Role of plasma renin activity and the renal nerves in the natriuresis of L-NMMA infusion in rats.

The objective of this study was to determine the effect of N(G)-monomethyl-L-arginine (L-NMMA) infusion on plasma renin activity (PRA) in the presence or absence of the renal nerves in normotensive Wistar-Kyoto (WKY) rats and Okamoto spontaneously hypertensive rats (SHR). All rats were unilaterally nephrectomized two weeks before the acute experiment. On the day of the experiment, acute renal denervation (Dnx) of the remaining kidney was performed in one group of WKY rats (Dnx-WKY; n= 10) and one group of SHRs (Dnx-SHR: n=7). The renal nerves were left intact in a group of WKY rats (Inn-WKY; n=8) and SHRs (Inn-SHR; n=9). After a control clearance period, L-NMMA was administered i.v. (15 mg/kg bolus followed by 500 microg/kg/min infusion) and another clearance period of 20 min was taken. In all experimental groups L-NMMA infusion resulted in a significant natriuresis. L-NMMA infusion increased fractional excretion of sodium (FE(Na)) to a greater extent in the Inn-SHR than in the Inn-WKY (delta FE(Na) = 5.23+/-0.87% vs delta FE(Na) = 2.87+/-0.73% respectively; P=0.05), PRA did not change in the SHR with the infusion of L-NMMA. However, in the Inn-WKY group, the natriuresis of L-NMMA infusion was associated with a tendency for lower PRA levels as compared to a group of time control Inn-WKY rats. In Dnx-WKY, the natriuresis of L-NMMA infusion (delta FE(Na) = 4.60+/-0.52%) was associated with a significantly lower level of PRA (4.26+/-1.18 ng AI/ml/hr) as compared to a group of time control Dnx-WKY rats (9.83+/-1.32 ng AI/ml/hr; P<0.05). In the Dnx-SHR, the natriuretic response to L-NMMA infusion was significantly attenuated by renal denervation (delta FE(Na) = 2.36+/-0.34%) and PRA was unchanged. In conclusion, the natriuretic effect of systemic inhibition of nitric oxide (NO) synthesis was associated with decreased PRA in the Dnx-WKY suggesting that a potential interaction exists between NO and the renal nerves in the modulation of PRA in the normotensive WKY rat. Whereas, the natriuretic effect of L-NMMA infusion in the SHR in the presence and absence of the renal nerves, were independent of changes in PRA.

Mechanism underlying diuretic effect of L-NAME at a subpressor dose.

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 N(omega)-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 microg. kg(-1). min(-1) in Sprague-Dawley rats (N = 8), which did not alter mean arterial pressure or glomerular filtration rate, significantly increased urine flow rate (U(v); from 78.2 +/- 12.7 to 117.1 +/- 14.9 microl/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 microM, 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.

Intrarenal serotonin, dopamine, and phosphate handling in remnant kidneys.

BACKGROUND: Serotonin (5-HT) and dopamine (DA) are intrarenal autocrine/paracrine substances that regulate phosphate reabsorption. The present studies explored intrarenal serotonin and DA metabolism and the implications for phosphate homeostasis in rats with remnant kidneys, a model for renal failure. METHODS: The intrarenal productions of serotonin and DA were determined from measurements of renal interstitial fluid (microdialysate) and urine in rats with remnant or intact kidneys. In clearance studies, the effects of infusion of methiothepin, a serotonin receptor antagonist, or gludopa, a renal selective DA precursor, on phosphate and sodium excretion were determined in rats with a remnant or intact kidneys. RESULTS: Renal interstitial serotonin (5-HT, 3.4 +/- 0.9 pg/min) was fourfold higher than DA (0.6 +/- 0.1 pg/min) in remnant kidneys. Conversely, urinary excretion of serotonin was fourfold less than DA in rats with a remnant kidney (5-HT 0.4 +/- 0.02 vs. DA 1.5 +/- 0.1 ng/min). Infusion of methiothepin or gludopa significantly increased the fractional excretion of phosphate (FE(Pi)) in rats with a remnant kidney from 54 +/- 3 to 67 +/- 7% (P < 0.05) and from 36 +/- 10% to 51 +/- 13% (P < 0.05), respectively. CONCLUSION: We conclude that serotonin preferentially accumulates in the renal interstitium, whereas DA exits primarily via the tubular lumen. Phosphate excretion is increased by both the acute infusion of the serotonin receptor antagonist and the infusion of gludopa, suggesting that both serotonin and DA modulate phosphate excretion in rats with remnant kidneys.

