Renomedullary Interstitial Cell Endothelin A Receptors Regulate BP and Renal Function

Significance Statement

The functional significance of renomedullary interstitial cells, which are uniquely and abundantly expressed in the renal inner medulla, is largely unknown. In vitro studies have demonstrated that endothelin A receptors regulate multiple aspects of renomedullary interstitial cell function. Using a novel mouse model with inducible renomedullary interstitial cell–specific endothelin A receptor gene targeting, the authors found that compared with control mice, mice lacking endothelin A receptors in renomedullary interstitial cells exhibited reduced BP, enhanced natriuresis and diuresis, increased endogenous natriuretic and diuretic factor production, and reduced medullary transporter expression. These studies identify a role for renomedullary interstitial cells in vivo in regulating renal function under physiologic conditions.

Abstract

Background

The physiologic role of renomedullary interstitial cells, which are uniquely and abundantly found in the renal inner medulla, is largely unknown. Endothelin A receptors regulate multiple aspects of renomedullary interstitial cell function in vitro.

Methods

To assess the effect of targeting renomedullary interstitial cell endothelin A receptors in vivo, we generated a mouse knockout model with inducible disruption of renomedullary interstitial cell endothelin A receptors at 3 months of age.

Results

BP and renal function were similar between endothelin A receptor knockout and control mice during normal and reduced sodium or water intake. In contrast, on a high-salt diet, compared with control mice, the knockout mice had reduced BP; increased urinary sodium, potassium, water, and endothelin-1 excretion; increased urinary nitrite/nitrate excretion associated with increased noncollecting duct nitric oxide synthase-1 expression; increased PGE₂ excretion associated with increased collecting duct cyclooxygenase-1 expression; and reduced inner medullary epithelial sodium channel expression. Water-loaded endothelin A receptor knockout mice, compared with control mice, had markedly enhanced urine volume and reduced urine osmolality associated with increased urinary endothelin-1 and PGE₂ excretion, increased cyclooxygenase-2 protein expression, and decreased inner medullary aquaporin-2 protein content. No evidence of endothelin-1–induced renomedullary interstitial cell contraction was observed.

Conclusions

Disruption of renomedullary interstitial cell endothelin A receptors reduces BP and increases salt and water excretion associated with enhanced production of intrinsic renal natriuretic and diuretic factors. These studies indicate that renomedullary interstitial cells can modulate BP and renal function under physiologic conditions.

The presence of renomedullary interstitial cells (RMICs) in the renal inner medulla has long been recognized, but the physiologic relevance of these cells remains largely unknown. RMICs form a ladder-like arrangement with their cell bodies oriented perpendicular to the long axis of the inner medulla.¹ Scanning electron microscopy,² confocal imaging,³ and multiphoton microscopy⁴ indicate that RMICs primarily contact, or at least are in very close proximity to, thin limbs and vasa recta; a close interaction with collecting ducts has not been as clearly demonstrated. This anatomic configuration suggests that RMICs might serve a number of functions. The ladder-like arrangement and high density of RMICs in the inner medulla may provide structural support, particularly in the papilla, and could limit interstitial compartment solute and water diffusion. Cultured RMICs can contract^5,6; although this has never been demonstrated in vivo, the possibility exists that RMIC contraction could affect medullary blood flow (MBF) or even tubule fluid flow. In this regard, measurements of renal interstitial hydraulic pressure after direct renomedullary interstitial volume expansion suggest that the medullary interstitium may contract.⁷ RMICs produce hyaluronan, a key component of inner medulla extracellular matrix, which may regulate fluid and/or solute excretion.⁸ RMICs have been suggested to produce a variety of natriuretic and/or vasodepressor factors, including (albeit never proven) medullipins or related compounds,^9,10 nitric oxide (NO; according to cell culture),^11,12 and (most clearly demonstrated) cyclooxygenase-2 (COX-2)–derived PGE₂ (PGE₂).^13–15 With regard to this latter point, it is notable that increasing BP was associated with a substantial reduction in RMIC granule volume (the granules are highly lipophilic and are thought to contain, among other factors, PGs¹²). Further, RMIC-specific knockout (KO) of COX-2 altered BP; however, interpretation of the physiologic relevance is complicated because these mice developed papillary necrosis.¹⁵

Consideration of how RMIC function might be modulated could provide clues as to their physiologic role. Virtually all of the studies examining this issue have used cultured RMICs and have demonstrated binding and/or elicited cell signaling by arginine vasopressin (AVP),^6,16 atrial natriuretic peptide,¹⁷ TNF,¹¹ IFN-γ,¹¹ hydrostatic pressure,¹⁴ bradykinin,¹⁸ angiotensin II,^18,19 and endothelin-1 (ET-1).^5,19–23 Of these factors, ET-1 regulation of RMIC biology has been the most extensively studied. ET-1 modulates a variety of RMIC functions, including cell contraction,⁵ PGE₂^13,23 and NO²¹ production, intracellular calcium concentration,²² cell proliferation and extracellular matrix accumulation,²¹ and hyaluronan production.¹⁹ In almost every study where receptor subtype was examined, endothelin A receptors (ETAs) mediated the effects of ET-1 on RMICs.^20–22 On the basis of these considerations, we hypothesized that alteration of RMIC ETAs in vivo would uncover a role for ET-1 in modulation of RMICs in particular, and a role for RMICs in the regulation of renal function in general. To accomplish this, we herein report the use of a recently developed system that allows RMIC-specific targeting of the EDNRA gene in the kidney in vivo, and describe the physiology of this novel mouse model.

