Abstract

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 (AT1) receptors. However, losartan, a selective AT1 receptor antagonist, ameliorated IR-induced renal insufficiency in the pigs. Blockade of TRPV4 channels attenuated Ang II-evoked receptor-operated Ca2+ 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.

Introduction

Due to immaturity, neonates are at high risk of acute kidney injury (AKI) caused by therapeutic agents, hypovolemia, sepsis, and ischemia [1–4]. The incidence of AKI among sick neonates has been estimated to range between 18 and 24% and is associated with high mortality rates [1,2]. Renal injury in the perinatal period is also a risk factor for developing cardiovascular and kidney diseases in later life [1,2].

Renal ischemia–reperfusion (IR) injury remains a significant cause of AKI in newborns [1,2]. Early renal vasoconstriction in ischemic AKI results in a rapid decline in kidney function, including regional perfusion, glomerular filtration, and urine production [5–7]. Mechanisms that underlie post-ischemic renal hypoperfusion in neonates are poorly understood, but possibilities include alterations in the production, expression, function, or regulation of vasoactive compounds and their mediators within the kidneys [5–7].

The cellular mechanisms that control vascular tone include transmembrane fluxes of ions such as Na+, K+, Cl, and Ca2+ via ion channels, the pore-forming proteins in perivascular nerves, endothelial cells, and smooth muscle cells (SMCs) [8]. Foremost ion channels that regulate vascular resistance include the transient receptor potential (TRP) cation channels [9]. The TRPV sub-family of TRP channels control diverse functions in the kidney, such as excretion, osmoregulation, and epithelial Ca2+ transport [10–12]. Despite voluminous work in other vascular beds [13,14], TRPV channel function and regulation in resistance size renal vessels are poorly understood. In a recent study, we demonstrated that, unlike TRPV1–3, TRPV4 channels are predominantly expressed in renal vascular SMCs and contribute to myogenic renal autoregulation in neonatal pigs [15]. However, evidence suggests that TRPV4 channels mediate polymodal Ca2+ signaling pathways. TRPV4-dependent aortic and pulmonary vasoconstriction have been reported [16–18]. By contrast, local intracellular ([Ca2+]i) signals, including Ca2+ sparks and sparklets generated by activation of smooth muscle and endothelial cell TRPV4 channels triggered cerebral and mesenteric vasodilation in adult rodents [19–21]. Studies have also shown that TRPV4 can mediate store-operated Ca2+ (SOC) or receptor-operated Ca2+ (ROC) entry (ROCE). For example, SOC entry (SOCE) was diminished in aortic endothelial cells from TRPV4 knockout mice and in TRPV4 siRNA-treated human umbilical vein endothelial cells [22]. By contrast, TRPV4 mediate acetylcholine- and ATP-induced ROCE, but not SOCE, in mouse dermal vascular endothelial and ciliated tracheal cells, respectively [23,24]. Thus, organ regional and cell type heterogeneity may influence TRPV4-mediated [Ca2+]i signaling and downstream physiological functions.

Inhibition of TRPV4 channels alleviated brain and myocardial IR injury in adult mice and rats [25–28]. Genetic ablation and pharmacological inhibition of TRPV4 have also been shown to attenuate chemical- and ventilation-induced acute lung injury in adult mice [29–33]. TRPV4 channel knockout aggravated tubular injury induced by renal IR following unilateral nephrectomy in adult mice [34]. Whether TRPV4 contributes to post-ischemic alterations in renal vascular resistance (RVR) and microcirculation remains unknown.

Amplified renin–angiotensin signaling promotes inflammation, fibrosis, and vascular dysregulation in AKI [35]. Administration of angiotensin II (Ang II) to lambs and neonatal pigs reduced renal blood flow (RBF) [36,37]. Hence, an increase in Ang II biosynthesis during the early phase of renal IR may increase RVR via [Ca2+]i-dependent mechanisms in renal vascular SMCs. Pretreatment of human umbilical vein endothelial and rat hypothalamic cells with Ang II increased TRPV4-dependent [Ca2+]i elevation [16,38]. Similarly, Ang II amplified TRPV4 agonist-induced contraction of mouse aorta [16]. In cerebral artery SMCs, Ang II stimulates local [Ca2+]i elevation via TRPV4 channels [39]. However, the pathophysiological relationship between Ang II and TRPV4 in renal vascular bed is unclear. In the present study, we used a preclinical large animal model to examine whether: (i) TRPV4 channel expression and activity are altered in resistance size renal vessels of neonates subjected to renal IR, (ii) pharmacological inhibition of TRPV4 channels alleviates IR-induced renal insufficiency in neonates, and (iii) TRPV4-dependent intrarenal vascular effect of Ang II contributes to IR-induced renal insufficiency in neonates.

Methods

Animals

All experimental animal procedures were approved and performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of the University of Tennessee Health Science Center (UTHSC). Full-term neonatal domestic pigs (male; 3–5 days old; Nichols Hog Farm, Olive Branch, MS) were housed and used for experiments in approved locations within the UTHSC.

Renal hemodynamics measurement and IR induction

Neonatal pigs were anesthetized, intubated, mechanically ventilated, and acutely instrumented (femoral artery and vein and ureteral catheters) as we have previously described [15,37,40]. Total RBF, renal cortical perfusion, and mean arterial pressure (MAP) were acquired using a flowmeter (Transonic Systems Inc., Ithaca, NY), laser-Doppler (Perimed, Jarfalla, Sweden), and physiological pressure transducer (ADInstrument, Colorado Spring, CO), respectively [15,37,40]. All recordings were acquired simultaneously using the PowerLab data acquisition system and LabChart Pro software (ADInstrument).

