Low serum magnesium is associated with decreased graft survival in patients with chronic cyclosporin nephrotoxicity

Ryan Holzmacher1, Christina Kendziorski2, R. Michael Hofman1, Jonathan Jaffery1, Bryan Becker1 and Arjang Djamali1

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



   Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Background. Hypomagnesaemia is a common side effect of cyclosporin A (CsA) therapy. Animal studies suggest that magnesium (Mg) supplementation inhibits chronic CsA nephropathy.

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 (1.67–1.9) mg/dl or 0.74 (0.68–0.78) mmol/l] and a normal Mg group [n = 31, 2.2 (2.0–2.4) mg/dl or 0.9 (0.82–0.98) 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 (36.3–64.3) ml/min vs 37.8 (25.2–47.3) 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



   Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Despite tremendous recent advances in transplantation, chronic renal allograft dysfunction remains a major cause of graft loss [1]. Immune and non-immunological factors continue to affect allograft survival [1,2]. These observations come at a time when there is increased emphasis on examining rates and mechanisms of disease progression in transplantation [3]. Calcineurin inhibitors (CNIs), e.g. cyclosporin A (CsA) or tacrolimus, play a significant role in the pathogenesis of disease progression in kidney as well as non-kidney solid organ transplantation [1,2,4–6].

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 [9–14]. This has raised the hypothesis that Mg wasting or deficiency might contribute to chronic CsA nephrotoxicity [9,15–17]. Interestingly, Mg supplementation for CsA-treated animals leads to a reduction in the DNA-binding activity of activator protein 1 and nuclear factor-{kappa}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.



   Patients and methods
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Patient selection and CSA toxicity
We performed a retrospective study of all available kidney allograft biopsies performed for a rise in serum creatinine levels (n = 3150 biopsies) at our institution between 1990 and 2002. As the study was aimed to determine the effect of serum Mg levels on kidney allograft outcomes in patients with chronic CsA nephrotoxicity, the following additional selection criteria were included: available serum total Mg levels at the time of biopsy, biopsies performed after the sixth post-transplant month, CsA-based immunosuppression and histopathological diagnosis consistent with chronic CsA-induced nephropathy. This diagnosis was characterized by tubular atrophy, striped interstitial fibrosis between the medulla and the medullary rays of the cortex, and arteriolar hyaline changes consisting of endothelial swelling, areas of smooth muscle lesion±necrosis and nodular hyaline protein deposition. These biopsies were then reviewed and semi-quantitative assessment of pathological findings was performed based on a scale of 0–3 (0 = normal, 1 = mild, 2 = moderate and 3 = severe lesions).

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 Kaplan–Meier 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 Cockcroft–Gault 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 Kolmogorov–Smirnov probability test was used to determine the normal distribution of numerical data. Two sided t-test or Mann–Whitney 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 (25–75% 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.



   Results
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Baseline characteristics
Serum total Mg levels at the time of biopsy were available for 320 individuals. Sixty of these individuals had the diagnosis of chronic CsA nephrotoxicity on biopsy. The low (n = 29) and normal (n = 31) Mg groups were then determined based on the median Mg level in all 60 patients (see the following section on serum Mg levels). There was a relatively homogenous distribution of baseline characteristics between the two groups. However, there were significantly fewer male patients in the low Mg group (45 vs 65 P<0.05). Interestingly, serum creatinine levels at the time of biopsy were significantly lower in this group (2.2±0.2 vs 2.9±0.2 mg/dl, P<0.05). There were no significant differences in age, race, ethnicity, transplant type, native kidney disease, prevalence of post-transplant hypertension, time to biopsy, delayed graft function, acute rejection episodes, proteinuria and anaemia (Table 1). All patients had received induction immunosuppression with antibodies (OKT3, thymoglobin or anti-interleukin-2 receptor antibodies) and a CsA-based maintenance regimen. No significant differences in immunosuppressive drugs were observed between the two groups (data not shown). Notably, CsA daily dose and levels at the time of biopsy were not significantly different between the groups (7.5±2.5 vs 8.2±3.6 mg/kg/day and 227±30 vs 248±64 ng/ml in the low and normal Mg groups, respectively, Table 1). Mean arterial pressure (96.2±4.8 vs 98.2±5.6 mmHg) and urine protein:creatinine ratios (1005±206 vs 920±110 mg/g) were not significantly different between the two groups (Table 1). Similarly, serum Na (140±4 vs 138±3 mmol/l), bicarbonate (19±4 vs 20±5 mmol/l), albumin (3.9±1.5 vs 3.8±2.2 mg/dl), uric acid (9.5±4.2 vs 8.9±5.8 mg/dl) and Ca (8.2±2.9 vs 7.8±4.3 mg/dl) levels were not significantly different between the two groups (data not shown in the table). There were no patients taking scheduled Mg supplements in either group. Eight and nine patients were on scheduled dose diuretics in the low Mg and normal Mg groups, respectively. In the low Mg group, four patients were on 30±11 mg a day of furosemide and four patients were on 31±12.5 mg a day of hydrochlorothiazide. One patient was also on 12.5 mg a day of spironolactone. There was no significant difference between the two groups in the class or daily dose of diuretics (Table 1). Seven patients in each group were on either an ACEI or an ARB. The average daily doses of lisinopril and losartan were 10±7 (n = 4) vs 11±5.5 mg (n = 5), and 66.6±28 (n = 3) vs 50 mg (n = 2) in the low Mg and normal Mg groups, respectively (NS) (data not shown in the table).


