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Renal Dopamine Production and Release in the Rat: A Microdialysis Study
Renal DA Production and Release in Rat
873
4. Logvinenko, N. S., Dulubova, I., Fedosova, N., Larsson, S. H., Nairn, A . C., Esmann, M., Greengard, P., Aperia, A. (1996).Phosphorylation by protein kinase C of serine-23 of the a-l subunit of rat Na+, K+-ATPaseaffects its conformational equilibrium. Proc. Nutl. Acad. Sci. U.S.A. 93, 9132-9137. 5 . Nowicki, S., Chen, S., Aizman, O., Cheng, X., Li, D., Nowicki, C., Nairn, A,, Greengard, P., and Aperia, A. 20 HETE activates protein kinase C. Role in regulation of rat renal Na+, K' ATPase 1. Clin. Invest. 99, 3224-1230. 6. Meister, R., Bean, A. J., and Aperia, A. (1993).Catechol-0-methyltransferase mRNA in the kidney and its appearance during ontogeny. Kidney Int. 11, 726-733.
Robert M. Carey,* Zhi-Qin Wang," Helmy M. Siragy," and Robin A. Feldert Departments of Medicine* and tPathology University of Virginia Health Sciences Center Charlottesville, Virginia 22908
Renal Dopamine Production and Release in the Rat: A Microdialysis Study The renal dopaminergic system plays an important role in the regulation of blood pressure, sodium homeostasis and kidney function. Proximal tubule cells, rich in aromatic amino acid decarboxylase, can take up circulating and/ or filtered L-dopa for decarboxylation to dopamine (DA). Most of the DA appearing in urine is of proximal tubule origin. However, the extent to which the urinary excretion of DA reflects the amount of the amine that has been synthesized in tubule cells, and the fate and outflow of newly formed DA from the proximal tubules remains unknown. D1-like receptors are localized in renal blood vessels, the juxtaglomerular apparatus, the proximal tubule (both apical and basolateral), cortical collecting duct, and medullary thick ascending limb. D2-like receptors are described in the endothelial and adventitial layers of renal vasculature and the glomerulus. DA generated intrarenally has a welldocumented natriuretic effect, and infusion of DA antagonists decreases sodium excretion independently of hemodynamic changes. Because DA is synthesized, stored, and released within the kidney in close proximity to its target site(s), it is believed that DA produced by the renal proximal tubule serves as an intrarenal paracrine hormone mediating diuresis and natriuresis. The precise mechanism by which DA exerts its cell-to-cell action is not fully understood. In the present study, renal interstitial (RIF) DA (by in vivo microdialysis techAdvances in Pharmacology. Volume 42 Copyright D 1998 by Academic Press. All riglit5 01 reproduction in any form reserved i o s 4 - 3 ~ ~ $9 z8m o
874
Robert
M. Carey et a/.
nique) and urinary DA excretion (UDAV) were investigated in anesthetized rats on either normal (0.28% NaC1, NS) or high (4.0% NaCI, HS) sodium balance and in response to acute administration of a DA prodrug, y-L-glutamyl-L-dopa (gludopa). Responses of renal sodium excretion and intrarenal blood flow distribution to gludopa also were examined. Microdialysis probes were constructed as previously described (1,2). Studies of in vitro recovery of DA showed that the best relative recovery was observed with a perfusion rate of 1 pl/min and was 92.7 2 2.0%, compared with 73.9 t 4.9% and 44.3 t 3.3% at 3 and 5 pVmin, respectively. In vivo renal microdialysis was performed in anesthetized Sprague-Dawley rats (1,2). In vivo recovery of i.v. infused [3H]inulin has demonstrated that [3H]inulin appearing in the RIF sample is 2% of urinary [3H]inulin (2),indicating that the dialysate is not significantly contaminated by renal tubule fluid. Using a gradient equilibrium dialysis (2), the theoretically derived point (4.1pglpl) at which there was no net transmembrane flux was similar to the interstitial DA concentration (2.9pg/pl) measured directly during normal salt intake. Urine flow rate (W)and sodium excretion (UN,V)in HS (n = 9 ) were greater than in NS (n = 9)rats (W 7.2 2 0.6 vs 3.8 2 0.3 pVmin, p < .01; UNaV497 2 66 vs 265 ? 27 nmovmin, p < .01).UDAVincreased in HS compared with NS rats (601 t 68 vs 420 ? 37)pg/min, p < .05). In contrast, RIF DA was significantly lower in HS than NS rats (1.3 t 0.4 vs 3.7 t 0.5 pg/min, p < .01).Basal UDAV and RIF DA from rats with prior bilateral renal denervation (n = 10)were similar to rats with intact renal innervation (n = 14)(UDAV 476 2 30 vs 394 t 28 pg/min, p > 0.5; RIF DA 2.9 t 0.2 vs 3.4 t 0.3 pg/min, p > .05). UV tended to be higher in rats with prior bilateral renal denervation (4.12 0.2 vs 3.4 i- 0.2 pl/min, p = .05) while UNa was not significantly different between the two groups (286 2 13 vs 265 2 16 nmoVmin, p > .05). In rats with intact renal innervation, i.v. injection of gludopa at 3, 5, and 7.5 n m o k g (n = 7 in each group) produced a larger increase in UDAVthan RIF DA. Only the highest dose of gludopa (7.5nmol/kg), which resulted in a 7.3-foldincrease in UDAVand 1.7-fold increase in RIF DA, was associated with significant diuresis and natriuresis (W 3.4 i- 0.4 vs 7.4 t 0.5 purnin, p < .O1; UN,V 265 ? 