Magnesium decreases urine supersaturation but not calcium oxalate stone formation in genetic hypercalciuric stone-forming rats

Abstract

Background/Aims:

Hypercalciuria is the most common identifiable risk factor predisposing to CaOx stone formation. Increased oral magnesium intake may lead to decreased CaOx stone formation by binding intestinal Ox leading to decreased absorption and/or binding urinary Ox to decrease urinary supersaturation. This study assessed the effect of oral magnesium on 24-hour urine ion excretion, supersaturation, and kidney stone formation in a genetic hypercalciuric stone-forming (GHS) rat model of human idiopathic hypercalciuria.

Methods:

When fed the oxalate precursor, hydroxyproline, every GHS rat develops CaOx stones. The GHS rats were fed a normal calcium and phosphorus diet with hydroxyproline to induce CaOx, were divided into three groups of ten rats per group: control diet with 4.0 g/kg MgO, low MgO diet (0.5 g/kg), and high MgO diet (8 g/kg). At 6 weeks, twenty-four-hour urines were collected, and urine chemistry and supersaturation were determined. Stone formation was quantified.

Results:

The GHS rats fed the low and high Mg diets had a significant reduction and increase, respectively, in urinary Mg compared to those fed the control diet. Dietary Mg did not alter urine Ca excretion while the low Mg diet led to a significant fall in urinary Ox. Urine supersaturation with respect to CaOx was significantly increased with low Mg, whereas urine supersaturation was significantly decreased with high Mg. There was no effect of dietary Mg on stone formation within 6 weeks of treatment.

Conclusion:

Dietary magnesium decreases urine supersaturation but not CaOx stone formation in GHS rats.

INTRODUCTION

Kidney stones (nephrolithiasis) are a major public health challenge since they can lead to severe pain, frequent hospitalizations and recurrent procedures, infections, and even chronic kidney disease [1]. The annual incidence of kidney stones exceeds 1 per 1,000 persons in developed countries, with a lifetime risk of ~7% in women and ~11% in men [2]. After an initial episode of nephrolithiasis, 60–80% of patients form at least one recurrent stone. Humans most commonly form calcium oxalate (CaOx) stones [3]. Idiopathic hypercalciuria (IH), excessive urine calcium excretion with no clearly demonstrable cause, is the most common metabolic abnormality found in humans with nephrolithiasis [4]. The increase in urine Ca leads to increased supersaturation (SS) with respect to the Ca-containing solid phases, principally CaOx and Ca hydrogen phosphate (CaP), which increases the probability for nucleation and growth of crystals into clinically significant stones.

To study hypercalciuria and stone formation in man, we generated a genetic hypercalciuric stone-forming (GHS) rat model by screening Sprague-Dawley (SD) rats for hypercalciuria. We used the rats with the highest urine Ca to breed the next generation, followed by subsequent selection and inbreeding of their most hypercalciuric offspring, repeating the selection for over one hundred generations. Compared with their parental SD rats, the GHS rats are hypercalciuric, excreting 8–10 times as much urine Ca as control rats [5]. The degree of hypercalciuria has continued to increase with successive generations, although at a much slower rate than with earlier generations. All GHS rats form CaP kidney stones when fed a normal Ca diet [6]. The addition of hydroxyproline, an Ox precursor, to the diet of GHS rats results in the universal formation of CaOx stone formation [7]. The pathophysiology of the hypercalciuria in the GHS rats closely parallels that of humans with IH; these rats have increased intestinal Ca absorption [8, 6], decreased renal Ca reabsorption [9], and increased bone resorption [10], leading to increased urine Ca excretion and CaP stone formation [6], as well as a decrease in bone mineral density [11, 12]. We have also shown that hypercalciuria in the GHS rats is polygenic [13], as it is in humans [14].

