Proton-Pump Inhibitor and Tacrolimus use is Associated with Hypomagnesemia in Connective Tissue Disease: A Potential Pathogenic Link with Renal Deterioration and Recurrent Infections

Background: Low levels of serum magnesium (Mg) perturb renal tubular cell function and, lymphocyte resulting in renal deterioration and an imbalance in mononuclear cells. Here, we investigated the inuence of hypomagnesaemia in patients with connective tissue disease (CTD). Methods: We retrospectively evaluated CTD patients who visited our hospital during 2019 with available serum Mg levels. Patients were divided into two groups, those with or without hypomagnesemia (<1.8 mg/dL) and compared by rate of hospitalization for severe infection and cumulative renal deterioration. They were also compared by fractions of lymphocytes, and natural killer (NK) and dendritic cell (DC) subsets as measured by uorescence-activated cell sorting (FACS) analysis. Results: Among 284 patients, hypomagnesemia was detected in 63 (22.2%). Multivariate analysis revealed that use of proton-pump inhibitors (PPIs) (OR 1.48, p=0.01) and tacrolimus (TAC) (OR 6.14, p<0.01) was independently associated with hypomagnesaemia. Renal deterioration rate was signicantly higher in TAC and/or PPI users with hypomagnesemia (p=0.01). Hospitalization rate for severe infection was also higher in patients with hypomagnesaemia (p=0.04). FACS analysis showed lower counts for CD8+ T cells, CD19+ B cells, NK cells, and DC in hypomagnesaemia (p=0.03, p=0.02, p=0.02, and p=0.03, respectively). Conclusions: Use of TAC and PPIs may be associated with hypomagnesaemia and lead to poor renal outcomes and severe infection in CTD. FEMg, fractional excretion of magnesium; TAC, tacrolimus; PPI, proton pump inhibitor.

The aim of this study was to investigate the prevalence of hypomagnesemia and its clinical impact on patients with CTD in relation to PPIs and TAC.
This study was approved by the Ethics Committee of Keio University School of Medicine. Blood samples for ow cytometric analysis were obtained after the subjects gave written informed consent, as approved by the Institutional Review Board.

De nition
Hypomagnesemia was de ned as a serum Mg concentrations < 1.8 mg/dL [38]. Renal deterioration was de ned as a greater than 30% decline in serum creatinine levels from baseline [39]. Fractional excretion of Mg (FEMg, %) was calculated with the following formula: FE Mg = (U Mg *P cr )/(0.7*P Mg *U Cr )*100 [40], where U and P refer to the urine and plasma concentrations of Mg and creatinine (Cr), respectively. Serum Mg concentration was multiplied by 0.7, as only about 70% of circulating magnesium is free and ltered across the glomerulus. Normal limit of FEMg is de ned as less than 2% [40].
TAC measurement TAC concentration in fresh whole blood samples collected 12 hours after the last TAC administration was measured by the TACR Flex Dimension immunoassay method using a Dimension EXL analyzer (Siemens Healthcare Diagnostics, Tokyo, Japan) [17].

Flow cytometry
Blood samples at the time of serum Mg measurement from our cell bank of RA patients treated with methotrexate (MTX) monotherapy were analyzed with uorescence-activated cell sorting. Samples were stained with antibodies (BD Biosciences and BioLegend; Table S1) and xed by Phos ow Lyse/Fix Buffer (BD Bioscience). Flow cytometric analysis was conducted on an LSRFortessa™ X-20 (Becton Dickinson) and analyzed by FlowJo ver.10 (FlowJo LLC). Phenotypes of immune cell subsets were de ned based on the Human Immunology Project protocol (Table S2) [41]. Mean numbers of each immune cell phenotype were compared.

