Alicia McDonough - US grants

The overall aim of this proposal is to understand how sodium transport, in particular transport by the sodium pump, is regulated in the proximal and distal nephron. In both regions the sodium and potassium dependent adenosine triphosphatase (NaK-ATPase), also known as the sodium pump, drives the active reabsorption of sodium. Regulation of renal NaK-ATPase is important for maintenance of fluid, electrolyte and metabolic homeostasis. NaK-ATPase is regulated by thyroid hormone in the proximal tubule, mineralocorticoids in the distal tubule (either directly or indirectly through intracellular Na+ and/or K+), and glucocorticoids in both sections. The most likely hypothesis is that both synthesis and degradation are subject to regulation. Specifically, the effects of the suspected regulators (thyroid hormone, glucocorticoids, aldosterone, and intracellular Na+ and K+) on transcription, translation, synthesis and degradation of the alpha catalytic subunit of the NaK-ATPase will be studied. Primary cultures of kidney proximal tubules will be used to study proximal nephron and Madin Darby canine kidney cells (MDCK) for distal nephron. Regulation of transcription will be studied with a cDNA to the alpha subunit, and regulation of synthesis and degradation will be studied by immunoprecipitation of labeled alpha subunit from cells fractionated into defined membrane populations by analytical cell fractionation.

Although recognized for decades that regulation of salt transport is crucial for maintenance of normal blood pressure, very little is understood about sodium transporter regulation during natriuresis or generation of hypertension. A rapid increase in blood pressure inhibits salt and fluid reabsorption in the proximal tubule (PT). This response is critical to the operation of tubuloglomerular feedback (TGF) by providing an error signal (increased delivery of NaCl) to the macula densa to sustain vasoconstriction in the afferent arteriole. The response also contributes to pressure natriuresis, which is altered in hypertension. The thick ascending limb of the loop of Hen1e (TALH) is involved in the feedback response to the extent that it does not completely reabsorb the increased load leaving the PT. The objective of this proposal is to define the cellular mechanisms responsible for the natriuresis that occurs in response to an acute increase in arterial pressure, and to determine whether the mechanisms are altered in pre- hypertensive rats. We have established that an increase in blood pressure rapidly decreases PT Na,K-ATPase activity and alpha and beta subunits' abundance in basolateral membranes and decreases apical sodium hydrogen exchangers associated with a redistribution of both sodium transporters to putative internal membranes. These results demonstrate the feasibility of dissecting the cellular mechanisms of pressure natriuresis. We will apply techniques of subcellular fractionation, immunocytochemistry, fluid phase endocytosis, and molecular biology to answer the following: Aim 1. Is the rapid decrease in PT sodium transport that accompanies an acute increase in arterial pressure due to a redistribution of apical and basolateral sodium transporters to endosomal pools, and is the response sodium transporter specific or a generalized internalization of surface membranes? Aim 2. Does the TALH respond to a pressure challenge and/or increased volume flow by regulating surface expression of sodium transporters? Aim 3. Is the response of PT and TALH to acute pressure challenge blunted in prehypertensive rats? We will study the fructose fed rats, and the Milan Hypertensive Strain rats (mutation in adducin) to test the hypothesis that altered abundance of sodium transporters, their distribution or interaction with cytoskeleton may blunt the response to an acute pressure challenge. Accomplishing the aims will fill gaps in our understanding of how altered ion transport is involved in the generation and maintenance of hypertension (salt sensitivity) and will identify components involved in the response that can be tested as "candidate genes" that may be involved in the disease process.

