1

Research

Title:

Low magnesium in food is an abettor of calcium and magnesium accumulation in renal tissue, and

dyslipidemia? Observations in men with idiopathic recurrent calcium urolithiasis responding to a low

magnesium containing test meal with different degree of unrecovered magnesium in urine, and male

rats fed a magnesium-deficient diet. With a re-appraisal of kidney calcification and stone

pathophysiology. Second Revised Edition.

Author:

Paul Otto Schwille

Institutions:

Mineral Metabolism and Endocrine Research Laboratory, Departments of Surgery and Urology,

University of Erlangen-Nürnberg, Germany

Running head: Magnesium status and renal calcium stone formation.

Key words: Low food magnesium, abettor, magnesium retention, calcium urolithiasis of humans,

dyslipidemia, nephrocalcinosis of rats,

Address for correspondence:

Paul O. Schwille, M.D.

5, Finkenweg,

D-91080 Uttenreuth, Germany

Phone: +49-9131-59790,

Fax: +49-9131-533331,

E-mail: p.o.schwille@web.de

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Preface

Since completion oft the 1st online publication (Key Words: Low food magnesium, abettor;

http://www.opus.ub.uni-erlangen.de/opus/volltexte/2012/3603/) additional literature references were

identified fitting into the concept of IRCU as a magnesium-deficiency related disorder of calcium-

overloaded cells. Therefore, in this 2nd edition and adherent APPENDIX the number of citations and

text were enlarged and several additional figures included, together nourishing the idea that IRCU is

an outgrow of a primary pathology of blood vasculature.

Abstract

Background: The role of Mg in pathophysiology of IRCU is incompletely understood and would

benefit from animal models. Aims: To elucidate whether 1) Mg nutrition in IRCU impacts on minerals

and lipids in blood, minerals in urine, 2) dietary Mg deficiency of rats can serve as model for renal Ca

and Mg accumulation. Procedures: 1. Adult male patients (clinical trial 1; n = 88) underwent a

standardized laboratory program (urine and blood collections, intake of a TM with low Mg (type 1);

from Mg retention, viz. Mg not appearing in urine, and stratification Mg retention ≥50% (H) or <50%

(L), Mg nutrition was extrapolated. Clinical trial 2 (27 patients, 16 healthy controls): TM type 2

(suboptimal Mg, normal Ca and Na content) and type 1 were separately ingested. 2. Male Sprague-

Dawley rats were fed normal (n = 12) or Mg-deficient (n = 11) diet over 111 days; Mg, Ca, Pi were

determined in dry tissues (three kidney regions, artery, bone), urine and blood. Analyses: Established

techniques were used. Key findings: In trial 1, mean Mg in 24 h urine of all patients was low relative

to recommended Mg intake per day, decreased in subset H (n = 22) vs. L (n = 66) (p = 0.0015);

volume, Ca, pH, Na, UA, MDA, Ca salt supersaturation were decreased in postprandial urine of H vs.

L; there was dyslipidemia (decrease of HDL-CH, increase of VLDL-CH, TGL, total lipids, Lp(a)) and

increase of uricemia. Determinants of Mg retention (multivariate correlations) were urine Ca, volume

(negative), serum total CH + TGL (positive), explaining 40% of variation. In trial 2 TM type 2 vs. TM

type 1 evoked increase of Mg and Na retention in IRCU (+70% and +30%, resp.), but decrease of Na

retention in controls (-45%). Rats fed low Mg diet exhibited hypomagnesemia (total, ionized),

hypercholesterolemia, higher mean urine MDA and Pi per unit body weight, increase of Mg in renal

medulla, Ca in all kidney regions, decrease of bone Mg, undetectably low Mg in aorta, Conclusions:

1) In IRCU assessment of Mg retention from a meal allows to gauge Mg nutrition, renal contribution

to Mg, Ca and Na homeostasis. 2) Mg undernutrition is an effector of dys- and hyperlipidemia (IRCU,

rats), renal Ca and Mg accumulation (rats). 3) From present and earlier work of our and other

laboratories in this area (see appending additional files) a Mg deficit and lipid excess related vascular

etiology of IRCU is suggested.

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Introduction

Intrarenal accumulation of calcium (Ca) and deposition as Ca phosphate (CaPi) can present as

nephrocalcinosis (NC) in humans and rats, in the latter species mostly caused by dietary magnesium

(Mg) deficit [1-6]. Nutritional Mg deficit also has been observed in survey-like studies of idiopathic

recurrent Ca urolithiasis (IRCU) of humans [7-9], a disease without clinically detectable NC, but

studies linking Ca stone formation and food Mg deficit more directly are not available. This situation

is somewhat surprising, considering that the idea of a link between Ca urolithiasis and Mg metabolism

is old [10], that in IRCU of males aged around 30 years a decrease of Mg in serum and erythrocytes

has been documented [11] and, more generally, Ca stone formation occurs on the basis of pre-existing

CaPi containing renal interstitial plaques (so-called Randall’s plaques) [12, 13] that is reduced by Mg

supply with food [14]. An increasing renal cortico-medullary-papillary Ca gradient is characteristic for

both humans with IRCU [15] and the Mg-deficient rat [2, 4]; a similar but less expressed tissue Mg

gradient was reported for the rat model [4], whereas in IRCU robust data for renal Mg are lacking

(according to Pub-Med search).