Effect of inhibition of MAO and COMT on intrarenal dopamine and serotonin and on renal function.

The purpose of the present investigation was to study the effects of inhibition of monoamine oxidase (MAO) and/or catechol-O-methyltransferase (COMT), enzymes involved in the degradation of dopamine (DA) and serotonin (5-HT), on intrarenal DA and 5-HT, as reflected in the renal interstitial fluid (RIF) microdialysate and urine, and on renal function. Inhibition of MAO selectively increased RIF 5-HT from 3.16 +/- 0.38 to 8.03 +/- 1.83 pg/min (n = 7, P < 0.05), concomitant with decreases in mean arterial blood pressure and glomerular filtration rate (2.09 +/- 0. 18 to 1.57 +/- 0.22 ml/min, n = 7, P < 0.05). Inhibition of COMT significantly increased RIF DA (3.47 +/- 0.70 to 8.68 +/- 1.96 pg/min, n = 9, P < 0.05), urinary DA (2.00 +/- 0.16 to 2.76 +/- 0.26 ng/min, n = 9, P < 0.05), and absolute excretion of sodium (6.42 +/- 2.00 to 9.82 +/- 1.62 micromol/min, n = 10, P < 0.05). Combined inhibition of MAO and COMT significantly increased RIF DA, urinary DA, and urinary 5-HT, which was accompanied with increases in urine flow rate, and absolute (3.03 +/- 0.59 to 8.40 +/- 1.61 micromol/min, n = 9, P < 0.01) and fractional excretion of sodium. We conclude that inhibition of MAO selectively increases RIF 5-HT. COMT appears to be more important than MAO in the metabolism of intrarenal DA. Physiological increases in intrarenal DA/5-HT induced by inhibition of their degrading enzymes are accompanied with significant alterations of renal function.

Role of adenosine in contrast media-induced acute renal failure in diabetes mellitus.

Increased release of renal adenosine and stimulation of renal adenosine receptors have been proposed to be major mechanisms in the development of contrast media-induced acute renal failure (CM-ARF). Patients with diabetes mellitus or preexisting renal disease who have reduced renal function have a markedly increased risk to develop CM-ARF. This increased risk to develop CM-ARF in patients with diabetes mellitus is linked to a higher sensitivity of the renal vasculature to adenosine, since experimental studies have shown increased adenosine-induced vasoconstriction in the kidneys of diabetic animals. Furthermore, recent evidence suggests that administration of adenosine receptor antagonists reduces the risk of development of CM-ARF in both diabetic and nondiabetic patients. The purpose of this review is to discuss the role of adenosine in the development of CM-ARF, particularly in the kidneys of diabetic patients, and to evaluate the therapeutic potential of adenosine receptor antagonists in the prevention of CM-ARF. Selective adenosine A1 receptor antagonists may provide a therapeutic tool to prevent CM-ARF in patients with diabetes mellitus and reduced renal function.

Production and functional roles of nitric oxide in the proximal tubule.

A significant role for nitric oxide (NO) in proximal tubule physiology and pathophysiology has been revealed by a series of in vivo and in vitro studies. Whether the proximal tubule produces NO under basal conditions is still controversial; however, evidence suggests that the proximal tubule is constantly exposed to NO that might include NO from nonproximal tubule sources. When challenged with a variety of stimuli, including hypoxia, the proximal tubule is able to produce large quantities of NO. In vivo studies generally indicate that NO inhibits fluid and sodium reabsorption by the proximal tubule. However, the final effect of NO on proximal tubular reabsorption appears to depend on the concentration of NO and involve interaction with other regulatory mechanisms. NO regulates Na(+)-K(+)-ATPase, Na(+)/H(+) exchangers, and paracellular permeability of proximal tubular cells, which may contribute to its effect on proximal tubular transport. Enhanced production of NO, perhaps depending on macrophage type inducible NO synthase, participates in hypoxic/ischemic proximal tubular injury. In conclusion, NO plays a fundamental role in both physiology and pathophysiology of the proximal tubule.

