Transdermal Measurement of Glomerular Filtration Rate in Mechanically Ventilated Piglets

Transdermal measurement of glomerular filtration rate (GFR) has been used to evaluate kidney function in conscious animals. This technique is well ...

College of Medicine University of Tennessee Health Science Center

Angiotensin II (ANG-II) receptors (AGTRs) contribute to renal physiology and pathophysiology, but the underlying mechanisms that regulate AGTR function in glomerular mesangium are poorly understood. Here, we show that AGTR1 is the functional AGTR subtype expressed in neonatal pig glomerular mesangial cells (GMCs). Cyclodextrin (CDX)-mediated cholesterol depletion attenuated cell surface AGTR1 protein expression and ANG-II-induced intracellular Ca(2+) ([Ca(2+)]i) elevation in the cells. The COOH-terminus of porcine AGTR1 contains a caveolin (CAV)-binding motif. However, neonatal GMCs express CAV-1, but not CAV-2 and CAV-3. Colocalization and in situ proximity ligation assay detected an association between endogenous AGTR1 and CAV-1 in the cells. A synthetic peptide corresponding to the CAV-1 scaffolding domain (CSD) sequence also reduced ANG-II-induced [Ca(2+)]i elevation in the cells. Real-time imaging of cell growth revealed that ANG-II stimulates neonatal GMC proliferation. ANG-II-induced GMC growth was attenuated by EMD 66684, an AGTR1 antagonist; BAPTA, a [Ca(2+)]i chelator; KN-93, a Ca(2+)/calmodulin-dependent protein kinase II inhibitor; CDX; and a CSD peptide, but not PD 123319, a selective AGTR2 antagonist. Collectively, our data demonstrate [Ca(2+)]i-dependent proliferative effect of ANG-II and highlight a critical role for lipid raft microdomains in AGTR1-mediated signal transduction in neonatal GMCs. 

Lipid rafts are required for signal transduction by angiotensin II receptor type 1 in neonatal glomerular mesangial cells.

Myogenic response, a phenomenon in which resistance size arteries and arterioles swiftly constrict or dilate in response to an acute elevation or reduction, respectively, in intravascular pressure is a key component of renal autoregulation mechanisms. Although it is well established that the renal system is functionally immature in neonates, mechanisms that regulate neonatal renal blood flow (RBF) remain poorly understood. In this study, we investigated the hypothesis that members of the transient receptor potential vanilloid (TRPV) channels are molecular components of renal myogenic constriction in newborns. We show that unlike TRPV1-3, TRPV4 channels are predominantly expressed in neonatal pig preglomerular vascular smooth muscle cells (SMCs). Intracellular Ca concentration ([Ca]) elevation induced by osmotic cell swelling was attenuated by TRPV4, L-type Ca, and stretch-activated Ca channel blockers but not phospholipase A inhibitor. Blockade of TRPV4 channels reversed steady-state myogenic tone and inhibited pressure-induced membrane depolarization, [Ca] elevation, and constriction in distal interlobular arteries. A step increase in arterial pressure induced efficient autoregulation of renal cortical perfusion and total RBF in anesthetized and mechanically ventilated neonatal pigs. Moreover, intrarenal arterial infusion of the TRPV4 channel blockers HC 067047 and RN 1734 attenuated renal autoregulation in the pigs. These data suggest that renal myogenic autoregulation is functional in neonates. Our findings also indicate that TRPV4 channels are mechanosensors in neonatal pig preglomerular vascular SMCs and contribute to renal myogenic autoregulation.

TRPV4 channels contribute to renal myogenic autoregulation in neonatal pigs.