Methods

Animal Care

All animal studies were conducted with the approval of the University of Utah and University of Southern California Animal Care and Use Committees in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Generation of Inducible RMIC ETA KO mice

RMIC ETA KO mice were generated as described in Figure 1A. Floxed ETA mice have loxP sites flanking exons 6–8 of the EDNRA gene and were originally obtained from Masashi Yanagisawa (University of Texas Southwestern).²⁴ Floxed ETA mice were bred with mice containing CreER2/IRES/GFP/Frt-NeoR-Frt knocked into exon 2 of the TNC gene (provided by C.-M.H. at Fudan University); the knockin inactivates the TNC gene and confers tamoxifen-inducible Cre expression under control of the tenascin-C promoter.²⁵ Most mice used in the studies were homozygous for the loxP-flanked EDNRA gene and homozygous for the tenascin-C-CreER2 (ETA^f/f;T/T, referred to as ETA KO or control); some mice were hemizygous for tenascin-C-CreER2 and homozygous for the loxP-flanked EDNRA gene (ETA^f/f;T/W) and are specifically indicated as such. Mice were injected with tamoxifen (160 mg/kg administered intraperitoneally) for five consecutive days at 3 months of age. Littermates of the same genotype and sex, but without tamoxifen treatment, were used as controls. All mice (1:1 male-to-female ratio) were studied at 4–7 months of age.

Figure 1.

EDNRA gene using tenascin-C-CreER2 markedly knocks down inner medullary ETA.  (A) Schematic of the targeting strategy. (B) Top: representative organ panel (n=3) demonstrating wild-type and recombinant bands; bottom: representative blot of kidney (cortex and inner medulla) recombination in control and ETA KO mice (n=3). (C) Relative expression of ETA (left) and ETB (right) mRNA in renal inner medulla from control and ETA KO mice. n=7–8 each group. Circles indicate males and squares indicate females in dot plots. *P<0.05 KO versus control on same Na⁺ diet. IM, inner medulla; IRES, internal ribosomal entry site; Neo, neomycin resistance gene.

Genotyping and Determination of EDNRA Gene Recombination and mRNA Expression

Genotyping PCR was performed using tail DNA. The ETA forward 5′-CCC​ATG​CTT​AGA​CAC​AAC​CAT​G-3′ and reverse 5′-GAT​GAC​AAC​CAA​GCA​GAA​GAC​AG-3′ primers yielded a 364-bp product from the floxed EDNRA gene and a 324-bp product from the wild-type allele; tenascin-C-CreER2 forward 5′-GGG​GGC​AAG​AAG​GCA​AAA​AT-3′, reverse1 5′-GTT​CTG​CGG​GAA​ACC​ATT​T-3′ and reverse2 5′-TCT​CGC​TTG​TGC​CTG​ATG​AT-3′ primers yielded a 430 bp product for tenascin-C-CreER2 and a 300-bp product for the wild-type TNC allele. To determine EDNRA gene recombination, DNA was isolated from a variety of organs, renal cortex, and renal inner medulla from ETA KO mice; PCR was performed using forward 5′-CCC​ATG​CTT​AGA​CAC​AAC​CAT​G-3′ and reverse 5′-CGC​TGT​TGT​ATA​TCC​AGT​ATC​AGG-3′ primers, which yielded a nonrecombined band of 1287 bp and a recombined band of 610 bp.

RNA was isolated from inner medulla from ETA KO and control mice. Reverse transcription was performed on 0.3 µg of total RNA with oligo(dt) and Superscript III reverse transcription according to the manufacturer’s protocol (Invitrogen, Grand Island, NY). The resulting complementary DNA was assayed for ETA and endothelin B receptor (ETB) mRNA expression using TaqMan Gene Expression Assay probes (ETA probe Mm01243722_m1, ETB probe Mm00432989_m1, and GAPDH probe Mm99999915_g1; Applied Biosystems, Carlsbad, CA). Endothelin receptor mRNA was normalized to GAPDH.

Histology and ETA Immunostaining

ETA KO mice were euthanized at 5–6 months of age. Kidneys were fixed in 10% buffered formalin phosphate overnight, embedded in paraffin, and 5-μm sections were obtained. Hematoxylin and eosin staining was performed. Deparaffinized and rehydrated tissue sections were treated with sodium citrate (pH 6.0), washed and blocked with 1% BSA in PBS for 1 hour, followed by overnight incubation with a primary rabbit antibody against ETA (see Supplemental Table 1). Tissues were washed and incubated with secondary antibody (A21428, Alexa Fluro 555 goat anti-rabbit IgG) for 1 hour at room temperature. Fluorescence images were captured using an Olympus microscope.

BP Determination

Mice were anesthetized with 2% isoflurane, implanted with radio transmitters with the catheter in the carotid artery, and allowed 7 days recovery. Mice were fed a normal Na⁺ diet (2920×; Teklad Global Diets, Madison, WI) for 7 days, followed by a low Na⁺ diet (0.03% Na⁺; TestDiet, St. Louis, MO) for 7 days, and then a high Na⁺ diet (#5001, 8% added NaCl; TestDiet) for 7 days. BP was recorded by telemetry (TA11-PAC10; Data Sciences International, St. Paul, MN). Mice were not disturbed during the BP recording period. BP readings were taken every 10 minutes throughout the day and the average of the entire day’s values was used for each data point. BPs over consecutive 4-hour periods throughout the day were analyzed to detect circadian BP rhythm differences between genotypes.

