Knox, Franklyn G.

In rats with streptozotocin (STZ)-induced diabetes, the renal vasoconstrictor effect of adenosine is enhanced. We investigated the role of nitric oxide (NO) in the renal vascular response to exogenous and endogenous adenosine in control and STZ diabetic rats. Exogenous adenosine (0.01–100 nmol) injected into the abdominal aorta decreased renal blood flow (RBF) in a dose-dependent manner to a much greater extent in STZ rats than in control rats ( P < 0.001). Inhibition of NO synthesis with Nω-nitro-l-arginine (l-NNA, 30 μmol/kg iv) and with renal perfusion pressure controlled potentiated the adenosine-induced renal vasoconstriction to a significantly greater extent in control rats than in STZ rats. In control rats,l-NNA shifted the dose-response curve of exogenous adenosine-induced RBF reductions to the left by a factor of 32 [half-maximal effective dose (ED50), from 5.5 to 0.17 nmol adenosine, n = 6] and in STZ rats only by a factor of 4.6 (ED50, from 0.32 to 0.07 nmol adenosine, n = 6). The renal response to endogenous adenosine was assessed by the magnitude of the postocclusive reduction of RBF (POR) after a 30-s renal artery occlusion. POR was markedly enhanced in STZ rats (−67.8 ± 3.8%, P < 0.001) compared with control rats (−38.8 ± 4.3%).l-NNA markedly enhanced POR in control rats but did not increase POR in STZ rats. These findings demonstrate a greater potentiation of the adenosine-induced renal vasoconstriction in the presence ofl-NNA infusion in control rats compared with STZ rats. We conclude that the increased vasoconstrictor sensitivity of the diabetic renal vasculature to adenosine is caused by a defective NO-dependent renal vasodilation of the afferent arteriole in diabetic rats.

The diuretic effects of nitric oxide (NO) synthase inhibitors administered at subpressor dose in rats are controversial, and the tubular segments involved are not known. In the present study, we examined the effect of Nω-nitro-l-arginine methyl ester (l-NAME) at a subpressor dose on renal interstitial NO and cGMP activity and on renal tubular segmental reabsorption of fluid in the rat. Intravenous infusion of l-NAME at 1 μg · kg−1· min−1in Sprague-Dawley rats ( N = 8), which did not alter mean arterial pressure or glomerular filtration rate, significantly increased urine flow rate (Uv; from 78.2 ± 12.7 to 117.1 ± 14.9 μl/min, P < 0.05). Paradoxically, this effect of l-NAME was concomitant with significant increases in nitrite/nitrate (from 10.79 ± 1.20 to 16.50 ± 2.60 μM, P < 0.05) and cGMP (from 0.65 ± 0.09 to 0.98 ± 0.18 nM, P < 0.05) concentrations in renal cortical microdialysate as well as the nitrite/nitrate concentration in the medullary microdialysate. Micropuncture studies in the superficial nephron revealed that l-NAME significantly increased the flow rate (from 8.3 ± 0.9 to 12.2 ± 1.2 nl/min, P < 0.05) and fractional delivery of fluid to the distal tubule, but not those in the late proximal tubule. In conclusion, l-NAME, at the subpressor dose used in this study, increased renal nitrate/nitrite and cGMP and inhibited fluid reabsorption in tubular segments between the late proximal tubule and the distal tubule of superficial nephrons.