Better understanding of the possible role played by low dietary Mg and other components of diet in

regulation of renal tissue Ca, Mg and other minerals in IRCU is further complicated by

overconsumption of lipids [16, 17] and the unknown role of lipids in pathogenesis of crystals and

stones [18, 19]. In rats fed a diet rich in cholesterol (CH) and triglycerides (TGL) but normal with

respect to Mg content, an increased renal cortico-medullary-papillary gradient of Mg and Ca not only

is preserved but even exaggerated, NC and CaPi stones form and document that both pathologies can

co-exist [2, 20]. On the other hand, IRCU patients reduce stone recurrence once Mg supplementation

of food has been instituted [14, 21], despite only marginal inhibitory effects of urinary Mg upon

crystallization of urinary Ca and oxalate (Ox) to CaOx [22], implying that once ambient Mg declines

the kidney tissue needs repletion of Mg stores, are formation of plaques and stones [13] to remain

under control, a task solved via inhibition of CaPi crystal maturation towards less soluble

hydroxyapatite (HAP) [23, 24]. As markers of disturbed mineral and bone metabolism in IRCU fasting

magnesiuria and magnesiemia were proposed [11, 25], but the gain of information for Mg in IRCU

pathophysiology remained limited. From these background informations we inferred that improved

knowledge of interplay of low Mg nutrition that leads to Mg "hunger", levels of blood lipids, renal

function and Mg appearing in simultaneously produced urine is indispensable, and should at best be

made transparent by means of a non-invasive laboratory procedure.

Among available methodologies quantitation of Mg hunger appeared to meet these requirements:

Assessment of the fraction Mg that is orally ingested as component of a test meal (TM) but not

recovered in postprandial urine ("retained" by the body) [26]. Because intestinal Mg absorption is

normal in normo- and hypercalciuric IRCU [27, 28], this kind of testing Mg status [26] might bring to

light low Mg status and possible association with so far etiologically inexplicable aspects of IRCU;

among the more prominent of the latter are changes of reactive oxidative species (ROS) [29, 30],

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sodium (Na) retention [31] and rise of blood pressure [8], together potentially acting as momentum of

all stone-forming processes (ASFP). To broaden the basis of the role of nutritional Mg in research of

IRCU etiology, rats were rendered Mg-deficient by dietary means, with post mortem analysis of Mg in

urine, blood, kidney and bone (the body’s major Mg storage site).

The present tripartite work, comprising two clinical trials and one experiment with rats, addresses

several questions: 1) Is there an IRCU subset detectable showing a high degree of Mg retention from a

TM containing very low Mg; if yes, is there alteration of Ca-uria, urine volume and saturation with

stone-forming salts, blood lipids and signs of oxidant/antioxidant imbalance? 2) When probing Mg

hunger in IRCU by intake of a TM with either definitively low or only marginally low Mg content,

does Mg retention deviate from non-stone-forming controls, depending on the amount of Mg ingested;

if yes, is variation of Mg retention a harbinger also of alteration of Ca and Na retention? 3) Is feeding

rats a low Mg diet followed by alteration of Mg, Ca, Pi in kidney, aorta, bone, as well as lipids in

blood, and signs of ROS excess? Because IRCU is a disorder with increasing incidence and prevalence

in Europe [32, 33] and abroad [34, 35], causing enormous financial burden to health-care providing

institutions, the observed data may be helpful for designing future projects.

Material and methods

1. Clinical studies, participants, procedures

The procedures described in the following were approved by the Ethics Committee of the University

of Erlangen Medical School; in addition, informed consent was obtained from all participants upon

prior written information about the necessity of examination under standardized laboratory conditions

at a fixed date and communication of program details [36, 37], including suspension of any medication

during the preceding two weeks. Other essentials of this program for outpatients were: collection of

24h urine on the day preceding examination while eating usual home food, withdrawal of fasting

venous blood, enhancement of diuresis (by drinking twice 200 ml deionized water), collection of 2h

fasting urine, intake of a test meal (TM), collection of 3h postprandial urine. The synthetic

carbohydrate-rich TM (Vivonex, Friesche Vlag, MA Leeuwarden, The Netherlands) was prepared to

deliver in 80 g dry weight: 1256 kJ (as glucose oligomers 67.9 g, fat 440 mg, amino acids 6.18 g), the

minerals (mM) calcium 25, Mg (two versions: one with 1.1 mM, designated very low Mg (VLMg),

another with 2.8 mM, designated low Mg (LMg)), Na (two versions: one with 19.5 mM, designated

normal Na (NNa), another with 6.1 mM, designated low-normal Na (LNa)); potassium 9.2, Pi (as

phosphorus) 5.4 were identical in both TM types. The meals (molar Ca/Pi 4.6) were taken as a

suspension in 300 ml deionized water (approx. 500 mOsm · l-1). Two trials (one cross-sectional, one

controlled) were performed.

