The endocytic adaptor ARH facilitates potassium conservation by regulating ROMK and BK

Abstract

Renal Outer Medullary K⁺ (ROMK) channels are essential for urinary potassium secretion, and their endocytosis prevents excessive K⁺ loss during dietary deficiency. The clathrin adaptor ARH (Autosomal Recessive Hypercholesterolemia) has been implicated in mediating ROMK internalization, yet its physiological significance remains unclear, as hypokalemia is not reported in patients with type 4 familial hypercholesterolemia (FH4) who lack functional ARH. To address this, we investigated potassium homeostasis in ARH knockout (KO) mice, a model of FH4. Despite conserving K⁺ during dietary restriction, ARH-KO mice exhibited exaggerated urinary K⁺ loss when challenged with hydrochlorothiazide, consistent with compensatory upregulation of the thiazide-sensitive sodium-chloride cotransporter (NCC). Immunoblotting revealed significantly higher ROMK and BKα protein levels in the renal cortex of ARH-KO compared to wild-type (WT) mice at matched plasma K⁺ concentrations. Because BKα contains NPXY motifs required for ARH binding, we confirmed ARH directly associates with BKα by co-immunoprecipitation. Under potassium-deficient conditions, ARH-KO mice showed impaired downregulation of apical ROMK and BKα, indicating ARH-dependent endocytosis. Interestingly, compensatory mechanisms differed by sex: female KO mice exhibited enhanced NCC abundance and phosphorylation, whereas male KO mice showed reduced ENaC cleavage and diminished BK auxiliary subunits relative to WT. These findings (i) establish ARH as a key regulator of ROMK and BKα trafficking in the distal nephron, (ii) reveal sex-specific compensatory mechanisms that preserve potassium balance, and (iii) underscore the delicate nature of K⁺ homeostasis upon ARH deletion, with maintained normokalemia at the expense of physiological trade-offs involving altered sodium handling.

Graphical Abstract

Introduction

The kidney maintains potassium (K⁺) homeostasis by adjusting K⁺ excretion to match dietary K⁺ intake. Renal function measurements, complemented by electrophysiological and kidney imaging studies, have shown ROMK-mediated K⁺ secretion in the late distal nephron, especially the late Distal Convoluted Tubules (DCT2) and Connecting Tubules (CNT) is a highly regulated (1-4). ROMK is chiefly regulated by controlling the number of channels on the apical membrane, increasing following high K⁺ intake and decreasing with consumption of a low K⁺ diet (5). Under hypokalemia or low K⁺ intake, ROMK apical abundance is reduced by clathrin-dependent endocytosis, stimulated by WNK (6-11), Src (12, 13) and MAPK kinases (14). We have previously shown that ROMK endocytosis is mediated by the clathrin-adaptor protein, ARH (8). Protein-Protein interaction and molecular reconstitution studies in COS-7 cells revealed that ARH directly binds ROMK and facilitates its recruitment to clathrin-coated pits (8). ARH acts as an endocytosis scaffold, simultaneously binding to clathrin, the AP2 adaptor and cargo through separate protein-protein interaction domains, allowing it to recruit membrane proteins, like ROMK, into clathrin coated pits (15-17). The PTB domain of ARH engages a NPXF motif in ROMK (8), a variant of the canonical endocytic motif (NPXY) that is present in other ARH targets such as LDLR (18-20) and Megalin (21). An unusual hydrophobic pocket in the PTB domain of ARH explains its distinctive ability amongst the family of PTB clathrin-adaptors to internalize proteins containing either NPxY or NPxF sequences (22). The physiological significance of ARH-dependent ROMK regulation in vivo has not yet been investigated.

Inactivating mutations in the ARH gene (Low-Density Lipoprotein Receptor Adaptor LDLRAP1) cause Autosomal Recessive Hypercholesterolemia (Type 4 familial Hypercholesterolemia, FH4) (18, 23), a rare monogenic disorder characterized by high levels of low-density lipoprotein cholesterol (LDL-C), tuberous and tendon xanthomas, and premature atherosclerosis. Analysis of human tissues not only revealed a high expression level of ARH RNA(18) and protein (23) in the liver, where ARH is required for the endocytosis of LDL receptors (15, 16, 18, 19, 24) but also in the kidney, where its physiological significance has not been fully understood (23). Studies in vitro suggested that ARH may control post-endocytic trafficking of megalin, a member of the LDL receptor family, in the proximal tubule, and ROMK endocytosis in the distal nephron. The ARH knockout mouse model of FH4 display the predicted increase in total cholesterol, secondary to a reduction in LDL clearance (16, 24, 25), however, renal potassium handling has not been described. Although emerging studies have associated ARH with kidney pathologies, including diabetic kidney diseases (26), and membranous nephropathy (27), predicted urinary potassium wasting and hypokalemia have not been reported in humans with FH4.

To explore mechanisms of renal potassium handling in FH4, we evaluated K⁺ balance, and the regulation of the main K⁺ exit channel in the distal nephron, ROMK and BK in mice lacking ARH (ARH-KO) compared to matched wild-type mice. We found that ARH-KO mice exhibit elevated ROMK and BKα protein levels and enhanced apical membrane localization in the distal nephron compared to wild-type control. Nevertheless, compensatory activation of thiazide-sensitive sodium-chloride cotransporter (NCC) prevented urinary potassium wasting. When NCC was inhibited using hydrochlorothiazide, ARH-KO mice exhibited increased potassium loss compared to WT. Further studies revealed a sex difference in the compensatory response. This study underscores the significant role of ARH-mediated ROMK trafficking in potassium homeostasis and points to a sex-dependent regulation of the K⁺-secreting machinery in the distal nephron.

