Maintaining Balance under Pressure: Hypertension and the Proximal Tubule Academic research paper on "Biological sciences"

Accepted Manuscript

Maintaining Balance under Pressure: Hypertension and the Proximal Tubule Alicia A. McDonough

PII: S2468-0249(16)30047-X

DOI: 10.1016/j.ekir.2016.06.008

Reference: EKIR 25

To appear in: Kidney International Reports

ÏfflÊfa Official Journal ^MHi) of the International mF Society of Nephrology

REPORTS

I Kidney International I Reports

Received Date: 26 May 2016 Revised Date: 22 June 2016 Accepted Date: 26 June 2016

Please cite this article as: McDonough AA, Maintaining Balance under Pressure: Hypertension and the Proximal Tubule, Kidney International Reports (2016), doi: 10.1016/j.ekir.2016.06.008.

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ISN Forefronts Symposium 2015

Maintaining Balance under Pressure: Hypertension and the Proximal Tubule Alicia A. McDonough

Department of Cell and Neurobiology, Keck School of Medicine of the University of Southern California

Running title: hypertension and the proximal tubule.

Correspondence:

Alicia A. McDonough, Ph.D.

Department of Cell and Neurobiology, Keck School of Medicine of USC 1333 San Pablo St, Los Angeles, CA 90033 phone: (323) 442-1238, fax: (323) 442-2411 email: mcdonoug@usc.edu

Sources of support: This work is supported by National Institutes of Health NIDDK grant R01DK083785 and AHA Grant in Aid Western States Affiliate 15GRNT23160003

Abstract

Renal control of effective circulating volume is key for circulatory performance. When renal Na+ excretion is inadequate, blood pressure rises and serves as a homeostatic signal to drive natriuresis to re-establish effective circulating volume (ECV). Recognizing that hypertension involves both renal and vascular dysfunction, this report concerns proximal tubule Na+/H+ exchanger 3 (PT NHE3) regulation during acute and chronic hypertension. NHE3 is distributed in tall microvilli (MV) in the PT where it reabsorbs a significant fraction of the filtered Na+. NHE3 redistributes, in the plane of the MV membrane, between the MV body, where NHE3 is active, and MV base where NHE3 is less active. High salt diet and acute hypertension both retract NHE3 to the base and reduce PT Na+ reabsorption independent of a change in abundance. The renin angiotensin system provokes NHE3 redistribution independent of blood pressure: the ACE inhibitor captopril redistributes NHE3 to the base and subsequent AngII infusion returns NHE3 to the body of the microvilli and restores reabsorption. Chronic AngII infusion presents simultaneous AngII stimulation and hypertension: NHE3 remains in the body of the MV, due to the high local AngII and inflammation, and exhibits a compensatory decrease in abundance, driven by the hypertension. Genetically modified mice with blunted hypertensive responses to chronic AngII infusion (due to lack of PT AngII receptors, IL-17A or IFN-g expression) exhibit reduced local AngII accumulation and inflammation and larger decreases in NHE3 abundance which improve the pressure natriuresis responses and reduces the need for elevated BP to facilitate circulating volume balance. Keywords: pressure-natriuresis, proximal tubule, Na+/H+ exchanger 3, angiotensin II, cytokines, trafficking.

Role of the kidneys in regulating effective circulating volume and blood pressure.

In 1909 Ernest Starling published his lectures on The Fluids of the Body.1 In this monograph, Starling outlines his thoughts on the relationship between body fluid balance, blood circulation, central venous pressure and cardiac performance. Importantly, renal control of the body fluid balance is viewed as key for circulatory and cardiac performance, "Change in the blood flow through the kidney may bring about alterations in the flow of urine quite irrespective of the composition of the blood or of the tissues. The occurrence of these two classes of phenomena seems to be determined by the fact that the kidney is a dual organ, and that while one part of it acts, so to speak, passively in response to force impressed upon it from without, another part, endowed with sensibility, reacts to external forces in a direction which may be opposed to these forces, but is in all cases the appropriate one for the welfare of the whole organism." 1. In 1963, Borst and Borst-de Geus2 discuss hypertension in light of Starling's theory of circulatory homeostasis and postulates that blood pressure rises as a homeostatic reaction to deficient sodium excretion, that is, pressure rises in order to re-establish sodium balance (at the expense of persistently elevated blood pressure).2 This response, known as pressure natriuresis, has been reviewed from various perspectives by many investigators.3 4-7 Figure 1 shows central blood pressure (BP) as a function of the effective circulating volume (ECV) and cardiac output (CO) as well as the kidneys' regulation of NaCl and H2O reabsorption. The kidney's decision to excrete NaCl and water is a function of mediators and controls, both extra-renal and intra-renal, that affect the rise in blood pressure, as well as the extravascular storage of sodium (discussed in an accompanying report in this series).8