Trial 1: From the available database with inclusion of several blood lipids 88 male IRCU patients, age

21 – 68 years, without overt NC from any cause, especially mild primary hyperparathyroidism (cases

with normo- or hypercalciuria but normal intact serum parathyroid hormone (PTH)), were recruited.

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On the day of laboratory examination ASFP was scored for the two preceding years as earlier

described [38], and kept within narrow limits. The ratio stones present/absent was 48/40, as judged

from a plain X-ray film of kidney-ureter-bladder region, in some cases complemented by additional

tomography. Creatinine in fasting serum was <120 µM · l-1. For absence of accompanying diseases

(and/or processes able to bias the data as presented herein), ethnicity, composition of stones (mainly

CaOx, and mature HAP or precursor forms), nature of crystals in fasting urine, readers are referred to

previous studies [36 – 39]. All patients received the VLMgNNa version of TM.

Trial 2: This pilot study, carried out as "Proof of Principle of Action" (PPA), aimed at unmasking

potential interaction of low dietary Mg with normal or low dietary Na in the presence of constant

dietary Ca. Randomly selected IRCU patients (n = 27; of similar age as in trial 1) and age-matched

male healthy individuals from medical personnel and students (n = 16) were compared. Both groups

received the VLMgNNa version of TM and, two weeks apart, the TM version LMgLNa, supplying more

but still sub-optimal Mg and less Na.

Definition of Mg retention: In IRCU with so far poorly defined Mg status the regulatory steps involved

in translocation of Mg from gut lumen to urine necessitate several assumptions: 1) Once individuals

are in Mg balance, the daily amount of Mg exported via urine roughly equals intestinally absorbed Mg

(approx. one third of Mg intake [27, 28]); 2) IRCU comprises at least two subsets, one with low,

another with high Mg hunger, differing in terms of urinary Mg per day; 3) in these two subsets the

high Ca content of TMs evokes a comparable degree of suppression of PTH secretion, hence

comparable postprandial renal tubular reabsorption of Ca and Mg, with the resulting degree of

calciuria forming the basis of classification as normo- or (idiopathic) hypercalciuria [36, 37]; 4) basal

Mg-uria per h in postprandial period is the same as during fasting, and any fraction of Mg as contained

in TM and absorbed intestinally is added to serum and filtered by renal glomeruli; 5) postprandial total

Mg-uria (basal plus increment) depends on Mg retained from Mg intake. On this basis Mg retention is

as following:

[Baseline Mg (µM per 2h) : 2] x 3 = Baseline Mg (µM per 3h);

Total Mg (µM per 3h) – Baseline Mg (µM per 3h) = Net Mg (µM per 3h);

Mg retention (µM) = Mg in TM (µM) – Net Mg (µM per 3h).

In trial 1 the arithmetic mean of Mg retention of all patients (see Table 1) was arbitrarily set as cut-off,

allowing to designate patients above the mean as subset H (high retention, synonymous low Mg

status), those below the mean as subset L (low retention, synonymous suboptimal or normal Mg

status). In trial 2 no cut-off was inserted.

2. Animals

Investigations described below were approved by the Ethics Committee of the German Law Against

Cruelties to Animals. Male Sprague-Dawley rats (bought from Charles River Wiga, Sulzfeld, FRG),

body weight approx. 220 g, were housed in Macrolon cages at 22 ± 1°C room temperature, relative

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humidity 60 – 65%, and exposed to 12h light – 12h dark rhythm. After 1 week of accommodation,

with feeding pelleted normal diet (C 1000, Altromin, Lage; FRG) and deionized water (content of Mg,

Ca, Na, chloride <1 µg · l-1) as drinking fluid, one group was ad libitum fed normal (Controls, n = 12)

diet, another group ad libitum fed Mg-deficient (MgD, n = 11) diet, supplying (per kg) 5.6 mM (137

mg) Mg; in both diets the molar Ca/Pi ratio was > 1.0. After 111 ± (SD) 3 days animals were housed

for 4 days in metabolic cages, maintaining access to diet and drinking fluid (see above); on the third

day urine was collected under paraffin oil (to prevent evaporation of CO2 and unspecific rise of

urinary pH). Following a 14h fasting period, blood was taken from abdominal aorta of anesthetized