Materials and Methods

Animal Models, Dietary Manipulation

Experimental protocols, including animal use, were approved by the Johns Hopkins University Animal Care and Use Committee. Mice were housed in a temperature-controlled room (19–22°C) with a 12h:12h light-dark cycle. ARH-KO mice (18) were obtained from Helen Hobbs​, University of Texas Southwestern in a hybrid (C57Bl/6 and 129Sv) background. These mice were crossed for 6 generations with wild-type mice with a C57BL/6 background, which contain one renin gene, mimicking humans (28). Genotype was determined by PCR performed on ear biopsies. Sex-matched ARH-KO and WT littermates were used for analysis. Mice were fed either a control or a potassium-deficient diet for two weeks. The electrolyte composition of the experimental diets used in this study is shown in (Table 1). Both diets were formulated in consultation with a Teklad-certified dietitian and were identical in caloric density and nutrient composition, differing only in their potassium chloride content.

Table 1:

Electrolyte composition of the experimental diets used in this study.

	Envigo Reference	% K+	% Na+	% Cl−	% Ca2+
Control	TD.190005	1	0.3	1.4	1
K+ deficient	TD. 88239	∼0	0.3	0.45	1

Hydrochlorothiazide Challenge

The primary goal of the HCTZ challenge was to evaluate sodium-dependent changes in K⁺ secretion that derive from the upregulation of NCC activity by low K diet (25651563, 25565204). To test this, the HCTZ challenge was performed in males, which exhibit similar NCC upregulation in WT and KO mice exhibit, providing a more controlled context to isolate and interpret differences in K⁺ exit. Adult males (WT and KO) were acclimated to the control diet (1% K⁺) for 3 days, and then provided with the K⁺-free diet for 2 weeks. The mice were challenged with thiazide. A stock solution (30 mg/mL) of Hydrochlorothiazide (HCTZ) was prepared by dissolving HCTZ (Sigma-Aldrich H2910) in Dimethyl sulfoxide (DMSO: Sigma-Aldrich D2438). Mice received a single intraperitoneal (IP) injection of HCTZ (20 mg/Kg of body weight) or equivalent vehicle (DMSO) volume as described previously. The kaliuretic response to HCTZ was evaluated in metabolic cages at 6h after injection. Urinary electrolytes and the urinary flow rate were measured. Because K⁺ excretion follows a circadian pattern with increased K⁺ excretion during the active phase (29), drug injection was performed at the start of the active (dark) phase.

Metabolic Cage, Blood, and Urine analyses

Mice were placed in metabolic cages (Nalgene) and allowed to adapt for three days as previously described (30, 31). Food and water were available ad libitum. Daily food and water consumption were measured, and urine samples were collected daily. In agreement with prior findings (30-34), measured water intake exceeded urinary volume by approximately 2.4- to 2.9-fold across all groups (Table 3), presumably reflecting the non-renal loss of water insensible losses from transepidermal and respiratory evaporation (35), and incomplete intestinal absorption of water (36). Additionally, technical factors, particularly water dribbling from the delivery spots, and urine evaporation may further contribute to this difference. Urine electrolytes were measured using the Diamond CareLyte Plus Electrolyte Analyzer. Urine osmolality was measured using a vapor pressure osmometer (Wescor-Vapro 5520). At the end of the experiment, blood and kidneys were collected. For these experiments, mice were anesthetized with an intraperitoneal injection of ketamine-xylazine (100 mg/kg ketamine and 10 mg/kg xylazine). Blood samples were obtained from the right common carotid artery using a 1-mL pipettor with a large bore tip containing heparin. Blood samples were used immediately for electrolyte analysis (i-STAT CG8+). Kidneys were removed, cortexes were immediately separated from medullas, snap-frozen in liquid nitrogen, and conserved at −80°C. Mice were euthanized by exsanguination.

Table 3:

Metabolic parameters of ARH-WT and KO males fed control diet or prolonged K⁺ deficient diet. Data from Body weight, 24h food and water intake, blood and urine analysis are presented. N=6 mice/group.

	ARH-WT	ARH-WT	ARH-KO	ARH-KO
	Control diet	K+ Deficient	Control diet	K+ Deficient
Body weight (g)	26.1 (+/0.5)	25.2 (+/−1.1)	29.8 (+/−0.8) #	28.7 (+/−0.5) #
Food intake (g/day)	2.6 (+/−0.6)	3.2 (+/− 0.5)	3.0 (+/−0.5)	3.0 (+/−0.2)
Water intake (mL/day)	3.8 (+/−0.5)	6.3 (+/−0.5) *	4.6 (+/−0.4)	7.3 (+/−0.9) *
24h Urine
Volume (mL)	1.5 +/− 0.3	2.6 (+/− 0.3) *	1.6 +/− 0.2	2.8 (+/− 0.4) *
24h Na+ (mmol)	360 (+/−21)	140 (+/−37) *	329 (+/−22)	139 (+/−19) *
24h K+ (mmol)	1139 (+/−82)	13 (+/−3.4) ***	972 (+/−80)	12 (+/−2.5) ***
24h Cl- (mmol)	1449 (+/−104)	154 (+/−41) ***	1314 (+/−107)	132 (+/−20) ***
Blood
Na+ (mmol/L)	148.5 (1.1)	150.4 (+/−3.8)	147.5 (+/−1.5)	148 (+/−1.4)
K+ (mmol/L)	4.2 (+/−0.1)	2.4 (+/−0.38)**	4.1 (+/−0.31)	2.1 (+/− 0.17)**
iCa2+ (mmol/L)	1.36 (+/−0.02)	1.34 (+/−0.04)	1.38 (+/−0.06)	1.32 (+/−0.06)
HCO3- mmol/L)	24.2 (+/−1)	24.6 (+/−1.2)	23.4 (+/−0.8)	24.7 (+/−1.2)
Glu (mg/dL)	489 (+/−46)	418 (+/−57)	451 (+/−31)	434 (+/−72)
Hct (%PCV)	36.5 (+/−1.5)	39.6 (+/−0.9)	36.3 (+/−2.1)	37 (+/−2.2)
Hb (g/dL)	12.4 (+/−0.5)	13.5 (+/−0.3)	12.3 (+/−0.7)	12.6 (+/−0.7)

Significance was tested using two way ANOVA, (*) indicates difference between diets, (#) indicates difference between genotypes.