Hypertension is the leading cause of stroke and cardiovascular diseases, affecting 30% of the adult population in Western cultures.9 BP can be elevated by vasoconstriction or by increasing effective circulating volume. Excess Na+ reabsorption raises ECV and BP, yet, according to Guyton, kidneys have the capacity via pressure natriuresis to excrete enough Na+ and volume to normalize BP in the face of expanded ECV.3 While, hypertension was classically viewed as a failure of pressure natriuresis, a recent discussion of the role of the kidney in the pathogenesis of hypertension10 concludes that for hypertension to become chronic there must be impairment of both renal output of salt and water as well as dysfunction of peripheral vascular tone, e.g. a failure of peripheral vasodilation due to arterial stiffness. Support for the latter is provided by recent studies illustrating a positive feedback loop wherein arterial stiffening leads to more arterial stiffening.11 While appreciating these complex interactions of body fluids, cardiac output, vascular stiffness and blood pressure, this report will focus on one aspect, the regulation of the renal proximal tubule Na+/H+ exchanger isoform 3 (NHE3) as a "case in point" mediator of the pressure natriuresis response, specifically regulation of the NHE3 trafficking and abundance, our understanding of how renal dysfunction resets the NHE3 regulation to higher pressures, and strategies that may be exploited to improve pressure natriuresis.

Proximal tubule NHE3 regulated by redistribution within the microvilli

As reviewed by Palmer and Schnermann, the proximal tubule reabsorbs 2/3 of the salt and water filtered at the glomerulus (120 ml/min) and NHE3 is the main Na+ transporter driving transcellular reabsorption in this region.12 The proximal tubule is a leaky epithelium well-built to reabsorb the ~ 80 ml/min of filtrate: as illustrated in the cross

section of the electron micrograph in Figure 2, the proximal tubule has an apical pole covered with a tall brush border of microvilli each scaffolded by an actin filament core bundled by villin. This specialization increases the surface area for reabsorption more than 30 fold.13 The apical microvilli contain water channels as well as many different transporters to reabsorb cations, anions and substrates from the filtrate; importantly, a significant fraction of the filtered salt and water is reabsorbed via a paracellular route by claudins.14

Membrane transporters and channels can be regulated by: 1) trafficking between plasma membrane and intracellular membranes, 2) altered total pool size, 3) covalent modifications such as cleavage or phosphorylation, or 4) protein-protein interaction. Once NHE3 is localized to the PT microvilli, there is scant in vivo evidence for regulated trafficking between microvilli and intracellular pools, rather, NHE3, localized to ordered lipid domains (rafts) in the microvilli redistribute between the body and the base of the microvilli, moving in the plane of the microvillar membranes, likely driven by the atypical molecular motor myosin VI.15-17 This redistribution from one location to another, rather than degradation and synthesis, facilitates rapid continuous adaptation to changing salt intake, effective circulating volume and/or blood pressure. Figure 2 illustrates the simple case of NHE3 regulation in the transition between normal and high salt diets in the absence of any change in blood pressure.18 Figure 2A illustrates that this natriuresis occurs without any change in NHE3 total abundance. Figure 2B shows cross sections of PT in a model and in an electron micrograph illustrating organization of dense apical microvilli. NHE3 redistribution along the proximal tubule microvilli is detected by co-labelling the actin bundling protein villin (red V) and NHE3 (green circle)

with specific antibodies. 18 The left half of the model represents a PT from a normal salt diet fed rat with villin and NHE3 co-localized in the microvilli yielding a yellow stain. The right half of the PT model represents a PT from a high salt diet fed rat wherein NHE3 is retracted to the base of the microvilli exposing red V in body of the villi and green/yellow at the base of the microvilli. Figure 2C demonstrates the redistribution of NHE3 from the body of the PT microvilli, in rats on 0.4% NaCl (left half-tubule), to the base of the microvilli, in rats on 4.0% NaCl (right half-tubule). Myosin VI, an atypical molecular motor implicated in the redistribution of NHE3 and Na+-Pi transporter (NaPi2) within the plane of the microvillar membrane,17,19 also redistributes from the body to the base of the microvilli during high salt diet, presumably "driving" the NHE3 (Figure 2D). As discussed below, when NHE3 is clustered at the base of the microvilli, its activity is predicted to be inhibited by unfavorable pH gradients.20 Recent studies have shown that when the excretory function of the kidney is chronically impaired by inhibiting nitric oxide synthase (NOS) activity, the resultant renal inflammation blunts the depression in sodium transport during high salt diet leading to a rise in blood pressure, that is, the renal dysfunction leads to salt-sensitive hypertension.21

Evidence for role of proximal tubule in acute pressure diuresis.