To induce renal IR, anesthetized neonatal pigs were subjected to 45 min bilateral renal pedicle occlusion using non-traumatic clamps followed by 3 h reperfusion. The control animals were subjected to the same surgical operation, but without renal pedicle occlusion. All animals were given isotonic fluid intravenously at a rate of 200 µl/min throughout the experiment and ventilated to maintain PCO2, PO2, and pH at ∼30 mmHg, >85 mmHg, and 7.4, respectively. Blood gas was measured using the GEM Premier 3000 Blood Gas Analyzer (Instrumentation Laboratory, Bedford, MA). The levels of hemoglobin, hematocrit, and red blood cells were determined with the LaserCyte Hematology Analyzer (IDEXX Laboratories, Inc Westbrook, ME).

Glomerular filtration rate determination

A one-compartment method with the FIT-GFR Inulin Kit (BioPhysics Assay Laboratory; BioPAL, Worcester MA) was used to determine the glomerular filtration rate (GFR). Venous blood samples were collected at 30, 60, and 90 min from the pigs after intravenous injection of GFR-grade inulin (5 mg/kg). Serum was prepared, and inulin concentrations measured using a plate assay (BioPAL). The GraphPad Prism software was used for a nonlinear regression plot to calculate the clearance of inulin according to the manufacturer’s instructions and as previously described [41–44].

Kidney injury bioassays

Liquid chromatography-tandem mass spectrometry was used to determine serum creatinine concentrations at the UAB/UCSD O’Brien Core Center for AKI Research at the University of Alabama at Birmingham. Blood urea nitrogen (BUN), urine Ang II, and urine neutrophil gelatinase-associated lipocalin (NGAL) were measured using colorimetric (Arbor Assays, Ann Arbor, MI), species-independent ELISA (Enzo Life Sciences, Farmingdale, NY), and pig NGAL ELISA (BioPorto Diagnostics, Hellerup, Denmark) kits, respectively.

Histopathologic analysis

Kidney sections processed for Hematoxylin and Eosin and Periodic acid–Schiff (PAS) staining were scored by a board-certified veterinary pathologist for histological damage associated with IR injury, including dilated tubules with loss of brush border, intratubular protein casts, and infiltration of inflammatory cells in the interstitium and tubular lumina. All slides were randomly intermixed and scored in a blinded manner for histological lesions: 0 absent, 1+ minimal or rare focal, 2+ mild, 3+ moderate, 4+ marked. Images were taken with a Nikon Ci microscope, 10× Plan Apo objective lens, DS-Fi2 camera and NIS-elements D (version 4.10) software (Nikon, U.S.A.) calibrated with a stage micrometer.

[Ca2+]i imaging and arterial diameter measurement

[Ca2+]i in intact afferent arterioles and changes in distal interlobular artery luminal diameter were studied using the fluorescence photometry (Ionoptix Corp., Milton, MA, U.S.A.) and pressure myograph systems (Danish Myo Technology, Aarhus, Denmark and Living Systems Instrumentation, St. Albans, VT), respectively as we have previously described [15,45,46]. Pial arterioles were also dissected from the cerebral cortical surface of the pigs and studied using the pressure myograph system.

Patch clamp electrophysiology

SMCs were freshly isolated from interlobular arteries as we have previously described [15]. Membrane cation currents (ICat) were recorded at room temperature in cells attached to a glass-bottom chamber using the conventional whole-cell configuration of the patch-clamp technique. Current recordings and data acquisition were made with the Axopatch 200B, Digidata 1440A, and pCAMP 10 software (Molecular Devices, Sunnyvale, CA). A current–voltage relationship was generated using a voltage ramp protocol (−120 to +100 mV) over 940 ms duration every 10 s, from a 0 mV holding potential. Whole-cell currents were filtered at 1 kHz and digitized at 5 kHz. The bath solution contained (in mM): 142 NaCl, 2 CaCl2, 6 KCl, 1 MgCl2, 10 HEPES, and 10 glucose (pH 7.4). The pipette solution contained (in mM): 120 Na-glutamate, 20 NaCl, 1 MgCl2, 1 EGTA, 10 HEPES, 4 Na2ATP (pH 7.2). TRPV4 channels were activated using a selective agonist 4α-Phorbol 12-13-dicaprinate (4α-PDD) [47,48].

Western immunoblotting

Tissues were homogenized in ice-cold RIPA buffer. The proteins were then separated by 4-20% ExpressPlus PAGE Gels (GenScript, Piscataway, NJ) as we have previously described [15,45].

Antibodies and reagents

Rabbit polyclonal anti-TRPV4 (AP18990a) [15], rabbit polyclonal anti-Ang II type 1 (AT1) receptor (SAB3500209) [49], and mouse monoclonal anti-β-actin (MA515739) [15] primary antibodies were purchased from Abgent Inc. (San Diego, CA), Sigma–Aldrich (St. Louis, MO), and Abcam (Cambridge, MA), respectively. HRP–conjugated anti-rabbit and anti-mouse secondary antibodies were purchased from Life Technologies. Unless otherwise specified, all reagents were purchased from Sigma–Aldrich (St. Louis, MO). HC067047 (HC) and RN1734 (RN) were purchased from EMD Millipore (Billerica, MA). Fura-2 AM, Pluronic F-127, ionomycin, and liberase blendzyme 1 were obtained from Life Technologies, AnaSpec (Fremont, CA), Cayman Chemical (Ann Arbor, MI), and Roche Life Science (Indianapolis, IN), respectively.

Statistical analysis

Statistical analysis was performed using the InStat statistics software (Graph Pad, Sacramento, CA). Data are presented as mean ± standard error of the mean. Paired and unpaired data were compared using the Student’s ttests. Fisher’s exact test was used to analyze the histologic data. All other multiple comparisons were performed using analysis of variance with the Student–Newman–Keuls test. Statistical significance implies a P-value <0.05.