^{* or #:}p value < 0.05

^**:p value <0.01

^***:p value <0.001

Tissues Lysate Preparation and Western Blot Analysis

Frozen tissues were homogenized in lysate buffer [200 mM sucrose, 20 mM HEPES, 0.5 mM CaCl2, and phosphatase and protease inhibitor (PCC1010, Sigma)] using a Beadbug device and 1.5 mM zirconium bead-prefilled tubes (Z763799–50EA, Sigma). Detergents (2% SDS, 0.5% Triton X-100, and 0.5% Tween 20) were added after homogenization to avoid foaming. After 1 hour of incubation with rotation at 4°C and 15 min centrifugation at 15,000 g, the supernatant was collected, and protein concentration was measured using a Pierce BCA protein assay (No. 23225, Thermo Scientific). 8%−16% Midi-PROTEAN TGX Stain-Free Protein Gels (No. #5678105, Bio-Rad) were used for protein separation, which was next transferred to nitrocellulose membranes. Membranes were blocked for 1 hour in 5% nonfat dry milk dissolved in Tris-buffered saline (TBS; pH 7.4, MFCD00132476, VWR) with 2% Tween 20 (No. 9005–64-5, VWR) and incubated overnight at 4 °C with the primary antibody. A list of the antibodies used in this study is presented in Table 2. After a 1 hour wash with TBS with 2% Tween 20, membranes were incubated for 2 hour at room temperature with HRP goat anti-rabbit IgG (1:5,000, No. 111–035-144, Jackson Immuno). After a 1 hour wash, the signal was developed using SuperSignal West Pico PLUS Chemiluminescent Substrate (No. 34579, ThermoFisher). Luminescence was detected using an Azur 300 imaging system (Azure Biosystems, Dublin, CA) over the linear range. Integrated band intensity was quantified using Fiji software and normalized to protein bands detected with the Stain-Free Protein Ge

Table 2:

A list of primary antibodies and dilutions used in this study

Antibody	Host	WB dilution	IF dilution
AQP2	Chicken		1/600	Wade, (37)
NCC	Guinea Pig		1/200	Wade/Welling, (38)
T53 NCC	Rabbit	1/2000		Phosphsolutions (p1311–53)
NCC	Rabbit	1/2000		Millipore (AB3553)
ROMK	Rabbit	1/4000	1/300	Welling/Wade , (3)
αENaC	Rabbit	1/500		Courtesy Johannes Loffing (39)
BKα	Rabbit	1/2000		Alomone Lab (APC-021)
BKα	Mouse		1/40	NeuroMabclone L6/60
BKβ1	Rabbit	1/2000		Alomone Lab (APC-036)
BKβ4	Rabbit	1/2000		Alomone Lab (APC-061)

Immunofluorescence Imaging and Quantification

Sections (5 μm thick) were prepared from paraffin-embedded kidneys, deparaffinized in hexane and absolute ethanol baths for 10 minutes each, and rehydrated in a graded ethanol series to distilled water. Epitope retrieval was obtained by 30 minutes of heating at low pressure in Trilogy solution (No. 920 P-04, Cell Marque). Sections were then incubated for 1 h with home-made blocking solution (PBS, 1% BSA, 50 mM glycine, and 0.2% Na-azide, pH 7.2). Antibodies prepared in the incubation medium (PBS, 1% BSA, 1% Tween 20, 0.35 M NaCl, and 0.2% Na-azide) were applied overnight at 4°C. A list of the antibodies used in this study is presented in Table 2. After 1 hour of washing (4 times) with high-salt wash buffer (incubation medium plus 0.5 M NaCl), sections were mounted in mounting medium (H-1000, Vector). Image acquisition was performed using a Zeiss LSM 700 confocal microscope. Images were acquired at a constant gain, contrast, pinhole size, and laser power with a calibrated photodetector. The fluorescence signal of a line scan across the apical membrane was quantified using Fiji plot profile analysis.

Co-immunoprecipitation of BKα and ARH

COS7 cells were transfected with constructs encoding either myc-BKα (Slo1), myc-ARH, or both, using Extreme Gene transfection reagent. After 48 hours, cells were harvested and lysed in HEENG buffer containing 1% Triton. Co-Immunoprecipitation (Co-IP) were performed with anti-BKα (NeuroMab, clone L6/60) and protein A/G agarose magnetic beads incubated with cell lysate; for negative controls, the primary antibody was replaced with IgG. Following overnight incubation, beads were washed three times with PBS and proteins were eluted at 42 °C in running buffer. Samples were resolved on a 10% SDS-PAGE gel, transferred to membrane, and probed with anti-myc (1:2000; rabbit secondary, 1:5000). For endogenous protein co-immunoprecipitation, homogenates from kidney cortex of adult male C57BL/6 mice were used. Immunoprecipitations were performed as described above, with anti-ARH as the IP antibody and anti-BKα for immunoblotting.