GFR Measurement

GFR was measured at baseline and after 2 days of high-salt intake. Mice were injected retro-orbitally with FITC-sinistrin (7.5 mg/100 g body wt; Mannheim Pharma and Diagnostics, Mannheim, Germany). The NIC-Kidney device (Mannheim) was used to detect fluorescence in the skin on the shaved back over 1 hour. GFR was calculated on the basis of the kinetics of fluorescence decay.

Metabolic Cage Studies

Chronic Dietary Salt Experiments

Mice were fed a normal Na⁺ diet for 5 days (0.3% Na⁺, Micro-Stabilized Rodent Liquid Diet LD 101; TestDiet), followed by a high Na⁺ diet for 7 days (3.2% Na⁺; TestDiet). On day 2 of normal and high Na⁺ diets, blood was collected for determination of plasma renin concentration (PRC) and plasma aldosterone. PRC was measured using an angiotensin I enzyme immunoassay (EIA) kit (S-1188; Peninsula, San Carlos, CA). Plasma aldosterone was measured by EIA (ADI-900–173; Enzo Life Sciences, Farmingdale, NY). Daily urine samples were collected using metabolic cages, centrifuged at 15,000 rpm for 15 minutes, and supernatants were stored at −80°C. Urinary Na⁺ and K⁺ were measured using the EasyVet analyzer (Medica, Bedford, MA). Urinary nitrite and nitrate (NOx), ET-1, PGE₂, and AVP were determined during normal Na⁺ intake and after 2 days of high Na⁺ intake. Urine NOx was analyzed using the nitrate/nitrite colorimetric assay kit (780001; Cayman Chemical, Ann Arbor, MI). Urine ET-1 was assayed in acidified samples applied to activated SPE (C-18) cartridges (WAT023590; Waters, Milford, MA), eluted with methanol/0.05 M ammonium bicarbonate (80/20 v/v), and measured using the Quantikine EIA kit (DET100; R&D Systems, Minneapolis, MN). Urine PGE₂ was determined by EIA (514010; Cayman Chemical). Urine AVP was determined by EIA (ADI-900–017A; Enzo Life Sciences). Renal inner medullary hyaluronan was measured at baseline and after 2 days of high Na⁺ intake, as previously described,¹⁹ using EIA (either K-1200; Echelon Biosciences, Salt Lake City, UT or DHYAL0; R&D Systems) and was related to protein content as measured using a Lowry-based method.

Water Deprivation and Loading Experiments

Mice were deprived of water for 24 hours followed by determination of urine volume and osmolality. For water loading studies, mice were given 3 days of a normal Na⁺ diet with 5% sucrose in drinking water. Urine volume and osmolality were determined daily. Plasma osmolality was determined after 2 days of water loading. Urinary NOx, ET-1, PGE₂, and AVP were determined after 2 days of high-water intake. Urine and plasma osmolality were measured using an Osmett II (Precision System, Natick, MA). Renal inner medullary hyaluronan was measured after 2 days of water loading.

Western Blot Analysis

Inner medullas were homogenized, protein was isolated, and immunoblotting was performed. Samples were homogenized in ice-cold buffer containing 250 mM sucrose, 10 mM triethanolamine, pH 7.6 with 100 μg/ml PMSF, 200 mM sodium orthovanadate, 200 mM sodium fluoride, and 1 mg/ml leupeptin. Total protein concentration was measured using the bicinchoninic acid protein assay kit (Pierce, Waltham, MA). Samples were diluted with Laemmli buffer, heated at 65°C for 15 minutes, and stored at −80°C in aliquots to avoid repeated freeze/thaw cycles. Proteins were separated using a 4%–12% bis-tris minigel (Invitrogen) and transferred onto a PVDF membrane. Membranes were blocked with 5% nonfat dry milk in tris-buffered saline with Tween 20 for 1 hour at room temperature. Membranes were incubated with specific primary antibodies overnight at 4°C. After washing with tris-buffered saline with Tween 20, membranes were incubated with horseradish peroxidase–conjugated secondary antibodies for 1 hour at room temperature.

Primary antibodies used were (see Supplemental Table 1 for predicted product size, species source, dilution, company source, and validation reference) against aquaporin-2 (AQP2); cyclooxygenase-1 (COX-1) and COX-2; epithelial Na⁺ channel (ENaC) α, β, and γ; nitric oxide synthase (NOS)-1 and -3; and GAPDH. Secondary horseradish peroxidase–conjugated antibodies were goat anti-rabbit IgG (1:2000; Abcam) and mouse anti-rabbit IgG (1:2000, sc-2357; Santa Cruz Biotechnology). Horseradish peroxidase was visualized using the Advance ECL System and Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, Piscataway, NJ). Images were obtained and quantified by ImageLab (Bio-Rad, Hercules, CA). All antibodies were initially tested for linearity by loading 5, 10, 20, and 35 µg of protein; linear results were obtained for all antibodies between 5 and 35 µg, so 20 µg of protein was loaded into each lane for all experiments. The one exception was the AQP2 antibody, which showed linearity between 2 and 10 µg of protein, so 3 µg was loaded into each lane. Normalizing to GAPDH was performed.

