more effective than nicardipine in countering glomerular injury. In the SHR, both afferent and efferent arterioles revealed luminal narrowing, accompanied by increased wall thickness in efferent arterioles. The dihydropyridine-type derivatives that were tested decreased the luminal narrowing of afferent arterioles. Lercanidipine and manidipine countered the luminal narrowing of efferent arterioles. Hydralazine had no effect on hypertension-dependent glomerular injury or vascular changes. The present data indicate that lercanidipine and manidipine vasodilate afferent and efferent arterioles in SHR. A vasodilatory activity on efferent arteriole, which is not induced by the majority of CAs, may represent an useful property in the treatment of hypertension complicated by renal disease.

Although transforming growth factor-beta (TGF-beta) has been shown to participate in regulating hormone synthesis and release, little is known about involvement of individual human TGF-beta isoforms, TGF-beta1, -beta2, and -beta3, in renin synthesis and release. We examined expression of these TGF-beta isoforms in a 50-year-old man with a Bartter's-like syndrome whose renal biopsy specimen showed hyperplasia of the juxtaglomerular apparatus (GA), mild mesangial hypercellularity, focal tubular atrophy, and interstitial fibrosis. Immunoreactivity for renin and marked expression of TGF-beta2 mRNA were noted in the glomerular arteriole of the JGA, whereas mRNA expression for TGF-beta1 was only slight and that for TGF-beta3 was still more faint. Expression of mRNAs for all 3 TGF-beta isoforms was increased in the fibrotic interstitium. This expression pattern suggests that TGF-beta2 may be involved in inducing renin synthesis and/or release in the glomerular arteriole of the JGA.

The current study determined the contribution of protein kinase-A (PKA) and protein kinase-G (PKG) to the vasodilation elicited by the N-methylsulfonimide analog of 11,12-epoxyeicosatrienoic acid (11, 12-EET). Experiments were performed, in vitro, using the juxtamedullary nephron preparation combined with videomicroscopy. The response of afferent arterioles to the sulfonimide analog of 11, 12-EET, was determined before and after inhibition of PKA, PKG, or guanylyl cyclase. Afferent arterioles, preconstricted with 0.5 micromol/L norepinephrine, averaged 18+/-1 microm (n=25) at a renal perfusion pressure of 100 mm Hg. Superfusion with 0.01 to 100 nmol/L of the 11,12-EET analog caused a graded increase in diameter of the afferent arteriole. Vessel diameter increased by 11+/-1% and 15+/-1%, respectively, in response to 10 and 100 nmol/L of the 11,12-EET analog. The afferent arteriolar response to 10 and 100 nmol/L of the 11,12-EET analog was significantly attenuated during inhibition of PKA with 10 micromol/L H-89 (n=7) or 5 micromol/L myristolated PKI (n=6), such that afferent arteriolar diameter increased by only 5+/-2% and 2+/-1%, respectively, in response to 100 nmol/L of the 11, 12-EET analog. In contrast, the afferent arteriolar vasodilatory response to the 11,12-EET analog was unaffected by PKG or guanylyl cyclase inhibition. In the presence of 200 micromol/L histone H2B (n=5) or 10 micromol/L ODQ (n=7), the afferent arteriolar diameter increased by 16+/-3% and 12+/-2%, respectively, in response to 100 nmol/L of the 11,12-EET analog. These results demonstrate that activation of PKA is an important mechanism responsible for the afferent arteriolar vasodilation elicited by the sulfonimide analog of 11,12-EET.

This study was designed to determine the influence of neuronal nitric oxide synthase (nNOS) in tubular flow-dependent regulation of afferent arteriolar diameter in hypertensive Sprague-Dawley rats that received 60 ng/min angiotensin II (Ang II) subcutaneously for 13 days. Systolic blood pressure of control and Ang II-infused rats averaged 122+/-2 (n=23) and 194+/-2 mm Hg (n=24). Afferent arteriolar responses to the nNOS inhibitor S-methyl-L-thiocitrulline (L-SMTC; 0.1 to 10 micromol/L) and the nonselective NOS inhibitor Nomega-nitro-L-arginine (L-NNA; 1 to 100 micromol/L) were assessed in vitro using the blood-perfused juxtamedullary nephron preparation. At a perfusion pressure of 160 mm Hg, afferent arteriolar diameters from control and Ang II-infused rats averaged 18.7+/-1.1 microm (n=8) and 18.1+/-1.1 microm (n=9), respectively, and decreased by 19. 9+/-1.5% and 11.8+/-1.1%, respectively, in response to 10 micromol/L L-SMTC. The L-SMTC-induced afferent arteriolar constriction was significantly greater in control than in Ang II-infused rats. In contrast, 100 micromol/L L-NNA constricted afferent arterioles similarly in both control (n=8) and Ang II-infused (n=7) rats. After transection of the loops of Henle to interrupt flow to the macula densa, the vasoconstrictor responses to L-SMTC but not to L-NNA were reversed. Increasing distal volume delivery by addition of 10 mmol/L acetazolamide to the blood perfusate significantly enhanced the afferent arteriolar constrictor responses to 10 micromol/L L-SMTC (34.5+/-4.8%, n=7) in normotensive rats. In contrast, in Ang II-infused rats, acetazolamide treatment did not enhance the responses to L-SMTC (n=8). These results indicate that chronic Ang II infusion reduces the ability of nNOS-derived nitric oxide to counteract the afferent arteriolar response to increased distal tubular flow.