Statistical Analysis

Data are presented as means ± SE. Each data point represents a different animal. Statistical analysis was performed using GraphPad PRISM 8. When single dependent variables were compared, the data were analyzed using an unpaired Student’s t-test. The influence of two different independent variables on one continuous dependent variable was assessed using two-way ANOVA. Sidak’s test was used for post-hoc analysis of multiple comparisons. The threshold of significance was P ≤ 0.05.

Results

ARH KO mice are more prone to urinary potassium loss

To assess the physiological significance of ARH deletion in K⁺ regulation, we analyzed K⁺ balance in ARH-KO and WT littermates under basal conditions and after prolonged K⁺ deficiency. WT and KO males were randomized to control diet (1% K⁺) or nominally K⁺-free (0% K⁺) diet for 2 weeks. As shown in Table 3, ARH-KO mice exhibit higher body weight than WT littermates under basal conditions and after prolonged K⁺ deficiency. The higher body weight in KO mice was not due to a difference in food intake but rather to altered lipid and insulin metabolism as previously described in this model (24, 37). ARH-KO mice maintain normal water balance, with water intake and urinary volume comparable to WT controls (Table 3). Prolonged K⁺ deficiency similarly led to polyuria and polydipsia in WT and ARH-KO mice, consistent with previous reports of hypokalemia-induced diabetes insipidus (30, 38, 39). We found plasma and urinary K⁺ excretion were conserved in ARH-KO mice under basal conditions and were reduced to a similar extent as WT mice after prolonged K⁺ deficiency (Table 3). Plasma K⁺ levels in KO females were also similar to WT females under basal conditions, and dropped to similar levels in both genotypes after a prolonged (2 weeks) challenge with KFD (Sup Fig1F). These data indicate that K⁺ balance is generally maintained in ARH deficiency.

Activation of the thiazide-sensitive sodium chloride cotransporter, NCC, provides compensatory mechanism to limit urinary potassium excretion in potassium deficiency by reducing distal sodium delivery necessary for Na⁺/K⁺ exchange. To test if this might prevent potassium wasting in the face of increased apical ROMK abundance in the distal nephron, we challenged male mice with hydrochlorothiazide, and compared the response of KO to WT (HCTZ). Acute HCTZ (6h) triggered a robust increase in urinary NaCl excretion that was similar between WT and KO mice (Fig 1A, B). The kaliuretic response, however, was greater in KO than in WT mice, indicating enhanced K⁺ exit in the KO mice (Fig 1C). Together, these data indicate ARH-KO mice are able to conserve K⁺ under dietary K⁺ deficiency but are prone to lose K⁺ under conditions that increase distal Na⁺ delivery.

Figure 1: Thiazide treatment results in urinary K⁺ loss in ARH-KO mice:

A-C) Urinary Na⁺, Cl⁻ and K⁺ excretion after 6h of HCTZ injection in WT and KO mice treated by K⁺-free diet for 2 weeks. Significance was analyzed using 2 way-ANOVA and Tukey’s multiple comparisons correction. **: p value <0.01, ***: p value <0.001, ns=non significant. N=5 mice/genotype.

ARH gene deletion results in altered ROMK regulation

Our previous studies revealed ARH directly interacts with ROMK to drive channel endocytosis (8). To understand the consequence of this on potassium-dependent ROMK regulation, we first examined ROMK protein abundance in kidney cortex preparations from ARH-KO and WT littermates fed a control diet or K⁺-free diet (KFD) for 2 weeks. Males and females were assessed separately. We found ROMK abundance was higher in the ARH-KO than in WT males under the control diet (Fig: 2A-C). A similar observation was made in females (Sup Fig 1A,B). When WT and KO males and females were fed the K⁺ deficient diet for prolonged period (2 weeks), both genotypes reduced cortical ROMK levels to similar extent (2D-F, Sup Fig1C-E). However, linear regression analysis of glycosylated ROMK versus plasma K⁺ concentration in males and females revealed that ROMK protein is more abundant in ARH KO than WT mice at any given blood K⁺ (Fig2 G). These observations indicate that ARH plays a role in basal ROMK turnover and its regulation by plasma K⁺.

Figure 2: ARH deletion impairs ROMK regulation:

A) WB analysis of ROMK expression in cortical samples from WT and ARH-KO mice under basal conditions.

B, C) Quantification of WB in A showing ROMK non glycosylated and Core-glycosylated bands (non+Core) in B, and fully glycosylated band (Glyco) in C.

D) WB analysis of ROMK expression in cortical samples from WT and ARH-KO under control diet (CtrD) and prolonged K⁺-free diet (KFD).

E,F) Quantification of ROMK non and Core-glycosylated bands in E, and fully glycosylated band in D.

G) Pearson’s correlation of fully glycosylated ROMK abundance in WT and KO mice and their respective plasma K⁺. Data from males and females WT and KO mice were combined. Note the intercepts of WT and KO are significantly different indicating ROMK expression at any given plasma K+ is higher in the ARH-KO compared to WT. Significance was tested using unpaired T-test in B and C, and two-way ANOVA with Sidak’s multiple comparisons correction in E and F. *: p value < 0.05, **: p value <0.01. ns=non significant. N=5 mice/group.