Statistical analysis
Continuous values are shown as median and interquartile range (IQR). Comparisons between two groups were performed with the Mann-Whitney U-test for continuous variables and the chi-squared test or Fisher's exact test for categorical variables. Four groups were compared by analysis of variance. Cumulative renal deterioration rates were analyzed by the Kaplan-Meier method with the log-rank test.
Correlations of two continuous variables were analyzed with the Spearman rank correlation coe cient. To identify independent parameters, binary logistic regression analysis was used with variables having a P-value < 0.005 in a previous univariate analysis as covariates. A P-value < 0.05 was de ned as statistically signi cant.

Relationship between hypomagnesemia and renal deterioration
As the use of TAC and PPI was the major cause of hypomagnesemia, we investigated the sequential renal function of patients treated with TAC and/or PPI (n = 173) from the initiation of these drugs until the last observation. When we divided the patients according to the presence of hypomagnesemia, the cumulative renal deterioration-free rates were signi cantly higher in patients with normal Mg (n = 124; 80.7%; observation period, 5.0 ± 2.9 years) than in those with hypomagnesemia (n = 49, 65.7%, observation period, 5.3 ± 3.4 years) (p = 0.007, Fig. 4). Of note, renal deterioration was not related with TAC use (renal deterioration-free at last observation, TAC users 84.5% and non-TAC users 90.1%, p = 0.34).

Effect of hypomagnesemia on immune cells
Among the 283 patients enrolled in this study, 17 patients who were also registered in another cohort of our university had information on peripheral blood mononuclear cells analyzed with FACS at the time Mg concentrations were measured. All 17 of these patients had RA and were treated with methotrexate alone. Six of these 17 patients had hypomagnesemia while the other 11 had normal Mg levels. These patients did not differ with regard to sex, disease duration, disease activity, or MTX dose (Table S3). In contrast, numbers of CD8 + T cells, CD19 + B cells, NK cells, and DC were signi cantly lower in the patients with hypomagnesemia than in the patients with normal Mg (p = 0.03, p = 0.02, p = 0.02, and p = 0.03, respectively, Fig. 5). Hospitalization due to infection was observed in 1 patient with hypomagnesemia (16.6%) and 1 with normal Mg (9.1%) (p = 0.64).