27 vs 711 2 120 nmoVmin, p < .01).Renal blood flow of the cortex and medulla recorded by a laser-Doppler flowmeter did not change in rats receiving gludopa at 7.5 nmoVkg (n = 6),while angiotensin 11 (100ng/kg/min) induced significant reduction in cortical (42.6%)and medullary (28.1Yo)blood flow, which gradually returned toward preangiotensin levels when the angiotensin infusion was stopped (n = 6).In rats with prior bilateral renal denervation (n = 5 in each group), gludopa at 7.5 nmol/kg produced a significant increase in UDAV (8.3-fold) and RIF DA (1.8-fold), accompanied by significant diuresis and natriuresis (W 3.9 2 0.2 vs 8.2 2 0.5 pVmin, p < .01;UN,V 289 t vs 778 ? 56 nmol/min, p < .01). In rats on a normal salt diet in the present study, renal interstitial fluid DA levels were much lower than UDAV, suggesting that intrarenally produced DA is released preferentially into the tubule lumen rather than in the peritubular space. Cellular mechanisms of renal DA secretion are not understood. DA immunoreactive granules and L-dopa-induced fluorescence are mainly concen-
Renal DA Production and Release in Rat
875
trated in the region of the apical membrane of proximal tubule cells. There may be lumenal organic cation transporters with high affinity for DA in porcine proximal tubule (LLC-PK1)cells. Our study confirmed that UrIAVincreases in response to chronic salt loading. The mechanism underlying the observed reduction in RIF DA with chronic salt loading is not apparent. RIF DA may derive from tubular outward transport of DA through the basolateral membrane. Histofluorescent and neurochemical findings also suggest the presence of dopaminergic neurons in the kidney, and adrenergic nerves may become dopaminergic under certain circumstances. Vagal afferents have been shown to stimulate renal release of DA and produce a neurogenically mediated natriuresis. Our data confirm that the main source of DA in the urine is nonneuronal, and further demonstrate that renal nerve activity does not contribute significantly to either urinary or interstitial fluid DA. During chronic salt loading, intrarenally produced DA is released preferentially into the luminal space, where it may act on apical DA receptors as an autocoid or paracrine factor. Significant natriuresis and renal vasodilation occurs both in the whole animal and humans and in the isolated perfused rat kidney following pharmacological increase of renal DA production by gludopa. In the present study, much smaller quantities of gludopa (7.5 nmol/kg) resulted in a physiological increase in UDAV(7.3-fold),accompanied by a slight increase in renal interstitial fluid DA (1.7-fold), and produced significant diuresis and natriuresis without detectable changes in intrarenal blood flow. These results also support preferential secretion of DA generated by proximal tubule cells into the tubule lumen. Our previous study in the uninephrectomized conscious dog demonstrated that blockade of the renal DA-1 receptor with intrarenal infusion of SCH 23390 produced a 50% decrease in urine flow rate and sodium excretion, but there were no renal hemodynamic changes accompanying the antinatriuresis ( 3 ) . A 5.2-fold increase in kidney DA content with gludopa administration was associated with decreased renal cortical brush border Na+/H+antiporter activity and significant natriuresis (4.9-fold) and diuresis (2.6-fold)without any change in glomerular filtration rate in anesthetized rats (4). Low-dose L-dopa infusion, which resulted in a five- to eightfold increase in urine DA excretion, had a natriuretic effect without any change in blood pressure or glomerular filtration rate in healthy subjects of a low-sodium diet (5).These results strongly support that intrarenal DA plays a role in the control of natriuresis through a tubule mechanism. The renal sympathetic nerve endings are closely related to the juxtaglomerular apparatus and proximal tubule cells. Whether the sympathetic nervous system influences intrarenal DA generation and regulates renal DA-mediated natriuresis and diuresis is unknown. Basal DA production (as reflected by UoAV and renal interstitial fluid DA) and UN,V and their responses to gludopa were similar between rats with and without prior renal denervation. These results demonstrate that intrarenal production of DA and its renal effects are not significantly influenced by renal sympathetic nerve activity. Our data demonstrate that renal interstitial microdialysis can be used to monitor DA levels in the renal interstitial fluid in anesthetized rats. DA produced in the kidneys is released preferentially into the tubule lumen and exerts a direct
876
Robert M. Carey et al.
