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Disruption of the endothelin A receptor in the nephron causes mild fluid volume expansion
© Stuart et al.; licensee BioMed Central Ltd. 2012
Received: 14 September 2012
Accepted: 27 November 2012
Published: 5 December 2012
Endothelin, via endothelin A receptors (ETA), exerts multiple pathologic effects that contribute to disease pathogenesis throughout the body. ETA antagonists ameliorate many experimental diseases and have been extensively utilized in clinical trials. The utility of ETA blockers has been greatly limited, however, by fluid retention, sometimes leading to heart failure or death. To begin to examine this issue, the effect of genetic disruption of ETA in the nephron on blood pressure and salt handling was determined.
Mice were generated with doxycycline-inducible nephron-specific ETA deletion using Pax8-rtTA and LC-1 transgenes on the background of homozygous loxP-flanked ETA alleles. Arterial pressure, Na metabolism and measures of body fluid volume status (hematocrit and impedance plethysmography) were assessed.
Absence of nephron ETA did not alter arterial pressure whether mice were ingesting a normal or high Na diet. Nephron ETA disruption did not detectably affect 24 hr Na excretion or urine volume regardless of Na intake. However, mice with nephron ETA knockout that were fed a high Na diet had mild fluid retention as evidenced by an increase in body weight and a fall in hematocrit.
Genetic deletion of nephron ETA causes very modest fluid retention that does not alter arterial pressure. Nephron ETA, under normal conditions, likely do not play a major role in regulation of Na excretion or systemic hemodynamics.
Endothelin-1 (ET-1) has been implicated in the pathogenesis of multiple disorders involving virtually every organ system, including congestive heart failure, pulmonary artery hypertension, atherosclerosis, cancer, autoimmune diseases, chronic kidney disease, and others . The adverse effects of ET-1 are in large part due to interaction with endothelin A receptors (ETA). ETA activation promotes generation of reactive oxygen species, insulin resistance, inflammation, vasoconstriction, cell proliferation, extracellular matrix accumulation, and cellular hypertrophy . In addition, multiple pre-clinical studies have demonstrated that ETA blockade substantially reduces end-organ injury in disease models involving all of the above disorders . Consequently, a large number of clinical trials have been conducted using ET receptor antagonists in heart failure, coronary artery disease, scleroderma, proteinuric renal disease, pulmonary artery hypertension, arterial hypertension, subarachnoid hemorrhage and other disorders . Unfortunately, despite such intense efforts, ET receptor blockers have been approved for only two indications: pulmonary artery hypertension and scleroderma digital ulcers .
There are several potential reasons for the failure of these trials, however an almost universal problem has been ET receptor antagonist-induced fluid retention. This fluid retention likely played a significant role in the failure of clinical trials using ET receptor blockers in patients with congestive heart failure. A large Phase III trial using avosentan, a relatively ETA-selective blocker, in patients with diabetic nephropathy was halted due to increased morbidity and mortality associated with drug-induced fluid retention . Atrasentan (a highly ETA-selective blocker) given to a large number of patients with prostate cancer doubled the risk of developing heart failure . While the doses of ET receptor blockers used in these trials were likely substantially higher than necessary, it is evident that ETA blockade-induced fluid retention is a significant problem potentially limiting this class of drug’s clinical utility.
A key question is how ETA blockade causes fluid retention. While ETA antagonism undoubtedly causes some fluid retention in response to vasodilation, this does not explain how patients could become fluid overloaded. A reasonable site for ETA blocker-induced fluid retention is the kidney. Studies in animals indicate that ETA blockade causes renal vasodilation (primarily afferent arteriole) , suggesting that a renal hemodynamic effect of these agents does not explain the fluid retention. The ETB receptor has been clearly demonstrated to inhibit thick ascending limb and collecting duct (CD) Na reabsorption, while the role of the nephron ETA receptor in modulating tubule Na and/or water transport remains uncertain . Collecting duct principal cell-specific disruption of ETA did not detectably affect blood pressure (BP) or Na excretion , however the role of ETA throughout the nephron in modifying these parameters has never been directly assessed in vivo. Consequently, the current study was undertaken, using a recently developed inducible gene targeting model, to determine whether ETA in the nephron can modulate BP or urinary Na excretion.
