Sympathetic nerve-derived ATP regulates renal medullary vasa recta diameter via pericyte cells: a role for regulating medullary blood flow?

Pericyte cells are now known to be a novel locus of blood flow control, being able to regulate capillary diameter via their unique morphology and expression of contractile proteins. We have previously shown that exogenous ATP causes constriction of vasa recta via renal pericytes, acting at a variety of membrane bound P2 receptors on descending vasa recta (DVR), and therefore may be able to regulate medullary blood flow (MBF). Regulation of MBF is essential for appropriate urine concentration and providing essential oxygen and nutrients to this region of high, and variable, metabolic demand. Various sources of endogenous ATP have been proposed, including from epithelial, endothelial, and red blood cells in response to stimuli such as mechanical stimulation, local acidosis, hypoxia, and exposure to various hormones. Extensive sympathetic innervation of the nephron has previously been shown, however the innervation reported has focused around the proximal and distal tubules, and ascending loop of Henle. We hypothesize that sympathetic nerves are an additional source of ATP acting at renal pericytes and therefore regulate MBF. Using a rat live kidney slice model in combination with video imaging and confocal microscopy techniques we firstly show sympathetic nerves in close proximity to vasa recta pericytes in both the outer and inner medulla. Secondly, we demonstrate pharmacological stimulation of sympathetic nerves in situ (by tyramine) evokes pericyte-mediated vasoconstriction of vasa recta capillaries; inhibited by the application of the P2 receptor antagonist suramin. Lastly, tyramine-evoked vasoconstriction of vasa recta by pericytes is significantly less than ATP-evoked vasoconstriction. Sympathetic innervation may provide an additional level of functional regulation in the renal medulla that is highly localized. It now needs to be determined under which physiological/pathophysiological circumstances that sympathetic innervation of renal pericytes is important.

Emerging key roles for P2X receptors in the kidney.

P2X ionotropic non-selective cation channels are expressed throughout the kidney and are activated in a paracrine or autocrine manner following the binding of extracellular ATP and related extracellular nucleotides. Whilst there is a wealth of literature describing a regulatory role of P2 receptors (P2R) in the kidney, there are significantly less data on the regulatory role of P2X receptors (P2XR) compared with that described for metabotropic P2Y. Much of the historical literature describing a role for P2XR in the kidney has focused heavily on the role of P2X1R in the autoregulation of renal blood flow. More recently, however, there has been a plethora of manuscripts providing compelling evidence for additional roles for P2XR in both kidney health and disease. This review summarizes the current evidence for the involvement of P2XR in the regulation of renal tubular and vascular function, and highlights the novel data describing their putative roles in regulating physiological and pathophysiological processes in the kidney.

Renal pericytes: regulators of medullary blood flow.

Regulation of medullary blood flow (MBF) is essential in maintaining normal kidney function. Blood flow to the medulla is supplied by the descending vasa recta (DVR), which arise from the efferent arterioles of juxtamedullary glomeruli. DVR are composed of a continuous endothelium, intercalated with smooth muscle-like cells called pericytes. Pericytes have been shown to alter the diameter of isolated and in situ DVR in response to vasoactive stimuli that are transmitted via a network of autocrine and paracrine signalling pathways. Vasoactive stimuli can be released by neighbouring tubular epithelial, endothelial, red blood cells and neuronal cells in response to changes in NaCl transport and oxygen tension. The experimentally described sensitivity of pericytes to these stimuli strongly suggests their leading role in the phenomenon of MBF autoregulation. Because the debate on autoregulation of MBF fervently continues, we discuss the evidence favouring a physiological role for pericytes in the regulation of MBF and describe their potential role in tubulo-vascular cross-talk in this region of the kidney. Our review also considers current methods used to explore pericyte activity and function in the renal medulla.

Fluorescence imaging of intracellular calcium signals in intact kidney tissue.

BACKGROUND: Intracellular calcium (Ca(2+)) plays an important role in normal renal physiology and in the pathogenesis of various kidney diseases; however, the study of Ca(2+) signals in intact tissue has been limited by technical difficulties, including achieving adequate loading of Ca(2+)-sensitive fluorescent dyes. The kidney slice preparation represents a model whereby three-dimensional tissue architecture is preserved and structures in both the cortex and medulla can be imaged using confocal or multiphoton microscopy. METHODS: Ca(2+)-sensitive dyes Rhod-2, Fura-red and Fluo-4 were loaded into tubular and vascular cells in rat kidney slices using a re-circulating perfusion system and real-time imaging of Ca(2+) signals was recorded by confocal microscopy. Kidney slices were also obtained from transgenic mice expressing the GCaMP2 Ca(2+)-sensor in their endothelial cells and real time Ca(2+) transients stimulated by physiological stimuli. RESULTS: Wide spread loading of Ca(2+) indicators was achieved in the tubular and vascular structures of both the medulla and cortex. Real time Ca(2+) signals were successfully recorded in different intracellular compartments of both rat and mouse cortical and medullary tubules in response to physiological stimuli (ATP and angiotensin II). Glomerular Ca(2+) transients were similarly recorded in kidney slices taken from the transgenic mouse expressing the GCaMP2 Ca(2+)-sensor. CONCLUSION: We present new approaches that can be adopted to image cytosolic and mitochondrial Ca(2+) signals within various cell types in intact kidney tissue. Moreover, techniques described in this study can be used to facilitate future detailed investigations of intracellular Ca(2+) homeostasis in renal health and disease.