tubule effect in the control of sodium excretion. Renal DA production and its renal effects were not significantly influenced by renal sympathetic nerve activity.
Acknowledgments The authors wish to thank Dawn M. Fultz and Nancy L. Howell for their skillful technical assistance. This work was supported in part by National Institutes of Health grant RO1-HL49575 to Dr. R. M. Carey.
References 1 . Siragy, H. M., and Carey, R. M. (1996).The subtype-2 “AT,) angiotensin receptor regulates renal cyclic guanosine 3‘, 5‘-monophosphate and AT, receptor-mediated prostaglandin El production in conscious rats. J . Clin. Invest. 97, 1978-1982. 2. Siragy, H. M., and Linden, J. (1996). Sodium intake markedly alters renal interstitial fluid adenosine. Hypertension 27, 404-407. 3. Siragy, H. M., Felder, R. A., Howell, N. L., Chevalier, R. L., Peach, M. J., and Carey, R. M. (1989).Evidence that intrarenal dopamine acts as a paracrine substance at the renal tubule. Am. J. Physiol. 257, F469-F477. 4. Jose, P. A,, Eisner, G. M., Drago, J., Carey, R. M., Felder, R. M. (1996).Dopamine receptor signaling defects in spontaneous hypertension. Am. J. Hypertens. 9, 400-405. 5. Barendregt, J, N. M., Muizert, Y.,van Nispen tot Pannerden, L. L. A. M., and Chang, P. C. (1995). Intrarenal production of dopamine and natriuresis following dopa and saline infusion in healthy human volunteers. J . Human. Hypert. 9, 187-194.
873
4. Logvinenko, N. S., Dulubova, I., Fedosova, N., Larsson, S. H., Nairn, A . C., Esmann, M., Greengard, P., Aperia, A. (1996).Phosphorylation by protein kinase C of serine-23 of the a-l subunit of rat Na+, K+-ATPaseaffects its conformational equilibrium. Proc. Nutl. Acad. Sci. U.S.A. 93, 9132-9137. 5 . Nowicki, S., Chen, S., Aizman, O., Cheng, X., Li, D., Nowicki, C., Nairn, A,, Greengard, P., and Aperia, A. 20 HETE activates protein kinase C. Role in regulation of rat renal Na+, K' ATPase 1. Clin. Invest. 99, 3224-1230. 6. Meister, R., Bean, A. J., and Aperia, A. (1993).Catechol-0-methyltransferase mRNA in the kidney and its appearance during ontogeny. Kidney Int. 11, 726-733.
Robert M. Carey,* Zhi-Qin Wang," Helmy M. Siragy," and Robin A. Feldert Departments of Medicine* and tPathology University of Virginia Health Sciences Center Charlottesville, Virginia 22908
Renal Dopamine Production and Release in the Rat: A Microdialysis Study The renal dopaminergic system plays an important role in the regulation of blood pressure, sodium homeostasis and kidney function. Proximal tubule cells, rich in aromatic amino acid decarboxylase, can take up circulating and/ or filtered L-dopa for decarboxylation to dopamine (DA). Most of the DA appearing in urine is of proximal tubule origin. However, the extent to which the urinary excretion of DA reflects the amount of the amine that has been synthesized in tubule cells, and the fate and outflow of newly formed DA from the proximal tubules remains unknown. D1-like receptors are localized in renal blood vessels, the juxtaglomerular apparatus, the proximal tubule (both apical and basolateral), cortical collecting duct, and medullary thick ascending limb. D2-like receptors are described in the endothelial and adventitial layers of renal vasculature and the glomerulus. DA generated intrarenally has a welldocumented natriuretic effect, and infusion of DA antagonists decreases sodium excretion independently of hemodynamic changes. Because DA is synthesized, stored, and released within the kidney in close proximity to its target site(s), it is believed that DA produced by the renal proximal tubule serves as an intrarenal paracrine hormone mediating diuresis and natriuresis. The precise mechanism by which DA exerts its cell-to-cell action is not fully understood. In the present study, renal interstitial (RIF) DA (by in vivo microdialysis techAdvances in Pharmacology. Volume 42 Copyright D 1998 by Academic Press. All riglit5 01 reproduction in any form reserved i o s 4 - 3 ~ ~ $9 z8m o