(Nembutal, Abbott, Lage; FRG) rats, centrifuged, plasma and serum stored at 4°C; the kidneys were

removed, longitudinally halved, shock frozen (in liquid nitrogen) and stored at -80°C. The right femur

was exarticulated, freed of soft tissue and stored at -30°C. From the renal cortical, medullary, papillary

regions specimens (30 – 100 mg) were excised, dried at 100°C until weight constancy, ashed at 450°C

in a furnace, then dissolved in 1,25 M HCl and stored at 4°C until analysis. Similarly prepared was

infrarenal aorta (2 – 3 cm). The femur was defatted in ethanol, dried (see above), and bone volume

assessed by Archimedian principle; bone, ashed at 800°C, was dissolved in 10 ml 6 M HCl and stored

at 4°C until analysis. Histology of kidney was not carried out (for Mg deficit-induced cortico-

medullary NC see earlier reports [2, 4]); histomorphometry of bone is under preparation (R.G. Erben,

P.O. Schwille).

3. Analyses, statistics and data presentation

Most analytes were routinely estimated by the Central Laboratory Services of the university hospital.

For several other analytes commercially available kits, including rat and human PTH and 1,25(OH)2D

(the dihydroxylated vitamin D metabolite), or established techniques were used: Serum total and

ultrafiltrable (using N2 pressure filtration) Mg, tissue Mg and Ca (atomic absorption

spectrophotometry), plasma ionized Mg (sensitive electrode), albumin (Alb) (immuno-nephelometry),

Ox in urine and tissue [40], Cit in serum, urine and tissue enzymatically (citrate lyase method), urinary

malonedialdehyde (MDA), a marker of peroxidation of lipids [41], serum non-esterified (free) fatty

acids [42]. Calculated were non-albumin-protein (N-Alb-P) as difference (total protein minus Alb), the

very low density lipoprotein-bound (VLDL) fraction of CH [43], creatinine clearance and fractional

Mg excretion (based on serum ultrafiltrable Mg), upper limit of undissociated uric acid (UA) in urine

(using pK1 5.35 as cut-off [44]). Urine supersaturation (relative to solubility) products (RSP) of CaOx,

CaPi (as brushite) and UA were read from nomogram [45]. Individual data in urine often followed

non-Gaussian distribution. The Wilcoxon or t-test was applied, as appropriate. From practical reasons

mean values are given throughout, occasionally without scatter measures. Several simple and multiple

regression analyses were calculated, using Mg retention as dependent variable. The software

STATISTICA (Stat-Soft, Tulsa; USA) was used. Appending to present work (APPENDIX) is

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supportive online material (additional files; AF 1 - 3), showing more details of published and

unpublished work.

Results

1. Clinical trial 1 (intake of TM type VLMgNNa)

Mg in fasting serum, and fasting, postprandial, daily urine: Table 1 shows that the H and L subsets of

IRCU (see Methods section) exhibit identical serum total and ultrafiltrable Mg, yet in subset H there is

increase of creatinine clearance (hence apparently higher load of Mg filtered by glomeruli into

proximal tubule), total Mg in fasting and baseline Mg in postprandial urine, but decrease of FE-Mg in

fasting and net Mg excretion in postprandial urine; as expected, Mg unrecovered from postprandial

urine is markedly increased, Mg excretion in corresponding urine per day decreased. These findings

indicate that once nutritional Mg supply per day is low, aberrant renal Mg handling is one

consequence.

In the three types of urine examined individual Mg excretion rates are roughly symmetrically

distributed, irrespective of classification as normo- or hypercalciuria (Fig. 1, A – C); however,

calciuria is increased over Mg-uria in postprandial and daily, but not fasting urine (Fig. 2).

Importantly, in IRCU in Germany, mean Mg-uria per day is lower than in non-stone forming

individuals, and in both populations markedly lower than expected from recommended daily intake

(Fig. 1, C; ALL); for further illustration mean values of Mg nutrition are given for a number of

geographic areas.

Other parameters in urine and serum, general features (Table 2, A, B, C): In fasting urine of subset H

vs. L there is decrease of pH and increase of RSP-UA (note that the mean value remains negative). In

contrast, in postprandial urine of subset H vs. L there is strong decrease not only of pH, but also of

volume, total protein, Alb, N-Alb-P, MDA, Ca, Na, UA, and increase of undissociated UA (can

undergo transformation into UA crystals), RSP-UA, RSP-CaOx. In fasting serum of subset H vs. L

there is no change of creatinine, and (data not shown) total protein, Alb, N-Alb-P, total and

ultrafiltrable Ca, Na, Pi, PTH, 1,25(OH)2D. However, also in serum of subset H vs. L, there is higher

mean total CH, slight but statistically significant decrease of heavy density-lipoprotein-bound

cholesterol (HDL-CH), increase of VLDL, triglycerides (TGL), sum of total CH and TGL, lipoprotein

(a) (Lp(a)); UA (an accepted antioxidant [54]) is increased, Cit (an accepted crystallization inhibitor

[55]) decreased. General features in H and L subsets were statistically indistinguishable.