In some experiments, inner medullary collecting ducts (IMCDs) were isolated for western blotting from normal Na⁺ and high Na⁺ diet ETA KO and control mice, as previously described.²⁶ Renal inner medullas were minced and incubated at 37°C for about 45 minutes under continuous agitation in 0.1% collagenase (type 1; Sigma) containing 0.01% DNase (type 1) in HBSS supplemented with 15 mM HEPES (pH 7.4). When a suspension containing predominantly single cells and individual tubules was obtained, 5% FBS was added to stop digestion and the digests were filtered through a 100-μm cell strainer to remove residual tissue. The tubule suspension was centrifuged at 1500 rpm for 5 minutes, and the cell pellet was resuspended in 10% BSA in HBSS, followed by an additional two centrifuge/washes with HBSS.

Freshly Isolated and In Vitro Superfused Renal Inner Medullary Tissue

Sox2Cre-GCaMP5/tdTomato transgenic mice (6–8 weeks, male, C57BL6 background), which specifically express the intensely green and calcium-sensitive fluorescent protein GCaMP5, and the calcium-insensitive tdTomato ubiquitously in all cell types including in RMICs, were generated by crossing mice constitutively expressing Cre recombinase under the control of the Sox2 promoter²⁷ and floxed GCaMP5G-IRES-tdTomato reporter mice²⁸ (both from the Jackson Laboratory, Bar Harbor, ME). Animals were anesthetized with a combination of ketamine (100 mg/kg) and xylazine (10 mg/kg), and the kidneys were perfused with cold PBS and freshly harvested. Approximately 300-µm-thick tubulovascular rays of the renal inner medulla (papilla) were hand dissected. The dissection and superfusion medium were prepared from sterile PBS with the addition of 300 mOsm/kg mannitol to create a hyperosmotic medium mimicking the interstitial environment of the renal medulla. The preparation was transferred to a tissue chamber and mounted with the help of a micromanipulator system (Vestavia Scientific, Vestavia, AL), as described before.^4,29 In some experiments, 10–300 nM ET-1 (Sigma) was added to the tissue superfusate.

Calcium Imaging of RMICs using Confocal Fluorescence Microscopy

Time-lapse optical sectioning of the freshly microdissected and in vitro superfused renomedullary tissue and ratiometric calcium imaging of RMICs were performed using a Leica TCS SP5 multiphoton confocal fluorescence imaging system (Leica Microsystems, Heidelberg, Germany) with a ×63 Leica glycerin-immersion objective (NA 1.3) powered by a Chameleon Ultra-II MP laser at 860–920 nm excitation (Coherent) and a DMI 6000 inverted microscope’s external nondescanned detectors with FITC (green channel, for detecting GCaMP5 signal) and TRITC (red channel, for detecting tdTomato signal) filters, as described before.^29,30 Ratiometric (GCaMP5/tdTomato) images were collected in time series (xyt, 2 seconds per frame) with the Leica LAS imaging software and using the same instrument settings (laser power, offset, gain of both detector channels). Fluorescence intensity (12 bit) was measured with the Leica LAS X imaging software’s quantification tools.

Statistical Analyses

Specific experiment sample sizes are indicated in the figure legends. The t test was used to compare differences in continuous parameters on the same day between genotypes (plasma osmolality). Differences in mRNA and protein expression, GFR, urine volume and osmolality (during dehydration), plasma aldosterone, PRC, urine AVP, urine ET-1, urine NOx, urine PGE₂, and mRNA expression on a normal and high Na⁺ diet between control and KO mice were analyzed by two-way ANOVA using genotype and treatment as model terms. Differences in systemic hemodynamics, body weight, food and water intake, and urine excretion of water, Na⁺ and K⁺ between control and KO mice were analyzed by two-way repeated measures ANOVA with Scheffe post hoc test using genotype and treatment as model terms. In the two-way ANOVA and two-way repeated measures ANOVA, genotype and sex as well as treatment and sex were also used as model terms. An interaction term was included in these models. Please note that the study was not adequately powered to detect sex differences; however, the individual data points by sex are reported where possible together with comments on any obvious trends or lack thereof. Data are shown as mean±SEM. P<0.05 was taken as significant.

Results

Specificity and Degree of EDNRA Gene Targeting in RMICs

Studies were initially conducted to determine if mice heterozygous and/or homozygous for the tenascin-C-CreER2 knockin (and homozygous for the floxed EDNRA gene) before tamoxifen treatment exhibited a distinct phenotype; the issue was not about leakiness of Cre expression, but rather related to partial or complete absence of tenascin-C per se (the knockin inactivates the TNC gene²⁵). In agreement with previous findings, mice heterozygous for tenascin-C-CreER2,²⁵ as well as mice homozygous for tenascin-C-CreER2, were born at the expected frequency, developed normally, and had no evident gross anatomic or renal histologic abnormalities (as assessed by hematoxylin and eosin staining) up to 8 months of age (the histology was completely normal so the images are not shown). These findings are also in agreement with previous studies observing no apparent effect of homozygous global tenascin-C KO on mouse birth rate, growth, or function,³¹ and with the finding that tenascin-C is expressed in extremely low amounts in normal adults.^25,32 In contrast, tenascin-C is induced by, and may play a role in, injury and/or inflammation (e.g., global tenascin-C KO impairs recovery from snake venom–induced GN³³). Thus, within the context of our model that utilizes adult mice not exposed to pathologic conditions, we felt that use of either homozygous or heterozygous tenascin-C-CreER2 knockin mice would not confound interpretation of the phenotype associated with targeting the EDNRA gene. Further, we chose to use homozygous tenascin-C-CreER2 knockin mice because initial studies showed a greater degree of renal inner medullary ETA mRNA knockdown as compared with heterozygotes (data not shown).