9513958 McDonough Potassium (K+) is the main salt found in intracellular fluid (ICF), while extracellular fluid (ECF) potassium is low, so there is a gradient of potassium across the cell membrane which is necessary for nerve and muscle excitability. Vertebrate animals must regulate extracellular fluid potassium concentration (ECF K+ ) within a narrow range. If K+ loss exceeds input over a period of days, ECF K+ falls, which is known as hypokalemia. The resulting increase in the K+ gradient across the cell membrane, if not corrected, can disrupt critical functions such as cardiac contractility (which can be fatal). During potassium deprivation, the skeletal muscle adapts by providing its K+ to the ECF to blunt and delay the fall in ECF K+. This important regulatory process is only poorly understood. In contrast, non-muscle cell types are adapted to maintain, not lose, intracellular K+ during K+ deprivation. The central aim of this proposal is to identify the specific muscle types that supply K+ to the ECF in hypokalemia and the underlying cellular mechanisms responsible for the shift and for the restoration response when K+ becomes available. Defining the target muscles and transporters are critical first steps toward understanding why a subset of muscles is specialized to lose K+ while others are spared in order to perform, different critical muscle functions. The loss of ICF K+ is accompanied by a decrease in number of sodium pumps, the molecules in the membrane that actively pump K+ into the cell from the ECF. This supports the hypothesis that the K+ loss is due to a decrease in active K+ influx rather than an increase in K+ exit from the cell. Sodium pumps, Na,K-ATPase, are composed of ( and ( subunits and muscle expresses (l, (2, (1, (2 subunit isoforms, thus, up to four possible (( combinations. The PI's laboratory has established that the isoforms are expressed in a muscle specific pattern. The PI has further established that abundance of (2 and (2 subunits are depressed in a muscle specific pattern. The PI aims to accomplish the following: 1) Test the hypothesis that the decrease in number of specific sodium pump isoforms is the underlying cellular mechanism causing shifts in K+ from a subset of muscles to ECF in hypokalemia. The PI will determine time course and muscle specificity of change in: ICF K+ , K+ influx, K+ efflux, and sodium pump subunit isoform abundance during low K+ diet and after restoring K+ in a panel of five muscles including white gastrocnemius where there is a 85% decrease in (2(2, to diaphragm where there is no change in (2(2 in hypokalemia. 2) Test the hypothesis that muscle sodium pump activity is regulated at multiple levels, both long term regulation of RNA and protein pool size, and acute regulation of existing pumps, during hypokalemia and K+ restoration. During K+ depiction and repletion determine the time course of change in ( and ( isoforms' mRNA vs. protein, and determine whether there is also acute regulation of Na,K-ATPase transport activity before there are changes in abundance of pumps. Significance: this fundamental, specialized, and poorly understood response of skeletal muscle to organism K+ deprivation is critical for survival of all vertebrates, including humans, during periods of fasting or famine, or when K+ loss is high (mineralocorticoids, prolonged exercise in the heat, diuretics). Accomplishing these aims will identify the location of the K+ reservoir and the cellular mechanisms responsible for tapping and repleting this reservoir. This information contributes to an understanding how a subset of muscles have specialized to lose K+ to buffer ECF K+ , thus sparing other muscles and organs to perform critical functions, and to development of assays for the signal(s) that mediate the response. All steps are requisite for defining the homeostatic loops responsible for adapting to altered K+ status. ***

Description: (Adapted from the applicant's abstract) - Previous results from the principle investigator's laboratory and other have shown that a rapid increase in mean arterial blood pressure inhibits salt and fluid reabsorption in the renal proximal tubule. The resulting increase in volume flow to the thick ascending limp of the loop of Henle provokes an increased sodium reabsorption and Na, k-ATPase activity to limit the increase in volume flow to the distal tubule and increase in Na-K-2Cl co- transporter. A defect in the proximal tubular or thick ascending limb of the loop of Henle (TALH) responses could be responsible for the reduced pressure natriuresis that is that is the hallmark of hypertension. The overall goal of the research is to test the hypothesis that the "downstream shift" of sodium reabsorption from proximal tubule (PT) to the TALH during acute hypertension is due to inhibition of PT sodium transporters, specifically NHE-3 and Na, K-ATPase), and that similar changes are evident in chronic genetic hypertension. There are three specific aims. Specific Aim 1 aims to determine the cellular mechanisms responsible for the decrease in proximal tubular sodium transport during acute hypertension. The principal investigator will investigate the mechanism of sodium pump inhibition and whether or not apical sodium transporters are themselves inhibited or move to sub-apical endosomal compartments. Specific Aim 2 is to determine the cellular mechanisms responsible for the increase in TALH sodium transport during acute hypertension Four questions will be addressed, namely, what is the mechanisms for sodium pump activation; is NHE-3 and/or NKCC2 activated or moved to the apical membrane; does a change in volume flow without hypertension activate sodium pump activity; and is cytochrome P450 arachidonic acid metabolism required for the response? Specific Aim 3 is to test the hypothesis that the cellular mechanisms responsible for the downstream shift in sodium reabsorption during acute hypertension are evident in genetic models of hypertension. For these studies, the principal investigator will use spontaneously hypertensive rats known to have defective proximal tubular sodium transport regulation, and Milan hypertensive rats known to have elevated thick ascending limp Na, K- ATPase.