Adenosine, a regulator of cardiovascular development and renal function, constricts renal afferent arterioles by inducing intracellular Ca concentration ([Ca]) elevation in smooth muscle cells (SMCs) via activation of its cognate A receptors (ARs). Mechanisms that underlie AR-dependent [Ca] elevation in renal vascular SMCs are not fully resolved. Whether AR expression and function in preglomerular microvessels are dependent on postnatal kidney maturation is also unclear. In this study, we show that selective activation of ARs by 2-chloro--cyclopentyladenosine (CCPA) does not stimulate store-operated Ca entry in afferent arterioles isolated from neonatal pigs. However, CCPA-induced [Ca] elevation is dependent on phospholipase C and transient receptor potential cation channel, subfamily C, member 3 (TRPC3). Basal [Ca] was unchanged in afferent arterioles isolated from newborn (0-day-old) pigs compared with their 20-day-old counterparts. By contrast, CCPA treatment resulted in significantly larger [Ca] in afferent arterioles from 20-day-old pigs. AR protein expression levels in the kidneys and afferent arterioles were unaltered in 0- vs. 20-day-old pigs. However, the TRPC3 channel protein expression level was ~92 and 78% higher in 20-day-old pig kidneys and afferent arterioles, respectively. These data suggest that activation of ARs elicits receptor-operated Ca entry in porcine afferent arterioles, the level of which is dependent on postnatal maturation of TRPC3 channels. We propose that TRPC3 channels may contribute to the physiology and pathophysiology of ARs.

Adenosine A receptor-operated calcium entry in renal afferent arterioles is dependent on postnatal maturation of TRPC3 channels.

Glomerular mesangial cell (GMC) proliferation and matrix expansion are pathological hallmarks of a wide range of kidney diseases, including diabetic nephropathy. Although the circulating level of peptide hormone urotensin II (UII) and kidney tissue expression of UII and UII receptors (UTR) are increased in diabetic nephropathy, it remains unclear whether UII regulates GMC growth and extracellular matrix (ECM) accumulation. In this study, we tested the hypothesis that UII-induced Ca signaling controls GMC proliferation and ECM production under normal and high glucose conditions. Mouse GMCs cultured under normal glucose conditions proliferated and synthesized ECM proteins in response to stimulation by mouse UII. UII-induced GMC proliferation and ECM protein synthesis were dependent on TRPC4 channel-mediated store-operated Ca entry (SOCE) and sequential activation of Ca/calmodulin-dependent protein kinase II (CaMKII) and Ca/cAMP response element-binding protein (CREB) transcription factor. Under high glucose conditions, GMCs synthesized UII. Moreover, proliferation and ECM production in high glucose-challenged GMCs were attenuated by selective UTR antagonist, TRPC4 channel blocker, and CaMKII and CREB-binding protein/p300 inhibitors. These findings indicate that UII-induced SOCE via TRPC4 channels stimulates CaMKII/CREB-dependent GMC proliferation and ECM protein production. Our data also suggest that UII synthesis contributes to GMC proliferation and ECM accumulation under high glucose conditions.

Urotensin II-induced store-operated Ca entry contributes to glomerular mesangial cell proliferation and extracellular matrix protein production under high glucose conditions.

Reactive oxygen species (ROS) modulate neuronal function, including plasticity and neurotransmitter biosynthesis and release. The cellular mechanisms that underlie redox modulation of neurotransmission are not fully resolved, but potential pathways include ROS-induced alterations in Ca signaling in nerve terminals. In this study, we show that cold-sensitive receptor TRPM8 is activated by pro-oxidant tert-butyl hydroperoxide (tBHP). Polymerase chain reaction, Western immunoblotting, and immunofluorescence indicated that TRPM8 channels are expressed in rat pheochromocytoma 12 (PC12) cells, a phenotypic model of sympathetic neurosecretion when differentiated with nerve growth factor. WS-12, a selective TRPM8 channel agonist, and tBHP increased intracellular Ca concentration in differentiated PC12 cells; an effect attenuated by AMTB, a selective TRPM8 channel blocker, and siRNA-mediated TRPM8 knockdown. Blockade of TRPM8 channels also reduced WS-12- and tBHP-evoked norepinephrine secretion from the cells. These data suggest that TRPM8 channels contribute to oxidant-induced neurotransmission in PC12 cells.

Oxidant-induced increase in norepinephrine secretion from PC12 cells is dependent on TRPM8 channel-mediated intracellular calcium elevation.