ETA KO mice showed widespread EDNRA gene recombination; within the kidney, recombination was evident in the inner medulla and not the cortex (Figure 1B), consistent with previous findings using tenascin-C-CreER2.²⁵ The magnitude and endothelin system specificity of EDNRA gene targeting in the inner medulla was assessed using real-time PCR for ETA and ETB receptors (Figure 1C). ETA KO mice had a marked decrease in inner medullary ETA mRNA on either a normal or high-salt diet, whereas inner medullary mRNA content for the ETB receptor was similar between ETA KO and control mice. Immunostaining for ETA was not successful in labeling any cells in the inner medulla, hence it is not possible to state with absolute certainty that only RMIC ETA was downregulated. However, given that tenascin-Cre-ER2 mice have consistently been shown to specifically target RMICs and not other cell types within the inner medulla,^15,25,34 it seems likely that the ETA knockdown within the kidney occurred selectively in RMICs.

Effect of RMIC ETA KO on BP and Fluid and Electrolyte Homeostasis

Systemic Hemodynamics and Renal Function

Systolic and diastolic BP and heart rate were similar between ETA KO and control mice on normal and low Na⁺ diets (Figure 2, A–C). In contrast, systolic and diastolic BP were lower in ETA KO compared with control mice on a high Na⁺ diet; heart rate was also lower after 2 days of high Na⁺ intake (Figure 2, A–C). Similar systemic hemodynamics were observed in tamoxifen-treated ETA^f/f;T/W mice (Supplemental Figure 1). GFR was similar between ETA KO and control mice on normal and high Na⁺ diets (Figure 2D). No sex differences were observed in any of the above parameters.

Figure 2.

RMIC ETA KO reduces blood pressure during high Na⁺ intake. Change the now second sentence to: Control and ETA KO mice were fed a normal Na⁺ diet for 5 days, a low Na⁺ diet for 7 days, and a high Na⁺ diet for 7 days. n=13–17 each data point for (A) systolic BP, (B) diastolic BP, and (C) heart rate. (D) GFR during normal and after 2 days of high Na⁺ intake in control and ETA KO mice (circles indicate males and squares indicate females); n=5–6 each group. *P<0.05 KO versus control on the same day.

Because differences in BP between ETA KO and control mice were most evident on a high Na⁺ diet, subsequent studies focused on normal (for comparison) and high Na⁺ intakes. Body weight and food intake were similar between ETA KO and control mice (Figure 3, A and B). In particular, body weight tended to be higher in ETA KO mice, but was not significantly different than controls (25.9±1.2 g control and 28.5±1.4 g ETA KO on day 2 of normal Na⁺ intake, P=0.16; 26.0±1.3 g in control and 28.7±1.4 g in ETA KO on day 2 of high Na⁺ intake, P=0.22). Water intake, urine volume, and urinary Na⁺ and K⁺ excretion were higher in ETA KO mice compared with controls on the first 4 days of high Na⁺ intake (Figure 3, C–F). No sex differences were observed in these metabolic cage studies. Comparable changes in water intake, urine volume, and urinary Na⁺ and K⁺ excretion were observed on the first 1–2 days of high Na⁺ intake in tamoxifen-treated ETA^f/f;T/W mice (Supplemental Figure 2). Given these findings in the ETA^f/f;T/W mice together with, as previously mentioned, the greater degree of inner medullary ETA mRNA knockdown in ETA KO mice, all subsequent studies were conducted using ETA KO mice.

Figure 3.

RMIC ETA KO increases urine volume, Na⁺ and K⁺ excretion during high Na⁺ intake. n=22 for each data point for (A) body weight, (B) food, and (C) water intake; (D) urine volume; (E) urine Na⁺ excretion (UNaV); and (F) urine K⁺ excretion (UKV). *P<0.05 KO versus control on the same day.

Endocrine and Local Factors

Plasma aldosterone (Figure 4A) and PRC (Figure 4B) were similar between ETA KO and control mice on a normal Na⁺ diet and were suppressed to a similar extent during high Na⁺ intake. Urinary AVP excretion was also similar between ETA KO and control mice on a normal Na⁺ diet and was increased to a similar degree during high Na⁺ intake (Figure 4C). In addition to these hormones, endogenous renal production of several key natriuretic factors was assessed (Figure 4, D–F). There were no differences in urinary ET-1, NOx, and PGE₂ excretion between ETA KO and control mice on a normal Na⁺ diet. High Na⁺ intake increased urinary ET-1 and NOx in both genotypes, but to a greater extent in ETA KO mice. Urinary PGE2 excretion was not different between ETA KO and control mice on a normal Na⁺ diet; it increased in ETA KO but not control mice during high Na⁺ intake. Finally, because ET-1 can modulate RMIC hyaluronan production in vitro,¹⁹ inner medullary hyaluronan content was assessed in ETA KO and control mice on normal and high Na⁺ diets. As shown in Figure 4G, no differences in hyaluronan content were observed between genotypes. No sex differences were observed in any of the above parameters.