Increases in renal interstitial hydrostatic pressure (RIHP) increase urinary sodium excretion (UNaV). Experimentally increasing RIHP by direct renal interstitial volume expansion (DRIVE) has been shown to decrease proximal tubule sodium reabsorption. The purpose of the present study was to investigate whether the renin-angiotensin system modulates the natriuretic response to DRIVE. Unilateral nephrectomy and implantation of two polyethylene matrices were performed 3 wk before the acute experiment. Fractional sodium excretion (FENa), RIHP, and glomerular filtration rate (GFR) were measured before and after DRIVE in control rats (n = 9) and in rats receiving the angiotensin II (ANG II) receptor antagonist, losartan potassium (10 mg/kg i.v.; n = 10). DRIVE was achieved by infusing 100 microliters of 2.5% albumin solution directly into the renal interstitium. GFR remained unchanged by DRIVE in both groups. In control animals, DRIVE significantly increased both RIHP (delta 3.8 +/- 0.5 mmHg) and FENa (delta 0.92 +/- 0.19%). In the losartan-treated group, RIHP (delta 2.8 +/- 0.4 mmHg) and FENa (delta 1.93 +/- 0.41%) also significantly increased. The natriuretic response to DRIVE was significantly enhanced during ANG II receptor blockade compared with control animals (delta UNaV/delta RIHP = 2.01 +/- 0.67 vs. 0.44 +/- 0.17 mu eq.min-1 x mmHg-1, respectively; P < 0.05). These results suggest that the blockade of angiotensin enhances the natriuretic response to increased RIHP during DRIVE.

Systemic inhibition of nitric oxide synthesis with NG-monomethyl-L-arginine (L-NMMA) increases renal perfusion pressure (RPP) and urinary sodium excretion. Increased RPP has been proposed as one of the mechanisms for the natriuresis caused by intravenous infusion of L-NMMA. We tested the hypothesis that increases in renal interstitial hydrostatic pressure (RIHP) are required for the natriuresis of L-NMMA infusion. Experiments were performed in four groups of Sprague-Dawley rats in which partial aortic clamping and/or bilateral renal decapsulation was performed to control RPP and RIHP. Infusion of L-NMMA (15 mg/kg bolus + 500 micrograms.kg-1 x min-1 continuous infusion) increased RPP (delta+ 14 +/- 1 mmHg), RIHP (delta+ 3.6 +/- 0.7 mmHg), and fractional excretion of sodium (FENa; delta 2.4 +/- 0.6%, P < 0.005). When RPP was prevented from increasing by controlling RPP with an adjustable clamp around the suprarenal aorta, RIHP and FENa did not significantly change. When only RIHP was held constant by bilateral renal decapsulation, FENa was not significantly increased (delta+ 0.68 +/- 0.36%, not significant), despite a significant rise in RPP (delta+ 18 +/- 2 mmHg, P < 0.001). Control of both RPP and RIHP prevented the increase in FENa. Thus, when renal interstitial pressure was controlled, the infusion of L-NMMA did not result in an increase in FENa. These results demonstrate that an increase in RIHP is a necessary component in the natriuresis due to systemic infusion of L-NMMA.

Nitric oxide (NO) reduces the molecular activity of Na + -K + -ATPase in opossum kidney (OK) cells, a proximal tubule cell line. In the present study, we investigated the cellular mechanisms for the inhibitory effect of NO on Na + -K + -ATPase. Sodium nitroprusside (SNP), a NO donor, inhibited Na + -K + -ATPase in OK cells, but not in LLC-PK 1 cells, another proximal tubule cell line. Similarly, phorbol 12-myristate 13-acetate, a protein kinase C (PKC) activator, inhibited Na + -K + -ATPase in OK, but not in LLC-PK 1 , cells. PKC inhibitors staurosporine or calphostin C, but not the protein kinase G inhibitor KT-5823, abolished the inhibitory effect of NO on Na + -K + -ATPase in OK cells. Immunoblotting demonstrated that treatment with NO donors caused significant translocation of PKCα from cytosolic to particulate fractions in OK, but not in LLC-PK 1 , cells. Furthermore, the translocation of PKCα in OK cells was attenuated by either the phospholipase C inhibitor U-73122 or the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one. U-73122 also blunted the inhibitory effect of SNP on Na + -K + -ATPase in OK cells. The phospholipase A 2 inhibitor AACOCF3 did not blunt the inhibitory effect of SNP on Na + -K + -ATPase in OK cells. AACOCF3 alone, however, also decreased Na + -K + -ATPase activity in OK cells. In conclusion, our results demonstrate that NO activates PKCα in OK, but not in LLC-PK 1 , cells. The activation of PKCα in OK cells by NO is associated with inhibition of Na + -K + -ATPase.