The classic acute pressure natriuretic protocol developed by Roman and Cowley22 and a typical response is illustrated in Figure 3A: in inactin anesthetized male rats, raising mean atrial blood pressure (BP) from 87 to 130 mmHg rapidly increases urine output more than 10 fold. 23,24 Since the pressure-natriuresis response is very large and rapid, the proximal tubule was identified as a good candidate region for natriuresis because it reabsorbs the bulk of the filtered load. In the mid-1980s, Chou and Marsh developed a

video-densitometric approach to analyze tubular flow in real time and found that acutely raising blood pressure rapidly increased end proximal tubule flow rate 50%.25,26 Since they also demonstrated this occurred without appreciable changes in GFR or RBF (due to autoregulation), they concluded that PT sodium transport was inhibited during acute hypertension, and that this response contributed not only to pressure diuresis but also to the autoregulation of glomerular filtration rate and renal blood flow (mediated by increasing salt delivery to the macula densa). This response confirmed Starling's assertions that, "The mechanisms, which determine the adaptation of the organism to changes in the total volume of its fluid content, must come into play with every rise or fall in the general blood pressure. 1

NHE3 regulated by redistribution within the microvilli during acute hypertension.

The renal natriuretic and diuretic responses to acute or chronic increases in BP are referred to very generally as pressure natriuresis. The responses to acute hypertension in the PT (Figure 3) are analogous to those observed during high salt diet (Figure 2). The pressure-natriuretic signals, discussed below, provoke the dynamic redistribution of apical transporters, including NHE3 and NaPi2,16 driven by molecular motors (myosin VI, IIA), and cytoskeleton-associated proteins, to the base of the PT microvilli (Figure 3B).17,19 The lipid raft-associated NHE3 remains at the base15 and the non-raft-associated NaPi2 is endocytosed, culminating in decreased Na+ transport activity and

7 27 28

increased PT flow rate.'2'20 Recently, with Brasen and Peti-Peterdi, we visualized the hypertension-stimulated redistribution of NHE3 to the base of the microvilli in vivo using two photon microscopy with the pH indicator BCECF.20 Mathematical modeling of this redistribution suggests that NHE3 clustering produces unfavorable pH microdomains

near the bottom of the brush border sufficient to inhibit NHE3 activity.20 This conclusion helps to explain how NHE3 redistribution can contributes to natriuretic responses during high salt diet and acute hypertension.

The signaling that provokes the rapid decrease in proximal tubule sodium and volume reabsorption, and retraction of NHE3 to the base of the microvilli in the face of autoregulated RBF and GFR appears to involve many layers of regulation by both intrinsic and extrinsic factors. Summarizing the findings of multiple labs, it appears that the initial natriuretic response is driven by rapid local generation of 20-HETE and NO, 2830 (perhaps involving non-autoregulating vasculature that senses the hypertension), and that the response in the proximal tubule involves the production of cGMP31 which plays a role in depressing Na+ transport. We found that clamping AnglI levels at a non-pressor level by co-infusion of both the angiotensin converting enzyme (ACE) inhibitor captopril and Angll before the acute hypertension protocol significantly blunted the pressure diuresis as well as the redistribution of NHE3 to the base of the microvilli (Figure 3C).32,33 The decrease in Angll is key to not only allow NHE3 redistribution to the base of the microvilli but also to sustain the response by reducing sodium transport in Angll sensitive regions all along the nephron, including the distal tubule.34 Not the focus of this review, but important to discuss in light of signaling, the medullary loop of Henle also clearly participates in pressure natriuresis during hypertension: medullary blood flow, NO and ROS participate as signals.35,36 A recent report from Crowley and colleagues suggests that inflammatory accumulation of lL-1 during hypertension activates loop of Henle Na+-K+- 2Cl- cotransporter (NKCC2) which blunts the natriuresis and raises blood pressure; eliminating lL-1 limits the blood pressure elevation by

reducing NKCC2 Na+ reabsorption.37 This study provides another example of how injury signals contribute to hypertension by blunting pressure natriuresis. In summary, regarding natriuretic signaling during hypertension, there is consensus that multiple signals have the potential to act all along the nephron, thus, the pressure natriuresis response is the sum of the prevailing natriuretic and anti-natriuretic influences.

