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We have demonstrated previously that paracrine signaling occurs between medullary thick ascending limb (mTAL) and the contractile pericytes of outer-medullary vasa recta (VR), termed “tubular-vascular cross-talk.” The aim of the current study was to determine whether tubular-vascular cross-talk has a functional effect on vasoconstrictor responses to angiotensin II and to determine whether this is altered in the Dahl salt-sensitive (SS) rat. Studies were performed on salt-resistant consomic SS.13 Brown Norway (BN) and SS rats using a novel outer medullary tissue strip preparation in which freshly isolated VRs within VR bundles were perfused either alone or in combination with nearby mTAL. In VRs from SS.13 bn rats, angiotensin II (1 µmol/L) increased VR bundle intracellular Ca 2+ concentration 19 ± 9 nmol/L (n=8) and reduced focal diameter in perfused VRs by −20 ± 7% (n=5). In the presence of nearby mTAL, however, VR bundle intracellular Ca 2+ concentration (−9 ± 8 nmol/L; n=8) and VR diameter (−1 ± 4%, n=7) in SS.13 bn rats were unchanged by angiotensin II. In contrast, in Dahl SS rats, angiotensin II resulted in rapid and sustained increase in VR bundle intracellular Ca 2+ concentration (89 ± 48 nmol/L, n=7; 50 ± 24%, n=8) and a reduction in VR diameter of (−17 ± 7%, n=7; −11 ± 4%, n=5) in both isolated VRs and VRs with nearby mTAL, respectively. In VRs with mTAL from SS13 bn rats, inhibiton of purinergic receptors resulted in an increase in VR bundle intracellular Ca 2+ concentration, indicating that purinergic signaling buffers vasoconstriction. Importantly, our in vitro data were able to predict medullary blood flow responses to angiotensin II in SS and SS.13 bn rats in vivo. We conclude that paracrine signaling from mTAL buffers angiotensin II vasoconstriction in Dahl salt-resistant SS.13 bn rats but not SS rats.

It has been reported previously that H + efflux via the Na + /H + exchange stimulates NAD(P)H oxidase-dependent superoxide (O 2 ·− ) production in medullary thick ascending limb. We have demonstrated recently that N -methyl-amiloride-sensitive O 2 ·− production is enhanced in the thick ascending limb of Dahl salt-sensitive (SS) rats, suggesting that H + efflux through Na + /H + exchangers may promote renal oxidative stress and the development of hypertension in these animals. In the current study we demonstrate, using selective and potent inhibitors, that inhibition of Na + /H + exchange does not mediate the ability of N -methyl-amiloride to inhibit thick ascending limb O 2 ·− production. To determine the mechanism of action of N -methyl-amiloride, we examined H + efflux and O 2 ·− production in SS and SS.13 BN thick ascending limbs of prehypertensive, 0.4% NaCl-fed rats. Tissue strips containing the medullary thick ascending limb were isolated from male SS and salt-resistant consomic SS.13 BN rats, loaded with either dihydroethedium or 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester, and imaged in a heated tissue bath. In Na + -replete media, activation of Na + /H + exchange using an NH 4 Cl prepulse did not stimulate thick ascending limb O 2 ·− production. In Na + -free media containing BaCl 2 in which Na + /H + activity was inhibited, an NH 4 Cl prepulse stimulated O 2 ·− production in medullary thick ascending limb renal tubular segments. This response was enhanced in medullary thick ascending limb of SS rats (slope Δethidium/Δdihydroethedium=0.029±0.004) compared with SS.13 BN rats (slope=0.010±0.004; P N -methyl-amiloride (slope=0.005±0.002 and 0.006±0.002 for SS and SS.13 BN , respectively). We concluded that only H + efflux through a specific, as-yet-unidentified, amiloride-sensitive H + channel promotes O 2 ·− production in the medullary thick ascending limb and that this channel is upregulated in SS rats.

