Materials and Methods s12

Materials and Methods

The plasmid pKAP2, a generous gift from Dr. Curt D. Sigmund of the University of Iowa, College of Medicine (Iowa, USA), contains the KAP promoter which is responsive to testosterone stimulation.1-4 The procedure for the generation of this plasmid has been described elsewhere. Oligonucleotides, restriction enzymes, and monoclonal anti-Myc tag antibodies were purchased from Invitrogen, Inc. (Burlington, Ontario, Canada). Losartan and perindopril were obtained from Dr. Ronald D. Smith (Dupont Merck, Wilmington, DE, USA) and Dr. Serge Carrière (Servier Amérique, Laval, Quebec, Canada), respectively. Placebo pellets or pellets containing 5 mg testosterone with a 21- day release schedule were purchased from Innovative Research of America (Sarasota, Florida, USA). Standard mouse chow (1% NaCl, Harlan Teklad 2018), and low salt diet chow (0.2% NaCl, Harlan Teklad 7034) were from Harlan Teklad, Inc., USA, and high salt diet chow (8% NaCl, TestDietTM) was from Modified Lab, USA. This project (H01033DGs) was approved by the Animal Care Committee of the Research Centre, Centre Hospitalier de l’Université de Montréal (CHUM).

Generation of KAP2-ARAP1 transgenic mice

cDNA encoding rat ARAP1 with Myc-tag at the 5’ terminus was inserted into pKAP2 at the Not I site. The final 17-kb KAP2-ARAP1 transgene was excised by digestion with Nde I and Spe I, purified by agarose gel electrophoresis, recovered by gel extraction, and then microinjected into 1-cell fertilized mouse embryos obtained from superovulated C57BL/6 X C3H mice. Founder mice were identified by Southern blot analysis of mouse tail genomic DNA digested with BamHI with a 0.5-kb BamHI fragment as probe. The positive transgenic founders were then crossed with wild-type C57BL/6 mice (Charles River, QC, Canada) to obtain the F1 generation.

The homozygous transgenic mice and their non-transgenic littermates used in these experiments were 8-20 weeks of age at the time of data collection. Non-transgenic, sex-matched littermates served as controls. The animals weighed approximately 22 g at 8 weeks, 25.5 g at 12 weeks, and 30 g at 20 weeks. All animals received standard mouse chow (1% NaCl) and water ad libitum, if not otherwise indicated. Their care in these experiments met the standards set forth by the Canadian Council on Animal Care, and the procedures utilized were approved by the Animal Care Committee of the CHUM. To determine when these animals became hypertensive, SBP of transgenic males at 8 and 12 weeks of age were measured, SBP was elevated at 12 weeks, but not 8 weeks of age. SBP at 12 weeks of age was 120 and 123 mmHg in lines 650 and 670, respectively.

Analysis of gene expression

Various tissues were harvested and snap frozen in liquid nitrogen. Total RNA was isolated by TRIzol (Invitrogen Inc.) after tissue homogenization. Before proceeding with reverse transcription, RNA was subjected to DNase I treatment to remove contaminating genomic DNA. Briefly, 20 to 30 mg of total RNA were treated with 2 units of DNase I/mg of total RNA in DNase I buffer containing 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 2 mM MgCl2 and 0.2 units of RNase inhibitor. The samples were incubated at 37oC for 30 min, after which RNA was extracted with phenol/chloroform, precipitated with ethanol, and then resuspended in RNase-free water. RNA was quantified and subjected to reverse transcription-polymerase chain reaction (RT-PCR) analysis as described previously.5 Briefly, 4 mg of DNase I-treated total RNA were taken to synthesize first-strand cDNAs with the Super-Script pre-amplification system. Then, 10 ml of cDNA and 0.4 mM of primers of rat ARAP1, Myc-tag and rat b-actin were added in a final volume of 50 ml PCR mixture (final concentration: PCR buffer (1X), dNTP (0.2 mM), MgCl2 (1.5 mM), and Taq polymerase (0.5 units) (Invitrogen Inc.), and amplified in a PTC-100 thermal cycler (MJ Research Inc., USA). The PCR conditions were: denaturation for 5 min at 95oC, then 25 cycles for 30 sec at 95oC, 1 min at 60oC and 45 sec at 72oC, followed by 5 min at 72oC. The rat ARAP1 antisense primer (5’-CTG TCT GGT TCA GGA TCC TGT TCT-3’) corresponding to the nucleotide sequences N+476 to N+500 from the starting codon of rat ARAP1 cDNA, the Myc-tag sense primer (5’-ATG GGC AGA GAA TTC GAA CAA AAG-3’), the b-actin forward primer and the reverse primer corresponding to the nucleotide sequences N+155 to N+179 of exon 3 (5’-ATG CCA TCC TGC GTC TGG ACC-3’) and N+115 to N+139 of exon 5 (5’-AGC ATT TGC GGT GCA CGA TGG-3’) of the b-actin gene, respectively, were deployed for PCR.