An intact kidney slice model to investigate vasa recta properties and function in situ.

BACKGROUND: Medullary blood flow is via vasa recta capillaries, which possess contractile pericytes. In vitro studies using isolated descending vasa recta show that pericytes can constrict/dilate descending vasa recta when vasoactive substances are present. We describe a live kidney slice model in which pericyte-mediated vasa recta constriction/dilation can be visualized in situ. METHODS: Confocal microscopy was used to image calcein, propidium iodide and Hoechst labelling in 'live' kidney slices, to determine tubular and vascular cell viability and morphology. DIC video-imaging of live kidney slices was employed to investigate pericyte-mediated real-time changes in vasa recta diameter. RESULTS: Pericytes were identified on vasa recta and their morphology and density were characterized in the medulla. Pericyte-mediated changes in vasa recta diameter (10-30%) were evoked in response to bath application of vasoactive agents (norepinephrine, endothelin-1, angiotensin-II and prostaglandin E(2)) or by manipulating endogenous vasoactive signalling pathways (using tyramine, L-NAME, a cyclo-oxygenase (COX-1) inhibitor indomethacin, and ATP release). CONCLUSIONS: The live kidney slice model is a valid complementary technique for investigating vasa recta function in situ and the role of pericytes as regulators of vasa recta diameter. This technique may also be useful in exploring the role of tubulovascular crosstalk in regulation of medullary blood flow.

Extracellular nucleotides affect pericyte-mediated regulation of rat in situ vasa recta diameter.

AIM: We hypothesized that extracellular nucleotides, established as being released from renal tubular epithelial cells, act at pericytes to regulate vasa recta capillary diameter. METHODS: A rat live kidney slice model and video imaging techniques were used to investigate the effects of extracellular nucleotides on in situ (subsurface) vasa recta diameter at pericyte and non-pericyte sites. In addition, RT-qPCR was used to quantify P2 receptor mRNA expression in isolated vasa recta. RESULTS: Extracellular ATP, UTP, benzylbenzyl ATP (BzATP) or 2-methylthioATP (2meSATP) evoked a significantly greater vasoconstriction of subsurface vasa recta at pericytes than at non-pericyte sites. The rank order of agonist potency was BzATP = 2meSATP > ATP = UTP. The vasoconstriction evoked at pericyte sites by ATP was significantly attenuated by the P2 receptor antagonists suramin, pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) (PPADS) or Reactive Blue-2 (RB-2). UTP-evoked vasoconstriction at pericytes was attenuated by suramin or RB-2 but not PPADS. Interestingly, suramin or PPADS, when applied in the absence of a P2 receptor agonist, evoked a weak but significant vasoconstriction of vasa recta at pericyte sites, suggesting tonic vasodilation by nucleotides. Significant levels of P2X(1, 3 and 7) and P2Y(4 and 6) receptor mRNA were detected in vasa recta. CONCLUSION: Extracellular nucleotides act at pericytes to cause vasoconstriction of in situ vasa recta. Pharmacological characterization, supported by RT-qPCR data, suggests that P2X(1 and 7) and P2Y(4) receptors mediate nucleotide-evoked vasoconstriction of vasa recta by pericytes. We propose that nucleotides released from renal tubular epithelial cells, in close proximity to vasa recta capillaries, are key in regulating renal medullary blood flow.

Diverse properties of store-operated TRPC channels activated by protein kinase C in vascular myocytes.