Several pharmacological therapies have been utilized to reduce CaOx stone recurrence, including potassium citrate and thiazide diuretics, alone or in combination [4, 15]. Dietary management to prevent recurrent stones is recommended [16], but there is little consensus regarding the effectiveness of specific dietary interventions. Some data suggests that increased oral magnesium (Mg) intake decreases recurrent calcium oxalate stone formation by lowering intestinal oxalate absorption and/or binding urinary oxalate. In this study, we tested the hypothesis that elevated Mg content in the diet would reduce urine SS and CaOx stone formation in GHS rats. We fed GHS rats a Mg free rodent powdered diet and 5% hydroxyproline, supplemented with either 4.0 g/kg MgO (control), 0.5 g/kg MgO (low Mg) or 8 g/kg MgO (high Mg). At the end of 6 weeks, urine chemistry, urine SS, and oxalate stone formation were evaluated.

MATERIALS AND METHODS

Study protocol

Eight-week-old GHS male rats from the 118th generation were randomly divided into three groups, 10 rats per group, and housed individually in metabolic cages. All rats were fed a fixed amount of a Mg free rodent powdered diet containing 1.2% Ca and 0.65% P (Envigo TD.90312) and 5% hydroxyproline, supplemented with either 4.0 g/kg MgO (control), 0.5 g/kg MgO (low Mg) or 8 g/kg MgO (high Mg). All rats had free access to deionized, distilled water. After six weeks on the control and experimental diets, each rat was weighed and 24-hour urine was collected over 4 days, with two collections in thymol for quantification of pH, uric acid, and chloride, and two collections in hydrochloric acid for all other measurements. Rats were then euthanized, their kidneys, ureters, and bladders were removed for x-ray imaging. All protocols were approved by the Institutional Animal Care and Use Committees of The University of Rochester and Thomas Jefferson University.

Urine chemistries

Urine Ca, magnesium (Mg), phosphorus (P), ammonium (NH₄), and creatinine were measured spectrophotometrically using a Beckman AU autoanalyzer (Beckman Coulter, Brea, CA). Urine potassium (K), chloride (Cl), and sodium (Na) were measured by ion-specific electrodes on the Beckman AU. Urine pH was measured using a glass electrode. Urine citrate (Cit), and sulfate were measured by ion chromatography using a Dionex ICS 2000 system (Dionex Corp., Sunnyvale, CA). Oxalate was measured enzymatically using oxalate oxidase. The net gastrointestinal absorption of alkali (GIAA) was calculated from the excretion of non-combustible cations minus the excretion of non-combustible anions [17]. All urine solutes were measured at 6 weeks on the control and experimental diets, and a mean value for each time period as well as an overall mean was calculated. All of these methods have been utilized previously [18–25].

Urine supersaturation

Urine supersaturation with respect to CaOx and CaP solid phases were calculated from solute measurements using the computer program EQUIL2 [26], as we have done previously [6, 7, 22–25, 27].

Kidney stone formation

Kidneys, ureters, and bladder were removed en bloc from each rat and imaged by x-rays (Faxitron, Tucson, AZ). The extent of kidney stone formation was based on a semi-quantitative measure of the lesions in the kidneys (scores: no stones, 0; mild, 1; moderate, 2; severe, 3; extensive, 4). Three observers blinded to the study scored all radiographs.

Statistical analyses

Urine analytes and stone formation scores were expressed as mean ± SEM, and were compared among the three groups (CTL, Low MgO, and High MgO) by ANOVA with Bonferroni correction (Statistica; StatSoft, Tulsa, OK). Statistical analysis was performed by Student’s t test using the SPSS Statistics 20 program (SPSS, Chicago, IL). Statistical significance was assigned at P < 0.05.

RESULTS

Urine analytes

The low Mg diet had ~12.5% MgO of the control diet, and the high Mg diet had ~ 200% MgO of the control diet. After six weeks on the designated diets, those fed the low Mg diet had a significant reduction in urinary Mg and those fed the high Mg diet had a significant increase in urinary Mg compared to those fed the control diet (Fig. 1). GHS rats fed the control diet excrete ~ 13 mg Ca per 24-hr (Fig. 1), which is consistent with previous findings [5]. Dietary Mg did not alter urine Ca excretion while the low Mg diet led to a significant fall in urinary Ox (Fig. 1).