ARH regulates ROMK apical localization specifically in the major K⁺-secreting segment

We analyzed ROMK cellular localization in ARH-KO and WT males fed a K⁺-deficient diet, using segment specific markers, namely NCC for distal convoluted tubules (DCT), AQP2 for collecting duct (CD). The segment which the more robust K⁺ secretion (DCT2/CNT) was identified by low to no NCC expression with no to low AQP2 expression. These analyses revealed altered ROMK regulation occurs largely in the DCT2/CNT, a major site of regulated ROMK expression (1-3, 40, 41). We found WT DCT2/CNT cells exhibits low levels of ROMK after consumption of a K⁺ deficient diet (Fig 3A), confirming previous observations (1-3). Contrasting with the near absence of ROMK in the DCT2/CNT of K⁺-deficient WT mice, ROMK was robustly expressed in ARH-KO DCT2/CNT cells (Fig 3A). Our analysis revealed that ARH-KO cells display a range of ROMK localization patterns, including continuous or patchy apical membrane expression, as well as complete absence from the apical membrane. To accurately capture this variability, we first categorized cells as either ROMK⁺ or ROMK^⁻ based on the presence of the channel on or near the apical membrane (Fig 3B). We found that 50% of KO DCT2/CNT cells express apical ROMK compared to only 7% of WT cells (Fig 3B). We then quantified membrane intensity specifically within the apical ROMK⁺ population (Fig 3C). Line scan measurements of ROMK intensity across the apical membrane revealed that apical membrane associated channel density in the ROMK⁺ KO cells was twice as high as in the ROMK⁺ WT cells (Fig 3C). Indeed, ARH deletion results in more apical ROMK⁺ cells in the KO than WT, with higher ROMK apical intensity in those cells. These observations are consistent with the requirement of ARH for efficient internalization and degradation of ROMK in the DCT2/CNT under K⁺ deficient conditions. By contrast ROMK expression and apical localization in the TAL, identified as medullary tubules with high ROMK intensity and negative AQP2-labelling, were unaffected by ARH deletion (Fig 2D, E). Thus ARH is involved in the regulation of ROMK, specifically in the major K⁺-secreting DCT2/CNT segment.

Figure 3: ROMK apical membrane internalization is blunted in the distal nephron upon ARH deletion:

A) Confocal immunofluorescence imaging of kidney cortex, showing ROMK (in green) in K⁺-deprived WT and KO DCT2/CNT identified by low NCC (in red) expression. Note the quasi-absence of apical ROMK from WT DCT2/CNT cells, while half of the KO DCT2/CNT cells exhibit ROMK along the apical membrane either in continuous (white arrowheads) or discontinued/patched pattern (orange arrowheads), only half of KO cells show absence of apical ROMK (yellow arrowheads).

B) Cell count showing the ratio of DCT2/CNT cells with internalized or apical ROMK, N= 400 cells/genotype from 4 WT and 3 KO.

C) Quantification of ROMK apical intensity in the cells expressing apical ROMK in WT and KO using a line transecting the apical membrane (5 µm long) from the lumen into the cytoplasm, red line. N= at least 75 cell/genotype from 4 WT and 3 KO.

D) ROMK staining in the thick ascending limb (TAL) in K⁺-deprived WT and ARH-KO mice (TAL segments was identified as medullary tubules with high ROMK expression and AQP2-negative staining).

E) Quantification of ROMK apical intensity in the TAL. N= at least 200 cell/genotype from 4 WT and 3 KO.

Significance in B was tested by unpaired 2-tailed T-test, significance in C and E was tested using two-way ANOVA with Sidak’s multiple comparisons correction. ***: p value <0.001, ns=non significant.

ARH co-immunoprecipitates with BKα

In addition to ROMK-mediated K⁺ secretion, Ca²⁺-and voltage-sensitive BK channels represent a major K⁺ secretory pathway in the distal nephron. The pore-forming α subunit (Slo1) assembles as a tetramer and is ubiquitously expressed, while tissue-specificity and functional diversity are conferred by auxiliary β and γ subunits (42). In the distal nephron, BKα is co-expressed with β1 in principal cells or with β4 in the DCT and intercalated cells (43). Cryo-EM structures of Slo1 in complex with β4 revealed that each Slo1 subunit contains one NPxY sequence, the canonical ARH recognition motif, located in the cytosolic domain (44). The functional significance of this motif, however, has not been investigated.

To explore whether ARH regulates BKα, we tested if ARH and BKα form a protein complex. COS7 cells were co-transfected with myc-BKα (Slo1) alone or together with myc-ARH, and co-immunoprecipitation was performed using anti-BKα. Immunoblotting with anti-myc revealed ARH co-immunoprecipitated with BKα (Fig 4A). This finding was validated in vivo using kidney cortex homogenates from WT mice, where endogenous ARH co-immunoprecipitated with endogenous BKα (Fig 4B). These data indicate that BKα and ARH form a protein complex in vivo suggesting a direct regulation of BKα by the clathrin adaptor ARH.

Figure 4: ARH co-immunoprecipitates with BKα in-vitro and in-vivo.

A) Western blot analysis of ARH-BKα interaction in COS7 cells transfected with constructs encoding myc-BKα (Slo1), myc-ARH, or both. Co-immunoprecipitation (IP) was performed using anti- BKα, and immunoblotting (IB) was carried out with anti-Myc, since both ARH and BKα were expressed as myc-tagged proteins.

B) Western blot analysis of endogenous ARH-BKα interaction in wild-type mouse kidney cortex homogenates. IP was performed with anti-ARH, followed by IB with anti-BKα. Input lysates were included to confirm protein expression.