874
Robert
M. Carey et a/.
nique) and urinary DA excretion (UDAV) were investigated in anesthetized rats on either normal (0.28% NaC1, NS) or high (4.0% NaCI, HS) sodium balance and in response to acute administration of a DA prodrug, y-L-glutamyl-L-dopa (gludopa). Responses of renal sodium excretion and intrarenal blood flow distribution to gludopa also were examined. Microdialysis probes were constructed as previously described (1,2). Studies of in vitro recovery of DA showed that the best relative recovery was observed with a perfusion rate of 1 pl/min and was 92.7 2 2.0%, compared with 73.9 t 4.9% and 44.3 t 3.3% at 3 and 5 pVmin, respectively. In vivo renal microdialysis was performed in anesthetized Sprague-Dawley rats (1,2). In vivo recovery of i.v. infused [3H]inulin has demonstrated that [3H]inulin appearing in the RIF sample is 2% of urinary [3H]inulin (2),indicating that the dialysate is not significantly contaminated by renal tubule fluid. Using a gradient equilibrium dialysis (2), the theoretically derived point (4.1pglpl) at which there was no net transmembrane flux was similar to the interstitial DA concentration (2.9pg/pl) measured directly during normal salt intake. Urine flow rate (W)and sodium excretion (UN,V)in HS (n = 9 ) were greater than in NS (n = 9)rats (W 7.2 2 0.6 vs 3.8 2 0.3 pVmin, p < .01; UNaV497 2 66 vs 265 ? 27 nmovmin, p < .01).UDAVincreased in HS compared with NS rats (601 t 68 vs 420 ? 37)pg/min, p < .05). In contrast, RIF DA was significantly lower in HS than NS rats (1.3 t 0.4 vs 3.7 t 0.5 pg/min, p < .01).Basal UDAV and RIF DA from rats with prior bilateral renal denervation (n = 10)were similar to rats with intact renal innervation (n = 14)(UDAV 476 2 30 vs 394 t 28 pg/min, p > 0.5; RIF DA 2.9 t 0.2 vs 3.4 t 0.3 pg/min, p > .05). UV tended to be higher in rats with prior bilateral renal denervation (4.12 0.2 vs 3.4 i- 0.2 pl/min, p = .05) while UNa was not significantly different between the two groups (286 2 13 vs 265 2 16 nmoVmin, p > .05). In rats with intact renal innervation, i.v. injection of gludopa at 3, 5, and 7.5 n m o k g (n = 7 in each group) produced a larger increase in UDAVthan RIF DA. Only the highest dose of gludopa (7.5nmol/kg), which resulted in a 7.3-foldincrease in UDAVand 1.7-fold increase in RIF DA, was associated with significant diuresis and natriuresis (W 3.4 i- 0.4 vs 7.4 t 0.5 purnin, p < .O1; UN,V 265 ? 27 vs 711 2 120 nmoVmin, p < .01).Renal blood flow of the cortex and medulla recorded by a laser-Doppler flowmeter did not change in rats receiving gludopa at 7.5 nmoVkg (n = 6),while angiotensin 11 (100ng/kg/min) induced significant reduction in cortical (42.6%)and medullary (28.1Yo)blood flow, which gradually returned toward preangiotensin levels when the angiotensin infusion was stopped (n = 6).In rats with prior bilateral renal denervation (n = 5 in each group), gludopa at 7.5 nmol/kg produced a significant increase in UDAV (8.3-fold) and RIF DA (1.8-fold), accompanied by significant diuresis and natriuresis (W 3.9 2 0.2 vs 8.2 2 0.5 pVmin, p < .01;UN,V 289 t vs 778 ? 56 nmol/min, p < .01). In rats on a normal salt diet in the present study, renal interstitial fluid DA levels were much lower than UDAV, suggesting that intrarenally produced DA is released preferentially into the tubule lumen rather than in the peritubular space. Cellular mechanisms of renal DA secretion are not understood. DA immunoreactive granules and L-dopa-induced fluorescence are mainly concen-
Renal DA Production and Release in Rat
875
trated in the region of the apical membrane of proximal tubule cells. There may be lumenal organic cation transporters with high affinity for DA in porcine proximal tubule (LLC-PK1)cells. Our study confirmed that UrIAVincreases in response to chronic salt loading. The mechanism underlying the observed reduction in RIF DA with chronic salt loading is not apparent. RIF DA may derive from tubular outward transport of DA through the basolateral membrane. Histofluorescent and neurochemical findings also suggest the presence of dopaminergic neurons in the kidney, and adrenergic nerves may become dopaminergic under certain circumstances. Vagal afferents have been shown to stimulate renal release of DA and produce