Studies were conducted to determine whether the diffusion of NO from the renal medullary thick ascending limb (mTAL) to the contractile pericytes of surrounding vasa recta was reduced and, conversely, whether diffusion of oxygen free radicals was enhanced in the salt-sensitive Dahl S rat (SS/Mcwi). Angiotensin II ([Ang II] 1 μmol/L)–stimulated NO and superoxide (O 2 ·− ) production were imaged by fluorescence microscopy in thin tissue strips from the inner stripe of the outer medulla. In prehypertensive SS/Mcwi rats and a genetically designed salt-resistant control strain (consomic SS-13 BN ), Ang II failed to increase either NO or O 2 ·− in pericytes of isolated vasa recta. Ang II stimulation resulted in production of NO in epithelial cells of the mTAL that diffused to vasa recta pericytes of SS-13 BN rats but not in SS/Mcwi rats except when tissues were preincubated with the superoxide scavenger TIRON (1 mmol/L). Ang II resulted in a greater increase of O 2 ·− in the mTAL of SS/Mcwi compared with SS.13 BN mTAL. The O 2 ·− diffused to adjoining pericytes in tissue strips only in SS/Mcwi rats but not in control SS-13 BN rats. Diffusion of Ang II-stimulated O 2 ·− from mTAL to vasa recta pericytes was absent when tissue strips from SS/Mcwi rats were treated with the NO donor DETA-NONOate (20 μmol/L). We conclude that the SS/Mcwi rat exhibits increased production of O 2 ·− in mTAL that diffuses to surrounding vasa recta and attenuates NO cross-talk. Diffusion of O 2 ·− from mTAL to surrounding tissue could contribute to reduced bioavailability of NO, reductions of medullary blood flow, and interstitial fibrosis in the outer medulla of SS/Mcwi rats.

It is well established that chronic immune system activation contributes to hypertension and kidney injury. Mitochondria carry hallmarks of their bacterial ancestry and thus have emerged as a significant source of inflammatogenic damage-associated molecular patterns (DAMPs). One of these hallmarks is that it still uses an N-formyl-methionyl-tRNA as an initiator of protein synthesis. Recently, we have observed that mitochondrial DAMPs are elevated in the circulation of SHR, and that mitochondrial N-formyl peptides (F-MIT) infusion in rats induces systemic inflammation and vascular dysfunction via formyl peptide receptor (FPR) activation. However, we do not know if FPR plays a role in kidney injury and hypertension. We hypothesized that F-MIT activate FPR and lead to intrarenal dysfunction in 12 week old male Wistar and SHR (n=4-8). Wistar rats were treated with F-MIT (0.02 mg/kg) or non-formylated peptide (control) for 6 h and intrarenal arteries (diameter >100 μm) were isolated. To exclude systemic effects of F-MIT, intrarenal arteries were also isolated from control rats and treated ex vivo with 100 nM F-MIT or non-formylated peptide. F-MIT treatment in vivo increased intrarenal arteries FPR protein expression (2.3-fold vs. control) and decreased β-arrestin 2 (protein that internalizes FPR upon activation) and phosphorylation of endothelial nitric oxide synthase (4-fold vs. control). These results were reproduced in isolated arteries incubated with F-MIT or control for 6 h ex vivo. Similarly, in intrarenal arteries from untreated SHR, we found that FPR protein expression was higher (1.5-fold vs. Wistar Kyoto, WKY) and β-arrestin 2 protein expression was decreased (2-fold vs. WKY). Interestingly, although treatment with hydrochlorothiazide (10-55 mg/kg/day) and reserpine (0.6-4.5 mg/kg/day) for 7 weeks attenuated the increase in blood pressure in SHR, anti-hypertensive therapy did not change FPR and β-arrestin 2 protein expression. Additionally, it was observed that the co-localization of FPR and β-arrestin 2 was decreased in intrarenal arteries from SHR. Overall, these data suggest that intrarenal arteries sense F-MIT. Also, FPR activation parameters following F-MIT treatment of normotensive rats are similar to those observed in SHR.

