Ulla Holtbäck

Activation/deactivation of renal Na+,K(+)-ATPase: a final common pathway for regulation of natriuresis.

Renal sodium metabolism, a major determinant of blood pressure, is regulated with great precision by a variety of endocrine, autocrine, and neuronal factors. Although these factors arc known to regulate sodium metabolism by affecting the rate of tubular sodium reabsorption, the molecular mechanisms by which they act are poorly understood, Na+,K+‐ATPase plays a pivotal role for sodium reabsorption in all tubular segments. The activity of this enzyme can be dynamically regulated by phosphorylation and dephosphorylation, Here we summarize both old and new evidence that several major substances believed to be involved in the regulation of sodium metabolism and blood pressure, i.e., the antidiuretic agents angiotensin II and norepinephrine, and the diuretic agents dopamine and atrial natriuretic peptide (ANP), may achieve their effects through a common pathway that involves reversible activation/deactivation of renal tubular Na+,K+‐ATPase. Regulation of Na+,K+‐ATPase activity was studied using a preparation of single proximal tubule (PT) segments, dissected from rat kidneys. Na+,K+‐ATPasc activity was stimulated by angiotensin II and the α‐adrenergic agonist, oxymetazoline, at physiological, nonsaturating Na+ concentrations. These stimulatory effects were blocked by dopamine and ANP as well as by their respective second messengers, cAMP and cGMP. They were also blocked by the specific protein phosphatase 2B inhibitor FK306, These results indicate that regulation of sodium excretion by norepinephrine, angiotensin II, dopamine, and ANP can be accounted for by a bidirectionally regulated intracellular protein phosphorylation cascade that modulates the activity of renal tubular Na+,K+‐ATPase.—Aperia, A., Holtbäck, U., Syrén, M.‐L., Svensson, L.‐B., Fryckstedt, J., Greengard, P. Activation deactivation of renal Na+,K+‐ATPase: a final common pathway for regulation of natriuresis. FASEB J. 8: 436‐439; 1994.

Negative Reciprocity between Angiotensin II type 1 and Dopamine D1 receptors in rat renal proximal tubule cells

Sodium excretion is bidirectionally regulated by dopamine, acting on D1-like receptors (D1R) and angiotensin II, acting on AT1 receptors (AT1R). Since sodium excretion has to be regulated with great precision within a short frame of time, we tested the short-term effects of agonist binding on the function of the reciprocal receptor within the D1R-AT1R complex in renal proximal tubule cells. Exposure of rat renal proximal tubule cells to a D1 agonist was found to result in a rapid partial internalization of AT1R and complete abolishment of AT1R signaling. Similarly, exposure of rat proximal tubule cells and renal tissue to angiotensin II resulted in a rapid partial internalization of D1R and abolishment of D1R signaling. D1R and AT1R were, by use of coimmunoprecipitation studies and glutathione-S-transferase pull-down assays, shown to be partners in a multiprotein complex. Na+-K+-ATPase, the target for both receptors, was included in this complex, and a region in the COOH-terminal tail of D1R (residues 397-416) was found to interact with both AT1R and Na+-K+-ATPase. Results indicate that AT1R and D1R function as a unit of opposites, which should provide a highly versatile and sensitive system for short-term regulation of sodium excretion.

Molecular determinants of sodium and water balance during early human development

The past decade has seen enormous progress in understanding the renal regulation of salt and water homeostasis. Most of the key transporters have been cloned, and their physiological importance has been revealed from studies of children with inherited diseases and from mutagenesis studies on a cellular level. We are beginning to understand the complexity with which the activity of these transporters is regulated by hormones. Studies on experimental animals have uniformly shown that the majority of renal salt and water transporters undergo profound changes in the postnatal period. There is generally a robust increase in the number of transporters expressed in a single tubular cell. Many of the transporters also shift their expression from one isoform to another with a somewhat different function. The short-term regulation of salt and water transporters, the key to a well-functioning homeostatic system, is often blunted in the early postnatal period. Taken together, these findings explain some phenomena well known in infants. The low urinary concentrating capacity can, for example, be at least partially attributed to immaturity of the expression of water channels, sodium losses in preterm infants to low expression of the energy generator for salt transport, Na(+),K(+)-ATPase, and the disposition to acidosis to immaturity of the Na(+)/H(+)exchanger. We propose that further studies on how these transporters are regulated will lead to the improved prevention and treatment of salt water balance disorders in infants.