Effect of gamma-L-glutamyl-L-dopa on phosphate excretion.

gamma-L-glutamyl-L-DOPA (gludopa) is a dopamine prodrug that is relatively specific for the kidney. Because dopamine is phosphaturic, the present study compared the phosphaturic effects of the infusion of equimolar doses of gludopa (n = 8), L-DOPA (n = 8), and gamma-L-glutamyl-L-tyrosine (glutyrosine, n = 6). Glutyrosine was used as a control to evaluate the effect of the glutamyl portion of gludopa on phosphate excretion. Sprague-Dawley rats (350 to 400 g) were anesthetized with 5-sec-butylethyl-2-thyobarbituric acid (Inactin; 100 mg/kg, IP) and underwent thyroparathyroidectomy. Clearances were taken during the infusion of normal saline vehicle, followed by the infusion of gludopa, L-DOPA, or glutyrosine, all infused at the rate of 10 nmol/kg bolus and 0.8 nmol/kg/min (iv). To determine the contribution of glutamyl derivative to phosphate excretion, gludopa or L-DOPA was infused in the presence of SCH23390, a DA-1 receptor antagonist. Gludopa infusion significantly increased dopamine excretion (from 1.9+/-0.2 ng/min to 17.0+/-3.9 ng/min, delta15.0+/-3.9 ng/min, P < .008) and fractional excretion of phosphate (from 2.6%+/-0.6% to 34.8%+/-1.8%, delta32.0%+/-1.6%, P < .001). L-DOPA infusion significantly increased dopamine excretion (from 1.4+/- 0.4 ng/min to 9.7+/-1.6 ng/min, delta8.3+/-1.5 ng/min, P < .001) and fractional excretion of phosphate (from 1.7%+/-0.6% to 8.2%+/-2.0%, delta6.4%+/-1.5%, P < .004). Glutyrosine infusion significantly increased fractional excretion of phosphate (from 2.8%+/-0.8% to 17.5%+/-5.2%, delta14.6%+/-4.8%, P < .03) without changing dopamine excretion (delta0.5+/-0.2 ng/min). Infusion of gludopa in the presence of SCH23390 increased fractional excretion of phosphate (from 5.7%+/-2.5% to 12.6%+/-3.5%, delta6.8%+/-2.3%, n = 6, P < .03), whereas SCH23390 completely blocked the phosphaturic effect of L-DOPA. We conclude that gamma-L-glutamyl-L-DOPA is more phosphaturic than L-DOPA in the rat because of the combined effects of dopamine and the glutamyl moiety.

Nitric oxide activates PKCalpha and inhibits Na+-K+-ATPase in opossum kidney cells.

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-PK1 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-PK1, 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 PKCalpha from cytosolic to particulate fractions in OK, but not in LLC-PK1, cells. Furthermore, the translocation of PKCalpha 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 A2 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 PKCalpha in OK, but not in LLC-PK1, cells. The activation of PKCalpha in OK cells by NO is associated with inhibition of Na+-K+-ATPase.

Dopamine enhances the phosphaturic effect of PTH during acute respiratory alkalosis.