Dahl salt-sensitive (SS) rats fed a high salt diet exhibit increased blood pressure and renal damage. Epithelial sodium channels (ENaC) in the aldosterone-sensitive distal nephron (ASDN) are responsible for the fine tuning of Na + absorption in the kidney and are regulated by various stimuli including hormones and growth factors. Members of the epidermal growth factor (EGF) family are important for maintaining transepithelial Na + transport. A combination of electrophysiological, immunohistochemical, biochemical in vitro and chronic in vivo studies were used to provide mechanistic insights into ENaC regulation by EGF and how changes in this pathway may contribute to salt-induced hypertension in SS rats. It was found that the ENaC inhibitor benzamil, administered through the drinking water (15 mg/L), significantly reduced the MAP from 173.3±15.0 to 127.0±10.1 mm Hg. ENaC expression (as measured by Western blotting and immunohistochemistry) and activity (single channel analysis in freshly isolated split open ASDN) were significantly enhanced in SS rats fed a high salt diet compared to SS rats fed a low salt diet and consomic SS.13 BN rats fed a high salt chow. Servo-controlled measurements in SS rats also revealed increased ENaC expression in the uncontrolled kidneys compared to controlled left kidneys. As assessed by ELISA assay, EGF level in the kidney cortex of SS rats fed a high salt diet was lower compared to rats on a low salt diet. To directly evaluate the role of EGF in the development of hypertension and its effect on ENaC activity, EGF (10 μg/h/kg) was intravenously infused and blood pressure was continuously monitored. Chronic infusion of EGF decreased ENaC activity ( NP o ; from 1.5±0.2 to 0.4±0.1), prevented the development of hypertension (from 184.0±8.9 to 149.0±9.6 mm Hg) and attenuated renal glomerular and tubular damage. Thus, in controversy to existing dogma, we provide an evidence of a critical role of ENaC in the ASDN in the development of salt-sensitive hypertension. Furthermore, these findings indicate that EGF deficiency in the renal cortex contributes to salt-sensitive hypertension and that in vivo EGF infusion prevents development of hypertension and kidney damage via downregulation of ENaC.

We have previously reported that H + efflux stimulates superoxide (O 2 - ) production in the medullary thick ascending limb of the loop of Henle (mTAL) in rats by activation of an as yet unidentified H + transport pathway. As HVCN1, a voltage gated H + channel associated with NADPH oxidase in immune cells, is also expressed in mTAL, we hypothesized that HVCN1 is responsible for H + efflux induced production of O 2 - in mTAL. In order to test this hypothesis we utilized HVCN1 -/- mice and wild type litter mates obtained from KOMP. To confirm that HVCN1 was functionally knocked out in HVCN1 -/- mice we stimulated the respiratory burst using PMA (100uM) in peritoneal macrophages extracted from HVCN1 -/- and WT mice and determined maximal O 2 - production using L-012 luminescence. Maximal O 2 - production during the respiratory burst was significantly lower in macrophages from HVCN1 -/- mice (23±4AU; n=6) compared to WT mice (92±3AU; n=5; p2 - sensitive dye dihydroethidium (DHE). Live mTAL were then imaged on a heated chamber attached to a fluorescent microscope and the ratio of ethidium (Eth) to DHE quantified using metafluor imaging software as an index of O 2 - production. O 2 - producing H + currents were isolated in 0 Na + , 100uM BaCl 2 media and cellular H + efflux stimulated using an NH 4 Cl (20mM) prepulse. Following removal of NH 4 Cl from the bath there was a significant increase in O 2 - production in mTAL from WT mice (Delta slope Eth/DHE 15.9±3.6 AU/sec; p-/- mice (delta slope 4.7±9.6; P=0.64 (n=6)). The response of WT mice was significantly greater than that of HVCN1 -/- mice (P2 - production in mTAL in response to H + efflux. These data suggest that HVCN1 may be a novel target to prevent renal oxidative stress.