Abstract
Introduction:
Aldosterone participates in the pathogenesis of calcineurin inhibitor nephrotoxicity (CIN), producing renal vasoconstriction and transforming growth factor beta (TGFß) expression. The objective of this study was to assess aldosterone polymorphisms and relationships to plasma aldosterone levels and the development of renal histological lesions in kidney transplant patients.
Material and methods:
Patients with kidney graft biopsy were divided according to the presence or absence of CIN. We determined aldosterone synthase (AS) -344 T/C and int 2 W/C gene polymorphisms and plasma aldosterone levels. Histological, biochemical and clinical variables were measured.
Results:
Calcineurin inhibitor (CI) levels were significantly higher in patients with the int 2 WW genotype than in patients with WC or CC genotypes. There was a greater degree of interstitial fibrosis in patients with int 2 CC genotype. No relationship was found between the different polymorphisms and a higher degree and/or frequency of CIN. There was also no relationship with plasma aldosterone levels.
Conclusion:
The frequency of the different polymorphisms studied was not related to plasma aldosterone levels or the development of CIN; however, the int 2 CC genotype was related to a greater degree of interstitial fibrosis, whereas the WW genotype was related to higher CI serum levels.
Keywords
Introduction
The benefits of calcineurin inhibitors (CI) are vast, both in the field of solid organ transplants and autoimmune diseases, nevertheless long-term use is limited due to potential side effects. From a clinical standpoint, nephrotoxicity is the most common as well as the most relevant side effect. 1 In a recent study, Nankivell and colleagues, showed that after 10 years of CI treatment, 100% of transplanted patients presented with nephrotoxicity. 2 Chronic nephrotoxicity develops with long-term CI treatment and is mainly characterized by arteriolopathy and tubulointerstitial fibrosis.3,4 Calcineurin inhibitor nephrotoxicity (CIN) is accompanied by activation of the renin-angiotensin system (RAS) and increased expression of transforming growth factor beta (TGFβ), which may be regulated by aldosterone. In experimental models of CIN, the blockade of aldosterone receptors has been associated with increased survival of experimental animals. 5 Aldosterone may promote development of CIN by vasoconstriction and induction of TGFβ expression. 6 The presence of polymorphisms in the aldosterone synthase (AS) gene can influence the activity of aldosterone.7 –9 Two polymorphic variations of this gene have been studied. The first one corresponds to a single nucleotide polymorphism called -344 T/C, which corresponds to the change of thymine/cytosine at the -344 position, altering the binding site for the steroidogenic factor 1 of the promoting 5’ transcriptional region. It is believed that this change alters the sensitivity of the gene for angiotensin II, causing an inappropriately high production of aldosterone as related to renin levels. 10 The -344T allele has been associated to an increased risk of hypertension.10,11 Another polymorphism called int 2 W/C, involves conversion of intron 2 of the AS gene, which is partially replaced by the corresponding intron of the CYP11B1 enzyme gene. Its presence has been associated with hypertension and a higher aldosterone-renin ratio.7,12 To the best of our knowledge, there has been no study to analyze the relationship between polymorphism variations of the SA gene and development of CIN.
Materials and methods
Subjects
Patients with renal transplantation and kidney graft biopsy were included between January 2000 and December 2009. All kidney graft biopsies were performed according to protocolized Nephrology Department guidelines. The first group included patients with histological diagnosis of CIN (n=24); the second group included patients in whom CIN (n=55) was dismissed. Clinical data were documented. The ethics committee of our institution approved the clinical protocol.