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Table 1. Baseline characteristics

 
Serum Mg levels, chronic CsA nephrotoxicity severity score, follow-up rate and follow-up interval
Serum Mg levels were not normally distributed in the study group (Kolmogorov–Smirnov normality test failed, with a skewness of 3.6 and probability <0.001, Figure 1). Median and 25–75% percentile values were 2 (1.8–2.2) mg/dl, corresponding to 0.82 (0.74–0.9) mmol/l, using the conversion formula Mg (mg/dl) x 0.4114 = Mg (mmol/l). Thus, median Mg levels were used as the cut-off value to determine the low Mg [Mg <2 mg/dl, median 1.8 (1.67–1.9) mg/dl or 0.74 (0.68–0.78) mmol/l, n = 29] and normal Mg [Mg ≥2 mg/dl, median 2.2 (2.02–2.4) mg/dl or 0.9 (0.83–0.99) mmol/l, n = 31] groups (Figure 1). Patients with normal graft function had Mg levels within the 1.7–2.3 mg/dl range, corresponding to 0.69–0.94 mmol/l (data not shown). Not surprisingly, serum Mg levels were significantly lower in the low Mg group (P<0.001). There was no correlation between serum Mg and CsA levels or between serum Mg and CCR1 levels. The average severity score of striped fibrosis (2.27±0.64 vs 1.9±0.76), arteriolar hyaline changes (2.4±0.6 vs 2.2±1.2) and tubular atrophy (2±0.8 vs 2.3±1.3) was not statistically different between the low Mg and normal Mg groups, respectively.



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Fig. 1. Serum magnesium levels at the time of biopsy in renal transplant recipients with chronic cyclosporin A (CsA) nephropathy. Median serum Mg levels and 25–75% percentile values are represented with the box-plot format.

 
The follow-up interval, defined as the time between CCR1 and CCR2, was distributed normally between the two groups (Kolmogorov–Smirnov test for normality passed with a probability value of 0.68 and 0.094 for the low Mg and normal Mg groups, respectively). This interval was not statistically different between the two groups: 62.8±7 months for the low Mg group and 69.1±8 months for the normal Mg group (NS). The rate of follow-up was 86% in the low Mg group and 81% in the normal Mg group (NS). Subsequent to the diagnosis of CsA nephrotoxicity, the drug dose was reduced in all patients (25±20 and 30±18% dose reduction in the low Mg and normal Mg group, respectively) and discontinued in two individuals per group (NS).

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 Mann–Whitney rank sum test and the t-test [44.3 (36.3–64.3) vs 37.8 (25.2–47.3) 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 (10–55.8) 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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Fig. 2. Low magnesium levels at biopsy were associated with a faster rate of disease progression in patients with chronic CsA nephropathy. (a) Box plots representing creatinine clearance (CCR1, median and 25–75% percentile values) at the time of biopsy. (b) Linear regression of CCR changes based on the normally distributed follow-up time between the two groups. Slopes are represented as median and 25–75% percentile values.

 
To determine the prevalence of hypomagnesaemia in a matched group of patients belonging to stage III chronic kidney disease but without biopsy evidence of CsA nephrotoxicity, a control cohort of individuals from the same pool of 260 patients was studied. Fifty-four individuals had both available serum Mg levels and a CCR1 level between 30 and 60 ml/min. Only nine patients (17%) had serum Mg levels lower than 2 mg/dl. These levels were significantly lower than the normal Mg group [1.8 (1.7–1.8) vs 2.5 (2.27–2.8) mg/dl, respectively, P<0.001]. However, there were no significant differences in the CCR1 [49 (47.7–52.4) vs 51.1 (45.8–55.9) ml/min] or CCR slopes (0.06±1.9 vs –1.6±0.8 ml/min/year) between these control groups. Altogether, these data suggest that low serum Mg levels are more prevalent in the presence of chronic CsA nephrotoxicity, and that the combination of hypomagnesaemia and CsA nephrotoxicity is associated with a more rapid loss of kidney function.

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 Kaplan–Meier 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) 0.4–1.4].



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Fig. 3. Reduced allograft survival in patients with chronic CsA nephropathy and low serum Mg levels at the time of biopsy. (a) A box-plot representation of follow-up times in the two groups. Median and 25–75% percentile values are shown. (b) The Kaplan–Maier survival curve of kidney allografts (top panel) and the number of censored cases and kidney allografts lost to ESRD and patient death (bottom panel).

 


   Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
In this small cohort, 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, similar to its effects in animal studies [9,15–17].

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 (60–70%) 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 [12–14].

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 [9,15–17,20]. 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 [12–14], 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.



   Acknowledgments
 
Parts of this work were supported by an AST/Fujisawa Fellowship Award (A.D.), and NIH KO8-DK067981 (A.D.), RO1-AI49285 (B.N.B.) and K24-DK616962 grants (B.N.B.).

Conflict of interest statement. None declared.



   References
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Received for publication: 31. 8.04
Accepted in revised form: 16. 3.05