The phosphaturic response to parathyroid hormone (PTH) is blunted during acute respiratory alkalosis. The objective of the present study was to determine the effect of dopamine on the blunted phosphaturic response to PTH during acute respiratory alkalosis. The phosphaturic response to PTH was determined in thyroparathyroidectomized (TPTX) normocapnic and respiratory alkalotic rats in the absence and presence of the infusion of exogenous dopamine (25 microg/kg/min) or of 3,4-dihydroxyphenylalanine (L-DOPA, 250 microg/kg/min) to increase endogenous dopamine synthesis. In normocapnic rats, PTH infusion (33 U/kg plus 1 U/kg/min) significantly increased the fractional excretion of phosphate (FE(Pi)), from 1.5%+/-0.5% to 28.4%+/-4.0%, (deltaFE(Pi) 26.9%+/-4.1%, n = 11, P<.05); in respiratory alkalotic rats, the increase was from 0.4%+/-0.1% to 11.4%+/-1.7% (deltaFE(Pi) 11.0%+/-1.8%, n = 13, P<.05). However, the phosphaturic response to PTH was attenuated in respiratory alkalotic rats (deltaFE(Pi) 26.9%+/-4.1% vs 11.0%+/-1.9%, P<.05). In normocapnic rats, in the presence of dopamine or L-DOPA infusions, PTH infusion significantly increased the FE(Pi) from 6.1%+/-2.3% to 33.4%+/-8.0% (deltaFE(Pi) 27.3%+/-7.0%, n = 5) and from 3.2%+/-0.6% to 32.5%+/-3.3% (deltaFE(Pi) 29.3%+/-3.2%, n = 7), respectively. In respiratory alkalotic rats, in the presence of dopamine infusion, PTH significantly increased the FE(Pi), from 0.6%+/-0.2% to 19.3%+/-3.3% (deltaFE(Pi) 18.7%+/-3.3%, n = 6); in the presence of L-DOPA infusion it increased from 1.0%+/-0.3% to 20.5%+/-2.8% (deltaFE(Pi) 19.5%+/-2.9%, n = 8, P<.05 as compared with PTH alone). Thus the phosphaturic effect of PTH that was attenuated in respiratory alkalotic rats was enhanced by stimulation of endogenous dopamine synthesis by the infusion of L-DOPA.

The paracellular permeability of opossum kidney cells, a proximal tubule cell line.

UNLABELLED: The paracellular permeability of opossum kidney cells, a proximal tubule cell line. BACKGROUND: The regulation of the unusually leaky paracellular pathway of the proximal tubule is poorly understood partially because of the lack of an appropriate in vitro cell model. In this study, we determined whether the paracellular permeability of opossum kidney (OK) cells would resemble that of the in vivo proximal tubule epithelium. METHODS: The parental and subclonal OK cells and, for comparison, LLC-PK1 cells were cultured on permeable Transwell supports. The apparent paracellular permeability coefficient (Papp) for the extracellular marker 3H-mannitol was determined. RESULTS: The Papp of OK cell sheets (12.17 x10-6 cm/sec) was remarkably close to the previously reported Papp of rat proximal tubules. The Papp of LLC-PK1 cells, another proximal tubule cell line, however, was approximately 20-fold lower than that of both OK cells and the in vivo proximal tubule. Phorbol 12-myristate 13-acetate, a protein kinase C activator, enhanced the Papp of OK cell sheets. The characteristic response of paracellular permeability to Ca2+ switch was demonstrated in OK cell sheets. Slight variations of Papp among several OK subclones were observed. Basal to apical Papp was uniformly higher than apical to basal Papp, independent of cell subtype. This rectification was attenuated by inhibition of active transport. CONCLUSIONS: OK cell sheets cultured on Transwell supports possess a leaky paracellular pathway resembling that of the proximal tubule epithelium in vivo.

Natriuretic response to increased pressure is preserved with COX-2 inhibitors.

Elevation of renal interstitial hydrostatic pressure (RIHP) by direct renal interstitial volume expansion increases sodium excretion. This natriuretic response is blunted by the nonspecific inhibition of the cyclooxygenase (COX) enzymes. The present study tested the hypothesis that the natriuretic response to increased RIHP during direct renal interstitial volume expansion is dependent on COX-1 but not COX-2. RIHP and fractional excretion of sodium (FE(Na)) were measured before and after direct renal interstitial volume expansion in control rats (n=7), rats infused with the COX-1 inhibitor piroxicam (n=6, 1.5 mg/kg), and rats infused with the COX-2 inhibitors NS-398 (n=5, 1.5 mg/kg) and meloxicam (n=6, 0.3 mg/kg). In control animals, direct renal interstitial volume expansion significantly increased RIHP (Delta2.3+/-0.5 mm Hg, P<0. 05) and FE(Na) (Delta1.1+/-0.3%, P<0.05). Likewise, in animals infused with NS-398 or meloxicam, direct renal interstitial volume expansion significantly increased RIHP (Delta1.8+/-0.6 mm Hg, P<0.05, and Delta1.7+/-0.3 mm Hg, P<0.05) and FE(Na) (Delta1.5+/-0.4%, P<0. 05, and Delta1.1+/-0.3%, P<0.05), respectively. In contrast, infusion of piroxicam significantly blunted the natriuretic response to direct renal interstitial volume expansion (DeltaFE(Na) 0.3+/-0. 2%), even though RIHP was increased (Delta1.9+/-0.6 mm Hg, P<0.05). Infusion of piroxicam but not NS-398 or meloxicam blunted the natriuretic response to increased renal interstitial hydrostatic pressure, suggesting that the natriuretic response to increased blood pressure may be preserved during inhibition of COX-2.