DNA extraction
Genomic DNA from whole blood containing ethylenediaminetetraacetic acid (EDTA) was isolated using the standard technique. 13
Determination of the CYP11B2 genotypes
Two polymorphisms in the AS gene were studied (-344 C/T located in the promoter region, and int 2 W/C located in the second intron). The -344C/T (rs1799998) single nucleotide polymorphism was genotyped using 5’ exonuclease TaqMan genotyping assays on a 7900HT Fast Real-Time Polymerase chain reaction (PCR) system, according to manufacturer’s instructions (Applied Biosystems, Foster City, USA). On the other hand, the Int 2 W/C polymorphism was detected by allele-specific PCR as previously reported. 10 Reactions were made separately, using primer 1 with reverse primer and primer 2 with reverse primer. Used primers were: (primer 1) 5’-TGGAGAAAAGCCCTACCCTGT-3’ and (reverse primer) 5’-AGGAACCTCTGCACGGCC-3’ for the wild type allele and (primer 2) 5’-CAGAAAATCCCTCCCCCCTA-3’ and (reverse primer) 5’-AGGAACCTCTGCACGGCC-3’ for the variant. PCR conditions were as follows: (a) 94°C for 2 min (b) 94°C for 1 min, 30 s at 66°C and 40 s at 72°C (repeated for 30 cycles), and (c) a final 7 min extension at 72°C. Amplifications were performed in a Perkin Elmer PCR System thermocycler, model 9700 from Applied Biosystems. A 1.8% agarose gel with ethidium bromide staining was visualized, using a UV Transilluminator.
Plasma aldosterone levels
Plasma aldosterone levels of a peripheral blood sample obtained at rest in the supine position were measured and analyzed using a radioimmunoassay method. All patients received a diet with a daily salt intake of 3–4 g.
Histological analysis
The kidney biopsy was taken according to the previously described standard technique; one to three cylindrical fragments of renal cortex, processed for light microscopy, were studied. Tissues were processed according to standard techniques and were embedded in paraffin to obtain 3 µm sections. Sections for light microscopy were stained with hematoxylin and eosin, periodic acid-Schiff (PAS), methenamine silver, and Masson’s trichrome. They were observed under the microscope at 200× and 400× magnification, in consecutive fields and graded histologically according to the Banff classification. Histopathological CIN criteria were presence of hyaline nodules and/or grade 2 or 3 arteriolopathy.
Statistical analysis
Results are stated as mean±standard deviation (SD) or proportions. Comparison of means between two groups was performed using the Student‘s t test for independent samples or its nonparametric alternative (U of Mann Whitney). Comparison of proportions between groups was performed using the chi-square (Χ2) test. Comparison of variables between study groups divided by genotype was performed with one-factor analysis of variance (ANOVA), using the Bonferroni test for multiple comparisons of means. Spearman’s Rho coefficient was used to search for associations. All probabilities were two-tailed and the level of significance was set at 0.05. Data were analyzed using the SPSS statistical package, version 15 for Windows.
Allele and genotypic frequencies of AS gene polymorphisms were determined by direct counting. Hardy-Weinberg equilibrium using the X2 test was assessed in each study group. Differences between groups were determined by using the Mantel-Haenzel X2 test, which combines 2×2 contingency tables. When any cell number being compared was less than five, Fisher’s exact test was used. Relative risk with a confidence interval of 95% was assessed as an odds ratio using the Woolf method.
Results
We studied a total of 79 patients who received a kidney transplant and immunosuppressive therapy, including a calcineurin inhibitor, at the Nephrology Department, National Institute of Cardiology in the period between January 2000 and December 2009. Patients were divided into two groups: group A included 55 patients with a renal graft biopsy that ruled out CIN, and group B included 24 patients with a CIN diagnosis. Biopsies (Figures 1 and 2) with any of the following histological criteria were considered positive for CIN: hyaline nodules and/or arteriolopathy grade 2 or 3. Of all the cases studied, 46 were men (58%) and 33 were women (42%). Group A consisted of 33 men (60%) and 22 women (40%) and group B included 13 men (54%) and 11 women (46%). Mean age was 30.3±10.5 years (range: 15–61 years) in all 79 patients; 29.2±9.9 years (range: 15–56 years) in group A and 33.3±12.2 years (range: 16–61 years) in group B. Clinical and demographic characteristics are shown in Table 1.
Renal parenchyma with a narrow zone of interstitial fibrosis with sclerotic glomeruli and atrophic tubules (top left of the picture) alternating with normal interstitial areas retaining the pattern ‘back to back’ of the tubules (bottom right of the picture). (10× Masson trichrome.)
Renal parenchyma with a central well-defined band of interstitial fibrosis and hypoperfused glomeruli. At the top and bottom of the band the parenchyma is conserved. (10× Masson trichrome.)
Clinical characteristics related to calcineurin inhibitor nephrotoxicity (CIN).