To delineate the microvascular role of cyclooxygenase-2 (Cox-2) in modulating tubuloglomerular feedback (TGF) signals and to determine its relationship to neuronal nitric oxide synthase (nNOS), afferent (AA) and efferent (EA) arteriolar diameters of rat kidneys were assessed using the blood-perfused juxtamedullary nephron technique. The Cox-2 inhibitor NS-398 (10 microM) did not alter AA diameters in untreated kidneys but significantly constricted AAs by 17.0 +/- 2.2% in kidneys treated with 10 mM acetazolamide, which enhances TGF-mediated AA constriction by increasing distal volume delivery. The NS-398-induced AA constriction was prevented after interruption of distal delivery by transection of the loops of Henle. The effect was selective for AAs since NS-398 did not influence EAs of untreated or acetazolamide-treated kidneys. Pretreatment with the nNOS inhibitor S-methyl-L-thiocitrulline (10 microM) prevented the NS-398-induced AA constriction observed during acetazolamide treatment. Although we previously demonstrated that acetazolamide treatment enhanced AA constrictor response to S-methyl-L-thiocitrulline, the enhancement by acetazolamide was inhibited by pretreatment with 10 microM NS-398 (16.4 +/- 1.9 and 15. 0 +/- 0.5% with and without acetazolamide, respectively, P > 0.05). These results indicate that, during increased activation of TGF-dependent vasoconstrictor signals, Cox-2 generates vasodilatory metabolites in response to increased nNOS activity and thus participates in the counteracting modulation of TGF-mediated AA constriction.

Impairment of autoregulation (AR) is associated with accelerated progression of chronic renal failure (CRF). As the bioavailability of insulin-like growth factor-I (IGF-I) is low in CRF, we investigated the effects of acute luminal application of 10 nM recombinant human IGF-I on AR in juxtamedullary (JM) afferent arterioles (AA) perfused in vitro with a blood solution [(approximately 30% hematocrit (HCT)]. Studies were conducted in AA from adult male rats three to four weeks after five-sixths nephrectomy (Nx) by either surgical excision (N = 7) or infarction (N = 5) of two thirds of the remnant kidney; controls (N = 6) had sham surgery. AA from both Nx groups exhibited marked hypertrophy and impaired AR responses (60 to 140 mm Hg perfusion pressure), features more pronounced in the infarction group. Responses to abluminal acetylcholine (10 microM) were similar in sham and excision groups but were significantly blunted in the infarction group. All groups vasodilated significantly after Ca-channel blockade (10 mM MnCl2). IGF-I restored AR in AA from both Nx groups (P < 0.05, analysis of variance) while it vasodilated AA from controls. These results suggest that IGF-I may protect the glomerulus from injury by maintaining autoregulatory control of renal blood flow, thereby slowing the progression of CRF.

The type 1 brain nitric oxide synthase (bNOS) isoform occurs in macula densa (MD) cells where it functions to vasodilate the afferent arteriole and blunt expression of tubuloglomerular feedback (TGF). Dietary salt restriction enhances bNOS expression, yet microperfusion studies with NOS inhibitors imply that it is functionally inactive. We thus assessed the hypothesis that reduced L-arginine (L-Arg) availability during low salt (LS) intake limits MD NO generation. Maximal TGF responses were recorded during Henle's loop perfusion with artificial tubular fluid (ATF). Microperfusion of L-Arg into the MD of LS, but not normal or high-salt (HS) rats blunted maximal TGF responses (8.0 +/- 0.4 to 6.0 +/- 0.5 mm Hg; N = 23; P < 0.01). Response to L-Arg was stereospecific, inhibited by coperfusion with monomethyL-L-arginine (L-NMA), and dependent on system y+ transport, because it was blocked by coperfusion with the competitors L-lysine or L-homoarginine. Absorption of [3H]-L-Arg from the perfused loop, via an L-Arg- or L-homoarginine-inhibitable process, was enhanced during HS. Salt restriction thus diminishes TGF attenuation by NO in the MD despite enhanced bNOS expression because of limited delivery and/or uptake of L-Arg via system y+. This defines a novel mechanism of renal microcirculatory adaptation to salt restriction via L-Arg-dependent changes in TGF.