Renin angiotensin system regulates PT NHE3 distribution and abundance

The renin angiotensin aldosterone system is the most powerful controller of ECV and BP, as recently reviewed by Rossier.38 AngII increases sodium reabsorption in the proximal tubule mediated by AT1 receptors. Building on the findings of the suppression of pressure natriuresis during the AngII clamp, we investigated the acute effects of adding or inhibiting AngII without changing blood pressure. Since angiotensin converting enzyme inhibitors (ACEI) are among the most popular drugs prescribed to lower blood pressure and slow the progression of renal and heart disease, it is key to understand how they regulates the PT NHE3. We addressed this issue by merging physiology and proteomics.39 Leong identified a dose of the ACEI captopril that, when infused for 20 min into anesthetized rats, did not change BP or GFR, but did significantly increase urine output. With the Yip lab, they demonstrated that this dose rapidly increased PT flow rate, evidence for ACEI suppression of PT Na+ reabsorption; Figure 4B shows that NHE3 is retracted to the microvillar base after 20 min ACEI treatment, evidence that basal AngII is responsible, at least in part, for the location of NHE3 within the microvilli at baseline.39 With the Klein lab, Leong applied a limited proteomic approach and discovered other brush border proteins that redistribute with

captopril include myosin Vl, dipeptidyl peptidase Vl, NHERF-1, ezrin, megalin, vacuolar H+-ATPase, aminopeptidase N, and clathrin.39

Starting with the ACEl infused rat, Riquier and Leong determined the effects of acute Angll infused at a rate that didn't alter blood pressure for another 20 minutes and found that the NHE3 completely returned to the body of the microvilli (Angll + captopril, Figure 4C)40; Yip and Leong had previously reported that Angll + captopril infusion also decreased PT flow rate, providing evidence that the NHE3 redistribution is anti-natriuretic.39 ln collaboration with the Girardi lab, we recently investigated the effects of a p-arrestin biased AT1 receptor agonist (TRV120023) on proximal tubule NHE3.41 Stimulating p-arrestin biased pathways promotes G-protein receptor internalization and desensitization and can activate G-protein independent responses.42 Perfusing proximal tubules in vivo with the TRV compound reduced bicarbonate reabsorption, a surrogate measure of NHE3 transport, and also moved NHE3 to the bottom of the microvilli.41 TRV can also prevent the effects of Angll activation: co-infusing the TRV compound with ACEl for 20 min blocks the effects of subsequent 20 min Angll infusion and redistributes the NHE3 to the base (Figure4D).

Experimental Angll hypertension involves infusing Angll continuously at a dose that is initially sub-pressor but eventually provokes hypertension. Using immunoblots of renal cortex, we determined in rats that after 3 days of Angll infusion, before BP increases, the total abundance of cortical NHE3 increases around 50% above baseline,43 and then by 14 days of Angll infusion, when BP is chronically elevated to 160 mmHg, the NHE3 abundance is depressed to 20% below baseline.35 Figure 4E shows a PT from a 14 day Angll infused rat with hypertension in which NHE3 is retained in the body of the

microvilli.35 Images in Figure 4A and E were collected with the same settings, demonstrating that the ratio of NHE3 to villin signal has decreased in the microvilli during AngII hypertension. Overall, these findings suggest that AngII "fixes" NHE3 in the microvilli and blunts redistribution, both acutely (Figure 3C) and chronically (Figure 4E). In lieu of redistribution, a compensatory decrease in NHE3 abundance in the microvilli becomes evident as blood pressure increases. The biphasic effects of AngII on proximal tubule reabsorption reported in rodents44 have led to the suggestion that the decreased NHE3 abundance may be due to high dose AngII inhibition of NHE3, rather than hypertension. However, this inhibitory effect is observed when AngII was directly applied to tubules at doses of (> 10 -7 mol/L), and not observed during systemic AngII infusion (where tubular AngII reaches the nmol/L range).45 Interestingly, human PTs studied in vitro only exhibit a stimulatory transport response to AngII. 46

Role of the PT NHE3 regulation in experimental AngII hypertension.

Blood vessels, kidney, and central nervous system are all implicated in the genesis of experimental hypertension, and T-cells may provide a key link. Animal models of chronic hypertension exhibit increased immune infiltration into the vascular adventitia and kidney.47-49 Mice and rats lacking T-lymphocytes exhibit blunted hypertensive responses to experimental hypertension, restored by adoptive transfer of T-cells.50,51 Evidence suggests that the initiating insult (whether angiotensin II infusion or other), increases NADPH oxidase mediated reactive oxygen species (ROS) generation which stimulates sympathetic nervous system activity (SNA) and norepinephrine release in tissues which can mediate tissue T-cell activation, producing local proinflammatory molecules (ROS, neoantigens, and cytokines).52,53 In the kidney, these processes are

reported to activate local accumulation of Angll even when systemic levels of Angll are very low.54,55 The local Angll is anti-natriuretic, produces ROS locally, and can attract more immune cells, creating a positive feedback loop that manifests as chronic inflammation and elevated blood pressure.49,52 ln rats, we determined that Angll infusion hypertension activates distal transporters and channels, specifically, increasing abundance and phosphorylation of NKCC2 and Na+- Cl- cotransporter (NCC) and/or activating proteolytic cleavage of epithelial sodium channels (ENaC). ln contrast, Angll hypertension suppresses proximal tubule and loop of Henle transporters including NHE3, NaPi2, medullary NKCC2, and medullary Na,K-ATPase.35 ln summary, the systemic Angll, inflammation and intrarenal production of Angll stimulate distal transporters and contribute to hypertension while the resultant hypertension counteracts the effects of the Angll and depresses proximal and loop of Henle Na+ transport facilitating pressure natriuresis.56