Figure 4.

RMIC ETA KO modulates regulators of urinary salt and water excretion. (A) Plasma aldosterone and (B) PRC were measured at baseline and after 2 days of high Na⁺ intake. (C) Urine AVP, (D) urine ET-1, (E) urine NOx, (F) urine PGE₂, and (G) inner medullary hyaluronan content were measured at baseline and after 2 days of high Na⁺ intake or 2 days of high-water intake. n=7–12 each data point. Circles indicate males and squares indicate females in dot plots. *P<0.05 versus baseline for same genotype; ^#P<0.05 KO versus control on same diet.

Western Blot Analysis

No differences were detected in protein levels for inner medullary ENaC-α, -β, or -γ between ETA and control mice fed a normal Na⁺ diet (Figure 5, A and C). In contrast, ENaC-α and ENaC-β protein levels were lower in ETA KO mice compared with controls when fed a high Na⁺ diet (Figure 5, A and C). Inner medullary COX-1 and -2, as well as NOS-1 and -3, protein levels were similar between ETA KO and control mice fed a normal Na⁺ diet (Figure 5, A and C). On a high Na⁺ diet, inner medullary NOS1, but not other COX or NOS isoforms, was increased in ETA KO compared with control mice (Figure 5, A and C). We were unable to reliably detect or quantitate COX or NOS isoform protein expression by immunostaining; however, to help localize alterations in their expression during high Na⁺ intake, Western blot analysis was performed in purified IMCDs from mice fed high Na⁺ (Figure 5, B and D). No COX2 was detected in IMCDs, confirming the relative purity of the preparation.³⁵ Interestingly, NOS-1 and -3 levels were not elevated in IMCDs from ETA KO mice compared with controls; because NOS1 levels were elevated in total inner medulla, this suggests that the increase in NOS1 is in noncollecting duct cells. In addition, COX1 levels were increased in IMCDs from ETA KO compared with control mice; because total inner medulla COX1 levels were similar between genotypes, this suggests that COX1 was predominantly elevated in the collecting duct in ETA KO mice.

Figure 5.

RMIC ETA KO regulates inner medullary ENaC, NOS and COX protein expression. The figure shows western blot analysis of ENaC, NOS-1 and -3, and COX-1 and -2 in control and ETA KO mice fed normal and high Na⁺ diets. (A) Representative Western blots for ENaC isoforms (left) and COX and NOS isoforms (right) in inner medulla from ETA KO and control mice fed a normal and high Na⁺ diet. (C) Densitometry of these proteins normalized to normal salt (top) or high-salt (bottom) controls (n=7–8 each group). (B) Western blot and (D) densitometry for COX and NOS isoforms in IMCDs from control and ETA KO mice fed a high-salt diet (no COX2 was detected in IMCDs) (n=6 each group). Numbers by Western blots refer to molecular size in kDa. Circles indicate males and squares indicate females in dot plots. *P<0.05 KO versus control.

Effect of RMIC ETA KO on Water Handling

Water Metabolism

Control and ETA KO mice had a comparable decrease in urine volume and an increase in urine osmolality in response to water deprivation (Figure 6, A and B). In contrast, ETA KO mice had a much greater increase in water intake and urine volume, and a decrease in urine osmolality, in response to water loading compared with control mice (Figure 6, C–E). There was no difference in plasma osmolality between ETA KO and control mice after 2 days of water loading (Figure 6F). Water metabolism was similar between males and females. Note that mice were not pair fed (to absolutely rule out differences in water intake driving differences in urine volume); such pair feeding for water loading is problematic.

Figure 6.

RMIC ETA KO increases urine volume and decreases urine osmolality in response to water loading. (A) Urine volume and (B) urine osmolality are shown during water deprivation. n=7–9 each data point; *P<0.05 KO versus baseline same genotype. (C) Water intake, (D) urine volume, and (E) urine osmolality are shown at baseline and during 3 days of water loading. n=9 each data point; *P<0.05 KO versus control on the same day. (F) Plasma osmolality on day 2 of water loading, n=6–7 each data point. Circles indicate males and squares indicate females in dot plots.

Endocrine and Local Factors

Urinary AVP was suppressed to a comparable degree in ETA KO and control mice after 2 days of water loading (Figure 4C). Water loading increased urinary ET-1 and PGE₂ excretion in both genotypes but to a greater extent in ETA KO mice (Figure 4, D and F). In contrast, and unlike the response to salt loading, urine NOx excretion did not change in either ETA KO or control mice in response to water loading (Figure 4E). No difference in inner medullary hyaluronan content after water loading was observed between genotypes (Figure 4G).

Western Blot Analysis

Inner medullary AQP2 protein content was lower in ETA KO as compared with control mice after 2 days of water loading (Figure 7, A and B). No differences were detected in urinary NOx excretion between genotypes during water loading and, not unexpectedly, no differences in inner medullary NOS1 or NOS3 protein expression were observed (Figure 7, A and C). Urinary PGE₂ excretion was elevated during water loading and this was associated with an increase in inner medullary COX2, but not COX1 protein (Figure 7, A and C).

Figure 7.