Tubuloglomerular feedback (TGF), which operates between the tubule and the parent glomerulus, is important to renal autoregulation and homeostasis of body fluid and electrolytes. The juxtaglomerular apparatus (JGA) has long been suggested as the anatomical site of TGF. To study the function of the JGA directly, we developed an in vitro preparation in which both the afferent arteriole (Af-Art) and macula densa (MD) of a microdissected rabbit JGA are microperfused simultaneously. We see that increasing the [NaCl] of the MD perfusate constricts the afferent arteriole in the segment close to the glomerulus. This constriction is blocked by furosemide, a loop diuretic known to inhibit TGF. On the other hand, microperfusion of Af-Arts alone showed the myogenic response to exist in the more proximal segments. Such an anatomical relationship between the myogenic response and TGF may enable the kidney to achieve its extremely efficient autoregulation.

Preglomerular responses to vasoactive agonists utilize calcium released from intracellular stores and activation of calcium influx pathways to elicit vasoconstriction. The current study was performed to determine the role of calcium release from intracellular stores on the afferent arteriolar response to increases in perfusion pressure. Experiments were performed, in vitro, using the blood perfused, juxtamedullary nephron technique combined with videomicroscopy. The response of afferent arterioles to 30 mm Hg increases in perfusion pressure was determined before and after depletion of intracellular calcium pools with a 10-minute preincubation with 1 micromol/L thapsigargin or 100 micromol/L cyclopiazonic acid. Afferent arteriolar diameter averaged 20.2+/-1.0 microm (n=19) at a control perfusion pressure of 100 mm Hg. Increasing perfusion pressure to 130 and 160 mm Hg reduced afferent caliber by 10.7+/-1.0% (P<.05 versus con) and by 24.7+/-1.6% (P<.05 versus diameter at 130 mm Hg); respectively. Thapsigargin significantly increased afferent diameter by 21+/-2% (n=6) at 100 mm Hg and prevented pressure-induced autoregulatory responses. Afferent diameter averaged 24.3+/-1.7, 24.5+/-1.8 and 24.3+/-1.8 microm at perfusion pressures of 100, 130 and 160 mm Hg; respectively. Cyclopiazonic acid treatment also inhibited autoregulatory behavior but did not alter resting vessel diameter. Afferent arteriolar diameter (n=6) averaged 21.4+/-1.9 microm at 100 mm Hg and 20.9+/-2.1 and 20.5+/-2.2 microm at 130 and 160 mm Hg; respectively. Additional studies were performed to assess the role of phospholipase C activity in pressure-mediated autoregulatory behavior of afferent arterioles. Step increases in perfusion pressure decreased afferent diameter by 10.7+/-3.8 and 21.7+/-4.1%; respectively. Administration of the phospholipase C inhibitor, U-73122, (5 micromoles/L) did not significantly alter baseline diameter but did attenuate the pressure-mediated vasoconstrictor response. Increasing perfusion pressure to 130 and 160 mm Hg reduced afferent diameter by only 6.5+/-1.5 and 10.0+/-2.0%; respectively. These data demonstrate that interruption of calcium mobilization with thapsigargin, cyclopiazonic acid, or phospholipase C inhibition markedly attenuates pressure-mediated afferent arteriolar vasoconstriction and suggests that autoregulatory adjustments in afferent arteriolar diameter involve calcium release from inositoltrisphosphate(IP3)-sensitive intracellular stores.