Interrelationships (Table 3): In ascending order Mg retention correlates significantly inversely with

urinary Na, total protein, Ca, volume, significantly directly with age, serum VLDL, urine pH, body

mass index, sum of serum total CH and TGL (A); as significant co-determinants of Mg retention

remain serum sum of total CH and TGL (positive), followed by urinary volume and Ca (negative),

together explaining approx. 40 per cent of variation of Mg retention (B).

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2. Clinical trial 2 (intake of TM types VLMgNNa and LMgLNa)

Unrecovered Mg, Ca, Na, (Fig. 3): This PPA trial reveals that unrecovered cations (given as mean per

cent of intake) are similar in IRCU and controls upon intake of TM VLMgNNa (the TM that is

extremely Mg-poor but normal with respect to Na): Mg is lowermost, Ca is higher, Na is highest,

exceeding intake (A); note that recommended Na intake per day is 65 - 70mM [56], corresponding to

approx. 4 gram Na chloride per day. In contrast, intake of TM LMgLNa (delivering a higher but still

suboptimal amount of Mg together with low-normal Na) evokes a substantial change of unrecovered

cations in both IRCU and controls: increase of Mg (IRCU, controls), no change of Ca (IRCU,

controls), further increase of Na in IRCU but dramatic decrease in controls (B).

3. Animal experiment

Substances in urine, serum, whole kidney, minerals in aortic wall, general features (Table 4, A - D): In

MgD vs. control rats there is decrease of urinary Mg, serum total and ionized Mg, viz. evidence of

systemically low Mg status; in addition there is increase of urinary total protein (due to both higher

Alb and higher N-Alb-P), decrease of MDA, Mg, Pi, Ox, no change of volume, pH, RSP-CaOx and

RSP-CaPi (brushite). In sharp contrast, mean urinary excretion rates per unit (100 g) body weight (see

Table 1; foot-note) of MgD rats show higher MDA and Pi, viz. reflect ROS excess induced lipid

peroxidation [41] and Mg deficit [57], respectively. Furthermore, in MgD rats there is decrease of

serum total protein, creatinine, free fatty acids, total phospholipids, no change of total Ca (increase

only when corrected for mean total protein), PTH and 1,25(OH)2D, but increase of total CH and LDL-

CH. Kidney Cit – an important small-molecular inhibitor of CaPi and other Ca salt crystallization

stages [55] – is approx. nine times as high in MgD as in control rats, but kidney Ox is unchanged.

Aortic wall Mg is undetectably low in both groups, the content of Ca and Pi statistically unchanged;

however, the mean molar Ca/Pi in MgD rats tends to rise (note that in vitro precipitation of Ca and Pi

starts at Ca/Pi 0.010 [58]). Food intake is statistically unchanged, yet body weight gain, final body

weight and food efficiency in MgD rats are drastically reduced.

Minerals in kidney, bone (Fig. 4): Control rats exhibit the well known [2, 4] renal cortico-medullary-

papillary gradients of Mg, Ca, Pi in this species. Similar mineral gradients are present in MgD vs.

control rats, with higher mean values of Ca and Pi in cortex, medulla, papilla, and significantly

elevated Mg in medulla. The mean molar Ca/Pi in controls is 0.25, 0.24, 0.64 in cortex, medulla,

papilla, respectively, slightly higher in MgD rats (0.31, 0.63, 0.83, for cortex, medulla, papilla,

respectively). This means that, if Ca and Pi precipitate, at Ca/Pi <1.0 non-crystallized (amorphous)

CaPi (ACP) should predominate in kidney tissue of both control and MgD rats, contrasting with HAP

in the papillary region of stone-forming pediatric patients [59], but in agreement with predominance of

ACP in plaques of adult nephrolithiasis patients [60]. In femur ash of control rats Mg is approx. one

sixtieth of Ca content, but in MgD rats femur Mg is decreased, Ca increased, Pi unchanged. These data

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give mean Ca/Pi around 1.60 (controls), indicative of Ca-rich HAP as the predominant crystallized

mineral, and Ca/Pi around 1.75 (MgD), indicative of carbonated HAP at lower ambient Mg, in

agreement with work in vitro [23].