Green tea polyphenol epigallocatechin gallate (EGCG), promotes vasodilation and reduces blood pressure, mechanisms of which are not fully resolved. Recent reports suggested that EGCG can activate heterologously expressed mouse and zebrafish TRPA1 channels. Activation of TRPA1 in sensory neurons triggers the release of calcitonin gene-related peptide (CGRP), a potent vasodilator. Whether CGRP-containing (CGRPergic) sensory nerves contribute to EGCG-induced reduction in vascular resistance remains unclear. In this study, we demonstrate that intravenous infusion of EGCG elevated the plasma level of CGRP in mice, an effect that was attenuated by TRPA1 channel blocker A-967079. EGCG-induced increase in mesenteric artery blood flow and reduction in mean arterial pressure were reversed by A-967079, CGRP receptor antagonist CGRP, and CGRP depletion in perivascular nerves. Moreover, EGCG stimulated TRPA1-dependent intracellular Ca elevation and CGRP release in a differentiated rat embryonic dorsal root ganglion/mouse neuroblastoma hybrid cell line. Together, these data suggest that EGCG-induced activation of TRPA1 channels in perivascular CGRPergic nerves decreases vascular resistance via Ca-dependent exocytosis of CGRP.

CGRPergic Nerve TRPA1 Channels Contribute to Epigallocatechin Gallate-Induced Neurogenic Vasodilation.

Renal vasoconstriction, an early manifestation of ischemic acute kidney injury (AKI), results in renal hypoperfusion and a rapid decline in kidney function. The pathophysiological mechanisms that underlie ischemia-reperfusion (IR)-induced renal insufficiency are poorly understood, but possibilities include alterations in ion channel-dependent renal vasoregulation. In the present study, we show that pharmacological activation of TRPV4 channels constricted preglomerular microvessels and elicited renal hypoperfusion in neonatal pigs. Bilateral renal ischemia followed by short-term reperfusion increased TRPV4 protein expression in resistance size renal vessels and TRPV4-dependent cation currents in renal vascular smooth muscle cells (SMCs). Selective TRPV4 channel blockers attenuated IR-induced reduction in total renal blood flow (RBF), cortical perfusion, and glomerular filtration rate (GFR). TRPV4 inhibition also diminished renal IR-induced increase in AKI biomarkers. Furthermore, the level of angiotensin II (Ang II) was higher in the urine of IR- compared with sham-operated neonatal pigs. IR did not alter renal vascular expression of Ang II type 1 (AT) receptors. However, losartan, a selective AT receptor antagonist, ameliorated IR-induced renal insufficiency in the pigs. Blockade of TRPV4 channels attenuated Ang II-evoked receptor-operated Ca entry and constriction in preglomerular microvessels. TRPV4 inhibition also blunted Ang II-induced increase in renal vascular resistance (RVR) and hypoperfusion in the pigs. Together, our data suggest that SMC TRPV4-mediated renal vasoconstriction and the ensuing increase in RVR contribute to early hypoperfusion and renal insufficiency elicited by renal IR in neonatal pigs. We propose that multimodal signaling by renal vascular SMC TRPV4 channels controls neonatal renal microcirculation in health and disease.

Pharmacological inhibition of TRPV4 channels protects against ischemia-reperfusion-induced renal insufficiency in neonatal pigs.