a neurogenically mediated natriuresis. Our data confirm that the main source of DA in the urine is nonneuronal, and further demonstrate that renal nerve activity does not contribute significantly to either urinary or interstitial fluid DA. During chronic salt loading, intrarenally produced DA is released preferentially into the luminal space, where it may act on apical DA receptors as an autocoid or paracrine factor. Significant natriuresis and renal vasodilation occurs both in the whole animal and humans and in the isolated perfused rat kidney following pharmacological increase of renal DA production by gludopa. In the present study, much smaller quantities of gludopa (7.5 nmol/kg) resulted in a physiological increase in UDAV(7.3-fold),accompanied by a slight increase in renal interstitial fluid DA (1.7-fold), and produced significant diuresis and natriuresis without detectable changes in intrarenal blood flow. These results also support preferential secretion of DA generated by proximal tubule cells into the tubule lumen. Our previous study in the uninephrectomized conscious dog demonstrated that blockade of the renal DA-1 receptor with intrarenal infusion of SCH 23390 produced a 50% decrease in urine flow rate and sodium excretion, but there were no renal hemodynamic changes accompanying the antinatriuresis ( 3 ) . A 5.2-fold increase in kidney DA content with gludopa administration was associated with decreased renal cortical brush border Na+/H+antiporter activity and significant natriuresis (4.9-fold) and diuresis (2.6-fold)without any change in glomerular filtration rate in anesthetized rats (4). Low-dose L-dopa infusion, which resulted in a five- to eightfold increase in urine DA excretion, had a natriuretic effect without any change in blood pressure or glomerular filtration rate in healthy subjects of a low-sodium diet (5).These results strongly support that intrarenal DA plays a role in the control of natriuresis through a tubule mechanism. The renal sympathetic nerve endings are closely related to the juxtaglomerular apparatus and proximal tubule cells. Whether the sympathetic nervous system influences intrarenal DA generation and regulates renal DA-mediated natriuresis and diuresis is unknown. Basal DA production (as reflected by UoAV and renal interstitial fluid DA) and UN,V and their responses to gludopa were similar between rats with and without prior renal denervation. These results demonstrate that intrarenal production of DA and its renal effects are not significantly influenced by renal sympathetic nerve activity. Our data demonstrate that renal interstitial microdialysis can be used to monitor DA levels in the renal interstitial fluid in anesthetized rats. DA produced in the kidneys is released preferentially into the tubule lumen and exerts a direct
876
Robert M. Carey et al.
tubule effect in the control of sodium excretion. Renal DA production and its renal effects were not significantly influenced by renal sympathetic nerve activity.
Acknowledgments The authors wish to thank Dawn M. Fultz and Nancy L. Howell for their skillful technical assistance. This work was supported in part by National Institutes of Health grant RO1-HL49575 to Dr. R. M. Carey.
References 1 . Siragy, H. M., and Carey, R. M. (1996).The subtype-2 “AT,) angiotensin receptor regulates renal cyclic guanosine 3‘, 5‘-monophosphate and AT, receptor-mediated prostaglandin El production in conscious rats. J . Clin. Invest. 97, 1978-1982. 2. Siragy, H. M., and Linden, J. (1996). Sodium intake markedly alters renal interstitial fluid adenosine. Hypertension 27, 404-407. 3. Siragy, H. M., Felder, R. A., Howell, N. L., Chevalier, R. L., Peach, M. J., and Carey, R. M. (1989).Evidence that intrarenal dopamine acts as a paracrine substance at the renal tubule. Am. J. Physiol. 257, F469-F477. 4. Jose, P. A,, Eisner, G. M., Drago, J., Carey, R. M., Felder, R. M. (1996).Dopamine receptor signaling defects in spontaneous hypertension. Am. J. Hypertens. 9, 400-405. 5. Barendregt, J, N. M., Muizert, Y.,van Nispen tot Pannerden, L. L. A. M., and Chang, P. C. (1995). Intrarenal production of dopamine and natriuresis following dopa and saline infusion in healthy human volunteers. J . Human. Hypert. 9, 187-194.