Two-Kidney, One Clip and One-Kidney, One Clip Hypertension in Mice

Mice

Male C57BL/6 mice (Iffa Credo) with a single renin gene and approximate weight of 20 g were used at 4 to 5 weeks of age. They were maintained on tap water and a regular rodent chow (2.5 mg Na⁺/g) ad libitum.

Clip

For clipping, U-shaped stainless steel clips were used (3×2×1 mm with a 2-mm-long cleft, Exidel SA). The width of the clip opening ranged from 0.04 to 0.18 mm.

Surgery

Mice were anesthetized by halothane inhalation (1% to 2% in oxygen). The kidney was exposed through a small flank incision, externalized, and carefully maintained with an ophthalmic chalazion forceps. For clipping, the renal artery of the left kidney was individualized over a short segment by blunt dissection, and a clip was placed close to the aorta. The kidney was then gently pushed back into the retroperitoneal cavity. For right nephrectomy, two ligatures were passed around the renal vascular pedicle and ureter and were tied. The kidney was removed without the adrenal gland. The muscle layer was sutured, and the skin incision was closed with surgical staples. A sham procedure, which included the entire surgery with the exception of artery clipping, was applied in control mice.

Blood Pressure and Heart Rate

The right carotid artery was exposed through a cervical incision and isolated by blunt dissection. A catheter, formed by a length of PE-10 tubing, was filled with a solution of glucose (5%) and heparin (300 IU/mL) and inserted into the vessel. A minute amount of 1% xylocaine was used to prevent spasm. Then, a ligature was tied around the artery, and the catheter was tunneled subcutaneously to exit at the back of the neck. The skin incision was closed with surgical staples. Mice were allowed 3 hours to recover from the anesthesia and then were placed in Plexiglas tubes to partially restrict their movements. Thirty minutes later, the arterial line was connected to a pressure transducer, and blood pressure and heart rate were recorded every 20 seconds for 15 minutes with a computerized data-acquisition system.¹⁴

Blood and Tissue Samples

After blood pressure measurements, 300 μL of blood was drawn through the arterial line into tubes containing EDTA. Plasma samples were frozen in liquid nitrogen and stored at −20°C. Mice were killed by neck dislocation under deep halothane anesthesia. The heart was removed and weighed without the atria. The heart and kidneys were stored at −70°C until further processing.

Histology

Cardiac weight index (CWI) was calculated as CWI=Heart Wet Weight (mg)/Body Weight (g). In some experiments, the thoracic aortas were collected and fixed in 4% formaldehyde. Aortic cross-sectional area was determined as follows. Hematoxylin-eosin–stained sections were analyzed with a laser-scanning confocal microscope (MRC 500 confocal imaging system, Bio-Rad) in the transmission mode. For each vessel, four determinations of the outer (R) and inner (r) diameters were obtained from two different sections. Since the heart and vasculature were not fixed at a constant pressure, cross-sectional area (CSA) was the only parameter determined and was calculated as CSA=π(R²−r²).

Plasma Renin Concentrations

Plasma renin concentrations were measured with a modification of a previously described microassay based on Ang I trapping by antibody.¹⁵ Briefly, aliquots of plasma samples were incubated for 15 minutes at 37°C in the presence of nephrectomized rat plasma as a source of renin substrate and a predetermined amount of a rabbit anti–Ang I polyclonal antiserum. The Ang I concentrations produced during the incubation period were determined with a sensitive radioimmunoassay.

Northern Blotting

Total RNA was purified from frozen tissues by the guanidine thiocyanate/cesium chloride gradient technique as described.¹⁶ Ten micrograms of total RNA was fractionated by electrophoresis on a 1.2% formaldehyde–agarose gel and transferred by capillarity on Hybond-N nitrocellulose membranes (Amersham). Kidney RNAs were hybridized with an [α-³²P]dCTP randomly labeled mouse renin probe (a kind gift of Dr K. Nakayama, University of Tsukuba [Japan]) for 12 hours at 42°C in the presence of 50% formamide. The intensity of the hybridization signals was quantified with an Instant Imager detector (Packard Instruments). The blot was then stripped and hybridized again with a mouse β-actin cDNA probe. Hybridization signals were similarly analyzed, and results are expressed as the ratio of the renin to the β-actin signal. A similar procedure was applied to RNAs extracted from cardiac tissues. These RNAs were hybridized successively to radiolabeled mouse ANP and GAPDH probes. The results are presented as the ratio of the ANP to the GAPDH signal.

Reverse Transcription–Polymerase Chain Reaction

One microgram total RNA was reversed transcribed into cDNA, and α-skeletal actin expression was detected by polymerase chain reaction (PCR) amplification with a set of primers, named SKAC/F1 (forward) and SKAC/B1 (backward), specific to the 3′ untranslated region of the α-skeletal actin RNA. Coamplification of the GAPDH RNA was carried out with the specific primers MGAP/F3 (forward) and MGAP/B1 (backward). One amplification cycle included denaturation at 95°C and annealing at 60°C for 1 minute each. A total of 20 cycles was performed. Preliminary experiments demonstrated linearity between signal intensity and the amount of total RNA present in the samples with the use of this number of cycles. A final 10-minute extension period at 72°C was also included. PCR products were separated on a 6% polyacrylamide gel. Since the reaction mixture contained [α-³²P]dCTP, it allowed quantitative analysis of the PCR products by means of an Instant Imager detector. Results are expressed as the ratio of the radioactivity associated with the α-skeletal actin–specific band to the GAPDH signal. The oligonucleotide sequences are as follows: SKAC/F1: 5′-TCTCTCCTCAGGACGACAATCGAC-3′; SKAC/B1: 5′-CCTTTCCACAGGGCTTTGTTTG-3′; MGAP/F3: 5′-TGTTCCAGTATGACTCCACTCACGG-3′; and MGAP/B1: 5′-AGCCCTTCCACAATGCCAAAG-3′.