Results

TRPV4 activation stimulated renal vasoconstriction and hypoperfusion in neonatal pigs

Selective TRPV4 agonist GSK1016790A (GSK) caused concentration-dependent constriction of renal distal interlobular arteries that were pressurized to 100 mmHg (Figure 1A,B). GSK-induced renal vasoconstriction was attenuated by selective TRPV4 channel blocker HC (Figure 1C,D). Another TRPV4 agonist, 4α-PDD, stimulated renal vasoconstriction in the pigs (Supplementary Figure S1). To investigate whether modulation of TRPV4 channels alters the diameter of extrarenal microvessels in neonatal pigs, we examined the effect of GSK on isolated pial arterioles that were pressurized to 40 mmHg. Supplementary Figure S2 shows that GSK reduced, while HC increased the lumen diameter of the pial arterioles by ∼8 and 19 µm, respectively. Next, we studied the effect of TRPV4 activation on renal perfusion. Intrarenal artery infusion of GSK caused a time-dependent reduction in cortical perfusion (Figure 1E). When compared with the control, significant hypoperfusion commenced ∼15 min following the start of GSK infusion (Figure 1E). GSK also elicited a time-dependent decrease in MAP, but a significant decrease in arterial pressure of the pigs started ∼40 min after the start of GSK infusion (Figure 1F). Both hypoperfusion and MAP reduction induced by GSK were abrogated by HC (Figure 1E,F).

TRPV4 activation stimulates renal vasoconstriction and hypoperfusion in neonatal pigs

Figure 1
TRPV4 activation stimulates renal vasoconstriction and hypoperfusion in neonatal pigs

(A) An exemplary trace and (B) bar charts (n=6) illustrating the concentration-dependent effect of GSK on the diameter of renal interlobular arteries isolated from neonatal pigs and pressurized to 100 mmHg. (C) traces and (D) bar charts (n=8, each) showing that HC (1 µM) inhibited GSK (10 nM)-induced renal vasoconstriction in neonatal pigs. (E and F) graphs summarizing changes in renal cortical perfusion (RCoP) and the MAP in neonatal pigs infused intrarenally with the vehicle (control; n=6) and GSK (3 µg/kg/min; n=5), HC (20 µg/kg/min; n=6), and HC + GSK (n=3). *P<0.05 vs. baseline (B); ¥P<0.05 vs. GSK; &P<0.05 vs. control (RCoP: 15–60 min; MAP: 40–60 min); #P<0.05 vs. GSK (RCoP: 15–60 min; MAP: 40–60 min).

Figure 1
TRPV4 activation stimulates renal vasoconstriction and hypoperfusion in neonatal pigs

(A) An exemplary trace and (B) bar charts (n=6) illustrating the concentration-dependent effect of GSK on the diameter of renal interlobular arteries isolated from neonatal pigs and pressurized to 100 mmHg. (C) traces and (D) bar charts (n=8, each) showing that HC (1 µM) inhibited GSK (10 nM)-induced renal vasoconstriction in neonatal pigs. (E and F) graphs summarizing changes in renal cortical perfusion (RCoP) and the MAP in neonatal pigs infused intrarenally with the vehicle (control; n=6) and GSK (3 µg/kg/min; n=5), HC (20 µg/kg/min; n=6), and HC + GSK (n=3). *P<0.05 vs. baseline (B); ¥P<0.05 vs. GSK; &P<0.05 vs. control (RCoP: 15–60 min; MAP: 40–60 min); #P<0.05 vs. GSK (RCoP: 15–60 min; MAP: 40–60 min).

Renal IR increased TRPV4 channel protein expression in preglomerular microvessels and TRPV4-dependent cation currents in renal vascular SMCs of neonatal pigs

To investigate the contribution of TRPV4 channels to IR-induced changes that occur in neonatal renal microcirculation, we subjected neonatal pigs to 45-min bilateral ischemia and 3 h reperfusion (Supplementary Figure S3A). The control animals were sham-operated. The concentrations of hemoglobin, hematocrit, and red blood cells were unaltered in neonatal pigs after 3 h IR (Supplementary Figure S3B–D). TRPV4 protein expression level was approximately two-fold higher in renal preglomerular microvessels (pooled interlobular arteries and afferent arterioles) that were isolated from IR- compared with sham-operated pigs (Figure 2A,B). By contrast, 45 min ischemia alone did not change TRPV4 expression in the vessels (Figure 2C,D). The basal ICat density was unaltered in renal vascular SMCs isolated from sham- and IR-operated neonatal pigs (Figure 2E,F). However, 4α-PDD-evoked ICat was larger in SMCs from IR compared with the sham group (Figure 2E,F).

Renal IR increases TRPV4 channel protein expression in preglomerular microvessels and TRPV4-dependent cation currents in renal vascular SMCs of neonatal pigs

Figure 2
Renal IR increases TRPV4 channel protein expression in preglomerular microvessels and TRPV4-dependent cation currents in renal vascular SMCs of neonatal pigs

(A and B) Western blot images and bar graphs showing TRPV4 channel protein expression levels in renal microvessels (pooled interlobular arteries and afferent arterioles) isolated from sham- and IR-operated neonatal pig kidneys (n=4 each). (C and D) Western blot images and bar graphs illustrating TRPV4 channel protein expression levels in renal microvessels isolated from neonatal pigs subjected to 45 min ischemia only (n=4 each). (E and F) traces and bar graphs (inward currents measured at −100 mV) indicating that control ICat density was unaltered in renal vascular SMCs isolated from sham (n=7)- and IR (n=7)-operated neonatal pigs; whereas 4α-PDD (10 µM) stimulated a larger ICat density in SMCs from IR compared with the sham group. *P<0.05 vs. Sham; #P<0.05 vs. Control.