In vascular smooth muscle, store-operated channels (SOCs) contribute to many physiological functions including vasoconstriction and cell growth and proliferation. In the present work we compared the properties of SOCs in freshly dispersed myocytes from rabbit coronary and mesenteric arteries and portal vein. Cyclopiazonic acid (CPA)-induced whole-cell SOC currents were sixfold greater at negative membrane potentials and displayed markedly different rectification properties and reversal potentials in coronary compared to mesenteric artery myocytes. Single channel studies showed that endothelin-1, CPA and the cell-permeant Ca(2+) chelator BAPTA-AM activated the same 2.6 pS SOC in coronary artery. In 1.5 mM [Ca(2+)](o) the unitary conductance of SOCs was significantly greater in coronary than in mesenteric artery. Moreover in 0 mM [Ca(2+)](o) the conductance of SOCs in coronary artery was unaltered whereas the conductance of SOCs in mesenteric artery was increased fourfold. In coronary artery SOCs were inhibited by the protein kinase C (PKC) inhibitor chelerythrine and activated by the phorbol ester phorbol 12,13-dibutyrate (PDBu), the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) and a catalytic subunit of PKC. These data infer an important role for PKC in activation of SOCs in coronary artery similar to mesenteric artery and portal vein. Anti-TRPC1 and -TRPC5 antibodies inhibited SOCs in coronary and mesenteric arteries and portal vein but anti-TRPC6 blocked SOCs only in coronary artery and anti-TRPC7 blocked SOCs only in portal vein. Immunoprecipitation showed associations between TRPC1 and TRPC5 in all preparations but between TRPC5 and TRPC6 only in coronary artery and between TRPC5 and TRPC7 only in portal vein. Finally, flufenamic acid increased SOC activity in coronary artery but inhibited SOCs in mesenteric artery and portal vein myocytes. These data provide strong evidence that vascular myocytes express diverse SOC isoforms, which are likely to be composed of different TRPC proteins and have different physiological functions.

Multiple activation mechanisms of store-operated TRPC channels in smooth muscle cells.

Store-operated channels (SOCs) are plasma membrane Ca2+-permeable cation channels which are activated by agents that deplete intracellular Ca2+ stores. In smooth muscle SOCs are involved in contraction, gene expression, cell growth and proliferation. Single channel recording has demonstrated that SOCs with different biophysical properties are expressed in smooth muscle indicating diverse molecular identities. Moreover it is apparent that several gating mechanisms including calmodulin, protein kinase C and lysophospholipids are involved in SOC activation. Evidence is accumulating that TRPC proteins are important components of SOCs in smooth muscle. More recently Orai and STIM proteins have been proposed to underlie the well-described calcium-release-activated current (ICRAC) in non-excitable cells but at present there is little information on the role of Orai and STIM proteins in smooth muscle. In addition it is likely that different TRPC subunits coassemble as heterotetrameric structures to form smooth muscle SOCs. In this brief review we summarize the diverse properties and gating mechanisms of SOCs in smooth muscle. We propose that the heterogeneity of the properties of these conductances in smooth muscle results from the formation of heterotetrameric TRPC structures in different smooth muscle preparations.

Endothelin-1 activates a Ca2+-permeable cation channel with TRPC3 and TRPC7 properties in rabbit coronary artery myocytes.

In the present work we used patch pipette techniques to study the properties of a novel Ca(2+)-permeable cation channel activated by the potent coronary vasoconstrictor endothelin-1 (ET-1) in freshly dispersed rabbit coronary artery myocytes. With cell-attached recording bath application of 10 nm ET-1 evoked cation channel currents (I(cat)) with subconductance states of about 18, 34 and 51 and 68 pS, and a reversal potential of 0 mV. ET-1 evoked channel activity when extracellular Ca(2+) was the charge carrier, illustrating significant Ca(2+) permeability. ET-1-induced responses were inhibited by the ET(A) receptor antagonist BQ123 and the phospholipase C (PLC) inhibitor U73122. The diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) also stimulated I(cat), but the protein kinase C (PKC) inhibitor chelerythrine did not inhibit either the OAG- or ET-1-induced I(cat). Inositol 1,4,5-trisphosphate (IP(3)) did not activate I(cat), but greatly potentiated the response to OAG and this effect was blocked by heparin. Bath application of anti-TRPC3 and anti-TRPC7 antibodies to inside-out patches markedly inhibited ET-1-evoked I(cat), but antibodies to TRPC1, C4, C5 and C6 had no effect. Immunocytochemical studies demonstrated preferential TRPC7 expression in the plasmalemma, whereas TRPC3 was distributed throughout the myocyte, and moreover co-localization of TRPC3 and TRPC7 signals was observed at, or close to, the plasma membrane. Flufenamic acid, Gd(3+), La(3+) and extracellular Ca(2+) inhibited I(cat) with IC(50) values of 2.45 microm, 3.8 microm, 7.36 microm and 22 microm, respectively. These results suggest that in rabbit coronary artery myocytes ET-1 evokes a Ca(2+)-permeable non-selective cation channel with properties similar to TRPC3 and TRPC7, and indicates that these proteins may be important components of this conductance.