RMIC ETA KO reduces inner medullary AQP2 and increases COX2 protein expression during high water intake. (A) Representative Western blots for AQP2, COX, and NOS isoforms in inner medulla from ETA KO and control mouse on day 2 of water loading. (B and C) Densitometry of AQP2 (B) and COX and NOS isoforms (C), n=6–8 each data point. Circles indicate males and squares indicate females in dot plots. *P<0.05 KO versus control.

Effect of ET-1 on RMIC Contraction and Calcium Responses

As described earlier, ET-1 contracted RMICs in vitro,⁵ suggesting that lack of ET-1–mediated RMIC contraction in ETA KO mice might be involved in the observed BP and salt and water homeostasis phenotypes. To evaluate this possibility, experiments were conducted to determine if ET-1 contracts RMICs in isolated inner medulla (it is not possible to assess this in vivo). RMICs did not produce calcium responses or obvious changes in cell shape in response to 10–300 nM ET-1 that would indicate cell contraction; in contrast, some collecting duct cells generated robust calcium responses (Figure 8). For an additional positive control, pipette-induced mechanical stimulation, a likely purinergic-mediated stimulus, was performed. As with ET-1 administration, robust calcium responses were observed in collecting duct cells, although RMICs showed no changes in calcium indicated by the lack of GCaMP5 signal (data not shown). These results suggest that ET-1 actions, at least on the basis of ex vivo studies, may not involve RMIC contraction.

Figure 8.

Exogenous ET-1 does not cause apparent RMIC contraction. Representative GCaMP5 fluorescence images of freshly dissected renomedullary tissue during ET-1 treatment are shown. Note a population of RMICs interconnecting various tubular and vascular segments (arrows). No changes are visible in GCaMP5 fluorescence intensity or cell shape in RMICs at the indicated time intervals (minutes:seconds) after ET-1 administration, whereas some collecting duct tubular cells generated robust calcium responses (GCaMP5 signal, arrowheads). Magnification, ×630.

Discussion

This study provides evidence supporting the notion that RMICs regulate BP and renal function under physiologic conditions. A previous study found that RMIC-specific COX2 KO in mice cause a hypertensive response to salt loading and then subsequent hypotension; however, these coincided with an increase in collecting duct and RMIC apoptosis, ultimately resulting in papillary necrosis.¹⁵ Our study suggests that because RMIC ETA KO mitigates salt retention and elevated BP in response to salt loading, RMIC ETAs normally exert an antinatriuretic and antidiuretic effect. Also, as ET-1 inhibits collecting duct Na⁺ and water reabsorption,³⁶ it may be that ET-1 acts on RMICs to counterbalance these effects. Inner medullary ET-1 largely derives from the collecting duct and is primarily secreted abluminally,³⁶ hence one possibility is that in states of particularly high IMCD ET-1 production (as occurs in salt or water loading³⁶), interstitial ET-1 rises to a level where RMIC ETAs are activated, which in turn activates mechanisms to dampen IMCD-derived ET-1–induced natriuresis and diuresis (see more detailed discussion below).

The lower BP, enhanced Na⁺ excretion, and reduced ENaC expression in response to salt loading in ETA KO, as compared with control, mice did not involve the studied circulating factors (AVP, aldosterone, and renin), but was associated with increased urinary excretion of several endogenous renal natriuretic factors (all of which can downregulate ENaC³⁷): ET-1, NO, and PGE₂. We did not specifically test whether each of these factors mediate the reduced BP, enhanced Na excretion, and/or ENaC downregulation (such experiments would be extremely problematic given that multiple potential factors were identified), nor do we know if other endogenous renal factors are involved. However, it remains important to consider how RMIC ETAs might modulate ET-1, NO, and PGE₂ (particularly in response to salt loading) as well as in which cell type(s) such modulation occurs. In addition, it is important to keep in mind that the salt-loaded mice are likely undergoing a saline diuresis (which can involve additional factors beyond simply eliminating sodium).

The collecting duct is the predominant renal source of inner medullary and urinary ET-1³⁶; RMIC ET-1 production has not been described. Because RMIC ETA KO increased ET-1 levels, it may be that RMIC ETA activation reduces collecting duct ET-1 production. How this is achieved is unclear; however, one possibility is that, if RMIC ETAs can modulate tubule fluid flow, then this could affect collecting duct ET-1 production (as well as K⁺ excretion and collecting duct production of NO and PGE2).³⁶ In addition, it has been reported that systemic ETA blockade markedly increases renal ET-1 production in response to big ET-1 infusion.³⁸ Finally, another possibility is that RMIC ETAs serve to clear inner medullary interstitial ET-1. Although possible, ETBs, and not ETAs, have been implicated in ET-1 clearance.³⁶

The increased urinary PGE₂ and NO excretion in salt-loaded ETA KO mice were unexpected because ET-1 stimulates RMIC PGE₂ and NO production in vitro.^13,21 We observed increased IMCD COX1 protein expression in salt-loaded ETA KO mice compared with controls, suggesting that the collecting duct is, at least in part, responsible for the increase in PGE₂. In contrast, inner medullary NOS1 protein levels were elevated in non-IMCD inner medulla; previous studies localizing NOS1 expression in mouse inner medulla suggest that the most likely source of the increased NOS1 in ETA KO mice is the vasculature.³⁹ As for ET-1, the mechanisms by which RMICs may regulate IMCD COX1 and vascular NOS1 are unknown. One possibility, as discussed earlier, is that RMICs might control MBF. If ET-1, via ETA, contracts RMICs, which then contract around vasa recta, then RMIC ETA KO could lead to increased MBF, which could promote natriuresis and diuresis and potentially increase PGE₂, NO, and even ET-1 production. Such changes in MBF in mice are very problematic to detect; however, we evaluated whether ET-1 had at least the potential to contract RMICs in ex vivo superfused inner medullary preparations. No effect of ET-1 on cell shape or intracellular calcium concentration was detected, suggesting that ET-1, via RMIC contraction, may not regulate MBF. However, given the limitations of the system, it is not possible to rule out the possibility that ET-1 can indeed contract RMICs and ultimately regulate MBF.