Fig. 1. Urine Ca, Ox and Mg in GHS rats fed diets containing different amounts of MgO.

Mg-free rat diets were supplemented with either 4.0 g/kg MgO (control), 0.5 g/kg MgO (low Mg) or 8.0 g/kg MgO (high Mg). Twenty-four hour urines were collected at 6 weeks for analysis of solute levels as described in Materials and Methods. Results are mean ± SEM for ten rats per group over three collections. *P < 0.05 versus control (CTL); °P < 0.05 versus low Mg. This figure was created with Biorender.com.

Changes in dietary Mg did not alter urine pH (Fig. 2). However, the low Mg diet increased urinary NH₄ and decreased urinary citrate (Cit), while the high Mg diet reduced urinary NH₄ and increased urinary Cit (Fig. 2). Urinary Cl and urinary P were not altered with the low Mg diet, but both fell with a high Mg diet (Fig. 3). Dietary Mg did not alter urinary volume. There were no differences in urinary sodium or potassium in any group (data no shown). The net gastrointestinal absorption of alkali (GIAA) was significantly lower for GHS rats on low Mg diet and significantly higher for GHS rats on high Mg diet (Fig. 4).

Fig. 2. Urine pH, NH₄ and Cit in GHS rats fed diets containing different amounts of MgO.

Mg-free rat diets were supplemented with either 4.0 g/kg MgO (control), 0.5 g/kg MgO (low Mg) or 8.0 g/kg MgO (high Mg). Twenty-four hour urines were collected at 6 weeks for analysis of solute levels as described in Materials and Methods. Results are mean ± SEM for ten rats per group over three collections. *P < 0.05 versus control (CTL); °P < 0.05 versus low Mg. This figure was created with Biorender.com.

Fig. 3. Urine Cl, P and Vol in GHS rats fed diets containing different amounts of MgO.

Mg-free rat diets were supplemented with either 4.0 g/kg MgO (control), 0.5 g/kg MgO (low Mg) or 8.0 g/kg MgO (high Mg). Twenty-four hour urines were collected at 6 weeks for analysis of solute levels as described in Materials and Methods. Results are mean ± SEM for ten rats per group over three collections. *P < 0.05 versus control (CTL); °P < 0.05 versus low Mg. This figure was created with Biorender.com.

Fig. 4. The gastrointestinal alkali absorption was differentially regulated by MgO.

Mg-free rat diets were supplemented with either 4.0 g/kg MgO (control), 0.5 g/kg MgO (low Mg) or 8.0 g/kg MgO (high Mg). Twenty-four hour urine collections were done at 6 weeks for analysis of solute levels as described in Materials and Methods. GI AA: gastrointestinal alkali absorption. Results are mean ± SEM for ten rats per group. *P < 0.05 versus control (CTL); °P < 0.05 versus low Mg. This figure was created with Biorender.com.

Urine supersaturation

Urine supersaturation with respect to CaP from three collections was not altered by either a low or high Mg diet (Fig. 5). Compared with the GHS rats on the control diet, low Mg significantly increased urine supersaturation, and high Mg significantly reduced urine supersaturation with respect to CaOx.

Fig. 5. Urine supersaturation (SS) of CaOx and CaP were differentially regulated by MgO.

Mg-free rat diets were supplemented with either 4.0 g/kg MgO (control), 0.5 g/kg MgO (low Mg) or 8.0 g/kg MgO (high Mg). Twenty-four hour urine collections were done at 6 weeks for analysis of solute levels as described in Materials and Methods. These values were used to calculate relative supersaturation and an overall mean of all three collections was calculated. Values for relative supersaturation are unitless. Results are mean ± SEM for ten rats per group. *P < 0.05 versus control (CTL); °P < 0.05 versus low Mg. This figure was created with Biorender.com.

Stone formation

Consistent with previous findings, the GHS rats on the control diet for six weeks developed kidney stones; however, alterations in dietary Mg did not alter stone formation in the GHS rats (Fig. 6).

