Pentosan polysulfate preserves renal microvascular P2X1 receptor reactivity and autoregulatory behavior in DOCA-salt hypertensive rats

Inflammation contributes to ANG II-associated impairment of renal autoregulation and microvascular P2X1 receptor signaling, but its role in renal autoregulation in mineralocorticoid-induced hypertension is unknown. Autoregulatory behavior was assessed using the blood-perfused juxtamedullary nephron preparation. Hypertension was induced in uninephrectomized control rats (UNx) by subcutaneous implantation of a DOCA pellet plus administration of 1% NaCl in the drinking water (DOCA-salt) for 3 wk. DOCA-salt rats developed hypertension that was unaltered by anti-inflammatory treatment with pentosan polysulfate (DOCA-salt+PPS) but was suppressed with “triple therapy” (hydrochlorothiazide, hydralazine, and reserpine; DOCA-salt+TTx). Baseline arteriolar diameters were similar across all groups. UNx rats exhibited pressure-dependent vasoconstriction with diameters declining to 69 ± 2% of control at 170 mmHg, indicating intact autoregulation. DOCA-salt treatment significantly blunted this pressure-mediated vasoconstriction. Diameters remained between 91 ± 4 and 98 ± 3% of control over 65–170 mmHg, indicating impaired autoregulation. In contrast, pressure-mediated vasoconstriction was preserved in DOCA-salt+PPS and DOCA-salt+TTx rats, reaching 77 ± 7 and 75 ± 3% of control at 170 mmHg, respectively. ATP is required for autoregulation via P2X1 receptor activation. ATP- and β,γ-methylene ATP (P2X1 receptor agonist)-mediated vasoconstriction were markedly attenuated in DOCA-salt rats compared with UNx ( P < 0.05), but significantly improved by PPS or TTx ( P < 0.05 vs. DOCA-salt) treatment. Arteriolar responses to adenosine and UTP (P2Y2 receptor agonist) were unaffected by DOCA-salt treatment. PPS and TTx significantly reduced MCP-1 and protein excretion in DOCA-salt rats. These results support the hypothesis that hypertension triggers inflammatory cascades but anti-inflammatory treatment preserves renal autoregulation in DOCA-salt rats, most likely by normalizing renal microvascular reactivity to P2X1 receptor activation.

Heme oxygenase induction attenuates afferent arteriolar autoregulatory responses

Heme oxygenases (HO-1, HO-2) catalyze conversion of heme to iron, carbon monoxide (CO), and biliverdin/bilirubin. We studied the effects of renal HO-1 induction on afferent arteriole (Aff-Art) autoregulatory responses to increases in renal perfusion pressure (RPP). Rats were treated with hemin and SnCl2 to induce HO-1, and Aff-Art autoregulatory responses were evaluated using the rat blood-perfused juxtamedullary nephron preparation. Renal HO-1 expression was significantly increased in hemin- and SnCl2-treated rats, while HO-2 was not altered. Aff-Art autoregulatory constrictor responses to increases in RPP from 100 to 150 mmHg were attenuated in hemin- and SnCl2-treated rats compared with control rats (+1.1 ± 3.3, n = 9 and +4.4 ± 5.3, n = 9 vs. −14.2 ± 1.5%, n = 10, respectively) ( P < 0.05). Acute HO inhibition with chromium mesoporphyrin (CrMP; 15 μmol/l) restored Aff-Art autoregulatory responses in hemin- and SnCl2-treated rats. Superfusing Aff-Arts from control rats with 100 μmol/l biliverdin did not alter autoregulatory responses; however, superfusion with 1 mmol/l CO significantly attenuated autoregulatory responses to increases in RPP from 100 to 150 mmHg (+3.3 ± 5.4 vs. −16.6 ± 3.8%, n = 6) ( P < 0.05). Acute soluble guanylate cyclase inhibition with 10 μmol/l ODQ restored Aff-Art autoregulatory responses in hemin-treated rats. Immunohistochemistry shows HO-2 to be expressed mainly in epithelial cells with weak staining in proximal tubules, interlobular arteries, and Aff-Arts. In hemin- and SnCl2-treated rats, HO-1 was induced in tubular epithelial cells but not interlobular arteries and Aff-Arts. We conclude that induction of renal HO-1 attenuates Aff-Art constrictor responses to increases in RPP via increasing CO production from tubular epithelial cells, suggesting that an augmented HO system in pathophysiological conditions modulates renal autoregulation.