MECHANISMS BY WHICH INTRARENAL DOPAMINE AND ANP INTERACT TO REGULATE SODIUM METABOLISM

Maintenance of a normal blood pressure requires a precise and fine-tuned regulation of salt metabolism. This is accomplished by a bidirectional regulation of renal tubular sodium transporters by natriuretic and antinatriuretic hormones. Dopamine, produced in the renal proximal tubular cells, plays an important role in this interactive system. Dopamine inhibits the activity of Na+,K+ATPase as well as of many important sodium influx pathways in the nephron. These effects of dopamine are particularly pronounced in situation of sodium loading. There is an abundance of evidence suggesting that the natriuretic effects of ANP are to a large extent mediated via renal dopamine 1 like receptors. The renal tubular dopamine 1 like receptors are, under basal conditions, mainly located intracellularly. ANP and its second messenger, cGMP, cause a rapid translocation of the dopamine 1 like receptors to the plasma membrane. This phenomenon may explain how ANP and dopamine act in concert to regulate sodium metabolism. Regulation of sodium metabolism and blood pressure is critically dependent on a normal function of the renal dopamine system. Hence, abnormalities in the interaction between dopamine and ANP may predispose to hypertension.

Recruitment of renal dopamine 1 receptors requires an intact microtubulin network

Abstract. Renal dopamine1 receptor (D1R) can be recruited from intracellular compartments to the plasma membrane by D1R agonists and endogenous dopamine. This study examines the role of the cytoskeleton for renal D1R recruitment. The studies were performed in LLCPK-1 cells that have the capacity to form dopamine from L-dopa. In approximately 50% of the cells treated with L-dopa the D1R was found to be translocated from intracellular compartments towards the plasma membrane. Disruption of the microtubulin network by nocodazole significantly prevented translocation. In contrast, depolymerization of actin had no effect. In control cells D1R colocalized with NBD-C6-ceramide, a trans-Golgi fluorescent marker. This colocalization was disrupted in L-dopa-treated cells. Tetanus toxin, an inhibitor of exocytosis, prevented L-dopa-induced receptor recruitment. L-Dopa treatment resulted in activation of protein kinase C (PKC). To test the functional effect of D1R recruitment, the capacity of D1R agonists to activate PKC was studied. Activation of D1R significantly translocated PKC-α from intracellular compartments to the plasma membrane. Disruption of microtubules abolished D1R-mediated – but not phorbol-ester-mediated – translocation of PKC. We conclude that renal D1R recruitment requires an intact microtubulin network and occurs via Golgi-derived vesicles. These newly recruited receptors couple to the PKC signaling pathway.

The Renal Dopamine System

Intrarenally formed dopamine induced natriuresis by inhibiting the activity of renal tubular Na/KATPase. This effect is mediated via a complex signal network, which includes inhibition of PP1 via the adenylyl cyclase-PKA-DARPP32 pathway and activation of PKC via the PLA2-arachidonic acid-20HETE pathway. The renal dopamine availability is a major determinant of the natriuretic effect of dopamine and is to a large extent modulated by the activity of COMT. The possibility that regulation of dopamine storage and release influences renal dopamine effects should be considered.

Prolactin and dopamine 1-like receptor interaction in renal proximal tubular cells

Prolactin is a natriuretic hormone and acts by inhibiting the activity of renal tubular Na(+)-K(+)-ATPase activity. These effects require an intact renal dopamine system. Here, we have studied by which mechanism prolactin and dopamine interact in Sprague-Dawley rat renal tissue. Na(+)-K(+)-ATPase activity was measured as ouabain-sensitive ATP hydrolysis in microdissected renal proximal tubular segments. Intracellular signaling pathways were studied by a variety of different techniques, including Western blotting using phosphospecific antibodies, immunoprecipitation, and biotinylation assays. We found that dopamine and prolactin regulated Na(+)-K(+)-ATPase activity via similar signaling pathways, including protein kinase A, protein kinase C, and phosphoinositide 3-kinase activation. The cross talk between prolactin and dopamine 1-like receptors was explained by a heterologous recruitment of dopamine 1-like receptors to the plasma membrane in renal proximal tubular cells. Prolactin had no effect on Na(+)-K(+)-ATPase activity in spontaneously hypertensive rats, a rat strain with a blunted response to dopamine. These results further emphasize the central role of the renal dopamine system in the interactive regulation of renal tubular salt balance.