Many mouse genetic models have been subjected to experimental Angll hypertension and several exhibit a blunted hypertensive response to Angll infusion. We tested the hypothesis that we could identify the locus of the blunting of the hypertension along the nephron: either reduced Angll stimulation of distal transporters or augmented pressure-natriuretic depression of transporter abundance. This section will focus specifically on the responses of NHE3. ln most cases, an augmented suppression of NHE3 abundance accompanied the lower blood pressure, consistent with the notion that local Angll "locks" NHE3 in the microvilli and counters the responses to hypertension.

Figure 5 summarizes the measurements of renal NHE3 abundance by immunoblot in wild type (WT) C57Bl/6J mice and genetically modified mice (KO) in response to 14 day

AngII infusion. The WT and KO samples were studied at the same time, and results are normalized to mean baseline results defined as 1.0. In the analyses of different sets of WT mice, we did not observe significant suppression of NHE3 abundance as we measured in rats,35 nor did we detect evidence for AngII stimulation of NHE3, thus, the counteracting influences of hypertension and AngII may balance at the baseline levels in these WT mice. We have not yet analyzed the NHE3 distribution in the microvilli, nor have we determined whether NHE3 is stimulated during short term AngII infusion. In mice with a specific genetic deletion of AT1R from the renal proximal tubule (ATIR KO), generated by Gurley and Coffman, the baseline levels of NHE3 were unaltered, yet during AngII infusion NHE3 abundance was decreased 40%, associated with a 20 mmHg lower BP. 57 In collaboration with the Harrison and Madhur groups we analyzed mice lacking the ability to synthesize the cytokine 1L-17A or the cytokine IFN-g during AngII hypertension. In both KO strains AngII decreased abundance of NHE3 (25-40%), myosin VI (25%) and NaPi2 (50%) associated with 20-25 mmHg lower BP and improved natriuretic response to saline infusion.58 A subsequent study led by the Madhur lab demonstrated that the IL-17A strain had little or no increase in urinary albumin or angiotensinogen during AngII infusion, suggesting that IL-17A may reduce intrarenal AngII. Interestingly, the study also demonstrated that IL-17A stimulated NHE3 expression in cultured kidney cells mediated by serum and glucocorticoid regulated kinase 1 phosphorylation. 59

With Gonzalez-Villalobos we investigated the sodium transporter responses in mice that were engineered to express normal systemic ACE but no kidney ACE (ACE 10/10).60 During AngII infusion, these mice filter and sense infused AngII but this AngII or

accompanying inflammatory cytokines cannot stimulate additional local intrarenal production of Angll. ln Angll infused ACE 10/10 mice BP was about 20 mmHg lower yet this was not accompanied by a fall in NHE3 abundance, rather, there was suppression of Angll activation of distal NKCC2 or NCC.60 This same strain was also subject to another distinct model of experimental hypertension caused by inhibition of nitric oxide synthase with the inhibitor Nw-nitro-l-arginine methyl ester hydrochloride (L-NAME). ln WT mice L-NAME raises BP 25 mmHg, suppresses systemic Angll and through local inflammation, stimulates intrarenal production of Angll. ln the ACE10/10 mice L-NAME did not raise BP but did significantly suppress NHE3 and NaPi2 abundance 50 and 30%, respectively.55 A third study in the ACE 10/10 mice demonstrated that this strain is resistant to salt sensitive hypertension: after washout of the L-NAME treatment, BP returns to normal in both genotypes, yet transporters remain lower in abundance in the ACE 10/10. When both genotypes were subsequently fed a high salt diet, the WT mice developed a salt sensitive rise of 20 mmHg in BP while the ACE 10/10 maintained baseline BP.21 This differential response can be attributed to the higher inflammation and local production of Angll in the WT, and lower NHE3 in the ACE 10/10. Taken together, the findings in these hypertension resistant mouse models suggest that elevated Angll, cytokines, and/or ROS maintain the NHE3 in the microvilli and blunt the redistribution or decreased abundance of NHE3 in response to sodium transport stimulation along the nephron. ln other words, in both Angll and L-NAME hypertension, elevated local Angll production "put the brakes" on pressure natriuretic adjustments by activating transporters; thus, a further increase in pressure, and perhaps a different signaling path, is required to decrease transporters.