The insulin-responsive glucose transporter, GLUT-4, is found primarily in adipocytes and skeletal muscle cells, where it is sequestered in a specialized recycling compartment, from which it can be recruited to the cell surface following insulin stimulation. Lower levels of GLUT-4 are also expressed in other tissues, including the kidney, where it is present particularly in cells of the afferent arteriole and juxtaglomerular apparatus (JGA). The exact nature of GLUT-4-containing compartments and their relationship to other regulated trafficking pathways in different cells are not yet well defined. The trafficking of GLUT-4 has been studied in different cells with regulated secretory pathways, and a recent study shows that, in cardiomyocytes, GLUT-4 is sorted and packaged into multiple regulated pathways (J. W. Slot, G. Garruti, S. Martin, V. Oorschot, G. Pshuma, E. W. Kraegen, R. Laybutt, G. Thibault, and D. E. James. J. Cell Biol. 137: 1243-1254, 1997). In the kidney, cells of the JGA synthesize and secrete their major product, renin, via a well-established, regulated, secretory pathway. These cells also express GLUT-4 and thus offer the potential to directly compare the localization and trafficking of GLUT-4 and renin in a unique cell type. The present study was undertaken to investigate the intracellular distribution of GLUT-4 in mouse kidney cortex and to determine whether GLUT-4 and renin are trafficked in the same or in separate regulated pathways. Ultrathin cryosections of mouse kidney were labeled by the immunogold technique and viewed by electron microscopy, demonstrating the distribution of GLUT-4 in cells of the JGA, afferent arteriole, and distal tubule. In granular cells of the JGA, renin was localized in secretory granules of the regulated secretory pathway, whereas GLUT-4 labeling in the same cells was found in a distinct tubulovesicular compartment located adjacent to the trans-Golgi network. We show that granular cells have separate, morphologically distinct compartments for the sequestration of renin and GLUT-4, providing evidence that there may be distinct pathways for the sorting and trafficking of these two proteins.

To characterize the effects on the rat renal preglomerular microvasculature of insulin-like growth factor I (IGF-I), experiments were performed using the in vitro blood-perfused juxtamedullary nephron preparation. IGF-I induced a reversible vasodilation of pre- but not postglomerular microvessels in a dose-dependent manner (10(-9)-10(-7) M). The IGF-I-induced vasodilation was similar in all preglomerular vascular segments: interlobular artery, 11.5 +/- 1.2% of control (n = 16); mid-afferent arterioles, 11.6 +/- 1.7% (n = 24); and juxtaglomerular afferent segments, 16.1 +/- 2.8% (n = 19). Renal autoregulatory capacity was not reduced by IGF-I. Pretreatment with the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester (10(-4) M) completely inhibited the vasodilatory response to IGF-I. IGF-I induced a rapid increase of NO concentration in intact renal microvessels, monitored by a NO-selective voltametric microelectrode. Pretreatment with the cyclooxygenase inhibitor indomethacin (10(-5) M) not only abrogated the IGF-I-induced dilation, but, moreover, IGF-I elicited a small but significant (approximately 10%) vasoconstriction in all preglomerular vessels. These results indicate that the renal vascular effects of IGF-I involve activation of two endogenous vasodilators (NO and vasodilatory prostaglandins). In addition, IGF-I may also release an undefined vasoconstrictor.

The present study compares cytosolic calcium concentration ([Ca2+]i) responses to angiotensin II (ANG II) of afferent (AA) and efferent arterioles (EA) by taking account of the localization and morphological differences of EA. In outer cortex, 1 nM ANG II induced smaller [Ca2+]i increases in thin EA than in AA[48 +/- 10 (n = 12) vs. 94 +/- 7 nM (n = 11); P < 0.001]. In inner cortex, two types of EA were considered, i.e., thin and muscular ones. The response to 1 nM ANG II was 35% lower in thin than in muscular EA (P < 0.05) but did not differ from that obtained with corresponding AA. In EA of the outer cortex, 1 microM nifedipine, a dihydropyridine blocker of voltage-operated channels (VOCC), did not affect calcium influx, which was suppressed by 1 mM NiCl2, a nonselective calcium entry blocker. In other arterioles, nifedipine inhibited by approximately 40% calcium entry, and remaining influx was blocked by NiCl2. These results indicate a relationship between the magnitude of [Ca2+]i responses, activation of dihydropyridine-sensitive VOCC by ANG II, and the muscular morphology in renal glomerular arterioles. They suggest that ANG II regulates differently local renal microcirculation. They do not, however, support the hypothesis of a greater sensitivity to ANG II of EA compared with the AA of a given nephron.

1. Extraglomerular mesangial cells are strategically positioned between the macula densa and the afferent arteriole. These cells form a syncytium and are connected with glomerular mesangial cells via gap junctions. The model of immunoablation of mesangial cells in anti-Thy-1 glomerulonephritis carries the promise for understanding the function of mesangial cells as potential transmitters of information between the macula densa and the afferent arteriole. 2. The above anatomical relations between structures in the juxtaglomerular apparatus predict several possible routes of information flow. This review charts some hypothetical routes. 3. Research into the messenger systems involved in the transfer of signals from the macula densa to mesangial cells and from mesangial cells to the afferent arteriole suggests several candidate molecules to function in this capacity. Mechanisms of action for each candidate are discussed. 4. The oscillating nature of the afferent signal and efferent function in the tubuloglomerular feedback system, as well as other discoveries, offer a fertile field for future studies.