Adenosine-induced renal vasoconstriction in diabetes mellitus rats: role of prostaglandins.

We investigated the role of prostaglandins in the renal vascular response to exogenous and endogenous adenosine in control and streptozotocin (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 prostaglandin synthesis with indomethacin (Indo; 10 mg/kg iv) potentiated the adenosine-induced renal vasoconstriction in control rats but not in STZ rats. In control rats, Indo shifted the dose response curve of exogenous adenosine-induced RBF reductions to the left by a factor of 10 (ED(50): from 5.5 +/- 0.51 to 0.55 +/- 0.07 nmol adenosine, n = 6, P < 0.001) and in STZ rats only by a factor of two (ED(50): from 0.32 +/- 0.03 to 0.16 +/- 0.02 nmol adenosine, n = 6, P > 0.05). The renal response to endogenous adenosine was assessed by the magnitude of the postocclusive reduction of RBF (POR), an adenosine-mediated phenomenon. POR was greater in STZ rats (-65.3 +/- 5.2%, P < 0.001) compared with control rats (-36.2 +/- 3.5%). Indo markedly enhanced POR in control rats (-20.3 +/- 3.7%) but not in STZ rats (-4.5 +/- 2.7%). Renal cortical and medullary PGE(2) microdialysate concentrations and urinary PGE(2) excretions were clearly not lower in STZ (cortex: 169 +/- 61 pg/ml; medulla: 640 +/- 88 pg/ml, urine: 138 +/- 25 pg/min) compared with control rats (cortex: 99 +/- 12 pg/ml; medulla: 489 +/- 107 pg/ml; urine: 82 +/- 28 pg/min). Indo significantly decreased renal cortical, medullary, and urinary excretion of PGE(2) in STZ and control rats. These findings demonstrate that the adenosine-induced renal vasoconstriction is increased in the presence of Indo in control rats but not in STZ rats. The observations suggest that the diabetic renal vasculature may have a diminished vasodilatory capacity in response to prostaglandins to counteract adenosine-induced renal vasoconstriction.

Chronic oral L-DOPA increases dopamine and decreases serotonin excretions.

Given the common pathways for uptake and synthesis for dopamine and serotonin, enhanced renal dopamine synthesis in response to increased substrate 3,4-dihydroxyphenylalanine (L-DOPA) is postulated to decrease renal serotonin synthesis. The present study compared the effects of chronic oral administration of L-DOPA on dopamine and serotonin excretion in vivo, with the effects of enhanced dopamine synthesis per nephron due to adaptation to reduced renal mass (RRM). Four groups of rats were studied: sham-operated rats and rats with RRM in the absence and presence of chronic oral L-DOPA. L-DOPA (2 mg. 100 g body wt(-1). day(-1)) for 6-14 days increased calculated dopamine synthesis per nephron in sham-operated rats from 2.0 +/- 0.3 (n = 7) to 13.6 +/- 1.8 pg. day(-1). nephron(-1) (n = 7, P < 0.05) and in rats with RRM from 6.1 +/- 1.3 (n = 7) to 39.3 +/- 5.2 pg. day(-1). nephron(-1) (n = 7, P < 0.05). Chronic oral L-DOPA concomitantly decreased serotonin synthesis per nephron in sham-operated rats (1.6 +/- 0.1 to 1.0 +/- 0.1 pg. day(-1). nephron(-1), n = 7, P < 0.05) and in rats with RRM (5.6 +/- 0.9 to 2.6 +/- 0.4 pg. day(-1). nephron(-1), n = 7, P < 0.05). Both serotonin and dopamine synthesis per nephron were increased in rats with RRM. In conclusion, chronic oral administration of L-DOPA enhances dopamine excretion and decreases serotonin excretion in normal rats and in rats with reduced renal mass. Both dopamine and serotonin excretions per nephron were elevated by renal mass reduction.