Figure 2
Renal IR increases TRPV4 channel protein expression in preglomerular microvessels and TRPV4-dependent cation currents in renal vascular SMCs of neonatal pigs

(A and B) Western blot images and bar graphs showing TRPV4 channel protein expression levels in renal microvessels (pooled interlobular arteries and afferent arterioles) isolated from sham- and IR-operated neonatal pig kidneys (n=4 each). (C and D) Western blot images and bar graphs illustrating TRPV4 channel protein expression levels in renal microvessels isolated from neonatal pigs subjected to 45 min ischemia only (n=4 each). (E and F) traces and bar graphs (inward currents measured at −100 mV) indicating that control ICat density was unaltered in renal vascular SMCs isolated from sham (n=7)- and IR (n=7)-operated neonatal pigs; whereas 4α-PDD (10 µM) stimulated a larger ICat density in SMCs from IR compared with the sham group. *P<0.05 vs. Sham; #P<0.05 vs. Control.

TRPV4 channel blockers mitigated renal IR-induced hypoperfusion in neonatal pigs

Both cortical perfusion and total RBF remained significantly low despite 3 h reperfusion (Figure 3A–D). Accordingly, RVR was significantly higher in IR- compared with sham-operated neonatal pigs (Figure 3E). Intrarenal artery infusion of TRPV4 channel blockers HC and RN for 20 min, starting at the time of reperfusion (Supplementary Figure S3A), reversed IR-induced renal hypoperfusion, and RVR increase in the pigs (Figure 3A–E). Moreover, changes in arterial pressure in sham- and IR-operated pigs were not significantly different (Figure 3F).

TRPV4 channel blockers mitigate renal IR-induced persistent hypoperfusion in neonatal pigs

Figure 3
TRPV4 channel blockers mitigate renal IR-induced persistent hypoperfusion in neonatal pigs

(AD) Exemplar traces and bar graphs (n=5 each) demonstrating changes in renal cortical perfusion (RCoP) and total RBF in IR-, HC + IR-, and RN + IR-operated neonatal pigs. Bar graphs summarizing (E) changes in RVR (n=5 each) and (F) MAP (n=5 each) in neonatal pigs subjected to renal IR and the effects of HC and RN. HC and RN (20 µg/kg/min) were infused intrarenally for 20 min starting at the time of reperfusion. NS, not significant; *P<0.05 vs. Sham; #P<0.05 vs. IR.

Figure 3
TRPV4 channel blockers mitigate renal IR-induced persistent hypoperfusion in neonatal pigs

(AD) Exemplar traces and bar graphs (n=5 each) demonstrating changes in renal cortical perfusion (RCoP) and total RBF in IR-, HC + IR-, and RN + IR-operated neonatal pigs. Bar graphs summarizing (E) changes in RVR (n=5 each) and (F) MAP (n=5 each) in neonatal pigs subjected to renal IR and the effects of HC and RN. HC and RN (20 µg/kg/min) were infused intrarenally for 20 min starting at the time of reperfusion. NS, not significant; *P<0.05 vs. Sham; #P<0.05 vs. IR.

Inhibition of TRPV4 channels attenuated renal IR-induced GFR reduction and AKI in neonatal pigs

IR caused ∼44% decrease in GFR in the pigs, and this effect was reversed by HC and RN (Figure 4A). Serum creatinine and urea nitrogen were significantly increased after 3 h reperfusion (Figure 4B,C). Urinary NGAL was elevated after 1 h and remained high until the end of reperfusion (Figure 4D). Treatment with HC and RN attenuated IR-induced GFR reduction and serum creatinine, BUN, and urinary NGAL increases (Figure 4A–D). Histopathological analysis of kidney sections from sham-operated neonatal pigs showed continuous red stain of proximal convoluted tubule brush border (Figure 5A). Intratubular protein casts and inflammatory cells were absent from the sham group (Figure 5A). IR caused tubular dilation, scattered protein casts, and inflammatory cell infiltration, which were abated in HC- and RN-treated neonatal pigs (Figure 5A–E).

TRPV4 inhibition attenuates renal IR-induced alterations in GFR and serum or urinary levels of predictive biomarkers of AKI in neonatal pigs

Figure 4
TRPV4 inhibition attenuates renal IR-induced alterations in GFR and serum or urinary levels of predictive biomarkers of AKI in neonatal pigs

(AD) Bar graphs summarizing GFR, serum creatinine, serum BUN, and urine NGAL in sham-, IR-, HC + IR-, and RN + IR-operated neonatal pigs (n=5 each). HC and RN (20 µg/kg/min) were infused intrarenally for 20 min starting at the time of reperfusion. *P<0.05 vs. Sham; &P<0.05 vs. 3 h IR; #P<0.05 vs. pre-ischemia (IR); ¥P<0.05: 1–3 h IR vs. 1–3 h Sham. Abbreviation: NS, not significant.

Figure 4
TRPV4 inhibition attenuates renal IR-induced alterations in GFR and serum or urinary levels of predictive biomarkers of AKI in neonatal pigs

(AD) Bar graphs summarizing GFR, serum creatinine, serum BUN, and urine NGAL in sham-, IR-, HC + IR-, and RN + IR-operated neonatal pigs (n=5 each). HC and RN (20 µg/kg/min) were infused intrarenally for 20 min starting at the time of reperfusion. *P<0.05 vs. Sham; &P<0.05 vs. 3 h IR; #P<0.05 vs. pre-ischemia (IR); ¥P<0.05: 1–3 h IR vs. 1–3 h Sham. Abbreviation: NS, not significant.

Histopathological scoring of IR injury in neonatal pigs

Figure 5
Histopathological scoring of IR injury in neonatal pigs

PAS staining of kidney sections showing: (A) normal kidney morphology with continuous red stain of proximal convoluted tubule (PCT) brush border in sham-operated neonatal pigs; (B) variable presence of stained brush border in PCT; tubular dilation; scattered protein casts (blue arrows); and inflammatory cells between and inside tubules (black arrowheads) in IR-operated neonatal pigs which were mitigated by HC and RN (C and D). (E) kidney injury scores in neonatal pigs subjected to renal IR and the effects of HC and RN. HC and RN (20 µg/kg/min) were infused intrarenally for 20 min starting at the time of reperfusion. #P<0.05 vs. IR; bar = 100 µm.