1. The balance of vascular tone of the afferent and efferent arteriole is a crucial determinant of glomerular haemodynamics. Despite their intimate anatomical relationship in the juxtaglomerular apparatus, the mechanisms that regulate afferent and efferent arteriolar tone are different. 2. In the afferent arteriole, two intrinsic mechanisms, the myogenic response and macula densa-mediated tubuloglomerular feedback (TGF) play a dominant role, maintaining the glomerular filtration rate (GFR) at a constant level over a wide range of renal perfusion pressure. Studies have shown that these two mechanisms are modulated by nitric oxide (NO). In addition, an interaction between TGF and angiotensin II (AngII) seems to be essential to maintaining GFR despite large variations in daily intake of salt and water. 3. In the efferent arteriole, neither myogenic response nor TGF seems to be important, while AngII is one major factor involved in the control of vascular resistance. In addition, recent studies have provided evidence that NO and prostaglandins produced by the glomerulus may control resistance of the downstream efferent arteriole. 4. As the early segment of the efferent arteriole resides within the glomerulus, various autacoid hormones produced by the glomerulus may reach and directly act on this segment, thereby controlling the glomerular capillary pressure. Thus, it would be important to understand the differences in the mechanisms operating at the afferent and efferent arteriole, as well as their alterations in various physiological and pathological conditions.

The spatial organization of autoregulatory responses (AR) was assessed in couples of afferent arterioles (AA), either grouped as anatomic pairs or branched sequentially along the same arcuate arterial branch (ArcB). With blood-perfused juxtamedullary nephron (JMN) preparations, AR were elicited by raising blood perfusion pressure from 60 to 120 mmHg and quantified by videomicroscopy as pressure-induced constrictions. Paired AA had unequal lengths (long-to-short ratio, 1.9 +/- 0.1; n = 36); however, no statistical difference in AR was found between long and short AA at juxtaglomerular or early AA (EAA) sites. Sequentially branched AA had the same length heterogeneity as paired AA (proximal-to-distal AA length ratio, 2.0 +/- 0.2; n = 30). However, AR exhibited a significant axial gradient, being higher in distal than in proximal AA or ArcB sites. In both AA branching patterns, EAA and nearby sites of the feed arteries had similar AR. Hence, our results are consistent with hemodynamic coupling in paired JMN. Around branching sites, AR are spatially organized in a way consistent with electrotonic vascular coupling.

Epoxygenase metabolites of arachidonic acid are produced by the kidney and have been implicated in the control of renal blood flow. This study examined the preglomerular actions of various epoxyeicosatrienoic acids (EET). By use of the in vitro blood-perfused juxtamedullary nephron preparation, interlobular and afferent arteriolar diameter responses to 5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET were determined. Diameters of interlobular and afferent arterioles preconstricted with 0.5 microM norepinephrine averaged 24 +/- 1 microns (N = 27) and 17 +/- 1 microns (N = 32), respectively, at a renal perfusion pressure of 100 mm Hg. Superfusion with 0.01 to 100 nM 11,12-EET caused graded increases in diameters of the interlobular and afferent arterioles. At a dose of 100 nM, 11,12-EET increased the diameters of the interlobular and afferent arterioles by 18 +/- 2% (N = 10) and 20 +/- 3% (N = 9), respectively. The vasodilatory response to 11,12-EET was stereoselective because 11,12(R,S)-EET but not 11,12(S,R)-EET increased the diameters of the interlobular and afferent arterioles. 14,15-EET had a much smaller effect and increased the diameters of the these vessels by 10%; 8,9-EET did not significantly affect vascular diameters. In contrast, 5,6-EET constricted the interlobular and afferent arterioles by 16 +/- 3% (N = 6) and 21 +/- 3% (N = 7), respectively. The corresponding diols, 5,6-DIHETE and 11,12-DIHETE, had no effect on diameters of the interlobular and afferent arterioles at concentrations up to 1 microM. The vasodilatory response to 11,12-EET was not affected by removal of the endothelium or by inhibition of cyclooxygenase with indomethacin. In contrast, the vasoconstrictor response to 5,6-EET was abolished by both removal of the endothelium or cyclooxygenase inhibition. The thromboxane/ enderoperoxide receptor inhibitor, SQ 29,548, resulted in a 60% attenuation of the afferent arteriolar vasconstriction to 5,6-EET. These results indicate that the preglomerular vasoconstriction to 5,6-EET is cyclooxygenase dependent and requires an intact endothelium, whereas the vasodilation to 11,12-EET is stereoselective and is the result of direct action of the epoxide on the preglomerular vascular smooth muscle.