Summary and future directions

Figure 6 provides a simplified overview of the connections between: 1) sodium transport stimulation by AngII, cytokines, ROS, and another important activator, renal sympathetic nerve stimulation (RSNA), 2) rise in effective circulating volume and blood pressure, and 3) suppression of proximal and loop sodium transporters during hypertension. The results presented in this report suggest that, ultimately, the magnitude of hypertension is determined by the strength of the blood pressure signal(s) required to reduce proximal nephron Na+ reabsorption enough to maintain effective circulating volume near baseline. The conclusion is not intended to ignore the importance of the neuro- and vascular aspects of hypertension, but to focus on renal sodium handling. Based on these findings, it is worth considering strategies to stimulate proximal tubule natriuresis as an approach to counteract the stimulatory influences of local AngII. Three candidate pathways appear promising.

AT2R mediated natriuresis: AT1R and AT2R share similar affinity for AngII yet AT2R stimulation counteracts the effects of AT1R by increasing bradykinin and NO release, reducing inflammation, promoting vasodilation and natriuresis.61 62 AngII I is the likely ligand for AT2R.63 The Denton lab showed that direct stimulation of AT2R with the selective agonist C21 increases natriuresis and diuresis without changes in GFR or RBF, evidence for tubular actions.62,64 The Carey group confirmed these findings in volume expanded female rats, also showing C21 may move NHE3 to the base of the microvilli, and that the natriuresis was dependent on NO and bradykinin. Denton has demonstrated that the lower BP and more sensitive pressure natriuresis observed in WT female rodents is associated with 4 fold higher AT2R mRNA;64 and that this sex

advantage disappears with age and in global AT2R KO mice.65 This fast growing area full of therapeutic potential61,62 has a large gap in knowledge about how AT2R signaling affects sodium transporters/channels and intrarenal RAS in males and females.

GLP1R mediated natriuresis: Glucagon like peptide-1 (GLP-1) is an incretin hormone constantly secreted from the intestine at low basal levels in the fasted state; plasma concentrations rise rapidly after nutrient ingestion. Upon release, GLP-1 exerts insulinotropic effects via a G protein-coupled receptor, stimulation of adenylyl cyclase, and cAMP generation. Although primarily involved in glucose homeostasis, GLP-1 can induce diuresis and natriuresis when administered in pharmacological doses in humans and rodents. The Girardi lab has defined the chronic effects of stimulation of the incretin receptor GLP-1 in kidney66,67 and discovered that GLP-1 has diuretic and natriuretic effects mediated by changes in both renal hemodynamics and by downregulation of PT NHE3 activity.67 Recently, the Girardi group demonstrated that endogenous baseline GLP-1 plays a significant role in regulating renal function. They blocked GLP-1 receptor (GLP-1R) with the antagonist exendin-9 in overnight-fasted anesthetized rats. Exendin-9-infused (30 min) rats exhibited reduced GFR, lithium clearance, urinary volume flow, and sodium excretion compared with vehicle-infused controls. NHE3 phosphorylation at a site associated with retraction to the base was also increased. 68 Collectively, these results provide novel evidence that GLP-1 is a physiologically relevant natriuretic factor that contributes to sodium balance, in part, via tonic modulation of sodium transport activity in the proximal tubule.

SGLT2 inhibitors. The sodium-glucose-cotransporter (SGLT) inhibitors used to treat diabetes directly target the proximal tubule, provoke natriuresis and diuresis and may

lower blood pressure.69 Interestingly, Pessoa et al recently reported that NHE3 activity is stimulated by luminal glucose, and that NHE3 co-localizes and may functionally interact with SGLT2 in the proximal tubule. Thus, it is possible that SGLT2 inhibition may inhibit NHE3 transport activity.70

Dopamine receptor (DR) mediated natriuresis. The intrarenal dopaminergic system is an important determinant of the BP set point: reduced dopamine (DA) signaling is associated with hypertension.71,72,73 There are 5 DR in the kidney: D1R and D5R (DllikeR) physically interact and their activation inhibits NHE3 and Na,K-ATPase,74 thus, counteracting the effects of Angll via AT1R.63 DllikeR activation increases salt and water excretion mediated by inhibition of PT NHE3, NaPi2 and Na,K-ATPase as well as TALH NKCC2, the proximal sites that can elicit pressure natriuresis.75 Additionally, DR activation is known to antagonize AT1R signaling, reduce intrarenal RAS components,63,75,76 and stimulate AT2R signaling.77 Numerous labs have provided evidence for functional complexes between Ang AT1R and DA D1R 78 in which activation of one attenuates the expression of the other. Likewise, AT2Rs may oppose AT1Rs by protein-protein interaction.63 Thus, effects of dopamine deficiency include AT1R activation and vice versa.

Determining how the pressor (AT1R) and natriuretic (AT2R) arms of the RAS, in parallel with the interacting dopaminergic system, regulate transporters and channels may fill important gaps in understanding the sexual dimorphism of blood pressure and provide new and sex specific therapeutic approaches to treat resistant hypertension.