Animal use assurance
All animal use and welfare adhered to the NIH Guide for the Care and Use of Laboratory Animals following protocol reviews and approval by the Institutional Laboratory Animal Care and Use Committees of the University of Utah Health Sciences Center.
Generation of inducible nephron-specific ETA knockout mice
Tail DNA was PCR amplified and the following primers were used for genotyping: ETA - F 5′-cccatgcttagacacaaccatg-3′ and R 5′-gatgacaaccaagcagaagacag-3- which yield a 364 bp product for the floxed EDNRA gene (includes loxP site) and a 324 bp product for the wild type EDNRA gene; Pax8-rtTA - F 5′-ccatgtctagactggacaaga-3′ and R 5′-catcaatgtatcttatcatgtctgg -3′ which yields a 600 bp product; and LC-1 - F 5′-tcgctgcattaccggtcgatgc-3′ and R 5′-ccatgagtgaacgaacctggtcg-3′ which yields a 480 bp product.
Screening for recombination
DNA from selected organs was PCR amplified to evaluate target organ recombination using primers spanning exons 6–8 in the EDNRA gene: F 5′-cccatgcttagacacaaccatg-3′ and R 5′-cgctgttgtatatccagtatcagg-3′. Recombination of the EDNRA gene yields a 610 bp product; the predicted size of the unrecombined wild type EDNRA gene is 1287 bp and, under the PCR conditions utilized, did not yield detectable amounts of product.
DNA from microdissected glomeruli, proximal tubules, thick ascending limbs and cortical collecting ducts was also analyzed for EDNRA gene recombination. Kidney slices were incubated with DMEM:F12 containing 2 mg/ml collagenase and 2 mg/ml hyaluronidase for 30 min, then the media replaced with ice-cold HBSS containing 15 mM HEPES and 1% FBS. Tubules were dissected free and individual nephron segments transferred to ice-cold HBSS + 15 mM HEPES; samples were frozen until analysis. DNA was isolated using a microDNA isolation kit (Qiagen, Valencia, CA).
Expression of mRNA
Total liver, and cortex and inner medulla from homozygous floxed ETA mice without the Pax8-rtTA or LC-1 transgenes (referred to hereafter as “control” mice) and ETA KO (DOX-treated iETA) animals were dissected, and RNA was extracted and reverse transcribed. The resulting cDNA was assayed for relative expression of ETA mRNA in control and ETA KO animals using the Taqman Gene Expression Assay (Applied Biosystems, Carlsbad, CA, ETA probe cat# Mm01243722_m1, GAPDH probe cat# Mm03302249_g1).
RNA from microdissected tubules from control and ETA KO mice was analyzed for ETB mRNA content. Tubules were obtained as described above, RNA isolated and reverse transcribed, and ETB mRNA determined using the Taqman probe cat# Mm00432989_m1, normalizing to GAPDH as described above.
Mouse kidneys were perfused with phosphate-buffered saline, then fixed for 1 day in 10% formalin and stored in ethanol. Kidneys were paraffin embedded and 4 μm sections obtained. Sections were deparaffinized on a Benchmark XT (Ventana Medical Systems, Sunnyvale, CA) using EasyPrep (Ventana), and Protease-2 (Ventana) digested for 8 min. Sections were incubated with a 1:100 dilution of rabbit anti-rat ETA (AER-001, Alomone Labs, Jerusalem, Israel) for 2 hr at 37°C, blocked with Sniper (BIocare Medical, Concord, CA) for 4 min, then incubated with a 1:100 dilution of goat anti-rabbit IgG (Sigma, St. Louis, MO) for 32 min. Goat IgG was detected using IView-DAB (Ventana) and sections were counterstained with hematoxylin for 8 min.
