Blood Pressure Measurement

The arterial blood pressure of conscious 12- to 14-week old male mice were measured by means of a right carotid artery catheter (300–500 μm outer diameter). After surgery (24–48 h), the animals were allowed to become familiar with their environment and, after stabilization, their arterial blood pressures were monitored continuously for at least 30 min by using a pressure transducer as previously described from our laboratory [5]. The animals were maintained on either a standard mouse chow (0.3% salt), low-salt diet (0.03% salt) or high salt diet (8%) for approximately 3 weeks.

In vitro Tubule Microperfusion

Proximal convoluted tubules from Cyp4a14 KO and wild-type mice (SV/129) were perfused in vitro as previously described [9,10,11]. Briefly, tubules were dissected in cooled (4°C) modified Hank's solution containing in mM: 137 NaCl, 5 KCl, 0.8 MgSO₄, 0.33 Na₂HPO₄, 0.44 KH₂PO₄, 1 MgCl₂, 10 Tris-HCl, 0.25 CaCl₂, 2 glutamine and 2 L-lactate. This solution was bubbled with 100% O₂ and had a pH of 7.4. Tubules were then transferred to a 1.2 ml thermostatically-controlled (38°C) bathing chamber and perfused with concentric glass pipettes. The perfusion solution contained in mM: 115 NaCl, 25 NaHCO₃, 2.3 Na₂HPO₄, 10 Na acetate, 1.8 CaCl₂, 1 MgSO₄, 5 KCl, 8.3 glucose and 5 alanine and had an osmolality of 290 mOsm/kg water. The bathing solution was designed to simulate plasma and contained in mM: 115 NaCl, 25 NaHCO₃, 2.3 Na₂HPO₄, 10 Na acetate, 1.8 CaCl₂, 1 MgSO₄, 5 KCl, 8.3 glucose and 5 alanine and also 6 gm/dl of albumin. The osmolality of the bathing solution was 290 mOsm/kg water. The perfusion and bathing solutions were bubbled with 95% O₂ and 5% CO₂ at 37°C and had a pH of 7.4. The osmolalities of the perfusion and bathing solutions were measured with a Wide Range Osmometer (Advanced Instruments, Model WDW, Norwood, Mass., USA) and adjusted to the desired osmolality by the addition of water or NaCl. The bathing solution was exchanged at a rate of 0.5 ml/min to keep the osmolality and pH constant.

Volume absorption (J_V; in nl/min · mm) was measured as the difference between the perfusion and collection rates and normalized per mm of tubule length. The collection rate was determined by timed collections using a constant volume pipette. Exhaustively dialyzed [methoxy-³H] inulin (New England Nuclear, Boston, Mass., USA) was added to the perfusate at a concentration of 50 μCi/ml so that the perfusion rate could be calculated. The tubule length was measured using an eyepiece micrometer.

Brush Border Membrane Vesicle Preparation

Animals were euthanized and kidneys were rapidly removed and immediately placed in ice-cold phosphate buffered saline (in mM: 137 NaCl, 2.7 KCl, 10.1 Na₂HPO₄, 1.7 KH₂PO₄, pH 7.4). The capsule was removed and the cortex dissected, minced and placed in 15 ml of an ice-cold isolation buffer (in mM: 300 mannitol, 16 HEPES, 5 EGTA, titrated to pH 7.4 with Tris containing protease inhibitors aprotonin (2 μg/ml), leupeptin (2 μg/ml), and phenylmethylsulfonyl fluoride (100 μg/ml). The minced cortex was then homogenized with a Power Gen 125 (Fisher Scientific, Pittsburgh, Pa., USA) homogenizer at 4°C. After addition of 230 μl of 1.0 M MgCl₂ to precipitate cell debris, the homogenate was shaken vigorously for 10 s, every 5 min for 20 min, as previously described [12,13,14]. Subsequently, the homogenate was centrifuged at 2,500 g for 15 min at 4°C. The supernatant was decanted, added to 230 μl 1.0 M MgCl₂, shaken vigorously for 10 s every 5 min for 20 min, and centrifuged for 15 min at 2,500 g at 4°C. The supernatant was then centrifuged at 48,400 g for 30 min at 4°C. The pellets were resuspended in 1.5 ml of ice-cold resuspension buffer (5 mM HEPES, pH 7.4; osmolality adjusted to 80 mOsm with D-mannitol) using 22 and 25 gauge needles. Protein was determined in the crude homogenate and brush border membrane vesicles (BBMV) using a BCA protein assay (Pierce, Rockford, Ill., USA). Alkaline phosphatase activity was used to determine the enrichment as described previously [12,13,14].