DESCRIPTION (Adapted from the Applicant's Abstract): Extracellular fluid (ECF) +} must be maintained within a narrow range. If ECF +] falls too low (hypokalemia), cell membranes hyperpolarize, and if ECF +] increases too much (hyperkalemia) cell membranes depobrize, both disrupt normal electrical excitability and can have life threatening cardiac effects. Kidneys and muscle work in concert to maintain ECF ]. During hypokalemia muscle ICF K is redistributed to buffer the fall in ECF }. During hyperkalemia K+ is pumped into muscle ICF until renal adjustments can occur. These important muscle specific homeostatic processes are only beginning to be understood at the molecular level. Evidence supports the hypothesis that K loss from muscle during hypokalemia results from decreased active K+ influx mediated by sodium pump (Na,KATPase, NKA) inhibition, and that K+ uptake during hyperktilemia is mediated by sodium pump activation. Our lab has established that during low K+ diet abundance of NKA subunits are depressed in an isoform and muscle specific manner: 60-95 percent fall in a2, not a 1. Using a novel K+ clamp technique, we recently showed that early in K+ restriction, prior to fall in a2, there is a severe blunting of both insulin stimulated K+ uptake, and of insulin stimulated redistribution of NKA ct2 type pumps from endosomes to the plasma membrane (PM). Evidence is mounting that the bumetanide sensitive Na,K,2C1 cotransporter also accounts for a component of muscle K+ influx and, thus, could play a role in potassium homeostasis. The overall aims are to determine the molecular mechanisms responsible for tapping muscle K+ stores during hypokalemia, for clearing excess plasma +] into the ICF store after K+ restoration, and to understand how these processes are altered in a set of clinically relevant paradigms. The contribution of both Na,K-ATPase isoforms and NKCCI in both red oxidative white glycolytic muscle will be studied with a compartmental analysis approach in which the following are assessed: whole body K+ uptake, muscle specific K+ transport, subcellular distribution and activity of K+ transporters, and pool size regulation of K transporter protein and mRNA levels. Aim 1 will test the hypothesis that the shift of K+ to ECF during K restriction is mediated by decreased plasma membrane (PM) expression of both NKA a2 and NKCC1 coupled to resistance to insulin stimulated K+ uptake, and that this process is altered in uremia accompanying chronic renal failure. Aim 2 will test the hypothesis that thyroid hormone or dexamethasone, both of which increase NKA cx2 (and perhaps NKCC 1), alter extrarenal control of K+ horneostasis. Aim 3 will test the hypothesis that the uptake of K+ from ECF to ICF during K+ restoration (following K+ restriction) is mediated by normalizing surface expression of both NKA a2 and NKCC1. Accomplishing these aims will identify the cellular mechanisms responsible for tapping and repleting the muscle K+ reservoir, which will, ideally, suggest strategies to manipulate muscle K stores in clinical settings.

