MATERIALS AND METHODS

Preparation of samples

Blood samples from renal transplant recipients undergoing treatment with cyclosporine A (mean steady state concentrations, 120–150 ng/ml) and healthy controls were processed within two hours. Lymphocytes from 20 renal transplant recipients and 25 healthy volunteers were isolated from 400 ml of blood for the aldosterone binding assays and subjected to subsequent Scatchard plot analysis as described by Shipman et al.⁹ For PCR analysis, 14 ml of blood was taken from 19 patients and 21 healthy volunteers. For PCR analysis, total RNA from 14 ml aliquots for each patient was isolated after hypotonic lysis of contaminating red blood cells using Trizol reagent (Gibco, Karlsruhe, Germany) and subsequent chloroform extraction. The remaining DNA was digested with 0.5 U/µl DNase I (Roche, Mannheim, Germany) for 30 minutes at 37°C. After phenol extraction and isopropanol precipitation, the RNA was dissolved in 50 µl RNA storage buffer (Ambion-Europe, Huntington, Cambridgeshire, UK), and 0.4 U/µl RNase inhibitor (Roche) was added. The amount and purity of the isolated RNA was determined by photometry (A₂₆₀/A₂₈₀). Aliquots of the RNA were stored at −20°C.

Preparation of the hMR internal competitor⁵

The 472 bp hMR PCR product, amplified by the hMR sense and antisense primers from total RNA of a healthy volunteer (fig 1) in 34 cycles, was cleaved by the EcoRI restriction enzyme, yielding a 113 bp fragment and a 359 bp fragment, which were cut out of the gel and purified with the QIAquick gel extraction kit (Qiagen, Hilden, Germany). Four step PCR amplification (fig 1) generated a 373 bp hMR product fused to the T7 promoter sequence, which permitted in vitro transcription in the T7 RiboMAX large scale RNA production system (Promega, Mannheim, Germany) for four hours at 37°C, according to the manufacturer’s protocol. The amount of competitor RNA was determined by photometry and the RNA was frozen at −70°C in aliquots of 10⁹ molecules/µl.

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Figure 1

In vitro synthesis of the 373 bp internal mineralocorticoid receptor (hMR) competitor by reverse transcription polymerase chain reaction and subsequent transcription.

cRT-PCR

Aliquots (0.5 µg) of RNA were subjected to reverse transcription in the presence of decreasing concentrations of competitor RNA molecules (usually 0.5 × 10²–10³) using a first strand cDNA synthesis kit (Roche) and 40pM hMR antisense primer. Subsequently, 30 µl of PCR mix (Gibco) and 40pM hMR sense primer were added to individual test tubes, which were then subjected to PCR for 38 cycles in a Personal cycler (Biometra, Göttingen, Germany). Cycling conditions consisted of 30 seconds of denaturation at 94°C, 30 seconds of annealing at 60°C, and 30 seconds of extension at 72°C. The primers recognised a 472 bp sequence unique to the coding region of the hMR gene (Genebank accession number 16801) and the 373 bp competitor RNA. Because of the narrow range of around 10²–10³ expressed hMR molecules, the patients’ RNA was also amplified with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers (sense, 5′-ACC ACA GTC CAT GCC ATC AC-3′; antisense, 5′-TCC ACC ACC CTG TTG CTG TA-3′) in the presence of GAPDH competitor RNA (Situs-Chemicals, Düsseldorf, Germany), yielding a 416 bp fragment from the patients’ RNA and a 386 bp fragment from the competitor RNA. The housekeeping gene GAPDH is constitutively expressed and is therefore well suited to correct for differences in the amount of RNA subjected to cRT-PCR analysis. Following RT-PCR, 5 µl aliquots of the PCR probes were subjected to electrophoresis in a 2% Metaphor agarose gel (Biozym, Hessisch-Oldendorf, Germany) for 2 hours at 100 V. After electrophoresis, the relative intensities of the ethidium bromide stained hMR and GAPDH DNA bands were evaluated by densitometry (Raytest, Straubenhardt, Germany). The number of hMR molecules was calculated by linear regression analysis of the logarithm of copies of RNA competitor versus the log ratio of hMR RNA_patient /hMR RNA_competitor and corrected for the definite number of 1 × 10⁵ GAPDH molecules.

Sequencing

The 472 bp hMR and the 373 bp competitor fragments were sequenced once using an ABI 310 genetic analyser (Applied Biosystems, Foster City, California, USA). Sequence data analysis was performed with Sequencer 4.0.5 (Gene Codes, Ann Arbor, Michigan, USA) and BLASTN software (kindly provided by the genome project Japan) to ensure the identity and purity of the amplified fragments.

Statistical analysis

Statistical analysis was performed using the Student’s t test for unpaired comparisons, with p < 0.05 being considered significant.