BMI: body mass index; BUN: blood urea nitrogen; CI levels: serum levels of calcineurin inhibitor; DBP: diastolic blood pressure; SBP: systolic blood pressure; UP: urinary protein.
Values are shown as mean±standard deviation (SD).
At the time the biopsy was performed, systolic and diastolic blood pressures were significantly higher in group B than in group A.
Post-transplant median follow-up for group A was 16 months (interquartile range (IQR) 25–75: 6–34) and for group B, 16.5 months (IQR 25–75: 4–25), (p=not significant (NS)).
Development of post-transplant hypertension was found in 71% of patients in group B and in 53% of patients in group A (p=NS). There was no significant difference between groups comparing the proportion of cases using antihypertensive drugs, including angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers.
Mean uric acid serum level for both groups was 7.2±2.1 mg/dl (range: 3.6–12.6 mg/dl), being significantly higher in group B. There was no significant difference in plasma aldosterone levels or in any of the other biochemical variables analyzed. From all patients tested, we were able to obtain blood samples to determine the genetic polymorphisms only in 54 patients: 40 in group A and 14 in group B.
For the -344 T/C polymorphism, we found four homozygous TT individuals (28.5%) in group B, compared to 22 (55%) in group A (p=NS). We found the CC genotype in three cases (21.4%) in group B and 9 cases (22.5%) in group A (p=NS). As to the TC heterozygous genotype, there were seven (50%) cases in group B and nine cases (22.5%) in group A (p=NS). Regarding the int 2 W/C polymorphism, we found seven homozygous WW individuals (50%) in group B compared to 19 (47.5%) in group A (p=NS). The CC genotype was present in one patient in group B (7.1%) and six patients in group A (15%) (p=NS). The WC heterozygous genotype was present in 6 cases in group B (42.8%) and 15 cases (37.5%) in group A (p=NS).
Clinical and biochemical variables were compared according to each genotype and results are shown in Tables 2 and 3. When comparing the different genotypes of the -344 T/C polymorphism, we found no significant differences in the analyzed variables. Regarding the int 2 W/C polymorphism, the patients expressing the wild WW homozygous genotype exhibited higher levels of CI at the time of graft biopsy, compared to cases with WC and CC genotypes. Likewise, aldosterone plasma levels, according to each genotype, were compared. In the case of the int 2 W/C polymorphism, a tendency for higher levels of aldosterone was found in cases with the CC genotype. Plasma aldosterone levels also tended to be higher in cases with the -344 TC genotype. No significant differences were found when comparing the frequency of post-transplant hypertension according to genotypes (Table 4).
Clinical and biochemical characteristics according to genotypes of -344 T / C polymorphism.
BUN: blood urea nitrogen; CI levels: serum levels of calcineurin inhibitor; DBP: diastolic blood pressure; SBP: systolic blood pressure, UP: urinary protein.
Values are shown as mean±standard deviation (SD).
Clinical and biochemical characteristics according to the genotypes of int 2 W/C polymorphism.
BUN: blood urea nitrogen; CI levels: serum levels of calcineurin inhibitor; DBP: diastolic blood pressure; SBP: systolic blood pressure; UP: urinary protein.
Values are shown as mean±standard deviation (SD).
Patients with post-transplant hypertension according to genotypes of -344 T/C and int 2 W/C polymorphisms.
Values represent percentage of patients with or without a diagnosis of post-transplant hypertension, grouped according to genotypes.
The presence of the different polymorphisms studied was not significantly related to the frequency and degree of CIN, however cases with the int 2 CC genotype showed a significantly higher degree of interstitial fibrosis compared to all other groups (Table 5).
Relationship between int 2 W/C genotypes and interstitial fibrosis.
Values represent the percentage of cases for each genotype according to the degree of interstitial fibrosis.
p<0.05 vs WC and WW.
Discussion
The study’s objective was to determine the frequency of -344 T/C and int 2 W/C polymorphisms and to assess whether there is any risk associated to the genotype for development of CIN in patients with a kidney transplant. Numerous research studies have assessed the relationship between AS gene polymorphisms and aldosterone synthesis and secretion in diseases in which this hormone has been implicated.7,10 –12 When assessing aldosterone plasma levels in this study, as related to the distribution of allelic and genotypic frequency in patients with and without CIN, no significant differences were found between groups. Nevertheless, we showed a tendency toward higher levels of aldosterone with C allele of the int 2 W/C polymorphism. Regarding the -344 T/C polymorphism, the TC genotype was related to a higher aldosterone level. However, in both cases, the difference did not reveal statistical significance, maybe due to the small sample size.