DESCRIPTION (provided by applicant): Blood pressure (BP) continuously fluctuates while RBF and GFR do not due to intra-renal adjustments including inhibition of PT reabsorption. This alters NaCI delivery to the macula densa and renin release and contributes to pressure natriuresis, thus, influences BP set point. We have previously established that the decrease in PT Na+ reabsorption is mediated by a retraction of transport competent Na+/H+ exchangers (NHE3) from the PT microvilli, that the response is chronically activated in the Spontaneously Hypertensive Rat (SHR), and that the converse is evident in Renal Injury (RI) hypertension where SNS activation moves NHE3 into the microvilli, potentially contributing to hypertension by counteracting BP mediated inhibition of PT Na+ transport. This progress sets the groundwork for addressing the molecular mechanisms governing NHE3 redistribution in distinct models of acute and chronic hypertension: the source and destination of NHE3, whether there are changes in NHE3 associated proteins and NHE3 activity/transporter en route, and the signals governing redistribution. Aim 1 tests the hypotheses that NHE3 retraction from the villi during acute hypertension involves a two step process within the apical surface membrane (1) from villi to intermicrovillar cleft, (2) then to intermicrovillar coated pits associated with a change in NHE3 interacting proteins, membrane domain properties, Na+/H+ exchanger activity/transporter, and coincident movement of myosin VI. Aim 2 tests the hypotheses that during chronic hypertension there are persistent shifts in NHE3 distribution within the apical domain that can be either compensatory, as in the SHR, or contributory, as in the chronic RI model both associated with distinct chronic changes in NHE3 associated proteins, domain properties and activity/transporter. This Aim also tests the hypothesis that these changes are reversed/normalized when BP is normalized. Aim 3 tests the hypotheses that Step 1 of NHE3 retraction is dependent on the intrarenal release of nitric oxide that a decrease in Ang II is important for Step 2, and that during acute RI driven by SNS activation NHE3 is recruited from the IMC and ICP regions to the microvilli, rather than by regulated exocytosis. Accomplishing these Aims will reveal how NHE3, the major high capacity renal Na+ transporter, is regulated in vivo by an acute change in blood pressure in the normal range and how it is regulated when BP is chronically elevated by genetics or injury induced SNS activation.

[unreadable] DESCRIPTION (provided by applicant): The Na-CI cotransporter (NCC) is expressed in the apical membrane of the distal convoluted tubule and is responsible for the reabsorption of 5% -10% of filtered Na and Cl. NCC is the target of the thiazide diuretics prescribed very frequently for treatment of hypertension, edema and heart failure. NCC abundance is highly regulated by: dietary NaCI restriction, aldosterone, estrogen and thiazide treatment, additionally, NCC is mutated in Gitelman's syndrome leading to salt wasting, hypokalemia and metabolic alkalosis, all confirming a homeostatic role of NCC abundance in regulating Na balance and blood pressure. We recently tested the hypothesis (in rats in vivo) that high salt diet provokes redistribution of DCT NCC from apical plasma membrane (PM) to sub-apical intracellular membrane pools (IM) and that salt restriction favors redistribution to the PM. Applying a density gradient centrifugation approach discovered that NCC redistributes to higher density membranes in response to high salt diet and to lower density membranes during salt restriction. By EM, sub-apical IM pools of NCC were evident and the ratio of NCC labeling in the IM to PM was higher in high salt diet than low salt diet where most all the NCC is in the PM, supporting our hypothesis. Our provocative preliminary results set the groundwork for addressing the molecular mechanisms regulating NCC subcellular distribution in vivo. In this R21 application we aim to fully confirm the hypothesis that NCC subcellular distribution is chronically regulated by salt diet and proceed to test the hypothesis that NCC distribution in PM vs. IM can be acutely regulated by adrenergic stimulation and/or acute hypertension. Our studies will break new ground in determining: the role of NCC in responding to adrenergic (antinatriuretic) and blood pressure (natriuretic) signals, the source and destination of NCC, the NCC associated proteins en route. In order to define NCC protein-protein interactions and how they change with apparent NCC redistribution in each model we need to generate reagents and methods to immunoprecipitate NCC along with associated proteins and along with NCC containing vesicles for subsequent candidate and proteomic analyses. This line of investigation aims to provide definitive evidence for regulated trafficking of NCC in vivo, identify potential mediators of the NCC trafficking, identify NCC trafficking defects in disease, and suggest alternative mechanisms to alter NCC therapeutically. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The Na-CI cotransporter (NCC) is expressed in the apical membrane of the distal convoluted tubule and is responsible for the reabsorption of 5% -10% of filtered Na and Cl. NCC is the target of the thiazide diuretics prescribed very frequently for treatment of hypertension, edema and heart failure. NCC abundance is highly regulated by: dietary NaCI restriction, aldosterone, estrogen and thiazide treatment, additionally, NCC is mutated in Gitelman's syndrome leading to salt wasting, hypokalemia and metabolic alkalosis, all confirming a homeostatic role of NCC abundance in regulating Na balance and blood pressure. We recently tested the hypothesis (in rats in vivo) that high salt diet provokes redistribution of DCT NCC from apical plasma membrane (PM) to sub-apical intracellular membrane pools (IM) and that salt restriction favors redistribution to the PM. Applying a density gradient centrifugation approach discovered that NCC redistributes to higher density membranes in response to high salt diet and to lower density membranes during salt restriction. By EM, sub-apical IM pools of NCC were evident and the ratio of NCC labeling in the IM to PM was higher in high salt diet than low salt diet where most all the NCC is in the PM, supporting our hypothesis. Our provocative preliminary results set the groundwork for addressing the molecular mechanisms regulating NCC subcellular distribution in vivo. In this R21 application we aim to fully confirm the hypothesis that NCC subcellular distribution is chronically regulated by salt diet and proceed to test the hypothesis that NCC distribution in PM vs. IM can be acutely regulated by adrenergic stimulation and/or acute hypertension. Our studies will break new ground in determining: the role of NCC in responding to adrenergic (antinatriuretic) and blood pressure (natriuretic) signals, the source and destination of NCC, the NCC associated proteins en route. In order to define NCC protein-protein interactions and how they change with apparent NCC redistribution in each model we need to generate reagents and methods to immunoprecipitate NCC along with associated proteins and along with NCC containing vesicles for subsequent candidate and proteomic analyses. This line of investigation aims to provide definitive evidence for regulated trafficking of NCC in vivo, identify potential mediators of the NCC trafficking, identify NCC trafficking defects in disease, and suggest alternative mechanisms to alter NCC therapeutically. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The Na+Cl- cotransporter (NCC) is expressed in the apical plasma membrane (APM) of the distal convoluted tubule (DCT). NCC inhibition provokes salt wasting and can lower BP, while NCC stimulation can raise BP: WNK kinases mutations increase APM NCC and activity, inactivating the WNK substrate SPAK kinase reduces NCC phosphorylation (NCCp) and BP. The renin angiotensin system (RAS) stimulates NCC activity via an AngII-WNK4-SPAK dependent pathway. We provided in vivo evidence that NCC redistributes out of the APM into subapical cytoplasmic vesicles (SCV) during high NaCl diet and ACE inhibition and redistributes into the APM from SCV during low NaCl diet and AngII infusion. We now show that NCCp increases with AngII treatment and decreases with high salt diet. AngII via AT1R stimulates NADPH oxidase (Nox), generating reactive oxygen species (ROS). We now show that ROS scavenging during AngII treatment blocks NCC trafficking and NCCp. Renal sympathetic nerve activity (RSNA) also plays a primary role in hypertension pathogenesis. We show that both RSNA and adrenergic agonists stimulate NCC trafficking to APM and increase NCCp. The overall aim of this proposal is to determine the molecular mechanisms responsible for integrated regulation of NCC in response to AngII and/or RSNA and the influence of dietary NaCl on these pathways. Our hypothesis is that AngII (via AT1R) and adrenergic agonists (via a1bAR) stimulate Nox generation of ROS and activates SPAK kinase which stimulates NCC accumulation in APM and NCCp. We postulate that