RESULTS

The construction of a competitor hMR RNA enabled the expression level of hMR in human peripheral blood leucocytes to be measured by quantitative RT-PCR. Sequence analysis of the PCR products revealed exclusive amplification of hMR RNAs. Figure 2 shows the cRT-PCR products of one patient and the subsequent evaluation by linear regression analysis. In addition to the determination of hMR mRNA molecules in 0.5 µg RNA, we correlated the number of receptor RNA molecules with 1 × 10⁵ GAPDH molecules, also determined by quantitative PCR. Because GAPDH is constitutively expressed in cells, this correlation corrects for differences in the photometric determination of RNA concentrations.

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Figure 2

Determination of expression of mineralocorticoid receptor (hMR) in human blood cells by competitor reverse transcription polymerase chain reaction with subsequent densitometry and evaluation. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TP, transplant.

In our study, we analysed 40 volunteers by this method (fig 3A). The renal transplant recipients (n  =  21) had significantly lower expression than the healthy control group (n  =  19) (p < 0.0012), although there was high individual variance. Longterm expression studies in healthy volunteers revealed that expression varied by a factor of four. Repeated cRT-PCRs using identical RNA probes after one to eight days of storage reflect the effect of storage and different enzyme batches on quantitative PCR analysis.

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Figure 3

Distribution of patients and healthy volunteers by the expression level of mineralocorticoid receptor (hMR) in their blood cells detected either by (A) quantitative reverse transcription polymerase chain reaction (cRT-PCR) or (B) conventional receptor binding studies. Median values are indicated for each group, and were significantly lower in patients undergoing immunosuppressive treatment, when investigated by cRT-PCR. The range of values labelled with “1” reflects the variation of hMR expression in a single RNA preparation as a result of storage for one to eight days and different enzyme batches analysed in four independent PCR reactions. The range of values labelled with “2” reflects the variance of hMR expression of one healthy volunteer in a longterm study covering two months.

Figure 3B shows the results of the radioreceptor assays on a cohort of 45 volunteers, different from those tested in the cRT-PCR assay. These assays also revealed a decreased number of receptor binding sites on the lymphocytes of graft recipients, but the difference between the healthy controls and the transplanted patients was not significant (p < 0.157). Because of the large volume of blood needed for each analysis, the assays could not be repeated, and no standard deviations were determined.

Comparison of the results of the cRT-PCR (fig 3A) and the radioreceptor assay (fig 3B) confirmed the different distribution of hMR molecules in the healthy and transplanted groups, although the number of molecules/0.5 µg RNA in the PCR analysis cannot be directly compared with the number of protein molecules/lymphocyte.

DISCUSSION

Immunosuppressive treatment after solid organ transplantation leads to hyperkalaemia and metabolic acidosis, possibly as a result of aldosterone resistance in approximately 75% of patients. This effect is not limited to kidney cells. Armanini et al described the downregulation of mineralocorticoid and glucocorticoid receptors by lymphocytes in patients suffering from anorexia nervosa.¹⁰ Glucocorticoid administration has also been shown to effect the number of glucocorticoid receptors on lymphocytes.⁹ In our present study, we examined whether this resistance correlated with the reduced expression of aldosterone receptors on blood cells in renal transplant recipients. The determination of free receptor binding sites on lymphocytes revealed 214 receptor molecules/lymphocyte compared with 328 molecules/lymphocyte in healthy controls (p < 0.157). This explains why symptoms of hypoaldosteronism are seen in transplant recipients despite normal aldosterone concentrations in the blood. Recently, Matsuda et al described the binding of cyclosporine A to immunophilins, which interact with the receptor in its non-functional status.¹¹ The formation of this complex might fix the receptor in the cytoplasm, thereby reducing the number of free receptor binding sites on the lymphocyte membrane. In addition, a decrease in the rate of transcription of the hMR gene could also explain the decreased number of hMRs on each lymphocyte. Indeed, further analysis by quantitative RT-PCR with hMR specific primers revealed decreased transcription of the gene in patients undergoing cyclosporine A treatment.

The mean number of hMR RNA molecules/0.5 µg RNA dropped from 468 molecules in untreated controls to 169 molecules in renal graft recipients (p < 0.002). The fact that the reduction was greater at the transcriptional level than at the protein level suggests that translation rates might be increased to compensate for the missing RNA molecules.

“It would be useful to find immunosuppressive drugs without side effects on mineralocorticoid receptor expression”

Our study further showed that both methods (the determination of receptor binding sites and quantitative PCR) are equally suitable for detecting the reduced number of aldosterone receptors on blood cells. Nevertheless, quantitative PCR—at least in laboratories where PCR technology is established—has some advantages, as listed in fig 4. Goerke et al described it as a robust technique, which is not susceptible to traces of PCR inhibitors in contrast to LightCycler RT-PCR.⁸ In particular, continuous control of hMR expression during the treatment of hyperkalaemia and metabolic acidosis is possible only with quantitative PCR, and it could be useful in the search for drugs that not only repair ion transport in the renal tube but also increase receptor expression on the cells. Furthermore, it would be useful to find immunosuppressive drugs without side effects on hMR expression.

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Figure 4

Comparison of the advantages and disadvantages of competitor reverse transcription polymerase chain reaction (cRT-PCR) and radioreceptor assay for the determination of mineralocorticoid receptor expression in blood cells.