Potassium channel contributions to afferent arteriolar tone in normal and diabetic rat kidney

We previously reported an enhanced tonic dilator impact of ATP-sensitive K+ channels in afferent arterioles of rats with streptozotocin (STZ)-induced diabetes. The present study explored the hypothesis that other types of K+ channel also contribute to afferent arteriolar dilation in STZ rats. The in vitro blood-perfused juxtamedullary nephron technique was utilized to quantify afferent arteriolar lumen diameter responses to K+ channel blockers: 0.1–3.0 mM 4-aminopyridine (4-AP; KV channels), 10–100 μM barium (KIR channels), 1–100 nM tertiapin-Q (TPQ; Kir1.1 and Kir3.x subfamilies of KIR channels), 100 nM apamin (SKCa channels), and 1 mM tetraethylammonium (TEA; BKCa channels). In kidneys from normal rats, 4-AP, TEA, and Ba2+ reduced afferent diameter by 23 ± 3, 8 ± 4, and 18 ± 2%, respectively, at the highest concentrations employed. Neither TPQ nor apamin significantly altered afferent diameter. In arterioles from STZ rats, a constrictor response to TPQ (22 ± 4% decrease in diameter) emerged, and the response to Ba2+ was exaggerated (28 ± 5% decrease in diameter). Responses to the other K+ channel blockers were similar to those observed in normal rats. Moreover, exposure to either TPQ or Ba2+ reversed the afferent arteriolar dilation characteristic of STZ rats. Acute surgical papillectomy did not alter the response to TPQ in arterioles from normal or STZ rats. We conclude that 1) KV, KIR, and BKCa channels tonically influence normal afferent arteriolar tone, 2) KIR channels (including Kir1.1 and/or Kir3.x) contribute to the afferent arteriolar dilation during diabetes, and 3) the dilator impact of Kir1.1/Kir3.x channels during diabetes is independent of solute delivery to the macula densa.

Interaction between endogenously produced carbon monoxide and nitric oxide in regulation of renal afferent arterioles

Heme oxygenases (HO-1 and HO-2) catalyze the conversion of heme to carbon monoxide (CO), iron, and biliverdin. CO causes vasorelaxation via stimulation of soluble guanylate cyclase (sGC) and/or activation of calcium-activated potassium channels. Because nitric oxide (NO) exerts effects via the same pathways, we tested the interaction between CO and NO on rat afferent arterioles (AAs) using the blood-perfused juxtamedullary nephron preparation. AAs were superfused with either tricarbonyldichlororuthenium (II) dimer, known as CO releasing molecule (CORM-2), 10 μmol/l CO solution, or 15 μmol/l chromium mesoporphyrin (CrMP, HO inhibitor). AAs were also superfused with 1 mmol/l Nω-nitro-l-arginine (l-NNA) to inhibit NO synthase (NOS) or 10 μmol/l 1 H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one to inhibit sGC, and then CrMP was superfused during NOS inhibition or sGC inhibition. Treatment with 150 and 300 μmol/l CORM-2 or with CO (10 μmol/l) significantly dilated AAs (22.0 ± 0.9 and 22.8 ± 0.9 vs. 18.3 ± 0.9 μm, n = 5, P < 0.05; and 26.0 ± 1.4 vs. 18.8 ± 0.7 μm, n = 5, P < 0.05). In untreated vessels, HO inhibition did not alter AA diameter (17.5 ± 0.7 vs. 17.2 ± 0.6 μm, n = 7, P > 0.05); however, during inhibition of NO production, which constricted arterioles to 14.6 ± 1.2 μm, n = 6, P < 0.05, concurrent HO inhibition led to further vasoconstriction (11.7 ± 1.6 μm, n = 6, P < 0.05). CORM-2 attenuated the l-NNA-induced vasoconstriction. Inhibition of sGC caused significant constriction (15.7 ± 0.4 vs. 18.8 ± 0.4 μm, n = 6, P < 0.05). HO inhibition during sGC inhibition did not cause further change in AAs (15.5 ± 0.7 μm, n = 6). We conclude that endogenously produced CO does not exert a perceptible influence on AA diameter in the presence of intact NO system; however, when NO production is inhibited, CO serves as an important renoprotective reserve mechanism to prevent excess afferent arteriolar constriction.