Visual deficits are a common concern among subjects with head trauma. Stem cell therapies have gained recent attention in treating visual deficits following head trauma. Previously, we have shown that adipose-derived stem cell (ASC) concentrated conditioned medium (ASC-CCM), when delivered via an intravitreal route, yielded a significant improvement in vision accompanied by a decrease in retinal neuroinflammation in a focal cranial blast model that indirectly injures the retina. The purpose of the current study is to extend our previous studies to a direct ocular blast injury model to further establish the preclinical efficacy of ASC-CCM. Adult C57BL/6J mice were subjected to repetitive ocular blast injury (rOBI) of 25 psi to the left eye, followed by intravitreal delivery of ASC-CCM (∼200 ng protein/2 μl) or saline within 2-3 h. Visual function and histological changes were measured 4 weeks after injury and treatment. In vitro, Müller cells were used to evaluate the antioxidant effect of ASC-CCM. Visual acuity, contrast sensitivity, and b-wave amplitudes in rOBI mice receiving saline were significantly decreased compared with age-matched sham blast mice. Immunohistological analyses demonstrated a significant increase in glial fibrillary acidic protein (a retinal injury marker) in Müller cell processes, DNA/RNA damage, and nitrotyrosine (indicative of oxidative stress) in the retina, while qPCR analysis revealed a >2-fold increase in pro-inflammatory cytokines (TNF-α, ICAM1, and Ccl2) in the retina, as well as markers for microglia/macrophage activation (IL-1β and CD86). Remarkably, rOBI mice that received ASC-CCM demonstrated a significant improvement in visual function compared to saline-treated rOBI mice, with visual acuity, contrast sensitivity, and b-wave amplitudes that were not different from those in sham mice. This improvement in visual function also was associated with a significant reduction in retinal GFAP, neuroinflammation markers, and oxidative stress compared to saline-treated rOBI mice. In vitro, Müller cells exposed to oxidative stress via increasing doses of hydrogen peroxide demonstrated decreased viability, increased GFAP mRNA expression, and reduced activity for the antioxidant catalase. On the other hand, oxidatively stressed Müller cells pre-incubated with ASC-CCM showed normalized GFAP, viability, and catalase activity. In conclusion, our study demonstrates that a single intravitreal injection of ASC-CCM in the rOBI can significantly rescue retinal injury and provide significant restoration of visual function. Our in vitro studies suggest that the antioxidant catalase may play a major role in the protective effects of ASC-CCM, uncovering yet another aspect of the multifaceted benefits of ASC secretome therapies in neurotrauma.

Mesenchymal stem cell secretome protects against oxidative stress-induced ocular blast visual pathologies.

Vasoactive endothelin (ET) is generated by ET converting enzyme (ECE)-induced proteolytic processing of pro-molecule big ET to biologically active peptides. HO has been shown to increase the expression of ECE1 via transactivation of its promoter. The present study demonstrates that HO triggered ECE1-dependent ET1-3 production in neonatal pig proximal tubule (PT) epithelial cells. A uniaxial stretch of PT cells decreased catalase, increased NADPH oxidase (NOX)2 and NOX4, and increased HO levels. Stretch also increased cellular ECE1, an effect reversed by EUK-134 (a synthetic superoxide dismutase/catalase mimetic), NOX inhibitor apocynin, and siRNA-mediated knockdown of NOX2 and NOX4. Short-term unilateral ureteral obstruction (UUO), an inducer of renal tubular cell stretch and oxidative stress, increased renal ET1-3 generation and vascular resistance (RVR) in neonatal pigs. Despite removing the obstruction, UUO-induced increase in RVR persisted, resulting in early acute kidney injury (AKI). ET receptor (ET)-operated Ca entry in renal microvascular smooth muscle (SM) via transient receptor potential channel 3 (TRPC3) channels reduced renal blood flow and increased RVR. Although acute reversible UUO (rUUO) did not change protein expression levels of ET and TRPC3 in renal microvessels, inhibition of ECE1, ET, and TRPC3 protected against renal hypoperfusion, RVR increase, and early AKI. These data suggest that mechanical stretch-driven oxyradical generation stimulates ET production in neonatal pig renal epithelial cells. ET activates renal microvascular SM TRPC3, leading to persistent vasoconstriction and reduction in renal blood flow. These mechanisms may underlie rUUO-induced renal insufficiency in infants.

Adebowale Adebiyi

Department of Physiology

College of Medicine University of Tennessee Health Science Center

Transdermal Measurement of Glomerular Filtration Rate in Mechanically Ventilated Piglets

Adebowale Adebiyi

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Physiology

College of Medicine University of Tennessee Health Science Center

Transdermal Measurement of Glomerular Filtration Rate in Mechanically Ventilated Piglets

Department of Physiology