INCREASED BLOOD PRESSURE AND LOSS OF ANP-INDUCED NATRIURESIS IN MICE LACKING DARPP-32 GENE

Atrial natriuretic peptide (ANP) is an important regulator of sodium metabolism and indirectly of blood pressure. Evidence has accumulated that ANP regulates sodium metabolism through a cascade of steps involving an increase in the level of cGMP, activation of cGMP-dependent protein kinase (PKG), and inhibition of renal tubular Na+,K+ -ATPase activity. One of the major substrates for PKG is DARPP-32. In the present study we observed that ANP does not induce natriuresis in mice that lack DARPP- 32. In contrast, there was a 4-fold increase in urinary sodium excretion following ANP administration to wild type mice. ANP as well as Zaprinast, a selective inhibitor of cGMP phosophodiesterase, inhibited renal Na+,K+-ATPase activity in wild type mice but had no such effect in mice lacking DARPP-32. Mean arterial blood pressure, measured in conscious animals, was significantly increased in DARPP-32 deficient mice as compared to wild type mice. The results confirm that DARPP-32 acts as a third messenger in the ANP signaling pathway in renal tissue and suggest an important role of DARPP-32 in the maintenance of normal blood pressure.

Cellular Response to Renal Hypoxia Is Different in Adolescent and Infant Rats

Immature renal tubules are more tolerant to ischemia than mature renal tubules. Here we compared the developmental pattern for some cellular responses evoked by hypoxia and reoxygenation in renal proximal tubules from 10- and 40-day-old rats. Redistribution of Na+-K+-ATPase from the plasma membrane was studied by confocal microscopy techniques in primary cultured renal proximal tubular cells. The developmental expression of Na+-K+-ATPase, μ-calpain and heme oxygenase-1 was measured by RT-PCR techniques in rat renal cortex. In response to hypoxia Na+-K+-ATPase redistribution from the plasma membrane was almost 2-fold increased in cells isolated from mature kidneys compared with cells isolated from immature kidneys. Reoxygenation resulted in a complete reestablishment of Na+-K+-ATPase in the plasma membrane in the immature but not in the mature cells. The dissociation of Na+-K+-ATPase from the plasma membrane was associated with a reduced activity and a reduced expression of Na+-K+-ATPase in the mature but not in the immature tubular cells. The expression of μ-calpain, a factor shown to induce ischemic injury to proximal tubular cells, was significantly lower in the immature compared with the mature kidney, whereas the expression of heme oxygenase-1, a factor shown to protect from renal ischemic injury, was significantly higher in the immature kidney. The results help to explain the increased tolerance of the immature kidney to injury caused by ischemia and reperfusion.

Prevention of apoptosis averts glomerular tubular disconnection and podocyte loss in proteinuric kidney disease

There is a great need for treatment that arrests progression of chronic kidney disease. Increased albumin in urine leads to apoptosis and fibrosis of podocytes and tubular cells and is a major cause of functional deterioration. There have been many attempts to target fibrosis, but because of the lack of appropriate agents, few have targeted apoptosis. Our group has described an ouabain-activated Na,K-ATPase/IP3R signalosome, which protects from apoptosis. Here we show that albumin uptake in primary rat renal epithelial cells is accompanied by a time- and dose-dependent mitochondrial accumulation of the apoptotic factor Bax, down-regulation of the antiapoptotic factor Bcl-xL and mitochondrial membrane depolarization. Ouabain opposes these effects and protects from apoptosis in albumin-exposed proximal tubule cells and podocytes. The efficacy of ouabain as an antiapoptotic and kidney-protective therapeutic tool was then tested in rats with passive Heymann nephritis, a model of proteinuric chronic kidney disease. Chronic ouabain treatment preserved renal function, protected from renal cortical apoptosis, up-regulated Bax, down-regulated Bcl-xL, and rescued from glomerular tubular disconnection and podocyte loss. Thus we have identified a novel clinically feasible therapeutic tool, which has the potential to protect from apoptosis and rescue from loss of functional tissue in chronic proteinuric kidney disease.