Figure 5
Histopathological scoring of IR injury in neonatal pigs

PAS staining of kidney sections showing: (A) normal kidney morphology with continuous red stain of proximal convoluted tubule (PCT) brush border in sham-operated neonatal pigs; (B) variable presence of stained brush border in PCT; tubular dilation; scattered protein casts (blue arrows); and inflammatory cells between and inside tubules (black arrowheads) in IR-operated neonatal pigs which were mitigated by HC and RN (C and D). (E) kidney injury scores in neonatal pigs subjected to renal IR and the effects of HC and RN. HC and RN (20 µg/kg/min) were infused intrarenally for 20 min starting at the time of reperfusion. #P<0.05 vs. IR; bar = 100 µm.

AT1 receptor activation is involved in IR-induced renal insufficiency in neonatal pigs

The concentration of Ang II in the urine of neonatal pigs subjected to renal IR was significantly higher than their sham-operated counterparts (Figure 6A). Western immunoblotting revealed that the protein expression level of renal vascular AT1 receptors in the pigs was unaltered by renal IR (Figure 6B,C). However, IR-induced reductions in renal cortical perfusion, total RBF, and GFR were attenuated by losartan, a selective AT1 receptor antagonist (Figure 6D,E). IR-induced increase in serum creatinine and BUN were also inhibited by losartan (Figure 6F–H).

AT1 receptor activation contributes to IR-induced renal insufficiency in neonatal pigs

Figure 6
AT1 receptor activation contributes to IR-induced renal insufficiency in neonatal pigs

(A) Bar graphs summarizing Ang II levels in the urine of neonatal pigs 3 h after sham and IR operations (n=5 each). To control for variations in urine flow rate and creatinine clearance, the urine concentration of Ang II was normalized to that of creatinine. (B and C) Western blot images and bar graphs showing AT1 protein expression levels in renal microvessels (pooled interlobular arteries and afferent arterioles) isolated from sham- and IR-operated neonatal pig kidneys (n=4 each). (DH) Bar graphs summarizing changes in renal cortical perfusion (RCoP), total RBF, GFR, serum BUN, and serum creatinine in IR (n=3)- and losartan + IR (n=4)-operated neonatal pigs. Losartan (1 mg/kg/min) were infused intrarenally for 20 min starting at the time of reperfusion. *P<0.05 vs. IR. Abbreviation: NS, not significant.

Figure 6
AT1 receptor activation contributes to IR-induced renal insufficiency in neonatal pigs

(A) Bar graphs summarizing Ang II levels in the urine of neonatal pigs 3 h after sham and IR operations (n=5 each). To control for variations in urine flow rate and creatinine clearance, the urine concentration of Ang II was normalized to that of creatinine. (B and C) Western blot images and bar graphs showing AT1 protein expression levels in renal microvessels (pooled interlobular arteries and afferent arterioles) isolated from sham- and IR-operated neonatal pig kidneys (n=4 each). (DH) Bar graphs summarizing changes in renal cortical perfusion (RCoP), total RBF, GFR, serum BUN, and serum creatinine in IR (n=3)- and losartan + IR (n=4)-operated neonatal pigs. Losartan (1 mg/kg/min) were infused intrarenally for 20 min starting at the time of reperfusion. *P<0.05 vs. IR. Abbreviation: NS, not significant.

Blockade of TRPV4 channels abrogated Ang II-evoked ROCE in neonatal pig renal vascular SMCs

Since renal IR elevated urinary Ang II and TRPV4 inhibition reversed renal insufficiency, we investigated whether TRPV4 is involved in Ang II-induced Ca2+ signaling in neonatal pig preglomerular microvascular SMCs. First, we stimulated SOCE with thapsigargin and examined whether thapsigargin-induced [Ca2+]i elevation is attenuated by TRPV4 inhibition. Thapsigargin increased [Ca2+]i in afferent arterioles incubated in a Ca2+-free solution containing EGTA (a Ca2+ chelator) and l-type Ca2+ channel blocker nimodipine (Figure 7A,B). In the continued presence of nimodipine, thapsigargin caused a further increase in [Ca2+]i when extracellular Ca2+ was restored (Figure 7A,B). Pretreatment of the arterioles with HC did not alter thapsigargin-induced [Ca2+]i elevation in the absence or presence of extracellular Ca2+ (Figure 7A,B). These data indicate that TRPV4 does not mediate SOCE in neonatal pig renal vascular SMCs.

Ang II-induced ROCE in neonatal pig afferent arteriolar SMCs is dependent on TRPV4 channels

Figure 7
Ang II-induced ROCE in neonatal pig afferent arteriolar SMCs is dependent on TRPV4 channels

(A) Traces and (B) bar graphs illustrating that HC (1 µM) did not alter thapsigargin (TG; 1 µM)-induced [Ca2+]i elevation in neonatal pig afferent arteriolar SMCs in the absence or presence of extracellular Ca2+ [n=7 and 5 for Control (TG) and HC + TG, respectively]. (C) Traces and (D) bar graphs showing that Ang II (10 µM) triggered [Ca2+]i elevation in neonatal pig afferent arteriolar SMCs in the absence and presence of extracellular Ca2+ and that HC (1 µM) had no effect on Ang II-induced [Ca2+]i store release but partially reversed Ang II-induced [Ca2+]i elevation in the presence of extracellular Ca2+. (E) Traces and (F) bar graphs demonstrating the effect of HC (1 µM) on Ang II (10 µM)-evoked [Ca2+]i elevation in neonatal pig afferent arteriolar SMCs in which SR Ca2+ stores had been depleted by TG (1 µM) and l-type Ca2+ channels blocked by nimodipine (1 µM); n=7 each. The zero Ca2+ bath solution was supplemented with Ca2+ chelator EGTA (0.1 mM). *P<0.05, 0 vs. 2 mM Ca2+; #P<0.05 vs. control.