dietary salt independently stimulates ROS generation via Nox. Aim 1. What is the role of NADPH oxidase and SPAK in AngII stimulated NCC phosphorylation and/or redistribution to APM? Are these effects influenced by dietary salt? Aim 2. Do RSNA or adrenergic agonists stimulate DCT NCC activity? Are NADPH oxidase stimulation and/or SPAK phosphorylation requisite? How is this regulation affected by dietary salt? By AngII? Methods. The aims will be examined in rats treated acutely or chronically with agonists and inhibitors of RAS, NADPH oxidase, RSNA and altered salt diets. Mouse knockout models of SPAK, p47phox, and alpha1b adrenoreceptors will be examined in parallel to define the roles of these regulatory pathways or intermediates in NCC regulation. Distribution of NCC, NCCp, SPAK and SPAKp will be examined by both subcellular fractionation and immunoblots and immunofluorescence and immuno-EM. Renal function, oxidative stress and BP will be measured routinely and NCC activity measured using a thiazide diuretic test. Accomplishing the aims will establish integrated effects of major BP regulating signals on DCT NCC cellular distribution, NCCp and activity, providing novel insights into homeostatic set point regulation of ECFV and BP by the DCT and, ideally, indicating strategies for the development of therapies to treat resistant hypertension and/or edema based on inhibiting multiple pathways that regulate DCT NCC activity.

? DESCRIPTION (provided by applicant): Hypertension is the leading cause of stroke and cardiovascular diseases, affecting 30% of the adult population. Excessive Na+ input can acutely raise effective circulating volume (ECV) and BP but normally the kidneys excrete enough Na+ and volume to normalize BP, a response termed pressure natriuresis. Thus, hypertension can be considered a failure of pressure natriuresis. In females the response is set to lower arterial pressures than in males and, until menopause, females exhibit lower BP than males. In the previous funding cycle we demonstrated how divergent signals are integrated to impact the overall renal response to hypertensive stimuli in males. We posit that these responses are substantially influenced by sex and present preliminary results demonstrating lower NHE3 and NaPi2, both retracted from proximal microvilli, along with a more rapid natriuretic response to a saline volume expansion in females compared to males. Our overall objectives are to determine how anti-natriuretic stimuli (AngII and L-NAME hypertension) and natriuretic pathways (dopamine, AT2R, GLP1R) are integrated during hypertension at the level of renal transporters and their regulators to balance Na+ output to input, and to use these findings to explain how natriuresis leads to lower pressures in females versus males. We will test the hypothesis that hypertension ensues when natriuretic pathways are inhibited due to blunting of the pressure natriuretic inhibition of transporters in the proximal nephron, and by activation of transporter/channels all along the nephron secondary to intrarenal renin angiotensin system (RSA) activation. We will test the hypothesis that females possess enhanced natriuresis due to lower fractional sodium reabsorption in the proximal nephron as well as reduced intrarenal AngII generation, and that inhibition of specific natriuretic pathways or ovariectomy will eliminate the advantage and identify sex dependent control points. Specific Aim 1 will characterize acute pressure natriuresis in females, then compare male and female responses to two distinct models of experimental hypertension. Mechanisms controlling transporters/channels and their regulators will be determined by assessing mRNA versus protein abundance, subcellular distribution, and intrarenal RAS components. Specific Aim 2 will determine how inhibition of candidate natriuretic pathways alters regulation of transporters/channels during experimental hypertension in females and males: 1) intrarenal dopamine production, 2) AT2 receptor stimulation, and 3) GLP1 receptor stimulation. The effects of negative reciprocity between dopamine and AngII receptors and signaling, well-established in males, will be extended to effects on transporters/channels in both sexes. Significance: Identifying physiologic mechanisms regulating sodium transporters and channels during hypertension that enable females to produce a natriuresis at a lower BP has the potential to advance the field significantly by identifying mechanisms that if enhanced could lower morbidity and mortality in males of all ages, and maintain protection in postmenopausal women.