Western blot analysis

To detect transgene products in various tissues, immunoprecipitation and Western blot analysis were carried out as described previously5. Briefly, 5 mg of 9E10 antibody were added to 800 mg of tissue lysate. Immunocomplexes were separated by 10% SDS-PAGE, and protein expression was visualized by enhanced chemiluminescence detection (Amersham Biosciences).

Gene expression of the RAS components, aquaporins, ENaC and sodium-hydrogen exchanger (NHE3) in KAP2-ARAP1 transgenic mice

To explore possible mechanisms of hypertension development, we examined mRNA expression of the RAS components, aquaporins 1, 2 and 7 (AQP1, AQP2, AQP7), the 3 subunits of ENaC, and NHE3 in the whole kidneys of non-transgenic and KAP2-ARAP1 male mice at 20 weeks of age. Total RNA was purified, and RT-PCR was performed as described above, except that the conditions of annealing temperature and the number of cycles for each gene were different (Table 1). The primer set for each gene is also shown in Table 1. b–actin expression was used as internal control to normalize mRNA expression of these genes. As shown in Figure 1A, ACE and AT1A mRNA expressions were significantly increased by 1.6-fold in the kidney of KAP2-ARAP1 transgenic mice compared to non-transgenic animals (n = 5, p < 0.05), whereas no change of renin, AT1B, ACE2 and AOGEN mRNA expressions in the kidney was observed. These results suggest that the development of hypertensive phenotype in the transgenic mice may be linked to intrarenal RAS activation. As shown in Figure 1B, a-ENaC mRNA expression was significantly increased by 1.6-fold in kidney of KAP2-ARAP1 transgenic mice compared to non-transgenic mice (n = 5, p < 0.05) whereas no difference in AQP1, 2 and 7, b- and g-ENaC, and NHE3 mRNA expression between KAP2-ARAP1 and non-transgenic mice was observed. These results indicated that activation of ENaC, but not aquaporin 1, 2 and 7, or NHE3, may contribute to the development of hypertension in KAP2-ARAP1 mice.

Blood pressure measurement

For the tail-cuff method, the mice were trained for 1 week, and SBP was recorded for an additional 5 days. For the radiotelemetry, the mice were anesthetized, and then surgically implanted with a TA11PA-C10 radiotelemetry in the left carotid artery for direct BP measurement. The mice were given 7 days to recover, after which BP was recorded continuously for 3 days as baseline values. All data were collected and stored with Dataquest ART, and a telemetry analyzer (JCL Consultants Inc.) was used for data analysis. Mean SBP was determined by averaging all acquired data into 12-h blocks paralleling the light-dark cycle.

Trunk blood was collected in chilled tubes for serum separation for the measurement sodium, potassium, chloride, and urea levels (Biochemistry Laboratories, CHUM-Hôtel-Dieu). Sodium, potassium, chloride, and urea concentrations in urine also were assayed by the Biochemistry Laboratories of the CHUM. Serum hematocrit was quantified by the standard method. No significant changes in sodium, potassium, chloride and urea levels in serum and urine between transgenic and non-transgenic mice was observed.

Measurement of Kidney hypertrophy in transgenic females

To examine the phenotype of kidney hypertrophy observed in male KAP2-ARAP1 mice, 6 female KAP2-ARAP1 transgenic mice and non-transgenic female littermates at 12 weeks of age were implanted with placebo or 5 mg testosterone pellet for 21 days. At the end of treatment, the mice were anesthetized with isoflurane, then sacrificed by cervical dislocation. Body, brain, kidney, heart and spleen weight were recorded. As seen in Table 2, although the kidney/body weight ratio was significantly increased in the female transgenic and non transgenic mice treated with testosterone compared to that treated with placebo, but the ratio was not significantly changed between female transgenic and non-transgenic mice treated with testosterone. These results confirm a previous report that testosterone itself could induce proximal tubular hypertrophy in female mice.6

References

1.  Ding Y, Sigmund CD. Androgen-dependent regulation of human angiotensinogen expression in KAP-hAGT transgenic mice. Am J Physiol Renal Fluid Electrolyte Physiol. 2001; 280: F54-F60.

2.  Ding Y, Davission RL, Hardy DO, Zhu L-J, Merrill DC, Catterall JF, Sigmund CD. The kidney androgen-regulated protein promoter confers renal proximal tubule cell-specific and highly androgen-responsive expression on the human angiotensinogen gene in transgenic mice. J Biol Chem. 1997; 272: 28142-28148.