Figure 7
Ang II-induced ROCE in neonatal pig afferent arteriolar SMCs is dependent on TRPV4 channels

(A) Traces and (B) bar graphs illustrating that HC (1 µM) did not alter thapsigargin (TG; 1 µM)-induced [Ca2+]i elevation in neonatal pig afferent arteriolar SMCs in the absence or presence of extracellular Ca2+ [n=7 and 5 for Control (TG) and HC + TG, respectively]. (C) Traces and (D) bar graphs showing that Ang II (10 µM) triggered [Ca2+]i elevation in neonatal pig afferent arteriolar SMCs in the absence and presence of extracellular Ca2+ and that HC (1 µM) had no effect on Ang II-induced [Ca2+]i store release but partially reversed Ang II-induced [Ca2+]i elevation in the presence of extracellular Ca2+. (E) Traces and (F) bar graphs demonstrating the effect of HC (1 µM) on Ang II (10 µM)-evoked [Ca2+]i elevation in neonatal pig afferent arteriolar SMCs in which SR Ca2+ stores had been depleted by TG (1 µM) and l-type Ca2+ channels blocked by nimodipine (1 µM); n=7 each. The zero Ca2+ bath solution was supplemented with Ca2+ chelator EGTA (0.1 mM). *P<0.05, 0 vs. 2 mM Ca2+; #P<0.05 vs. control.

Despite the absence of extracellular Ca2+ and the presence of EGTA and nimodipine, Ang II caused [Ca2+]i elevation which was further increased following extracellular Ca2+ re-addition (Figure 7C,D). In the absence of extracellular Ca2+, HC did not change Ang II-induced increase in [Ca2+]i, but partially reduced Ang II-induced [Ca2+]i elevation after the restoration of extracellular Ca2+ (Figure 7C,D). In another set of experiments, we used thapsigargin to deplete afferent arteriolar sarcoplasmic reticulum (SR) Ca2+ store and blocked l-type Ca2+ channels by nimodipine before Ang II treatment (Figure 7E). Ang II triggered [Ca2+]i elevation in SR-depleted afferent arterioles, indicating a ROCE mechanism (Figure 7E,F). Ang II-evoked ROCE was essentially abrogated by HC (Figure 7E,F). Together, these data suggest that Ang II stimulates ROCE in neonatal pig renal vascular SMCs via TRPV4 channels.

Inhibition of TRPV4 attenuated Ang II-induced renal vasoconstriction and hypoperfusion in neonatal pigs

Next, we investigated whether TRPV4 contributes to Ang II-induced neonatal renal vasoconstriction. Ang II reduced the luminal diameter of pressurized distal interlobular arteries (Figure 8A,B). Pretreatment of the vessels with HC inhibited Ang II-induced renal vasoconstriction (Figure 8A,B). Intrarenal artery infusion of Ang II reduced cortical perfusion and RBF (Figure 8C–F). Ang II also increased the MAP and RVR in the pigs (Figure 8G–I). Pretreatment of the pigs with HC inhibited Ang II-induced reduction in cortical perfusion and RBF and elevation in RVR (Figure 8C–F,I). However, HC did not modulate Ang II-induced increase in arterial pressure (Figure 8G,H).

Inhibition of TRPV4 attenuates Ang II-induced renal vasoconstriction and hypoperfusion in neonatal pigs

Figure 8
Inhibition of TRPV4 attenuates Ang II-induced renal vasoconstriction and hypoperfusion in neonatal pigs

(A) Traces and (B) bar graphs showing Ang II (100 nM)-induced reduction in luminal diameter of pressurized (100 mmHg) neonatal pig renal interlobular arteries in the absence and presence of HC (1 µM) pretreatment. (CH) Traces and bar graphs demonstrating Ang II (10 µg/kg/min; 5 min)-induced changes in renal cortical perfusion (RCoP), total RBF, and MAP in the absence and presence of HC (20 µg/kg/min; 30 min) pretreatment. (I) Bar graphs summarizing Ang II-induced changes in RVR in the absence and presence of HC pretreatment. Both Ang II and HC were administered intrarenally. n=5 each; *P<0.05 vs. Control. Abbreviation: NS, not significant.

Figure 8
Inhibition of TRPV4 attenuates Ang II-induced renal vasoconstriction and hypoperfusion in neonatal pigs

(A) Traces and (B) bar graphs showing Ang II (100 nM)-induced reduction in luminal diameter of pressurized (100 mmHg) neonatal pig renal interlobular arteries in the absence and presence of HC (1 µM) pretreatment. (CH) Traces and bar graphs demonstrating Ang II (10 µg/kg/min; 5 min)-induced changes in renal cortical perfusion (RCoP), total RBF, and MAP in the absence and presence of HC (20 µg/kg/min; 30 min) pretreatment. (I) Bar graphs summarizing Ang II-induced changes in RVR in the absence and presence of HC pretreatment. Both Ang II and HC were administered intrarenally. n=5 each; *P<0.05 vs. Control. Abbreviation: NS, not significant.

Discussion

In the present study, we demonstrate that in neonatal pigs, (i) pharmacological activation of TRPV4 channels constricted renal preglomerular microvessels and induced renal hypoperfusion; (ii) forty-five minutes of bilateral renal ischemia followed by 3 h reperfusion increased the functional expression of TRPV4 channels in preglomerular microvessels; (iii) TRPV4 channel blockers attenuated IR-induced increase in RVR and renal insufficiency; (iv) TRPV4 channel inhibition suppressed renal IR-induced alterations in the serum or urinary levels of the predictive biomarkers of AKI; (v) renal IR increased urinary concentration of Ang II; (vi) AT1 receptor antagonist reversed IR-evoked renal insufficiency; (vii) pharmacological inhibition of TRPV4 channels reduced Ang II-induced increase in RVR and hypoperfusion by blocking ROCE. Our data suggest that SMC TRPV4-mediated renal vasoconstriction contributes to the onset of ischemic AKI in neonates.