ARH deletion results in altered BKα regulation in the distal nephron

To determine whether BK channel abundance is altered in ARH-KO kidneys, we examined expression of the BKα, and its auxiliary β1, and β4 subunits by western blotting. In WT kidneys, BK subunit levels were unchanged under K⁺ deficiency, indicating that regulation likely occurs through trafficking or post-translational modifications. In contrast, ARH-KO mice of both sexes showed elevated BKα expression under control diet but suppressed expression under K⁺ deficiency (Fig 5A,B; Sup Fig 1G,H), suggesting a role for ARH in BKα regulation in vivo. Moreover, ARH-KO males exhibited reduced BKβ1 expression under both dietary conditions and reduced BK β4 expression specifically under K⁺ deficiency (Fig 5A,C,D), whereas ARH-KO females displayed no adaptive changes in β1 or β4 expression (Sup Fig1G-J).

Figure 5: BKα abundance and cellular localization is altered in ARH-KO mice.

A) WB analysis of BKα, β1 and β4 subunits in WT and ARH-KO males under control diet (CtrD) and prolonged K⁺-free diet (KFD).

B-D) quantification of WB in A.

E, F) Confocal immunofluorescence imaging of kidney cortex (E) and outer medulla (F), showing BKα (in magenta) in K⁺-deprived WT and KO distal nephron identified by AQP2 co-staining (Cyan). Note BKα is more abundant in intercalated (AQP2-negative) than principal (AQP2-positive) cells. Note greater BKα abundance at the apical membrane (white arrowheads) in KO mice.

G,H) Quantification of BKα cellular intensity in AQP2-positive and AQP2-negative cells respectively using a line transecting the apical membrane (5 µm long) from the lumen into the cytoplasm (green line in E). N= at least 40 cell/genotype from 3 WT and 3 KO.

Because BKα is expressed in multiple cell types across the nephron (45, 46), we asked whether ARH specifically regulates BKα in the K⁺-secreting distal nephron. To address this, we analyzed BKα cellular localization in ARH-KO and WT males fed a K⁺-deficient diet, using AQP2 co-staining. Confocal imaging revealed BKα is localized in the plasma membrane and cytosol; and is higher in the intercalated compared to principal cells (Fig 5E-H), confirming previous data (47). Compared to WT mice, BKα membrane abundance was higher in AQP2-positive KO cells (Fig 5E-G), while AQP2-negative KO cells exhibit mostly increase in cytosolic BKα (Fig 5E,F,H).

Taken together, these findings indicate that ARH regulates BKα abundance and membrane localization.

Males and females compensate differently for ARH deletion

Given the ability of ARH-KO mice to conserve K⁺ despite increased apical ROMK and BKα, we searched for potential compensatory mechanisms. Western blot analysis revealed NCC expression and phosphorylation were similar between WT and ARH-KO males under basal conditions (Fig 6A-C). After prolonged K⁺ deficiency, total and phosphorylated NCC are dramatically increased to a similar extent in ARH-KO and WT males (Fig 6A-C). KO females exhibit normal NCC regulation under basal conditions, but increase NCC expression and phosphorylation to a higher extent than WT females when challenged with prolonged K⁺ deficiency (Fig 6G-I).

Figure 6: Males and females differently regulate NCC and ENaC to compensate for ARH deletion.

A) WB analysis of total (T NCC) and phosphorelated NCC (pNCC) expression in WT and ARH-KO males under control diet (CtrD) and prolonged K⁺-free diet (KFD).

B,C) quantification of WB in A.

D) WB analysis of full length (FL) and cleaved (Cl) aENaC expression in WT and ARH-KO males under CtrD and KFD. The red star indicates the non-specific band detected by αENaC antibody as described (66).

E,F) quantification of WB in D.

G) WB analysis of total T NCC and pNCC expression in WT and ARH-KO females under under CtrD and KFD.

H,I) quantification of WB in G.

J) WB analysis of full length and cleaved aENaC expression in WT and ARH-KO females under under CtrD and KFD.

K,L) quantification of WB in G.

Significance was tested using two way ANOVA, *: p value < 0.05, **: p value <0.01, ***: p value <0.001, ns=non significant. Every lane represents different mouse.

We then analyzed αENaC and found ARH-KO males exhibit reduced αENaC cleavage compared to WT mice under basal conditions with no difference in αENaC full length (Fig 6D-F). After prolonged K⁺ deficiency, αENaC cleavage was reduced to a similar extent in both genotypes (Fig 6D-F). In contrast to males, αENaC expression and cleavage were conserved in ARH-KO females under a control diet, and they decreased similarly after prolonged K⁺ deficiency (Fig 6J-L). The more robust NCC phosphorylation in females and the reduced ENaC cleavage in males are different strategies aiming to facilitate K⁺ conservation.

Together, these analyses indicate that ARH interact the regulate the cellular trafficking of ROMK and BK. Increased expression of the channels in ARH null mice is paralleled by compensatory mechanisms that prevent potassium loss. Sex-dependent differences in compensatory responses and mechanisms were observed.

Discussion

Our previous work demonstrated that ARH binds the NPXF motifs of ROMK, targeting the channel for endocytosis. Here, we extend these observations by showing that a similar mechanism regulates BKα through its NPXY motifs. This finding is compelling because ROMK and BKα belong to two distinct potassium channel families with fundamentally different structures, gating, and regulatory mechanisms. ROMK is an inward rectifier K⁺ channel (Kir), whereas BKα is a Ca²⁺- and voltage-activated K⁺ channel (Slo). Beyond the conserved K⁺ selectivity filter sequence (TVGYG) present in nearly all K⁺ channels, these two families share little sequence similarity (5, 48, 49). It is therefore more plausible that the NPXY and NPXF motifs, in BK and ROMK respectively, were independently selected during evolution to enable convergent regulation by common trafficking mechanisms. Consistent with this idea, both ROMK and BK channels are modulated by common mechanism including dietary K⁺ intake (1, 3, 50, 51) and its accompanying anion (4, 52). At the molecular level, the surface abundance of both channels is regulated by endocytosis, a process stimulated by WNK4, which reduces channel activity (13, 53-56). SGK1, by phosphorylating and inhibiting WNK4, opposes this effect, thereby enhancing channel activity under conditions of high K⁺ intake (13, 55, 57, 58). ARH has been shown to facilitate WNK1-mediated ROMK endocytosis (8), but whether ARH also participates in BK endocytosis in WNK-dependent manner remains to be determined.