3.  Sigmund CD. Genetic manipulation of the renin-angiotensinogen system: targeted expression of the renin-angiotensin system in kidney. Am J Hypertension. 2001; 14: 33S-37S.

4.  Lavoie JL, Bruse-Lake KD, Sigmund CD. Increased blood pressure in transgenic mice expressing both human renin and angiotensinogen in renal proximal tubule. Am J Physiol Renal Fluid Electrolyte Physiol. 2004; 286: F965-F971.

5.  Guo DF, Tardif V, Ghelima K, Chan JSD, Ingelfinger RJ, Chen XM, Chenier I. A novel angiotensin II type 1 receptor-associated protein induces cellular hypertrophy in rat vascular smooth muscle and renal proximal tubular cells. J Biol Chem. 2004; 279: 21109-21120.

6.  Wolf G. Cellular mechanisms of tubule hypertrophy and hyperplasia in renal injury. Miner Electrolyte Metab. 1995; 21: 303-316.

Figure legends:

Figure I. Densitometric analysis of RAS components (A), sodium channels and aquaporins mRNA expression (B) in whole kidneys of KAP2-ARAP1 transgenic and non-transgenic animals by RT-PCR method. (n = 5, *p < 0.05).

Table I. Primer set and PCR conditions of gene expression.

Sequences of primer set and PCR conditions for various gene expressioin this study.______

Gene Sense primer Anti-sense primer PCR conditions

NHE3 5'-ACCACGTCCAGGATCCATACA-3' 5'-CACGAAGAAGGACACTATGCC-3' 60 oC; 30 cycles

a-ENaC 5'-TCGCCCGCTCTAGAGGAAGAA-3' 5'-GTGAGTAGCCGGCAGAGAGTT-3' 56 oC; 25 cycles

b-ENaC 5'-TACAGTCCCTGCACCATGAA-3' 5'-CCACCCAGGTTAGAGAGCAG-3' 57 oC; 29 cycles

g-ENaC 5'-CCGAGAAATGGTTGCTGAAT-3' 5'-GTGTCTGTGAGCTGGGATGA-3' 57 oC; 29 cycles

AQP1 5'-ATGGCCGTGAAATCAAGAAGAAG-3' 5'-CGACTAGGGAGGAGGTGATGCCC-3' 63 oC; 30 cycles

AQP2 5'-GTGGCTGCCCAGCTGCTGGG-3' 5'-AGCTCCACCGACTGCCGCCG-3' 60 oC; 20 cycles

AQP7 5'-CCACAATGGCCGGTTCTGTG-3' 5'-CCAATCTCTAAGAACCCTGT-3' 56 oC; 30 cycles

AOGEN 5'-CCACGCTCTCTGGATTTATC-3' 5'-CAGACACCGAGATGCTGTTG-3' 56 oC; 35 cycles

ACE 5'-AGGAGTTTGCAGAGGTCTGG-3' 5'-GGAAGCAGACCTTGCCAGTG-3' 56 oC; 30 cycles

ACE2 5'-ATGAAGAACAGTCTAAGACTGCCC-3' 5'-TCCTGGCTCAAGTAATAAGC-3' 55 oC; 35 cycles

AT1A 5'-CTGAAGCCAGTACCAGCTCTG-3' 5'-ACCACAAAGATGATGCTGTAG-3' 55 oC; 25 cycles

AT1B 5'-AGAGACCAGACAAGACACGCA-3' 5'-ACCACAAAGATGATGCTGTAG-3' 56 oC; 35 cycles

Renin 5'-CTACACACTCAGCAGTACGG-3' 5'-GCCAGCTTGGCTTGGCCTAG-3' 58 oC; 30 cycles____

Table II. Organs/body weight in transgenic females.

Body weight, brain, heart, kidney, and spleen/body weight of female transgenic

and non-transgenic mice with or without testosterone implantation at 12 weeks of age.___

Mice BW Br/BW He/BW Ki/BW Sp/BW

N.T., Placebo 19.26 + 1.65 15.01 + 1.42 5.28 + 0.27 12.37 + 1.03 4.09 + 0.61

N.T., Testosterone 20.43 + 1.81 14.32 + 1.08 5.71 + 0.37 16.62 + 1.12* 3.89 + 0.43

KAP2-ARAP1, Placebo 20.01 + 1.71 15.38 + 1.35 5.97 + 0.63 13.01 + 1.04 4.65 + 0.58

KAP2-ARAP1, Testosterone 20.98 + 1.93 14.35 + 1.03 5.88 + 0.44 16.97 + 1.36* 4.13 + 0.43

BW, Body weight (g); Br, brain; He, heart; Ki, kidney; Sp, spleen (mg/g). *P < 0.001 vs placebo.

Figure I