Fig. 6. MgO did not affect kidney stones and calcification.

At the conclusion of the 6-week study the extent of kidney stones and calcification were quantified by three observers as described in Materials and Methods. Results are mean ± SEM for ten rats per group. This figure was created with Biorender.com.

DISCUSSION

Kidney stones are common, painful, and frequently recur, with considerable morbidity and healthcare cost. Dietary interventions may reduce the risk of stone formation. Among dietary supplements, magnesium is thought to influence stone formation either by decreasing oxalate absorption by binding oxalate in the intestine or decreasing CaOx supersaturation by binding urinary Ox [28]. Magnesium intake by adults in the US appears to be lower than recommended [29]. Urinary Mg is a surrogate for dietary Mg intake, and stone formers have lower urinary Mg than healthy people [30], suggesting that oral Mg supplementation may reduce urinary risk factors for stone recurrence. Magnesium supplementation in healthy volunteers given an oxalate load decreased intestinal absorption of oxalate and urinary Ox [28, 31]. A randomized clinical trial in 164 stone formers demonstrated that 400 mg MgO with concomitant 25 mg vitamin B6 supplementation reduced urine Ox [32]. Another possible mechanism of Mg may involve direct interactions between Mg and Ca ions in the stone formation process, i.e., Mg competes with Ca-Ox binding and forms magnesium oxalate complexes, which are slightly soluble, thereby preventing the CaOx stone formation [33, 30]. The effects of Mg on reducing renal stones in recurrent stone formers, however, had contradictory results [34, 35, 30].

In this study, we utilized GHS rats to determine the effects of oral supplementation of MgO, which is commonly prescribed to help reduce urinary oxalate excretion in stone formers. We found that a high Mg diet did not alter urinary Ox but had a significant effect on decreasing urinary CaOx supersaturation. This result is not surprising. The GHS rats were not fed a high oxalate diet to increase urinary oxalate excretion. Rather they were fed an oxalate precursor, hydroxyproline, which is metabolized to oxalate. Thus, the increased dietary Mg would not have the opportunity to bind excess dietary oxalate. In this study, the increased urinary Mg almost certainly bound urinary oxalate, derived from the added hydroxyproline, resulting in a significant decrease in urinary CaOx supersaturation. As humans derive at least 15% of their urinary oxalate from metabolism of dietary oxalate precursors, such as hydroxyproline [36], increased dietary Mg may be beneficial in lowering human urinary CaOx supersaturation. However, a six-week study showed that the GHS rats fed the low and high Mg diets had no effect on kidney stone formation. Urine supersaturation appears to be the best measure available clinically to predict stone recurrence [37]. In this study, it is possible that six weeks on the MgO diet is not long enough to see an effect on stone formation.

Our data demonstrated that GHS rats fed low Mg diet had significantly lower urinary Mg and those fed the high Mg diet had significantly increased urinary Mg compared to those fed the control diet. The urinary pH trended higher in GHS rats fed high Mg diet, but the difference did not reach statistical significance. The urinary NH₄, Cl, and P were significantly lower in GHS rats fed high Mg diet, whereas their amounts were numerically higher in GHS rats fed low Mg diet and the differences did not reach statistical significance. The urinary Cit was significantly lower in GHS rats fed low Mg diet and higher in those fed with high Mg diet. These changes all suggest a difference in net diet acid-base balance. Mg is provided as an alkaline salt (MgO), the changes induced are quantified by the GIAA which was lower in GHS rats on low Mg diet and higher in GHS rats on high Mg diet.

In summary, the results of our study demonstrated that MgO significantly affects urine supersaturation with respect to CaOx. The addition of MgO significantly reduces urine CaOx supersaturation. Additional, long-term studies will be required to test the potential consequences of dietary magnesium on CaOx stone formation in the GHS rats and most importantly, clinical trials in human stone formers will be necessary to determine if additional dietary Mg will reduce recurrent CaOx stone formation.

DATA AVAILABILITY STATEMENT

The data underlying this article are available in the article.