Protein Abundance of Na/H Exchanger (NHE3) in Brush Border Membrane

BBMV protein was denatured and separated on a 7.5% polyacrylamide gel as previously described (50 μg/lane) [12,13,14]. The separated proteins within the gel were then transferred to polyvinylidone difluoride membrane overnight at 140 mA at 4°C. The blot was blocked with Blotto (5% nonfat milk, 0.05% Tween 20, and PBS [pH 7.4]) for 1 h, and then a primary antibody to rat NHE3 (gift from O. Moe, University of Texas Southwestern Med Center, Dallas, Tex., USA) was added at a 1:750 dilution and incubated for 2 h at room temperature on a shaker. β-Actin antibody (Sigma Chemical Co. St. Louis, Mo., USA) was added at a 1:15,000 dilution. The blot was washed with PBS containing 1% Tween, and then the secondary horseradish perioxidase-conjugated anti-rabbit immunoglobulin (for NHE3 antibody) and anti-mouse immunoglobulin (for β-actin antibody) were added at 1:10,000 dilution for 1 h in Blotto at room temperature. The blot was subsequently washed with PBS containing 1% Tween, and enhanced chemiluminescence was used to detect the bound horseradish perioxidase-conjugated antibody (Amersham Life Sciences, Inc., Arlington Heights, Ill., USA).

Afferent Arteriolar Response to Angiotensin II and Endothelin

Experiments were performed on male wild-type SV129 mice and Cyp4a14 KO mice weighing an average of 29.5 ± 0.81 and 30.8 ± 1.15 g, respectively. Mice were anesthetized with a combination of thiobutabarbital (Inactin; 100 mg/kg i.p.) and ketamine (Ketaset; 10 mg/kg i.p.) and a midline abdominal incision was made. The right renal artery was cannulated via the superior mesenteric artery, and the kidney was immediately perfused with Tyrode's solution containing 6% albumin and a mixture of L-amino acids [15]. All protocols were conducted in the juxtamedullary microvascular preparation perfused with the cell-free Tyrode's solution containing 6% albumin. The Tyrode's solution was stirred continuously in a closed reservoir that was pressurized by a 95% O₂/5% CO₂ tank. The kidney was removed from the mouse and maintained in an organ chamber at room temperature throughout the isolation and dissection procedure. The juxtamedullary microvasculature was isolated for study as previously described [15]. The organ chamber was then warmed, and the tissue surface was continuously superfused with Tyrode's solution containing 1% albumin at 37°C. Renal artery perfusion pressure, measured at the tip of the cannula, was set to 100 mm Hg.

Determination of afferent arteriolar diameter was accomplished using transillumination videomicroscopy as previously described [15]. The tissue was transilluminated and the focused image converted to a video signal by a high-resolution Newvicon camera. This video signal was electronically enhanced and recorded on videotape for later analysis. Afferent arteriolar inside diameters were measured at 15 s intervals using a digital image shearing monitor. The image shearing device is accurate to within 0.2% of the screen width or 0.2 μm and measurement reproducibility is within 0.5 μm.

After a 20-min equilibration period, baseline diameter measurements of the afferent arteriole were made. Angiotensin II (0.1–100 nM) or endothelin-1 (0.1–10 nM) was then administered in increasing concentrations and diameter changes monitored. Steady-state diameter was attained by the end of the second min and the average diameter of the third min of each treatment period was utilized for statistical analysis.

Proximal Convoluted Tubule Transport

The volume absorption rate of the proximal convoluted tubules (PCTs) from control and Cyp4a14 KO mice was determined by in vitro microperfusion. Figure ​Figure11 shows the volume absorption rates in PCTs from the Cyp4a14 KO and wild-type mice. As can be seen, the rate of transport is about 50% higher in the Cyp4a14 KO mice (p < 0.05). The length of the perfused tubule segments was not different between the 2 groups (wild-type 1.1 ± 0.1; Cyp4a14 KO 0.9 ± 0.1, p = NS). Thus, there was a significant increase in the volume absorption rate in the Cyp4a14 KO mice that could contribute to the development of hypertension.

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Fig. 1.

The volume absorption rates of PCT from wild-type (SV/129) mice and Cyp4a14 KO mice. Volume absorption was significantly higher in the Cyp4a14 KO mice than in the wild-type control mice ( p < 0.05).

NHE3 Expression

Most of the sodium transport in the proximal tubule is mediated by sodium proton exchange. The principal protein that performs this function is NHE3. We then determined the expression of NHE3 in the brush borders of proximal tubules from wild-type and Cyp4a14 KO mice using an immunoblot as previously used in our laboratory [6]. The antibody for NHE3 was a gift from Dr. Orson Moe and has been previously used in our laboratory [6].

Figure ​Figure22 shows the results of the Western blot for NHE3 and β-actin. Densitometry showed that the abundance of NHE3 was significantly higher in the samples obtained from the Cyp4a14 KO mice as compared to the wild-type controls. The expression of β-actin was not significantly different between the 2 groups. Thus, increased expression of NHE3 appears to be responsible for the increased volume absorption rate in the Cyp4a14 KO mice.

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Fig. 2.

Immunoblots of the brush border membrane NHE3 expression. The apical membranes of the proximal tubules from Cyp4a14 KO mice have a higher expression of NHE3 than the wild-type control mice.

This work was supported by National Institutes of Diabetes and Digestive and Kidney Diseases Grant DK 38226 (R.Q., J.I. and J.C.) and an American Heart Association Established Investigator Award to J.I. We wish to thank Laurel Johnson for her able secretarial assistance. Current E-Mail address for Dr. Chakravarty: sumanachak@iict.res.in.