1 Department of Medicine and 2 Department of Biostatistics, University of Wisconsin, Madison, WI, USA
Correspondence and offprint requests to: Arjang Djamali, MD, 3034 Fish Hatchery Road, Suite B, Madison, WI 53713, USA. Email: axd{at}medicine.wisc.edu
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Abstract |
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Methods. To determine if low Mg levels correlate with true CsA-induced nephrotoxicity in humans, we examined kidney transplant biopsy records at our centre for all transplant biopsies performed between 1990 and 2002. We simultaneously reviewed the medical records to determine whether serum Mg levels were checked at the time of biopsy. Those individuals with histologically proven CsA nephrotoxicity were studied.
Results. Serum total Mg levels were available for 320 patients, 60 of whom were diagnosed with chronic CsA-mediated nephropathy. Patients were divided in two groups, a low Mg [n = 29, 1.8 (1.671.9) mg/dl or 0.74 (0.680.78) mmol/l] and a normal Mg group [n = 31, 2.2 (2.02.4) mg/dl or 0.9 (0.820.98) mmol/l, P<0.05] based on the median Mg level in the entire cohort (2 mg/dl or 0.82 mmol/l). Both groups were analysed for disease progression and graft loss using the slope of creatinine clearance (CCR) and multivariate analyses. Although the CCR at the time of biopsy was greater in the low Mg group [44.3 (36.364.3) ml/min vs 37.8 (25.247.3) ml/min, P<0.05), the decline in graft function was faster in this group (8.9±3.5 vs 1±2.7 ml/min/year; P = 0.02) compared with the normal Mg cohort. Using Cox proportional hazards analyses, the adjusted graft survival was significantly reduced in the low Mg group 5 years after biopsy.
Conclusions. Our study demonstrates that low serum Mg levels were associated with a faster rate of decline in kidney allograft function and increased rates of graft loss in renal transplant recipients with chronic CsA nephropathy. This suggests that hypomagnesaemia could potentiate CsA-mediated nephropathy.
Keywords: allograft; cyclosporin; kidney; magnesium; outcomes; transplantation
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Introduction |
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CsA, the first clinically used CNI, has several forms of nephrotoxicity [4,7]. It has been implicated as an aetiological agent inducing thrombotic microangiopathy. It can also mediate acute nephrotoxicity, illustrated by haemodynamic events mainly secondary to afferent arteriole vasoconstriction, without permanent structural injury. Finally, CsA can lead to a chronic form of allograft injury, characterized by progressive and irreversible renal interstitial fibrosis and arteriolar hyaline changes [4,7].
A number of different strategies have been attempted to stave off chronic CsA nephrotoxicity. Clinically, these include dose reduction or CsA withdrawal [8]. However, the profound beneficial effects of CNIs in preventing acute rejection often limit these approaches. Therefore, interest continues in identifying other means to use these drugs without incurring their detrimental kidney effects. Hypomagnesaemia and renal magnesium (Mg) wasting are common findings in CsA-treated animals and in human kidney transplant recipients [914]. This has raised the hypothesis that Mg wasting or deficiency might contribute to chronic CsA nephrotoxicity [9,1517]. Interestingly, Mg supplementation for CsA-treated animals leads to a reduction in the DNA-binding activity of activator protein 1 and nuclear factor-B [15]. It may further inhibit local inflammation by decreasing osteopontin and monocyte chemoattractant protein-1 (MCP-1) expression [16]. Such findings suggest that Mg may limit chronic injury in the setting of CsA therapy. The first step in defining whether hypomagnesaemia plays a role in CsA-mediated nephrotoxicity is to validate the association between low Mg levels and chronic CsA nephrotoxicity in humans. We undertook an examination of our kidney transplant population to answer that question.
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Patients and methods |
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Patients were then subdivided into two groups: low Mg and normal Mg based on the median serum Mg level in the entire study group. A control cohort of patients with similar kidney allograft function, CsA-based therapy and available serum Mg levels, but without histopathological diagnosis of chronic CsA nephrotoxicity, was also included.
Laboratory measurements were performed at our core laboratory facility using the methylthymol blue (serum Mg), kinetic alkaline picrate (serum creatinine), Abbott Cell-Dyn (haematocrit), liquid chromatography/mass spectrometry (whole blood CsA) and pyrogallol red (urine protein) methods.