In a study with hypertensive subjects, Mulatero and colleagues analyzed the frequency of the -344 T/C polymorphism and its relationship with aldosterone plasma levels, finding that a stronger expression of the C allele was related to higher levels of this hormone. 7 In another study performed in individuals with high blood pressure, Lim and colleagues analyzed the aldosterone/renin ratio and its relationship to AS gene polymorphisms, describing how the presence of the T allele in the -344 T/C polymorphism and the C allele in the int 2 W/C polymorphism was accompanied by a higher aldosterone/renin ratio. 14 In contrast, Tamaki and colleagues reported that the C allele in the -344 T/C polymorphism was associated with a higher aldosterone/renin ratio in Japanese patients. 15
On the other hand, Nejatizadeh and colleagues studied the relationship of AS gene polymorphisms, plasmatic activity of renin and serum levels of aldosterone with the risk for developing hypertension. 16 They found the T (-344 T/C) C (int 2 W/C) was a risk haplotype for the development of hypertension, as well as for increased serum aldosterone levels and higher aldosterone/renin ratios. In this study, no relationship was found between higher levels of aldosterone and development of post-transplant hypertension.
It is also important to point out that in our study we observed higher uric acid serum levels in the CIN group. Recent studies have considered the role of uric acid in endothelial dysfunction; with plasmatic levels of over 6 and 9 mg/dl, causing cellular aging and death, respectively. Implicated mechanisms are activation of local oxidative stress and over-regulation of some RAS components. 17 These effects can be reduced through pharmacological blockade of the RAS. Because hyperuricemia is considered an independent cardiovascular risk factor, as well as a factor implicated in progression of kidney diseases, 18 there is a need to perform more studies to assess this aspect, with the possibility of considering it as an early marker of vascular damage associated with CI administration.
When assessing renal histological alterations related to CI toxicity, we did not find any relationship between the frequencies of different genotypes and a higher degree of CIN defined by the presence of hyaline nodules and/or arteriolopathy. However, when we analyzed the degree of interstitial fibrosis, it was discovered that patients with the conversion homozygous genotype (CC) of int 2 W/C showed a greater degree of interstitial fibrosis, compared to the WW and WC genotypes (p=0.03). In this case, 42.9% and 14.3% of the patients with CC genotype presented grade II and grade III interstitial fibrosis. This result suggests that the main pro-fibrotic pathways may be dependent on other mediators different from aldosterone, such as TGFβ. However, this association should be made with caution due to the small sample size studied and because 28.6% of the patients with CC genotype showed no fibrosis. Additional studies in a large number of patients could help to define the true role of this marker in interstitial fibrosis. It also should be mentioned that in cases with the CC genotype of int 2 W/C polymorphism, we observed a tendency for higher diastolic blood pressure and proteinuria at the time the biopsy was taken, higher levels of serum creatinine a year after graft biopsy, a lower glomerular filtration rate at 37 months of follow-up, as well as an adverse metabolic profile manifested by a higher body mass index and serum glucose (data not shown).
Likewise, we found that the presence of the CC genotype of int 2 W/C polymorphism was related in a non-significant manner with a higher degree of tubular atrophy and arteriolopathy. This suggests that patients with this genetic profile could be at greater risk for accelerated deterioration of renal function, which should be assessed in future studies. Regarding the int 2 W/C polymorphism, the CC genotype was related to a higher degree of interstitial fibrosis and the WW genotype was related to higher CI serum levels. The frequency of the specific polymorphisms studied was not related to plasma aldosterone levels or development of CIN. However, our data are preliminary due to the study sample size and additional studies in a larger number of individuals and in other populations could help define the true role of these polymorphisms as risk factors for developing CIN.
Footnotes
Acknowledgements
The authors are grateful to the study participants. Institutional Review Board approval was obtained for all sample collections.
Conflict of interest
None declared.
Funding
This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.