Metabolic cage studies
Control and iETA mice were given free access to water and a normal salt (0.3% Na) diet for 3 days. On the third day, mice were placed in metabolic cages (Hatteras Instruments, Cary, NC) for 24 hr with free access to food and water and urine collected. Tail vein blood (50 μl) was obtained at the end of the 24 hr period. Mice were then fed a high salt (3.2% Na) diet for 7 days with metabolic cage studies performed on the third and seventh days of the high Na diet. Subsequently, both control and iETA mice were given DOX and, after the treatment and recovery period (~2 weeks), the dietary procedures and metabolic cage studies were repeated.
Urine Na was determined on an EasyVet Analyzer (Medica, Bedford, MA). Urine and blood creatinine was determined using the QuantiChrom Creatinine Assay Kit (BioAssays Systems, Hayward, CA).
Blood pressure monitoring
Blood pressure was monitored in control and iETA mice by radiotelemetry (TA11-PAC10, Data Sciences International, St. Paul, MN) with catheters inserted into the right carotid artery. The mice were allowed to recover for 1 week after surgery. BP and heart rate and were monitored continuously. Mice were fed normal and high Na diets as described under the metabolic studies, and these were repeated after DOX administration. The BP studies were conducted separately from the metabolic cage studies, using different mice, in order to minimize handling of animals during hemodynamic recordings.
Fluid retention analysis
The iETA mice were fed a normal Na diet for 7 days, followed by a high Na diet for 7 days. This time schedule was used to insure maximal stability of mice, i.e., giving them a full week on each diet to adjust their body fluid volume. Mice were then treated with DOX or vehicle after which the normal and high Na diets were repeated. On the 7th day of each diet, body weight was determined. A 20 μl blood sample was obtained for determination of hematocrit. Body compartment fluid volume was then determined by impedance plethysmography as previously described . Briefly, mice were anesthetized and measured for length and width. Four needles were inserted under the skin at the base of the tail, the intercept between the front of the ears and the longitudinal midline, and 0.5 cm from these sites toward the tip of the tail and the nose, respectively. Leads from the needles were attached to the ImpediVet BIS1 system (ImpediMed, San Diego, CA) that analyzes whole body bioimpedance data to determine total body water (TBW), extracellular fluid volume (ECF) and intracellular fluid volume. A resistance coefficient equal to 10% of that for rats was used for all mice studies.
Data are presented as mean percent of control or as absolute values, either one ± SE. Data were compared using one- or two-way ANOVA as indicated. The criterion for significance was p < 0.05.
Confirmation of inducible nephron-specific EDNRAgene knockout
Effect of nephron ETA KO on blood pressure and urinary Na and water excretion
Fluid retention analysis in iETA mice
Effect of nephron ETA KO on nephron ETB mRNA
ETB mRNA content was determined in proximal tubules (PT), thick ascending limbs (TAL) and cortical collecting ducts (CCD) from iETA mice treated with vehicle or DOX. ETB mRNA levels were detected in the following order: CCD > TAL > PT. The deltaCT, defined as cycle number at which ETB is first detected minus the cycle number at which GAPDH is first detected (so the lower the deltaCT, the more ETB mRNA is present), was 11.22 ± 0.27 for PT, 9.44 ± 0.32 for TAL, and 7.67 ± 0.19 for CCD, N = 12 each nephron segment. Nephron ETA KO did not significantly affect ETB mRNA in CCD (99 ± 8% of control), TAL (76 ± 19% of control) or PT (79 ± 13% of control) wherein 6 different tubules from 3 mice in each group were analyzed.