The coregulation of ROMK and BK by shared molecular pathways likely provides the kidney with a coordinated and partially redundant system for K⁺ secretion. This ensures that excretion is adjusted in parallel across different nephron cell type (principal and intercalated cells), tightly matching systemic K⁺ and acid-base status to enhances homeostatic precision and minimizes the risk of dyskalemia.

Our study provides insight into the lack of reported hypokalemia in individuals with Type 4 familial Hypercholesterolemia (FH4), who are ARH deficient. We found ARH-KO mice exhibit a normal range of plasma K⁺ as WT mice because several elements counterbalance the increased apical membrane ROMK and BK densities in the distal nephron. Potassium secretion is influenced by a multitude of factors besides ROMK activity (59), especially distal delivery of sodium (60) and the activity of the Epithelial Sodium Channel, ENaC (1). Sodium delivery and ENaC activity must be adequate to provide a favorable electrochemical gradient for electrogenic sodium-potassium exchange, mediated by ENaC and ROMK. In recent years, a potassium-dependent pathway that regulates sodium-delivery has been described. Coined the potassium-switch, the pathway, comprised of basolateral potassium channels, Kir4.1/5.1 (61-63), WNK-SPAK kinases (64) and K⁺-regulated phosphatases (65), adjusts the activity of the thiazide sensitive sodium-chloride cotransporter in the distal convoluted tubule. It allows small physiological changes in blood potassium (65–68) to be transmitted to phosphorylation of NCC. In settings of K⁺ deficiency, the pathway activates NCC to limit Na⁺ delivery, and safeguard against urinary K⁺ loss. Our observations revealed involvement of switch-activation in limiting expected K⁺ loss in ARH KO mice, especially in the setting of dietary K⁺ deficiency. Indeed, when the contribution of NCC activation is unmasked with thiazide, ARH KO mice exhibit increased kaliuresis compared to the WT counterparts.

Sex-specific compensatory mechanisms were observed, adding to the growing appreciation of sex-specific K⁺ transport mechanisms in the kidney (69-73). Both Male and Female mice activate NCC, but to different extents. Female ARH KO mice showed greater increases in NCC protein and phosphorylation than their WT counterparts or male KO. NCC does not contain the endocytic motif required for ARH binding, making it unlikely that ARH directly controls NCC endocytosis, which is Clathrin-dependent (74). Given NCC upregulation was detected only in female mice, which are more susceptible to acute hypokalemia than males (30), a more plausible explanation is that an acute drop in plasma K⁺ triggers NCC phosphorylation in ARH-KO females, which in turn quickly corrects potassium levels by reducing distal Na⁺ delivery and limiting urinary K⁺ excretion. Because NCC phosphorylation stabilizes NCC by inhibiting its ubiquitination (74), NCC protein levels increase. This signal amplification positive feedback provides a high gain in the system, which limits the development of hypokalemia despite increased ROMK and potassium deprivation.

Male, but not ARH KO female mice, use an ENaC-driven strategy to limit urinary K⁺ loss in the face of increased ROMK channel density in the distal nephron. Specific proteases increase ENaC open-probability by removing inhibitory peptides in the channel’s extracellular domain (75). Proteolytic processing of ENaC was uniquely reduced in male ARH KO mice consuming the control diet but was similarly decreased with dietary K⁺ depletion in male and female WT and ARH KO mice. The decrease in ENaC activity is expected to reduce the electrical driving force for K⁺ secretion, limiting urinary potassium excretion; as a result, KO males can maintain appropriate K⁺ excretion without requiring changes in upstream Na⁺ delivery as females do. The proteolytic processing of the ARH-client, Megalin, depends on ARH and post-endocytic trafficking (76). However, ENaC is not expected to interact with ARH, and further studies will be required to elucidate the mechanism by which ARH affects ENaC proteolysis.

Under K⁺-replete conditions, only males exhibit an adaptive response (ENaC-mediated), while females initiate an enhanced K⁺ conservation response (NCC-mediated) only after exposure to K⁺-deficient conditions. These findings indicate that males exhibit an anticipated response to K⁺ loss, while females display a delayed activation of K⁺-conserving mechanisms, pointing to sex differences not only in the compensatory transporters involved but also in how male and female kidneys sense and respond to disruptions in K⁺ secretion. This may reflect a sex-specific regulatory threshold for activating K⁺-conserving pathways. Interestingly, in an independent study, specifically designed to explore the sex differences in K⁺ handling, with head-to-head males and females comparison, we also observed male kidneys develop more efficient strategies for K⁺ conservation than female kidneys (Abstract: Jung HJ, Physiology 2024, 39-S1–1825). Additionally, our previous work in WT mice demonstrated sex-dependent remodeling of the distal convoluted tubule (DCT), with females showing higher NCC cellular density and greater structural plasticity; as female DCT elongates more the male DCT in response to loop diuretics (PMID: 32924546). This female-specific adaptation may underlie a preferential reliance on NCC as a primary defense mechanism against dysregulated K⁺ handling.