Role of nitric oxide in intrarenal hemodynamics in experimental diabetes mellitus in rats.

The role of nitric oxide (NO) in the regulation of the intrarenal microcirculation in streptozotocin (STZ)-induced diabetes mellitus in rats is not clear. We examined renal cortical and papillary hemodynamics in STZ rats and determined the effects of systemic inhibition and stimulation of NO synthesis. Renal blood flow in cortical (QCC), and inner medullary ascending (QAV) and descending (QDV) vasa recta capillaries was measured by fluorescence videomicroscopy in STZ Munich-Wistar rats and nondiabetic control rats. Ten days after STZ injection (80 mg/kg ip), basal QCC and QDV were significantly greater in STZ rats (n = 16) compared with control rats (n = 15). Infusion of N(G)-monomethyl-L-arginine (L-NMMA, 15 mg/kg bolus, 500 microg. min(-1). kg(-1) iv) decreased Q(CC) (-41%), QAV (-38%), and QDV (-37%) in control rats (n = 6) and to a significantly greater magnitude than in STZ rats (n = 7), Q(CC) (-14%), QAV (-20%), and QDV (-25%). Coinfusion of L-arginine (L-Arg, 1 mg. kg(-1). min(-1) iv) with L-NMMA increased Q(CC) to a significantly greater extent (P < 0.01) in control rats compared with STZ rats. In subsequent studies, infusion of L-Arg alone increased QCC (+50%), QAV (+16%), and QDV (+11%) in control rats (n = 5) but had no effect in STZ rats (n = 5). These results show that the response of renal cortical and papillary capillary blood flow to both inhibition and stimulation of NO synthesis is attenuated in the early onset of STZ-diabetes mellitus rats compared with control rats.

Nitric oxide reduces the molecular activity of Na+,K+-ATPase in opossum kidney cells.

BACKGROUND: Nitric oxide (NO) directly inhibits fluid and solute reabsorption in the proximal tubule. In the present study, we investigated the effect of NO on the Na+, K+-ATPase of opossum kidney (OK) cells, a proximal tubule cell line, and its mechanisms. METHODS: Na+,K+-ATPase activity in the membrane fraction of OK cells was measured as the ouabain-sensitive ATP hydrolytic activity. The enzyme unit number on intact cells was measured by ouabain-binding assay. RESULTS: Incubation with 0.5 mM sodium nitroprusside (SNP), a NO donor, for two hours inhibited the catalytic activity of the membrane-associated Na+,K+-ATPase in OK cells to 65.5 +/- 9.7% of control (N = 6, P < 0.05 vs. control). This effect of SNP was concentration- and time-dependent. The NO scavenger hemoglobin blunted, while another NO donor spermine NONOate (5 microM) mimicked this effect of SNP. At all concentrations and time points tested, SNP did not alter the molecular number of Na+,K+-ATPase on intact OK cells, indicating that NO inhibited the molecular activity of Na+,K+-ATPase. The soluble guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo-[4, 3-a]quinoxalin-1-one (ODQ), blunted the inhibitory effect of SNP on the Na+,K+-ATPase activity. An exogenous cGMP analog similarly inhibited the Na+,K+-ATPase activity. Neither lipid soluble antioxidants vitamin E/probucol or thiol group compound DL-dithiothreitol (DTT) altered the inhibitory effect of SNP on the Na+,K+-ATPase activity. CONCLUSIONS: NO inhibited the molecular activity of the Na+,K+-ATPase of the OK proximal tubule cell line probably via cGMP-dependent mechanisms.

Nitric oxide enhances paracellular permeability of opossum kidney cells.