Unlike cerebral and mesenteric arteries of adult rodents [20,21,50], activation of TRPV4 channels by its selective agonists constricted renal interlobular arteries isolated from neonatal pigs. Previous evidence suggests that TRPV4 activation can trigger vasoconstriction, which may be dependent on vascular bed type, generation of vasoactive mediators, or vessel size. For example, agonist- and hypoxia-induced pulmonary vasoconstriction were mediated by TRPV4 channels [17,18]. A TRPV4 agonist stimulated mouse aortic vasoconstriction through the release of prostanoids [16,51]. Activation of TRPV4 channels relaxed the main pulmonary arteries but increased intra-pulmonary vascular resistance in mice [52]. To investigate whether TRPV4-mediated vasoconstriction in neonatal pigs is restricted to the renal microvessels, we examined the effects of a TRPV4 agonist on pial arterioles that had developed spontaneous myogenic tone. Surprisingly, GSK constricted neonatal pig pial arterioles. These data suggest that interspecies differences in TRPV4-mediated vasoregulation may exist. Also, since TRPV4 is mostly expressed in neonatal pig renal vascular SMCs compared with endothelial cells [15], it is possible that vasoconstriction via SMC TRPV4 activation predominates over endothelial cell-dependent vasodilation. TRPV4 protein expression levels are higher in adult pig kidneys and preglomerular microvessels when compared with neonatal pigs [15]. Thus, organ maturation/age-related changes in TRPV4-mediated vascular reactivity require further investigations.

It is noteworthy that 1 h intrarenal artery infusion of GSK in neonatal pigs caused time-dependent renal hypoperfusion and reduction in arterial pressure. However, the onset of GSK-induced hypoperfusion occurred earlier than MAP reduction. Conceivably, the decrease in the MAP was due to the effects of residual GSK as it reaches the systemic circulation. Indeed, intravenously administered GSK has been shown to cause a dose- and time-dependent reduction in cardiac output and arterial pressure in dogs [53].

Strong evidence suggests critical roles for microvascular dysregulation in ischemic AKI [5–7]. Since activation of TRPV4 in vitro and in vivo stimulated renal vasoconstriction in neonatal pigs, we reasoned that amplified TRPV4 function may contribute to hypoperfusion in neonatal ischemic AKI and that targeting these channels could mitigate renal insufficiency. The serum or urinary levels of creatinine, BUN, and NGAL were all increased in IR-operated pigs. Unlike serum creatinine and BUN, a significant increase in urinary NGAL was observed as early as 1 h following reperfusion, supporting previous reports that urinary NGAL is an effective predictor of early AKI in neonates [54–57]. RVR was increased, and both cortical perfusion and total RBF in the pigs did not return to basal levels during 3 h reperfusion. Collectively, these data suggest that 45 min of bilateral renal ischemia and 3 h of reperfusion elicits hypoperfusion and AKI in neonatal pigs. Renal IR, but not ischemia alone, significantly increased TRPV4 protein expression in preglomerular microvessels of the pigs, indicating that the reperfusion phase altered the channel expression. IR-induced increase in TRPV4 channel protein expression level is supported by patch clamp electrophysiology data demonstrating that activation of TRPV4 channels caused larger cation currents in renal vascular SMCs isolated from IR- compared with sham-operated neonatal pigs. Pharmacological inhibition of TRPV4 channels by HC and RN reversed IR-induced renal insufficiency and injury in the pigs. Hence, our data suggest a link between up-regulated TRPV4 channel function in renal preglomerular microvasculature and IR-induced AKI in neonatal pigs.

A recent study reported that adult TRPV4 knockout mice were more prone to renal tubular damage elicited by left kidney IR following right kidney nephrectomy [34]. The reason for the seemingly conflicting findings between the study by Mannaa et al. [34] and our current work could be due to differences in species, age, or renal insults. We investigated renal insufficiency induced by bilateral renal IR in neonatal pigs, but in the previous study, the impact of TRPV4 ablation on IR-induced alterations in renal vasoregulation and injury in the absence of nephrectomy was not determined [34]. Since TRPV4 controls kidney function, complete ablation of the channels may result in basal renal changes that aggravate renal tubular injury in the remnant kidney after unilateral nephrectomy [34,58,59].

The possible mechanisms by which pharmacological inhibition of TRPV4 protects against IR-induced renal insufficiency include attenuation of renal vasoconstriction caused by vasoactive mediators. Ang II, one of such mediators, has been demonstrated to reduce RBF and increase RVR in conscious chronically instrumented lambs and anesthetized neonatal pigs [36,37]. Renal IR increased kidney tissue and urine, but not plasma levels of Ang II in adult rats, indicating elevated biosynthesis of Ang II within the kidneys [60–62]. Here, we show that the urinary concentration of Ang II was elevated in neonatal pigs subjected to renal IR, corroborating the studies in rats [60–62]. AT1 receptors mediate Ang II-induced alterations in renal hemodynamics during early postnatal period [37,63–66]. AT1 receptor expression was reduced in the kidneys of adult rats subjected to IR [60–62]. By contrast, we did not observe alterations in AT1 receptor protein expression in renal microvessels from IR-operated neonatal pigs, suggesting potential species- or maturational-dependent changes in AT1 receptor expression in ischemic AKI. The reported IR-induced decrease in AT1 receptors in rat kidneys [60–62] may also have occurred outside the vascular system. Inhibition of Ang II receptors, angiotensin-converting enzyme, and renin have been shown to protect against renal IR injury in adult rats [62,67,68]. Here, post-ischemic administration of losartan ameliorated IR-induced renal insufficiency in neonatal pigs. Together, our findings suggest that an early increase in renal Ang II and the ensuing activation of AT1 receptors contribute to renal IR-induced kidney dysfunction in neonatal pigs.