Discussion

Mg retention in humans – Approach to assessment of Mg nutrition

There is an urgent need for cost-saving methods effectively assessing Mg deficiency, either by already

existing or newly developed biomarkers such as enzymes and cytokines [61, 62]. In present work we

present Mg retention from Mg in TM VLMg-NNa as a rather simple procedure applicable by ambulatory

clinical units; however, broad-scale efforts in testing this procedure are needed to validate its

usefulness. According to presented data, in Germany and USA [63] Mg in urine per day is lower than

anticipated from recommended Mg intake, and even lower-than-normal in IRCU patients living in

various regions of Germany (Fig. 1) and parts of Asia [64]. Survey-like informations include

recommended daily intake of Mg by males and females, active workers and pensioners, residents

living in varying geographic areas under various conditions, and extraordinary challenges such as

pregnancy, etc.; not surprizingly, there is uncertainty among physicians as to whether a mild urinary

Mg abnormality in IRCU may be clinically detectable [65], and which complaints and diseases need

dietary supplementation of Mg in excess of Mg eaten with food under the prevailing conditions of

living [66]. Moreover, Mg deficiency is often misdiagnosed because accessible markers of Mg status

(Mg in bone, blood, cells, kidney, renal handling of Mg, Mg in urine) are ill-defined or require

specialized knowledge for installment and interpretation [66]. Thus, in IRCU, a Mg paradox exists: Is

the state of Mg solely dependent on Mg nutrition, viz. without renal loss, or does it reflect some

combination (see increase of fasting Mg-uria in subset H, Table 1, Fig. 1, A)? To interpret this,

knowledge of Mg regulation at the level of cells is indispensable. For example, low nutritional Mg

status interferes with transcription of genes coding for proteinaceous renal tubular apical membrane

channels [67, 68]: Depending on degree of pre-existing damage to DNA and/or structure/function of

these channel proteins (see below) and a varying degree of damage exerted to different anatomic

nephron sites, maintenance of adequate tubular Mg reabsorption or Mg loss via urine may prevail. In

other words: Long-term primary Mg undernutrition during the day, resulting into Mg deficit, ROS

excess and damage to cell membrane phospholipids and proteins [69, 70] of functionally highest

developed vascular endothelium and nephron sites (glomeruli, cortex, medulla) may manifest as

fasting increase of filtered load of Mg in proximal tubule during fasting (Table 1) and followed by Mg

overflow to distal tubule with only insufficient attenuation of Mg reclaim, ending up in elevated Mg

excretion in fasting urine. Noteworthy, use of endogenous creatinine clearance, a suboptimal

glomerular filtration marker, can mask this picture; therefore, the reduction of FE-Mg (Table 1, subset

H) should be apparent rather than real. Mg content of renal tissue, especially the papillary region, is

unknown in IRCU, except one early report [71]; these authors conclude that Mg is unchanged,

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according to comparison of 11 IRCU patients with 11 healthy controls. This report, and numerous

others, while befitting the theory that in daily urine Ca dominates over Mg, contrasts with present

observation that hypercalciuria is not ubiquitous during a daily cycle (Fig. 2). One explanation may be

stronger increase of urinary Mg during fasting (Fig. 1, A, subset H) and preceding night hours due to

pre-existing low Mg status induced tissue damage [70].

Modelling of diet in the rat – Light is shed on IRCU etiology?

Rats fed a Mg-deficient diet develop, in addition to NC [1, 2, 4], elevation of renal tissue Ca and Mg

(Fig. 4), elevated serum total CH and LDL-CH (Table 4) and impairment of bone structure (increase of

osteoclasts, decrease of osteoblasts) [72]. In rats fed a CH- and lipid-rich diet, NC and CaPi stones

develop in a renal tissue environment that is rich in neutral lipids and phospholipids [20]; also in this

setting, cortico-medullary-papillary Mg gradient and cortical Ca content as well as renal tissue mean

molar Ca/Pi are elevated and high, respectively (Ca/Pi is 0.8 in lipid-fed vs. 0.2 in control rats), and

intra-bone redistribution of Ca (on the basis of 45Ca movement) starts at bone Mg content that is only

borderline lower-than-normal [20]. Although unproven, such dietary conditions may mediate a shift of

Ca, Mg and Na from bone [73] via blood plasma to blood cells [29, 74], blood vessel walls [75] and, at

renal level, combine with microvascular lipids, lipoproteins [76, 77] and blood Ca to the increasingly

discussed functional axis "bone - blood vasculature - kidney" [78]. If this axis exists it may be a

feature of kidney that is primarily lipid-laden and/or threatened by Mg deficit induced dyslipidemia

(Table 4), forcing the organ to allow for ACP (Ca/Pi <1.0) formation; because the rise in tissue Ca is

paralleled by increase of tissue and cell Mg (see APPENDIX) renal Ca/Pi is prevented from rising

towards HAP (Ca/Pi ≥1.6) formation as in bone (Fig. 4). Unexpectedly, NC with hyper- and

dyslipidemia and CaPi stones [20] contrasts with NC and hyperlipidemia but no stones as exhibited by