To dissociate the renal effects from the systemic effects of angiotensin II blockade, the present study was designed to determine the effects of systemic and renal interstitial infusion of the specific angiotensin II (ANG II) receptor antagonist, losartan, on blood pressure and sodium excretion in rats fed a low-, normal, or high-sodium diet. Fractional sodium excretion (FENa) and mean arterial pressure (MAP) were measured in rats before and during systemic infusion of losartan (10 mg/kg) or renal interstitial infusion of losartan (3 mg/kg) by means of a chronically implanted matrix. In rats fed a low- or normal sodium diet, systemic infusion of losartan markedly decreased MAP (delta -21 +/- 2, delta -10 +/- 2 mmHg, respectively; P < 0.05) with an accompanying fall in FENa (delta -0.10 +/- 0.05, delta -0.91 +/- 0.40%, respectively; P < 0.05). In contrast, preferential blockade of renal ANG II with renal interstitial losartan infusion resulted in an increase in FENa (delta 0.13 +/- 0.04, delta 0.95 +/- 0.45%, respectively; P < 0.05) and no significant change in MAP. In rats fed a high-sodium diet, both systemic and renal interstitial infusion of losartan increased FENa (delta 1.90 +/- 0.26, delta 1.40 +/- 0.56%, respectively; P < 0.05). Although systemic infusion of losartan decreased MAP (delta -4.4 +/- 0.6 mmHg, P < 0.05) in rats fed a high-sodium diet, the reduction in MAP was much less than that in rats fed a low- and normal sodium diet.(ABSTRACT TRUNCATED AT 250 WORDS)

We tested the hypothesis that dopamine (DA) acts as an autocrine/paracrine regulator of Na(+)-Pi symport in proximal tubules, using opossum kidney (OK) cells as an in vivo model. Both DA and parathyroid hormone (PTH) increased adenosine 3',5'-cyclic monophosphate (cAMP) and inhibited Na(+)-gradient-dependent uptake of 32P but not that of L-[3H]-alanine. Incubation of OK cells with L-dopa, a DA precursor, resulted in accumulation of DA (7.4 nM), a ninefold increase of cAMP in the medium, and an inhibition (-10%) of Na(+)-Pi uptake. Carbidopa, an inhibitor of aromatic-L-amino acid decarboxylase, prevented the formation of DA from L-dopa, the increase in cAMP, and the inhibition of Na(+)-Pi cotransport. Pi-replete OK cells produced more DA (+15%) from L-dopa than Pi-deprived cells; however, the endogenous DA inhibited Na(+)-Pi cotransport both in Pi-deprived and in Pi-replete cells. Thus OK cells can synthesize DA from L-dopa in a quantity sufficient to elicit both the maximum DA-stimulated cAMP accumulation and inhibition of Na(+)-Pi cotransport in the same cell population. Our data, obtained on an in vitro system, support the hypothesis proposing that DA generated in proximal tubular cells can modulate, via cAMP, the Na(+)-Pi symport in the same or adjacent cells. If present in the kidney, this pathway might represent an autocrine/paracrine system that can contribute to regulation of renal Pi homeostasis.