Demographic data were reported on age, gender, ethnicity, native kidney disease, type of renal transplant, serum creatinine and creatinine clearance (CCR) at the time of biopsy, as well as haematocrit levels, proteinuria and use of diuretics, angiotension-converting enzyme inhibitors (ACEIs) or angiotension receptor blockers (ARBs). Patients were considered lost to follow-up when the final outcome was unknown or if there was a failure to present at their last clinic appointment, or review of medical records failed to reveal if they had reached end-stage renal disease (ESRD), died or moved. The study was performed in accordance with the University of Wisconsin Health and Human Subjects Committee and HIPAA guidelines.
Disease progression and outcomes
Outcomes were defined by patient and kidney survival rates and presented by KaplanMeier survival curves, as previously described [3]. Patient and kidney survival time was defined as the time interval between T1 (time of the biopsy) and T2 (time of last visit, ESRD or patient death). The CockcroftGault formula: CCR = (140 age) x weight (kg)/serum creatinine (mg/dl) x 72, multiplied by 0.8 in female individuals was used to estimate kidney function as defined previously [3]. Creatinine clearance 1 (CCR1) was determined using the serum creatinine at the time of biopsy. Creatinine clearance 2 (CCR2) was calculated using the serum creatinine at the last follow-up appointment, ESRD or patient death. The slope of the CCR was determined using the two time points, as a measure of disease progression. Outcome measures included differences in disease progression, patient survival and kidney survival during the follow-up time interval (between T1 and T2).
Statistical analysis
The KolmogorovSmirnov probability test was used to determine the normal distribution of numerical data. Two sided t-test or MannWhitney rank sum test were used to compare parametric and non-parametric numerical data, respectively. Fisher's exact test was performed to analyse nominal data. Patient and kidney survival rates, as well as the relative risk of death and kidney failure, were assessed using Cox proportional hazards models adjusting for gender, CCR, age, mean arterial blood pressure, serum calcium, sodium, bicarbonate, albumin and uric acid levels. Relative risks were expressed as the probability of death/kidney failure in the low Mg compared with the normal Mg group. A likelihood ratio test was used to test for significance. The Spearman rank order test was utilized to determine the correlation between serum Mg levels, CCR1 and CsA levels. Non-parametric data were reported as median and (2575% percentiles), whereas parametric data were noted as mean±SE. Data analysis was carried out in S-PLUS (MathSoft 1997) and SigmaStat (SPSS 3.0). A P-value of 0.05 was considered significant.
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Results |
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Creatinine clearance and disease progression
Calculated creatinine clearance values at the time of biopsy (CCR1) were significantly higher in the low Mg group compared with the normal Mg group as determined by both the MannWhitney rank sum test and the t-test [44.3 (36.364.3) vs 37.8 (25.247.3) ml/min, corresponding to mean (±SE) values of 49.9±3.6 and 38.3±2.9 ml/min, respectively, P = 0.01, Figure 2a]. CCR2 levels, however, were similar in both groups [42 (1055.8) and 37± 5.1 ml/min in the low Mg and normal Mg groups, corresponding to mean (±SE) values of 37.2±4.6 and 37±5.1 ml/min, respectively]. As CCR1 levels were between 30 and 60 ml/min, all studied individuals could be placed in stage III of chronic kidney disease based on the National Kidney Foundation classification of chronic kidney disease [18]. However, the rate of disease progression characterized by the slope of CCR between CCR1 and CCR2 was significantly faster in the low Mg group compared with the normal Mg group (8.9±3.5 and 1±2.7 ml/min/year, respectively, P = 0.02). Figure 2b depicts linear regression of CCR changes based on the normally distributed follow-up time between the two groups.
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Outcomes
Patient and kidney outcomes were determined using multivariate regression analyses adjusting for gender, CCR, age, mean arterial blood pressure, serum calcium, sodium, bicarbonate, albumin and uric acid levels. These analyses revealed no difference in overall patient survival (90.5 vs 91.5%, NS, Figure 3a). However, consistent with the observed rates of disease progression, kidney survival was significantly lower in the low Mg group (P<0.05 by the likelihood ratio test, Figure 3b). The results remained unchanged when the Wald test or Efficient Score tests were utilized. Figure 3b depicts the KaplanMeier survival curves and the number of kidneys lost to ESRD or patient death in each group. The number of censored patients is also represented. The adjusted relative risk (RR) of graft loss was 35% higher in the low Mg compared with the normal Mg group [RR = 0.65, confidence interval (CI) 0.41.4].