Discussion
In our study, hypomagnesemia was observed in approximately one-fth of patients with CTD and was associated with renal deterioration and hospitalization due to severe infections. The development of hypomagnesemia might have been caused by the use of TAC and PPIs.
Our study showed a high renal deterioration rate in patients with hypomagnesemia. This nding is consistent with previous studies which reported the association of hypomagnesemia with incident CKD [42], a decline in eGFR in CKD patients [43], and progression to end-stage renal disease in diabetic nephropathy [44]. Laecke et al investigated 1,650 patients with CKD with a median follow-up of 5.1 years and reported that a 1-mg/dL decrease in baseline serum Mg was associated with a yearly decrease in eGFR of 5.1%. Another report in Japanese patients with diabetic nephropathy (n = 144) showed that patients with hypomagnesemia were twice as likely to progress to end-stage renal disease compared to those with a normal range. The pathogenic mechanism of hypomagnesemia related to renal deterioration is not fully understood, but hypomagnesemia is considered to damage renal tubules. In one study, incubation of tubular epithelial cells in low-Mg medium increased the rate of apoptosis, whereas this effect was signi cantly suppressed when Mg concentration was increased [45].
In our study, 22.2% of patients with CTDs showed serum magnesium levels below 1.8 mg/dL. Low levels were associated with a high hospitalization rate due to severe infection. An association of hypomagnesemia and recurrent infection has been reported. Patients with X-linked XMEN, a hereditary immune de ciency syndrome in which dysfunction of the magnesium channel MAGT1 in T lymphocytes leads to a low intra-lymphocytic free magnesium concentration, suffer from recurrent infection [46]. Hypomagnesemic rats were shown to die earlier than control rats when injected with intravenous Escherichia coli endotoxin, whereas magnesium supplementation improve survival [47]. In a clinical report on kidney transplantation, low serum Mg was associated with an increased hazard of infection, and every 0.1 mg/dL reduction in serum magnesium below 2.0 mg/dL increased the hazard ratio by 15% [48]. Hypomagnesemia decreases T cell numbers, activation, and the cytotoxicity of CD8 + T cells and NK cells [49]. Although this study did not examine lymphocyte function, we did identify a decrease in the number of CD8 + T cells, CD19 + B cells, NK cells, and DC in RA patients with hypomagnesemia. Taken together, the decreased function and number of mononuclear cells caused by hypomagnesemia may be associated with the impaired immune function in hypomagnesemia.
Hypomagnesemia in patients with CTD was signi cantly associated with the use of TAC and PPIs in our study. These ndings are consistent with the action of these drugs, namely with TAC's interference with Mg-reabsorption from urine and PPIs interference with Mg absorption from the intestines [11][12][13]. Of the two drugs, TAC's interference on systemic Mg transportation is much stronger than that of PPIs, given that the reabsorption of Mg in the kidneys can handle 20-fold greater amounts of dietary Mg than absorption from the intestines [1]. In fact, our study showed that patients using TAC had lower Mg concentrations than those using PPIs, but that the combination use of PPIs and TAC did not show any additional lowering effect on Mg concentration than TAC use alone. In our study, the discontinuation of PPI increased Mg levels in patients without TAC, suggesting that hypomagnesemia caused by PPIs is reversible. We therefore recommend the monitoring of serum Mg levels in patients treated with PPIs, and consideration of the discontinuation when hypomagnesemia emerges. As for patients treated with TAC, Mg levels did not change when PPIs were stopped. We speculate that this is due to the far stronger effect of TAC than PPIs on lowering serum Mg levels. However, while we cannot conclude that serum Mg concentration would increase after TAC discontinuation because no patient discontinued TAC in our study, we can say that TAC dose should be reduced by monitoring TAC concentration to as low as possible to prevent hypomagnesemia, given our ndings that TAC concentrations were negatively correlated with serum Mg concentrations.
Our study has several limitations. First, it is a retrospective, single-centered cohort with a small sample size. This could have caused a degree of selection bias. Second, serum magnesium levels were measured cross-sectionally. Changes in magnesium levels over the period of observation of renal function were therefore unclear, which weakened the discussion about the relationship between hypomagnesemia and renal deterioration. Third, PPI was discontinued at the discretion of the attending physicians, which may have resulted in a degree of selection bias. Con rmation of our ndings will require a multi-center prospective study.

Conclusions
The use of TAC and PPIs was associated with hypomagnesaemia and led to poor renal outcomes and severe infection in patients with CTDs. The lowest possible dose of TAC should be prescribed in the management of CTDs, and the need for PPIs should be periodically reassessed. Availability of data and materials

List of abbreviations
The datasets analyzed during the current study are available from the corresponding author upon reasonable request. Comparison of magnesium level and fraction excretion of magnesium by drug use. All patients were divided into 4 groups according to tacrolimus and proton pump inhibitor use. Comparison of serum Mg levels among the 4 groups isre shown (A). Dotted line indicates the normal limit of magnesium level (1.8 mg/dL). Comparison of FEMg in patients with hypomagnesemia is shown (B). Dotted line indicates the normal limit of FEMg (2.0%). FEMg, fractional excretion of magnesium; TAC, tacrolimus; PPI, proton pump inhibitors.

Figure 3
Serial change in magnesium level after discontinuation of proton pump inhibitor. Magnesium level was signi cantly increased after discontinuation of PPI in patients without TAC (p=0.04) (A), but no signi cant difference was seen in FEMg (B). In patients with TAC, no change in magnesium level or FEM was observed after PPI discontinuation (C, D). FEMg, fractional excretion of magnesium; TAC, tacrolimus; PPI, proton pump inhibitor.

Figure 4
Cumulative renal deterioration-free rate. A signi cantly lower renal deterioration-free rate was observed in patients with hypomagnesemia compared to patients with normal Mg (p=0.007). Mg, magnesium; TAC, tacrolimus; PPI, proton pump inhibitor.