Disclosure statement: no relationships and no financial conflicts.

Acknowledgements. Arvid Maunsbach generated the proximal tubule electron micrograph in Figure 2. This work is supported by National Institutes of Health NIDDK grant R01DK083785 and AHA Grant in Aid Western States Affiliate 15GRNT23160003

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Figure Legends

Figure 1 Central blood pressure (BP) shown as a function of the effective circulating volume (ECV) and cardiac output (CO) as well as the kidneys' regulation of NaCl and H2O reabsorption, adapted from Starling 1 and Borst 2 The kidney's "willingness" to excrete NaCl and water when blood pressure rises is a key controller of the ECV and BP. This manuscript examines factors, both extrarenal and intrarenal, that reduce this willingness, in the proximal tubule (and mechanisms involved) leading to an elevation in blood pressure and activation of pressure natriuresis mechanisms in the proximal tubule that contribute to maintenance of ECV homeostasis.

Figure 2. Elevating salt intake from 0.4% to 4% does not change the total abundance of proximal tubule (PT) NHE3, but redistributes NHE3 to the base of the PT microvilli. In salt-resistant, animals this occurs without an elevation in blood pressure. A. Immunoblots of renal cortical homogenates from rats fed 0.4 or 4% salt diet.18 B. Cross section of PT shown in simple model and electron micrograph illustrating dense apical microvilli. NHE3 redistribution along the proximal tubule microvilli is detected by co-labelling the actin bundling protein villin (red V) and NHE3 (green circle) with specific antibodies (ref). Left half of the PT model represents PT at normal salt diet with villin and NHE3 co-localized yielding microvilli stained yellow. Right half of the PT represents PT during high salt diet with NHE3 retracted to the base of the microvilli exposing red V in body of the villi and green/yellow at the base of the microvilli. C. Co-labelling of PT from rats on 0.4% NaCl (left half-tubule) with both NHE3 and villin in body of microvilli, and 4.0% NaCl (right half-tubule) with NHE3

concentrated at the base of the microvilli. C. Myosin VI, an atypical molecular motor implicated in the redistribution of NHE3 and NaPi2 within the plane of the microvilliar membrane 17,19 is located in the body of the microvilli with 0.4% NaCl diet and redistributes to the base of the microvilli during 4% NaCl diet.

Figure 3. Acute hypertension produced by raising total peripheral resistance rapidly increases urine salt and volume output (pressure natriuresis/diuresis) and redistributes NHE3 to the base of the PT microvilli. A. In inactin anesthetized male rats, raising mean atrial blood pressure from 87-130 mmHg (BP) according to the protocol of Roman and Cowley 22 rapidly increases urine output more than 10 fold 23 B. Cross section model of PT as described in Figure 2. Co-labelling of PT from rats at baseline BP (left half-tubule) with both NHE3 and villin in body of microvilli, and at elevated BP (right half-tubule) with NHE3 concentrated at the base of the microvilli.16 C. When Angll levels are clamped by pre-infusion with an ACE inhibitor to prevent Angll production along with a constant infusion of Angll to maintain baseline BP, the redistribution of NHE3 to the base of the microvilli is significantly blunted 32 The findings suggest that a drop in local Angll may contribute to the NHE3 redistribution during acute hypertension, and that high local Angll can blunt pressure natriuresis.

Figure 4. Angiotensin II regulates NHE3 distribution in the proximal tubule microvilli. Renal cortex examined by co-labelling NHE3 (green) and villin (red) with specific antibodies as described in Figure 1. A. NHE3 in body of the microvilli in inactin anesthetized rat at baseline. B. lnfusing captopril for 20 min at a dose that does not lower BP redistributes NHE3 to the base of the microvilli 39 C. lnfusing captopril for 20 min followed by Angll infusion for 20 min, at doses that do not change blood

pressure, moves NHE3 from the base to the body of the microvilli 40 D. Co-infusing a p-arrestin biased Angll receptor (ATI R) specific agonist along with captopril for 20 min moved NHE3 to the base of the microvilli and blocked the effects of subsequent Angll infusion.41 E. Infusion Angll into rats for 14 days raised mean arterial pressure from 120 to 160 mmHg and decreases the total abundance of renal cortical NHE3 by 20% yet despite the hypertension, NHE3 is retained in the body of the microvilli. 35 lmages in panels A and E were collected with the same settings, demonstrating that the ratio of NHE3 to villin signal has decreased in during Angll.