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Discussion |
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Seventy to 80% of serum Mg is freely filtered at the glomerulus and most (up to 97%) is reabsorbed throughout the nephron [19]. The molecule is reabsorbed through both active and passive mechanisms, and the thick ascending limb represents the segment responsible for most (6070%) of the reabsorption [19]. Although the effects of CsA on Mg cellular transport mechanisms are not completely elucidated, decreased Mg reabsorption, urine Mg wasting and hypomagnesaemia have clearly been described in animal models [10,11] and renal transplant recipients receiving CsA [1214].
Whether hypomagnesemia per se contributes to CsA nephropathy, or whether Mg supplementation may lessen the CsA nephropathy is a point of debate. Mg supplementation can inhibit or even prevent renal fibrosis in experimental models of CsA toxicity [9,1517,20]. In an in vitro study of renal proximal epithelial cells, Carvalho et al. recently demonstrated that Mg supplementation attenuates direct CsA-mediated cellular toxicity [20]. Similarly, studies by Miura et al. highlighted the efficacy of Mg supplementation in reducing interstitial fibrosis, tubular atrophy, arteriolopathy and nephrocalcinosis in an animal model of CsA nephrotoxicity [9]. These investigators showed that the mRNA levels for fibrotic molecules (collagens type I and IV and fibronectin EIIIA) were significantly lower in Mg-treated animals, suggesting an antifibrotic role for Mg in this setting. Asai et al. compared the effects of Mg supplementation with ACE inhibition (benazepril 4 mg/kg orally by gastric tube), in the same experimental model [15], and noted that Mg supplementation alone significantly reduced interstitial fibrosis compared with CsA and the combination of CsA plus benazepril. Other studies have suggested that oral Mg supplementation can blunt CsA's fibrotic effects through the inhibition of interstitial inflammation, chemoattractant molecules (osteopontin and MCP-1) [16] and renal dopaminergic deficiency [17].
However, human data are limited. Although CsA-treated kidney transplant recipients clearly have a higher incidence and prevalence of hypomagnesaemia [1214], it is unknown whether low serum Mg levels affect disease progression and long-term kidney allograft outcome.
In this small cohort of patients, we were able to demonstrate that patient survival was not affected by low serum Mg levels. However, kidney allograft survival was significantly reduced in patients diagnosed with both hypomagnesaemia and chronic CsA nephrotoxicity. Our study may have suffered from a selection bias. Indeed, the two groups had significantly different gender distribution and creatinine clearance levels at the time of biopsy. More female subjects in the low Mg group may have contributed to decreased serum creatinine and Mg levels due to a lower muscular mass. Similarly, lower creatinine clearance levels in the normal Mg group may have contributed to higher Mg levels as serum Mg levels may rise with declining renal function. However, Cox proportional hazards analyses performed to adjust outcomes for these covariates found that gender, CCR, age, serum calcium, sodium, bicarbonate, albumin and uric acid levels had no significant impact on the observed results. Given that the median Mg level in the low Mg group was at 1.8 mg/dl (0.74 mmol/l), the current study emphasizes that low-normal Mg levels should not be overlooked in renal transplant recipients with chronic CsA nephrotoxicity. The low prevalence of hypomagnesaemia in patients without histopathological findings of CsA nephrotoxicity is an additional point supporting the importance of low serum Mg levels in these patients. Moreover, this may be applicable to all transplant patients, not just kidney transplant recipients. The epidemic of chronic kidney disease clearly affects non-kidney solid organ transplant recipients [6]. Hypomagnesaemia may be a factor also potentiating this process.
Several issues may relate directly to Mg measurement. Although data on ionized Mg levels were not available in our study, it has been shown that both total and ionized levels of Mg are decreased in CsA-treated renal transplant recipients [14]. Similarly, serial levels and urine Mg excretion rates in these patients would have added more light on the impact of Mg level fluctuations on outcomes. However, the retrospective nature of this study obviated this approach as Mg levels were not determined in all patients on a regular basis.
In summary, our study demonstrates that low serum Mg levels were associated with a faster rate of decline in kidney allograft function and increased rates of graft loss in renal transplant recipients with chronic CsA nephropathy. This suggests that hypomagnesaemia may potentiate CsA-mediated nephropathy.
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Acknowledgments |
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Conflict of interest statement. None declared.
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References |
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