Water loading elicited a marked polyuria and hyposthenuria in ETA KO mice compared with control mice. There were no genotypic differences in urinary AVP levels, suggesting that the exaggerated diuretic response was of renal origin. Total AQP2 levels were reduced in ETA KO mice after water loading; although not studied, it is possible that specific phosphorylation sites on AQP2 as well as AQP3 levels could be regulated by RMIC ETAs. Similar to salt loading, water loading induced a greater increase in urinary ET-1 and PGE₂ excretion in ETA KO, compared with control, mice; no increase in urinary NOx or NOS isoform expression was observed. The increased PGE₂ excretion in ETA KO mice was associated with elevated COX2 expression; because RMICs are the predominant source of inner medullary COX2, it suggests that somehow ETAs modulate RMIC COX2 during high-water intake. Normally, medullary COX2 expression is stimulated by dehydration, an effect that has been hypothesized to help preserve renal function under very hypertonic conditions.⁴⁰ However, because medullary ET-1 levels are elevated during water loading, it suggests that RMIC ETAs may function during water loading to reduce COX2 expression; in essence, to act, as in salt loading, as a brake on the diuretic effects of ET-1. How RMIC ETAs modulate COX2 during water loading is unknown, and why water loading elicits different natriuretic/diuretic factor responses than salt loading in ETA KO mice is unknown, but indicates that different and potentially complex mechanisms of action are involved.

A potential limitation of this study is that EDNRA gene recombination occurred in several organs. This was not unexpected because tenascin-C is expressed by adult stromal cells, albeit mostly at very low levels.^25,32 Although we cannot absolutely rule out that non-RMIC EDNRA gene targeting affected the observed renal phenotype, no evidence was observed that this was the case, e.g., AVP, aldosterone, and PRC were unaffected. Nonetheless, it is still possible that deletion of the EDNRA gene in nonrenal cell types could have had unanticipated effects. Another potential limitation was the use of the tenascin-C knockin that disrupted tenascin-C function. However, as stated earlier, global tenascin-C KO mice do not manifest a significant phenotype under physiologic conditions.³¹ Further, we observed no difference in BP or renal salt excretion phenotypes between tamoxifen-treated ETA^f/f;T/T and ETA^f/f;T/W mice. Finally, the possibility existed that somehow RMIC ETA KO would affect inner medullary structure, e.g., somewhat analogous to RMIC COX2 KO inducing RMIC apoptosis and eventual papillary necrosis.¹⁵ However, renal histology, including the inner medulla, was normal in ETA KO mice up to at least 8 months of age. In addition, because ET-1 has been reported to modulate cultured RMIC hyaluronan production,¹⁹ it was conceivable that changes in inner medullary hyaluronan content, which may affect kidney function,⁸ could play a role in the observed ETA KO phenotype. However, no effect of RMIC ETA KO was observed on inner medullary hyaluronan content during either normal or high-salt or -water intake.

In summary, this study demonstrates that RMICs in general, and RMIC ETAs in particular, are capable of regulation of renal function under physiologic conditions. We identified ENaC and AQP2 as RMIC targets; it is also possible that Na/K ATPase and even non-inner medullary transporters could be modulated. Further, it remains unclear if the natriuretic effects of RMIC ETA KO can be fully attributed to IMCD ENaC regulation as active salt transport by this nephron segment remains controversial. Surprisingly, RMIC ETA KO resulted in relatively enhanced hypotensive, natriuretic, and diuretic responses to high Na⁺ and/or high-water intake, implying that RMIC ETAs normally function to raise BP and promote fluid retention. Key issues remain to be determined, including the mechanisms by which RMICs regulate endogenous renal natriuretic and diuretic factors and ultimately water and electrolyte transporters/channels. Looking forward, one way to begin to assess this would involve attempts to isolate RMICs (the tenascin-C-CreER2 knockin also has tamoxifen-induced GFP expression) and, assuming enough cells can be obtained, perform RNA sequencing and/or proteomics on cells obtained from control and ETA KO mice. These and other studies are needed to uncover how this relatively unexplored cell type regulates renal function in health and disease.

Disclosures

All authors have nothing to disclose.

Funding

This research was supported by National Institutes of Health, National Heart, Lung, and Blood Institute grant P01 HL136267 (to Dr. Kohan) and Center for Scientific Review grant NIH S10 OD021833 (to the University of Southern California Multi-Photon Microscopy Core).

Supplemental Material

This article contains the following supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2020020232/-/DCSupplemental.

Supplemental Table 1. Primary antibodies for immunoblots and immunohistochemistry.

Supplemental Figure 1. Hemodynamics in ETA^f/f;T/W mice.

Supplemental Figure 2. Metabolic balance studies in ETA^f/f;T/W mice.