This study examined the effect of increases in renal interstitial hydrostatic pressure (PI) on sodium excretion (UNaV) utilizing a direct technique for increasing renal interstitial volume. PI was increased by renal interstitial volume expansion (RIVE) via injection of 50 microliters of a 2% albumin in saline solution into the renal interstitium through a chronically implanted interstitial catheter. RIVE resulted in a stable increase in PI (4.6 +/- 0.4 to 9.4 +/- 0.8 mmHg) that was sustained over a 30- to 40-min period without significant changes in renal blood flow or glomerular filtration rate. Increases in PI were associated with significant increases in urine flow (13.8 +/- 3.4 to 31.7 +/- 5.0 microliters/min) and UNaV (2.3 +/- 0.6 to 6.2 +/- 1.1 micro eq/min) and fractional excretion of Na (2.6 +/- 0.8 to 6.9 +/- 1.5%). To determine the importance of albumin in maintaining an elevated PI, the effects of renal interstitial injections of saline were compared with albumin in saline solution. Injection of 50 microliters of saline into the renal interstitium had no sustained effect on PI. Injection of 2% albumin in saline solution in the same group of rats resulted in significant elevations in PI and UNaV. These data indicate that direct increases in PI via renal interstitial volume expansion result in significant increases in UNaV, thus supporting a role for PI in controlling UNaV.

Previous studies have shown that renal interstitial volume expansion (RIVE) increases renal interstitial hydrostatic pressure and urinary sodium excretion. In the present study we investigated whether blockade of prostaglandin synthesis inhibits the increase in fractional sodium excretion induced by RIVE. Expansion of the renal interstitial volume was achieved by injecting 50 microliters of 2.5% albumin solution into a polyethylene matrix chronically implanted in the left kidney. Fractional sodium excretion (FENa), renal interstitial hydrostatic pressure (PI), and urinary prostaglandin excretion (UPGE2) were measured before and after RIVE in eight control, seven meclofenamate-treated, and eight indomethacin-treated rats. RIVE in the control animals resulted in significant increases in PI (delta + 4.2 +/- 0.8 mmHg), in FENa (delta + 1.02 +/- 0.27%), and in UPGE2 (% delta + 150 +/- 38%) without significant changes in glomerular filtration rate. Inhibition of prostaglandin synthesis with meclofenamate or indomethacin attenuated the natriuretic response and blocked the increase in UPGE2 associated with RIVE. In summary, direct increases in renal interstitial hydrostatic pressure increase UPGE2 and urinary sodium excretion. This natriuretic response is markedly diminished by inhibition of prostaglandin synthesis. These studies suggest that prostaglandin synthesis may have an important role in mediating the natriuretic effect of increased renal interstitial hydrostatic pressure during renal interstitial volume expansion.

The effect of prostaglandins (PGs) on proximal sodium reabsorption has not been fully defined. The objective of the present study was to determine the response of proximal tubular sodium reabsorption to infusions of arachidonic acid and specific PGs into the renal interstitium in rats. Renal interstitial infusions of arachidonic acid as well as the individual PGs, I2, E2, and F2 alpha, were employed to elevate the concentration of these PGs in the kidney. Infusion of 10(-4) M arachidonic acid elicited a marked increase of urinary excretion of 6-keto-PGF1 alpha (a stable metabolite of PGI2) from 260.1 +/- 52.7 to 507.4 +/- 129.5 pg/min (P less than 0.05) and a smaller increase of PGE2 from 18.4 +/- 11.2 to 25.9 +/- 10.9 pg/min (P less than 0.05). When micropuncture samples were obtained from superficial late proximal tubules, infusion of arachidonic acid increased the fractional delivery of sodium (FDNa) from 47.8 +/- 5.9 to 58.3 +/- 4.6% (n = 6, P less than 0.01). In the presence of indomethacin, arachidonic acid failed to augment FDNa. Infusion of 10(-5) M PGI2 also increased FDNa from 51.4 +/- 3.4 to 64.0 +/- 4.4% (n = 10, P less than 0.01). PGF2 alpha did not change FDNa and PGE2 decreased it from 53.1 +/- 5.4 to 37.4 +/- 3.3% (n = 8, P less than 0.01). In summary, the present study demonstrates that renal interstitial infusion of arachidonic acid decreases sodium reabsorption by the superficial proximal tubules possibly through the stimulation of PGI2 production.