MgD rats of present work. Evidently, NC is a sinister metabolic process [79]; to understand this type

of renal CaPi calcification as possible Ca stone precursor role necessitates more precise insight into the

nature of overload of tubular and interstitial cells with both Ca and Mg (see APPENDIX), whether free

ions, complexes, or protein binding of ions dominate, and whether in this setting the mechanism(s) of

action of Mg is Ca-antagonistic like in other disorders [80]. In humans with low bone Mg the degree

of retention of systemically administered Mg is enormous [46], and in rats there is a strong positive

correlation of femur Mg with food Mg over a wide range (n = 53, r = 0.90, p <0.001), developing

within 2 weeks after institution of a Mg-deficient diet [81]. Reasonably, to stimulate insight into the

role of Mg nutrition in IRCU pathophysiology studies of rat models with NC should include those

with feeding low dietary Mg alone, lipid-enriched food alone, and combination(s) [70].

Site and mode of renal Ca accumulation in IRCU – An ongoing enigma?

Regarding the anatomic site of origin of stones two theories concur. One, more recently proposed and

favoring tubular fluid as birthplace of Ca-containing crystals and stones, starts with nucleation of CaPi

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in the neutral or weakly acidic proximal nephron fluid, postulating heterogeneous (CaPi-induced)

nucleation of CaOx in the distal increasingly acidic and more Ca-rich (from CaPi dissolution)

nephron, ending up in plugging of tubular lumen by crystal aggregates and formation of microliths

[82]. Another theory, favoring pre-existing interstitial CaPi-containing plaques as basis of stone

formation [8, 9], attracts growing interest despite its neglecting the cause(s) of plaques. From present

work a primarily Mg malnutrition related theory may come to the forefront of IRCU pathophysiology:

These patients, when stratified according to "Mg retention" (Table 1), are more or less hungry for Mg

as contained in food, allowing in addition featuring of strata with respect to physico-chemical

supersaturation of urine, lipids and other biochemical variables in blood (Table 2, A and B); more

specifically, declining urinary Ca, volume, pH leave behind innocuous urine supersaturation (mean

RSP <1.0; Table 2, A), forcing to focus stone etiology research on renal cells and tissues. A so far

unreported finding is inappropriately high Na retention from food (Fig. 3), a risk factor for blood

pressure increase [56] and, together with lowered urine pH, a possible harbinger of a deeper cellular

defect (see below). Tissue co-localization of CaPi and CH, perhaps mutually inducing nucleation [83],

and a similar ratio free CH/esterified CH in blood and Ca stones [17, 19] indicate that intrarenal lipid

deposition potentially acts as tissue damaging factor [84, 85, 86], especially glomerular and tubulo-

interstitial ROS-mediated damage [87], in turn able to accumulate Ca and CaPi (see below). Thus,

although unproven to date for IRCU, low systemic Mg and high systemic and local (inside kidney)

lipid status may interact to bridge the gap between constituents of blood, tubular fluid and urine via

CaPi deposition inside intrarenal blood vessel walls and interstitium (note that during mineral

formation in bone such bridging is physiological [88]). Given CaPi solubility and ambient Mg

concentration are directly related [23, 24] also in vivo, renal tissue CaPi may represent Ca-poor ACP

or Ca-rich HAP depending on intrarenal Ca availability, an interpretation that is difficult to reconcile

with growth of plaque area along increasing, instead of decreasing, calciuria [89]. Viewed this way,

Mg hunger of varying degree together with dys- and hyperlipidemia may characterize IRCU as

chronic kidney disease [90] of which at least subsets have ramifications to pathologies (calcifications,

formation of plaques) of blood vasculature [91, 92]. For further informations (comments, illustrations,

references, together justifying re-appraisal of IRCU pathophysiology) readers are referred to

APPENDIX (AF 1-3).

Limitations

The present work deals with male individuals (patients, rats); therefore, none of the described

observations can be automatically transferred to female individuals. The observations in IRCU are

based on one single but strictly supervised laboratory examination, with Mg in daily urine being

allocated to Mg as observed in postprandial and baseline fasting urine. Although this strategy may

restrict scatter of individual data in urine and blood, considerable within-subject instability especially

of urine components [93] forces to caution when generalizing our findings. Isolated measurement of

12

MDA, the widely used marker of unsaturated fatty acid oxidation to lipid peroxides [41], is probably

not the "gold standard" [94], suggesting that gauging oxidant/antioxidant imbalance would benefit

from knowledge of additional markers. The measurement of total and ultrafiltrable Mg in IRCU

instead of total and ionized plasma Mg as in the rat experiment presumably led to underestimation of

blood Mg status. In IRCU and rats we had no access to quantitation of intracellular ionized and

complexed Mg and Ca, using an increasingly practiced methodology [95].