The first major finding in the current study is that nephron ETA can modulate volume homeostasis, albeit their normal physiological role appears to be very modest. An increase in body weight and fall in hematocrit indicate fluid retention after nephron ETA KO, however such fluid retention was too small to permit detectable changes in body fluid volume compartments or in urinary Na excretion. The latter measurements (body fluid compartments and urinary Na excretion) have rather high intrinsic variability, making detection of significant changes challenging. Nonetheless, at least on a high Na diet, total body water and extracellular fluid compartments tended to be increased in nephron ETA KO animals.
Nephron ETA KO causes mild fluid retention, suggesting that nephron ETA exerts a natriuretic effect. Previous studies have clearly implicated the ETB receptor in the proximal tubule, thick ascending limb, cortical collecting duct (CD), and inner medullary CD as mediating ET-1 inhibition of Na and/or water reabsorption . In contrast, CD-specific ETA KO mice do not manifest alterations in BP or UNaV (although subtle changes in volume regulation could have been missed) . Similarly, ETA blockers do not affect ET-1 inhibition of epithelial Na channel (ENaC) activity in isolated cortical CD  nor is ENaC activity in this nephron segment altered in CD ETA KO mice . However, there is data to suggest that renal ETA can exert a natriuretic effect. Mice with CD ETB KO are modestly hypertensive and retain Na , while combined CD ETA and ETB KO mice are significantly more hypertensive and retain more Na than mice with CD ETB KO alone . In addition, intra-renal medullary administration of ET-1 to rats deficient in ETB causes a natriuresis and diuresis and this effect is prevented by an ETA antagonist . Taken together, these studies suggest that ETB is the primarily mediator of ET-1 inhibition of nephron Na and water transport, but that ETA can also exert a natriuretic effect.
The mechanism(s) by which nephron ETA exerts a natriuretic effect and the precise sites in the nephron at which this occurs remain unknown. An intriguing possibility is that ETA might affect ETB activity as suggested by the finding that combined CD ETA and ETB KO mice retain more Na than mice with KO of either receptor alone . ETA/B heterodimerization has been described in vitro and such heterodimerization can affect ET receptor trafficking and potentially signaling [15, 16]. While the goal of the current study was to determine whether nephron ETA could affect renal Na handling, clearly additional studies are necessary to dissect out the mechanism(s) responsible for this effect.
It remains to be determined how ETA antagonists cause fluid retention, however the current studies suggest that blockade of nephron ETA may pay a role. Given that the ETA KO phenotype is very mild, it may be that blockade of ETA outside of the nephron may also be involved in ETA antagonist-induced fluid retention. Despite the disappointing results with earlier clinical trials, ETA blockers still hold promise for the treatment of a variety of diseases. As mentioned earlier, multiple pre-clinical and early phase clinical trials suggest that ETA blockers may be of therapeutic benefit in a wide variety of disorders . A recent phase IIA trial in patients with diabetic nephropathy using atrasentan in doses that caused only modest fluid retention showed significant reductions in albuminuria over an 8-week period ; further trials are planned to determine whether ETA blockade slows progression of diabetic kidney disease. Although the primary endpoint of reduction in office BP was not met, darusentan significantly reduced ambulatory BP in patients with resistant hypertension as compared to guanfacine . Trials are actively recruiting for patients to study the effect of ETA antagonists in myocardial infarction , cancer , and other disorders. Thus, determination of the sites and mechanisms responsible for ETA antagonist-induced fluid retention remains an important question.
Using an inducible Cre-based strategy, we found that disruption of ETA receptors in the nephron causes mild Na retention with no detectable effect on BP. These findings indicate that ETA receptors in the nephron, per se, are potentially involved, at least partially, in ETA antagonist-induced fluid retention. Key next steps involve determination of the sites and mechanism(s) of nephron ETA regulated Na transport, as well as how ETA blockers exert their fluid retaining effects.
The authors thank Sheryl Tripp in ARUP Laboratories (Salt Lake City, UT) for her assistance with sectioning and staining slides. This work was partially supported by funding from Gilead Sciences and Abbott Laboratories (to D.E.K.).
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