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.

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.

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.