Moreover, humoral-mediated adaptive responses are likely to contribute to maintaining K⁺ balance in ARH-KO mice, potentially in a sex-specific manner. Both systemic and intrarenal RAAS pathways play a pivotal role in potassium homeostasis, as evidenced by in vivo studies showing that kidney-specific deletion of components within either the aldosterone-mediated signaling pathway (31, 32, 77) or the intrarenal RAAS (78) leads to K⁺ imbalance. Furthermore, in vivo and ex vivo studies in rats have revealed sex differences in aldosterone responses to extracellular K⁺ and AngII stimulation (79), which may be linked to the higher expression of aldosterone biosynthetic enzymes observed in female adrenals (80).

Studies from human and animal models have associated ROMK function with blood pressure. Indeed, genetic studies have shown heterozygous loss-of-function mutations in ROMK (81) are associated with lower blood pressure and protection from the development of hypertension (82). Similarly, heterozygous deletion of ROMK in rats results in reduced blood pressure (83). Pharmacological inhibition of ROMK in Dahl salt-sensitive rats can prevent and reverse hypertension (84). The effect of ROMK on blood pressure has been attributed to its role in K⁺ recycling in the TAL, which facilitates NKCC2-mediated salt reabsorption (82, 83). Here we show increased ROMK function results in enhanced NCC response to hypokalemia in females. Given the established role of NCC in blood pressure, we speculate that factors that increase ROMK function in the distal nephron, such as gain-of-function mutations (85), may elevate blood pressure.

Our analysis show no significant differences in food intake across groups. Although the control and K⁺-deficient diets contain similar Na⁺ levels (Table 1), K⁺-deficient mice exhibit reduced urinary Na⁺ excretion, indicating Na⁺ retention, consistent with findings in both mice and humans. In mice, 10 days of low K⁺ intake leads to approximately 33% Na⁺ retention (86). In humans, reducing dietary K⁺ from 96 mmol/day to 16 mmol/day for 10 days results in the retention of ~270 mmol of Na⁺ (87), which is further exacerbated when K+ intake is reduced to 10 mmol/day (88). Notably, this occurs with little to no change in body weight (87, 88), likely due to non-osmotic Na⁺ storage in skin and skeletal muscle (89). Similarly, both WT and KO mice on a K⁺-deficient diet excrete less Cl⁻ compared to K⁺-replete controls, which is more likely due to the combined effect of lower Cl⁻ content in the K⁺-deficient diet (Table 1) and Cl⁻ retention induced by K⁺ deficiency (88). The observed Na⁺ and Cl⁻ retention is likely mediated by two mechanisms, a) Increased NCC abundance and phosphorylation, as demonstrated in the current study and supported by previous reports (31, 62, 67); and b) Low Cl⁻ intake as Cl⁻ deficiency activates the Cl/HCO3 exchanger (pendrin) (90, 91), which elicits a neutral NaCl reabsorption mechanism (92).

We found that downregulation of ROMK in prolonged K⁺ deficiency is not completely inhibited by ARH deletion, consistent with the operation of multitude of pathways that control ROMK abundance and apical surface density (93). For example, ROMK internalization can occur via Clathrin-independent Dynamin-dependent endocytosis (94). Targeting ROMK to Dynamin-dependent endocytosis has been reported to be directed by monoubiquitination (95, 96). The E3 ubiquitin ligase that ubiquitinates ROMK was identified as POSH (Plenty Of SH3) (96). The physiological significance of POSH-mediated ROMK regulation and the signaling pathway linking POSH to extracellular K⁺ remain to be identified. Additionally, it has been shown that ROMK membrane abundance is positively regulated by the anti-aging protein Klotho (97). Our recent study has shown that Klotho is co-expressed with ROMK in the distal nephron in humans and mice, where it is regulated by K⁺ intake (98). Given the life-threatening conditions associated with altered K⁺ balance and the key role that ROMK plays in this underlying mechanism, regulating ROMK apical abundance by multiple independent mechanisms is not surprising.

In summary, ARH is involved in regulating ROMK and BK apical abundance in the distal nephron. ARH KO mice are able to compensate increased ROMK and BK abundance by activating other K⁺ conservation mechanisms, which are different between sexes. In females, more prone to hypokalemia, the compensation for blunted ROMK and BK internalization involves increasing NCC, a significant player in salt-sensitive hypertension.

Limitations

Our study did not assess the potential compensatory role of K⁺ reabsorption in maintaining K⁺ balance in ARH-KO mice. Notably, the gastric H⁺/ K⁺-ATPase (HKA2) contributes to K⁺ reabsorption, as evidenced by increased transcript and protein abundance in the renal medulla of K⁺-deprived rats compared to K⁺-replete controls (99, 100). While renal K⁺ conservation was unaffected in HKA2-KO mice under K⁺-deprived conditions (101), more detailed investigations have shown that HKA2-mediated K⁺ reabsorption plays an important role in maintaining K⁺ balance under physiological conditions, including circadian fluctuations (102) and pregnancy (103). Thus, it is plausible that compensatory upregulation of HKA2 contributes to the stable K⁺ balance observed in ARH-KO mice.

Supplementary Material

https://doi.org/10.6084/m9.figshare.30311401

New and Noteworthy.

ROMK and BK, both regulated by the clathrin adaptor ARH, play essential roles in maintaining potassium balance. Given the life-threatening risks of dyskalemia, it is unsurprising that their activity is controlled by multiple mechanisms, though not without physiological costs. We found that impaired ARH-mediated ROMK and BK internalization triggers activation of alternative potassium-conserving pathways in a sex-specific manner. In females, who are more prone to hypokalemia, this compensation involves NCC upregulation, a key player in blood pressure regulation.