BACKGROUND: Nitric oxide (NO) has been shown to be a paracrine/autocrine regulator of proximal tubular transport. In this study, we investigated the effect of NO on the paracellular permeability of opossum kidney (OK) cells, a proximal tubule cell line that possesses a leaky paracellular pathway resembling that of the in vivo proximal tubule. METHODS: Paracellular permeability of OK cells cultured on permeable supports was measured as the apparent paracellular permeability coefficient (Papp) for 3[H]-D-mannitol. Changes in cell viability, cellular adenosine triphosphate (ATP) content, cGMP levels, and lipid peroxidation were assessed. RESULTS: Incubation with 2 mM sodium nitroprusside (SNP), an NO donor, for 24 hours significantly enhanced the Papp of OK cell sheets by 30.6 +/- 7.0% (N = 8, P < 0.05). This effect was largely blunted by hemoglobin, a NO scavenger. Cell viability was not compromised. This effect of SNP was concomitant with a moderate reduction of cellular ATP content, an increase in lipid peroxidation, and an increase in cellular cGMP levels. The antioxidant superoxide dismutase (SOD) significantly attenuated the effect of SNP on cellular ATP content and blunted the increase in Papp caused by SNP. A soluble guanylate cyclase inhibitor did not affect the effect of SNP on the Papp. CONCLUSIONS: NO enhances the paracellular permeability of OK cells possibly via mechanisms involving decreases in cellular ATP content.

Adenosine-induced renal vasoconstriction in diabetes mellitus rats: role of nitric oxide.

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 Nomega-nitro-L-arginine (L-NNA, 30 micromol/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 of L-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.

Effect of renal interstitial infusion of L-dopa on sodium and phosphate excretions.

It has been hypothesized that dopamine synthesized by the proximal tubule can act as a paracrine substance that regulates reabsorption by the proximal tubule. The present study was performed to study the effects of the stimulation of endogenous synthesis of dopamine by infusion of L-DOPA directly into the renal interstitium on sodium and phosphate excretions and to determine the roles of D1 and D2 receptors in the response. The infusion of L-DOPA (50 microg/kg/min) into the renal interstitium through an implanted matrix significantly increased the fractional excretion of sodium (FENa) from 1.0%+/-0.2% to 3.1%+/-0.6% and the fractional excretion of phosphate (FEPi) from 23%+/-3% to 36%+/-3%, P < .05, n = 10. The infusion of D1 receptor antagonist SCH23390 or SKF83566 (5 microg/kg/min) into the renal interstitium blocked the natriuretic (FENa 1.5%+/-0.2% to 1.9%+/-0.4%) and phosphaturic (FEPi 41%+/-3% to 41%+/-4%) effects of L-DOPA infusion. The infusion of the D2 receptor antagonist sulpiride at a rate of 4 microg/kg/min into the renal interstitium also attenuated the natriuretic (FENa 1.3%+/-0.3% to 1.6%+/-0.5%) and phosphaturic effects of L-DOPA infusion (FEPi 36%+/-5% to 39%+/-5%). We conclude that the renal interstitial infusion of L-DOPA increases sodium and phosphate excretions and that these responses are mediated by D1 and D2 receptors.

Effect of renal interstitial adenosine infusion on phosphate excretion in diabetes mellitus rats.

We previously demonstrated an increased sensitivity of the renal vasculature to adenosine (ADO) mediated via ADO A1 receptors in streptozotocin (STZ) diabetic rats. Because ADO stimulates P(i) reabsorption in the proximal tubule, the present study was performed to determine whether the sensitivity of the renal tubular system to the antiphosphaturic effect of ADO is enhanced in STZ rats. Clearance studies were performed, and ADO was infused into the renal interstitium via implanted matrices in STZ- and control (Con) rats to mimic the effects of endogenous ADO. Renal phosphate excretion was significantly increased in STZ rats (0.75 +/- 0.05 mumol/24 h) compared with Con rats (0.35 +/- 0.08 mumol/24 h), and fractional phosphate excretion (FEPi) tended to be higher in STZ rats (34.8 +/- 4.1%) than in Con rats (26.7 +/- 2.2%). Renal interstitial ADO infusion (5 mumol/h) was significantly more antiphosphaturic in STZ rats (FEPi decreased by 2.90 +/- 1.6%; P > 0.05), in which ADO only tended to decrease FEPi. To determine the role of ADO A1 receptors on P(i) excretion, the selective ADO A1 receptor blocker 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) was infused into the renal interstitium. DPCPX increased FEPi by 4.3 +/- 1.2% (P < 0.05) in the presence and 7.1 +/- 3.9% (P < 0.05) in the absence of ADO infusion in Con rats but had no effect on FEPi in STZ rats. In conclusion, STZ-diabetes mellitus enhances the antiphosphaturic effect of ADO by mechanisms unrelated to ADO A1 receptor stimulation.