Figure 5. In mouse genetic models that exhibit a blunted hypertensive response to AngII or L-NAME experimental hypertension, NHE3 abundance is significantly depressed. Models treated for 14 days with Angll infusion by osmotic minipumps include: PT AT1R knockout 57, mice with whole body knockout of lL17A 58, mice with whole body knockout of lFNg 58, and in mice with no kidney ACE (ACE 10/10). 60 lmmunoblots of a constant amount of renal homogenate are shown for each set of WT and KO mice infused +/- Angll. The last set shows the ACE 10/10 mice infused with the nitric oxide synthase inhibitor Nw-nitro-l-arginine methyl ester hydrochloride (L-NAME).55 These common findings in disparate models implicate a decrease in NHE3 abundance in the improved pressure natriuresis and blunted hypertension. Additionally, the findings suggest that elevated Angll, cytokines, and ROS prevent the redistribution and/or decreased abundance of NHE3 in response to chronic sodium transport stimulation.

Figure 6. Simplified overview of the connections between: sodium transport stimulation by AngII, cytokines, ROS, and another important activator, renal

sympathetic nerve stimulation (RSNA), rise in effective circulating volume and blood pressure, and suppression of proximal and loop sodium transporters during hypertension. The local activation of proximal tubule NHE3 by intrarenal AnglI, cytokines, reactive oxygen or RSNA can blunt the pressure natriuresis responses and raise blood pressure. Ultimately, the magnitude of hypertension is determined by the strength of the blood pressure signal required to reduce proximal nephron Na+ reabsorption (by NHE3 redistribution to the base of the microvilli or depressed abundance) in order to restore effective circulating volume. The signals connecting the hypertension to the anti-natriuresis are discussed in the text.

extravascular NaCl and H2O

extra-renal neuro-humoral controls

NaCl and H2O intake

EFFECTIVE CIRCULATING VOLUME CARDIAC OUTPUT

CENTRAL BLOOD PRESSURE

KIDNEYS

regulated NaCl and H2O output

regulated

NaCl and H2O reabsorption

Figure 1 Central blood pressure (BP) shown as a function of the effective circulating volume (ECV) and cardiac output (CO) as well as the kidneys' regulation of NaCl and H2O reabsorption.

Complete legends provided for all figures in ms. file following references cited.

Figure 2. Elevating salt intake from 0.4% to 4% does not change the total abundance of proximal tubule (PT) NHE3, but redistributes NHE3 to the base of the PT microvilli.

Complete legends provided for all figures in ms. file following references cited.

•ÎBPto 130 mmHg

5 10 15min

•in. M:;-iimi

baseline BP 87 mmHg

15 min

NORMAL BLOOD PRESSURE

HYPERTENSION

NHE3 • villin V

Figure 3. Acute hypertension produced by raising total peripheral resistance rapidly increases urine salt and volume output (pressure natriuresis/diuresis) and redistributes NHE3 to the base of the PT microvilli. Complete legends provided for all figures in ms. file following references cited.

ANGII CLAMP ACUTE

HYPERTENSION

♦ VW*

control

captopril

+ captopril

AngII + captopril + AT1R biased ß-arrestin agonist

14 days AngII hypertension

Figure 4. Angiotensin II regulates NHE3 distribution in the proximal tubule microvilli. Renal cortex examined by co-labelling NHE3 (green) and villin (red) with specific antibodies as described in Figure 1. Complete legends provided for all figures in ms. file following references cited.

WT AngII AT1R KO KO AngII citation 54

T . T_ _ _

1.0 ±0.07 0.74 ±0.09 1.0 ±0.11 0.58 ± 0.07*

WT AngII IL17-A KO KO AngII 55

NMN • M

1.0 ±0.05 0.95 ±0.04 1.0 ±0.07 0.62 ±0.05*

WT AngII IFN-g KO KO AngII 55

NNHMM h^MMM ■ ■ -

1.0 ±0.05 0.95 ±0.04 1.0 ±0.05 0.75 ±0.09*

WT AngII ACE10/10 ACE10/10 AngII 57

mmm——

1.0 ±0.09 0.99 ± 0.06 1.0 ±0.14 0.81 ±0.11

WT L-NAME ACE10/10 ACE10/10 L-NAME 52

«IMMMM i 4 ••

1.0 ±0.07 0.85 ±0.04 1.0 ±0.15 0.47 ±0.11*

Figure 5. In mouse genetic models that exhibit a blunted hypertensive response to AngII or L-NAME experimental hypertension, NHE3 abundance is significantly depressed. Complete legends provided for all figures in ms. file following references cited.

f Effective circulating volume and ^ blood pressure

hypertension

AngII, ROS, cytokines, RSNA

salt transport

AngII, ROS, cytokines, RSNA

Figure 6. Simplified overview of the connections between: sodium transport stimulation by AngII, cytokines, ROS, and another important activator, renal sympathetic nerve stimulation (RSNA), rise in effective circulating volume and blood pressure, and suppression of proximal and loop sodium transporters during hypertension. Complete legends provided for all figures in ms. file following references cited.