Src family kinase involvement in rat preglomerular microvascular contractile and [Ca2+]iresponses to ANG II

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 μM 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 Ca2+concentration ([Ca2+]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 (Δ = 293 ± 66 nM) and plateau (Δ = 23 ± 8 nM) increases in [Ca2+]i. In PVSMCs pretreated with PP2, baseline [Ca2+]iwas unaltered, but both the peak (Δ = 140 ± 22 nM) and plateau (Δ = 3 ± 2 nM) phases of the ANG II response were significantly reduced compared with untreated cells. PP3 did not alter [Ca2+]iresponses to ANG II. Immunoprecipitation and Western blot analysis confirmed that 100 nM ANG II increased phosphorylation of c-Src (at Y416) 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 [Ca2+]iresponse to the peptide.

Renal segmental microvascular responses to ANG II in AT1A receptor null mice

The relative contributions of AT1A and AT1B receptors to afferent arteriolar autoregulatory capability and afferent and efferent arteriolar responses to ANG II are not known. Experiments were conducted in kidneys from wild-type (WT) and AT1A−/− mice utilizing the in vitro blood-perfused juxtamedullary nephron technique. Direct measurements of afferent (AAD) and efferent arteriolar diameters (EAD) were assessed at a renal arterial pressure of 100 mmHg. AAD averaged 14.8 ± 0.8 μm for WT and 14.9 ± 0.8 μm for AT1A−/− mice. AAD significantly decreased by 7 ± 1, 16 ± 1, and 26 ± 2% for WT mice and by 11 ± 1, 20 ± 2, and 30 ± 3% for AT1A−/− mice (120, 140, 160 mmHg). AAD autoregulatory capability was not affected by the absence of AT1A receptors. AAD responses to 10 nM ANG II were significantly blunted for AT1A−/− mice compared with WT (−22 ± 2 vs. −37 ± 5%). ANG II (0.1–10 nM) failed to elicit any change in EAD for AT1A−/− mice. AAD and EAD reductions in ANG II were blocked by 1 μM candesartan. We conclude that afferent arteriole vasoconstrictor responses to ANG II are mediated by AT1A and AT1B receptors, whereas efferent arteriolar vasoconstrictor responses to ANG II are mediated by only AT1A receptors in the mouse kidney.

Interactions of adenosine A1 and A2areceptors on renal microvascular reactivity

Adenosine vasoconstricts preglomerular arterioles via adenosine A1receptors. Because adenosine also activates adenosine A2receptors, its overall renal vascular actions are complex and not fully understood. The present study was performed to determine the relative contributions of adenosine A1 and A2a receptors to the responsiveness of the renal microvasculature to adenosine. Afferent and efferent arteriolar diameters were monitored in vitro using the blood-perfused rat juxtamedullary nephron preparation. Basal afferent and efferent arteriolar diameters averaged 17.1 ± 0.5 ( n = 35) and 17.8 ± 0.5 ( n = 20) μm, respectively. Superfusion with 0.1 and 1 μmol/l adenosine did not significantly alter afferent and efferent arteriolar diameters; however, 10 μmol/l adenosine significantly reduced afferent and efferent arteriolar diameters (−8.2 ± 0.8 and −5.7 ± 0.6%, respectively). The afferent and efferent arteriolar vasoconstrictor responses to adenosine waned at a dose of 100 μmol/l, such that diameters returned to values not significantly different from control within 2 min. During adenosine A1 receptor blockade with 8-noradamantan-3-yl-1,3-dipropylxanthine (KW-3902: 10 μmol/l), 10 and 100 μmol/l adenosine significantly increased afferent diameter by, respectively, 8.1 ± 1.2 and 13.7 ± 1.3% ( n = 14) and efferent arteriolar diameter by 6.4 ± 1.3 and 9.3 ± 1.2% ( n = 8). The afferent and efferent arteriolar vasodilatory responses to adenosine in the presence of KW-3902 were significantly attenuated by addition of the adenosine A2a receptor antagonist 1,3-dipropyl-7-methyl-8-(3,4-dimethoxystyryl)xanthine (KF-17837: 15 μmol/l, n = 7 and 6, respectively). The addition of KF-17837 alone significantly enhanced afferent ( n = 15) and efferent ( n = 6) arteriolar vasoconstrictor responses to 1, 10, and 100 μmol/l adenosine. These results indicate the presence of adenosine A1 and A2a receptors on afferent and efferent arterioles of juxtamedullary nephrons, such that adenosine A2a receptor-mediated vasodilation partially buffers adenosine-induced vasoconstriction in both pre- and postglomerular segments of the renal microvasculature.