Determination of Clip Size Shown to Induce Hypertension

To determine the precise clip opening that would reduce renal perfusion and induce high blood pressure without producing renal infarction, we evaluated clips with lumen sizes ranging from 0.04 to 0.18 mm in the 2K1C model. Preliminary experiments demonstrated that sizes of 0.10 mm and smaller always resulted in renal infarction of the clipped kidney, whereas sizes of 0.13 mm and larger did not cause any kidney damage but were not able to induce hypertension (data not shown). Since high blood pressure was observed in most of the mice carrying clips with 0.11- or 0.12-mm openings, we initiated a second series of experiments to investigate the development of high blood pressure induced by these clips (Table 1⇓). The main difference between the two sizes consisted of the higher incidence of renal infarction in mice bearing the 0.11-mm clip than in those bearing the 0.12-mm clip. It is noteworthy that blood pressure was not elevated in mice with signs of infarction.

We then evaluated the effects of these two clip sizes on the blood pressure of uninephrectomized mice (1K1C model). Similar to what we observed in the 2K1C model, hypertension developed rapidly with 0.12-mm clips (Table 2⇓). Mice demonstrated an identical number of renal infarctions in this model. However, renal ischemia was lethal in this case because of the absence of a normal contralateral kidney. This was particularly evident in the group of mice bearing 0.11-mm clips in which most of the mice died 1 week after clipping. Tables 1⇑ and 2⇓ also show intra-arterial blood pressures in sham and clipped mice at different times after clipping. In the 2K1C model with 0.12-mm clips, a significant rise in blood pressure was observed 2 weeks after surgery, with no further increase at 4 weeks. In contrast, clipping of the artery in uninephrectomized mice not only resulted in an elevated blood pressure 2 weeks after surgery but the values continued to rise until 4 weeks. At 4 weeks, blood pressure in 1K1C mice was also considerably higher than observed in 2K1C mice. Heart rate appeared slightly increased in the 2K1C group, whereas it remained unchanged in the 1K1C model. Since the 0.12-mm clip was demonstrated to be the most suitable for our study, we used it to further characterize the two models in the experiments described below.

Renin Expression in the Kidneys and Plasma Renin Concentration

Because the 2K1C and 1K1C models differ in their dependence on an activated renin-angiotensin system during the chronic phase of hypertension, we measured renin expression in the kidneys of clipped mice as a means to evaluate renin synthesis. As expected, renin mRNA levels in the clipped kidney of 2K1C mice were greatly elevated compared with those measured both in the contralateral normal kidney and in kidneys from sham-operated mice (Fig 1A⇓). On the other hand, no activation of renin expression could be detected in clipped kidneys from uninephrectomized mice (Fig 1B⇓). Plasma renin concentrations were also measured 4 weeks after clipping. Fig 2⇓ demonstrates that plasma renin levels in 2K1C mice were significantly higher than those observed in control mice. In accordance with what was seen at the RNA level, plasma renin concentrations in 1K1C mice were not different from basal values. Plasma renin activity was also determined in these mice and found to be exactly correlated with the levels of circulating renin (data not shown).

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Figure 1.

Renin expression in kidneys of 2K1C and 1K1C mice. Renin mRNA levels were determined by Northern blot analysis 2 weeks (open bars) or 4 weeks (solid bars) after clipping. **P<.01 compared with matched control group (ANOVA). L indicates left; R, right.

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Figure 2.

Plasma renin concentrations in 2K1C and 1K1C mice. Active renin levels were determined 4 weeks after clipping. **P<.01 compared with matched control group (ANOVA).

Cardiovascular Hypertrophy and Cardiac α-Skeletal Actin and ANP Expression in Heart

To estimate the increase in cardiac mass, we calculated the cardiac weight index in 2K1C and 1K1C mice. The 1K1C mice demonstrated a remarkable development of cardiac hypertrophy, whereas only a modest, but still significant, increase in cardiac weight index was observed in 2K1C mice (Tables 1⇑ and 2⇑). Absolute heart weight followed the same pattern (data not shown). In both models, cardiac growth appeared maximal already 2 weeks after clipping. Expression of the α-skeletal actin gene, which is known to be activated during the development of cardiac hypertrophy, was also determined (Fig 3⇓). In 2K1C mice, α-skeletal actin gene expression in the heart did not appear to be different from that observed in control mice, whereas expression was strongly activated in 1K1C mice (Fig 3B⇓). Similarly, ANP expression was highly increased in ventricles from 1K1C mice and was not significantly different from control values in 2K1C mice (Fig 4⇓). To determine whether vascular hypertrophy was also present in 2K1C and 1K1C mice, we measured the cross-sectional area of the thoracic aortas. Fig 5⇓ demonstrates a significant increase in the aortic cross-sectional area in both models of hypertension.

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Figure 3.

α-Skeletal actin expression in heart of 2K1C and 1K1C mice. α-Skeletal actin mRNA levels were determined by semiquantitative reverse transcription−polymerase chain reaction 2 weeks (open bars) or 4 weeks (solid bars) after clipping. **P<.01 compared with matched control group (ANOVA).

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Figure 4.

Atrial natriuretic peptide (ANF in figure) expression in heart of 2K1C and 1K1C mice. ANP mRNA levels were determined by Northern blot analysis 4 weeks after clipping. **P<.01 compared with matched control group (ANOVA).

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Figure 5.

Aortic cross-sectional area in 2K1C and 1K1C mice determined 4 weeks after clipping. **P<.01 compared with matched control group (ANOVA).