TRPV4 may constitute SOC or ROC channels [22–24]. We show in this study that thapsigargin stimulates SOCE in neonatal pig afferent arterioles, consistent with previous studies in adult rats and newborn pigs [45,69]. Pretreatment of the arterioles with HC did not alter SOCE, indicating that although neonatal pig afferent arteriolar SMCs exhibit SOCE, it is not mediated by TRPV4 channels. Ang II triggered SOCE in afferent arteriolar SMCs. Ang II also elevated [Ca2+]i independently of SR Ca2+ store and l-type Ca2+ channels. These findings signify that comparable with adult rodents [70–74], Ang II promotes both SOCE and ROCE in neonatal preglomerular microvascular SMCs. However, unlike SOCE, TRPV4 mediates Ang II-induced ROCE in the cells. Thus, TRPV4 is an AT1-ROC channel in neonatal pig preglomerular vascular SMCs.

Pharmacological inhibition of TRPV4 channels by HC reduced Ang II-induced renal vasoconstriction in neonatal pigs. Accordingly, direct infusion of HC into the kidneys of the pigs attenuated Ang II-induced increase in RVR and reduction in RBF. By contrast, increased MAP elicited by acute intrarenal Ang II infusion was unaffected by HC. These findings indicate that TRPV4-dependent ROCE in preglomerular vascular SMCs contribute to Ang II-induced reduction in neonatal renal microcirculation, but not Ang II-induced increase in arterial pressure. Chronic infusion of Ang II to mice increased the MAP which was unaltered by genetic ablation of TRPV4 channels [75]. Apart from vasoconstriction, Ang II raises blood pressure by other mechanisms, including increased sodium and water retention and sympathetic nerve activity [76]. Hence, the lack of effect of TRPV4 inhibition/knockout on acute and chronic Ang II-induced increase in arterial pressure is a strong indication that TRPV4 channels contribute to the direct renal vascular, but not the pressor effect of Ang II.

Renal IR causes loss of renal autoregulation [77,78]. We have shown that efficient myogenic renal autoregulation in neonatal pigs requires TRPV4 channels [15]. Ang II-evoked increase in arterial blood pressure stimulated renal autoregulatory vasoconstriction in adult rats [79]. Whether Ang II-evoked increase in renal perfusion pressure triggers renal autoregulation in neonatal pigs is unclear. Hence, the impact of TRPV4 inhibition on renal IR-induced renal autoregulation failure requires additional studies.

In summary, findings in the present study suggest that SMC TRPV4-mediated vasoconstriction and the ensuing increase in RVR underlie early hypoperfusion and renal insufficiency elicited by renal IR in neonatal pigs. Since TRPV4 is also a mechanosensitive channel in neonatal renal vascular SMCs [15], we propose that multimodal signaling by renal vascular TRPV4 channels regulates neonatal renal microcirculation in health and disease.

Clinical perspectives

  • Renal vasoconstriction in ischemic AKI results in a reduction in RBF and a rapid decline in kidney function. Vascular mechanisms that mediate renal IR-induced renal insufficiency in neonates are unclear. Here, we used newborn pigs to examine whether renal vascular TRPV4 channel expression and function are altered in neonates subjected to renal IR.

  • We show that bilateral renal IR in pigs increased TRPV4 channel protein expression in renal microvessels. We also demonstrate that SMC TRPV4-mediated vasoconstriction and the ensuing increase in RVR underlie early hypoperfusion and renal insufficiency elicited by renal IR in neonatal pigs.

  • The findings in the present study provide new insights into the vascular mechanisms that underlie neonatal renal IR injury. TRPV4 could be a potential therapeutic target for amplified RVR in the early phase of neonatal AKI.

Acknowledgments

We thank Robert Read, DVM, Ph.D., DAVCP (TriMetis Life Sciences, Memphis) for histological analysis. We also thank the UAB-UCSD O’Brien Center for Acute Kidney Injury Research (NIH P30-DK079337) for the assistance with serum and urine creatinine measurements.

Funding

This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health [grant number R01DK101668 (to A.A.)].

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Author Contribution

H.S. performed and analyzed animal surgical, pressure myograph, Ca2+ imaging, and colorimetric assay/ELISA experiments. D.P.-N. performed and analyzed patch clamp electrophysiology and pressure myograph experiments. M.A.O. performed and analyzed hematological tests. A.A. conceived and designed the study, performed and analyzed Western blot experiments, and wrote the paper.

Abbreviations

     
  • AKI

    acute kidney injury

  •  
  • Ang II

    angiotensin II

  •  
  • AT1

    Ang II type 1

  •  
  • BUN

    blood urea nitrogen

  •  
  • GFR

    glomerular filtration rate

  •  
  • GSK

    GSK1016790A

  •  
  • HC

    HC067047

  •  
  • ICat

    membrane cation current

  •  
  • IR

    ischemia–reperfusion

  •  
  • MAP

    mean arterial pressure

  •  
  • NGAL

    neutrophil gelatinase-associated lipocalin

  •  
  • RN

    RN 1734

  •  
  • RBF

    renal blood flow

  •  
  • ROC

    receptor-operated Ca2+

  •  
  • ROCE

    ROC entry

  •  
  • RVR

    renal vascular resistance

  •  
  • SMC

    smooth muscle cell

  •  
  • SOC

    store-operated Ca2+

  •  
  • SOCE

    SOC entry

  •  
  • TRP

    transient receptor potential

  •  
  • UTHSC

    University of Tennessee Health Science Center

  •  
  • 4α-PDD

    4α-Phorbol 12-13-dicaprinate

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Supplementary data