Conclusions

In humans with IRCU a TM with low Mg content as part of a standardized laboratory program allows

to quantitate renal Mg retention as indirect marker of Mg nutrition. The so far elusive role of low

nutritional Mg status and lipid nutrition as modifiers of blood minerals and lipids needs

reconsideration as risk factor(s) of renal Ca stones. IRCU with high Mg retention, low fasting urine

Ca, volume, pH and Na impress as possible harbingers of an extraordinary complex network,

presumably initiated by etiologically uncertain systemic tissue damage that spans from minerals in

bone over those in blood and blood vasculature to renal handling, modulation of urinary excretion and

intrarenal CaPi deposition. From present and previous work of our laboratory and others (see

APPENDIX, AF 1 – 3) into etiology of renal calcifications in humans and rats malregulation of both

intrarenal Ca and Mg accumulation likely plays a Ca stone precursor role. There is an urgent need in

IRCU and rats with NC for reliable quantitative data of renal interstitial and cellular Mg, Ca and other

minerals, helping to identify mutualities, rule out species difference(s) in regulation of mineral and

lipid metabolism, and decide whether and in which form blood vessel dynamics is affected. A tentative

view of events in this "point of care to bench" study is presented (Fig. 4), hopefully benefitting future

clinical and experimental work.

Abbreviations in text: IRCU, idiopathic recurrent calcium urolithiasis; ASFP, all stone-forming

processes; NC: nephrocalcinosis; Ca, calcium; Mg, magnesium; P, phosphorus; Pi inorganic

phosphate; CaPi, calcium phosphate; Ca/Pi, molar ratio; HAP, hydroxyapatite; Ox, oxalate; CaOx,

calcium oxalate; ROS, reactive oxygen species; MDA, malonedialdehyde; Na, sodium; UA, uric acid;

Cit, citrate; TM, test meal; VLMgNNa, TM with very low Mg, normal Na content; LMgLNa TM with low

Mg, low Na content; N-Alb-P, non-albumin protein; Alb, albumin; RSP, relative supersaturation

product; CH, cholesterol; HDL-CH, heavy-density-lipoprotein-bound cholesterol; LDL-CH, low-

density-lipoprotein-bound cholesterol; VLDL-CH, very-low-density-lipoprotein-bound cholesterol;

TGL, triglycerides; Lp(a), lipoprotein a; H, proton; HX, hypoxanthine; XO, xanthine oxidase; AF,

additional file; NO, nitric oxide.

Competing interests: The author declares that he has no competing interests.

13

Acknowledgements

This work was supported by the Friedrich Bauer Foundation, Munich, the W. Sander Foundation for

the Advancement of Medical Sciences, Munich, grants from the "Universitätsbund Erlangen",

Erlangen, and the Deutsche Forschungsgemeinschaft, Bonn. We are grateful to Professor Dr. A. Sigel,

former head of the Erlangen University Department of Urology, Professor Dr. G. Hegemann, former

head of the Erlangen University Department of Surgery, for long-year support of our unit specializing

in clinical and experimental mineral metabolism, Dr. A. Schmiedl and Dr. U. Hermann for deep

engagement in medical care of patients and participation in animal experimentation, K. Schwille for

technical work.

14  

Table 1: Mg status of all IRCU patients (ALL) and as broken down into subsets with apparently high (H) or low (L) Mg retention (unrecovered in postprandial urine; for calculation see Methods section). S: serum; U: urine; n: number of patients. Mean values (SE); *: p<0.05; **: p<0.001 vs. L.

L H All n=66 n=22 n=88

Fasting S-Total Mg concentration; mM · l-1 0.87 (0.008) 0.86 (0.01) 0.87 (0.004) S-Ultrafiltrable Mg concentration; mM · l-1 0.66 (0.004) 0.66 (0.008) 0.66 (0.004) U-creatinine clearance; ml · min-1 105 (4) 116* (4) 107 (3) U-Total Mg excretion+; µM · 2h-1 263 (13) 321* (25) 275 (13) FE-Mg+; % 6.6 (0.3) 4.2* (0.2) 6.0 (0.3)

Postprandial U-Total Mg excretion; µM · 3h-1 771 (29) 504* (25) 704 (4) Baseline Mg excretion++; µM · 3h-1 392 (21) 483* (42) 413 (21) Net Mg excretion; µM · 3h-1 379 (25) 21** (21) 292 (25) U-Unrecovered (from Mg in TM+++); µM · 3h-1 684 (25) 1042** (29) 771 (25)

Daily U-Magnesium excretion; mM · 24h-1 4.3 (0.2) 3.4** (0.3) 4.1 (0.1)

+: In the authors’ laboratory Mg values in fasting urine of 12 healthy normals are <282 (mean 235) µM ·

2h-1 and FE-Mg is <5%, respectively. ++: Assuming no change of serum ultrafiltrable Mg and urine creatinine clearance vis-à-vis fasting period.

+++: 1063 µM

15 

Table 2: Parameters in urine (A.), serum (B.) and general features (C.) of the 88 IRCU patients as broken down into the subsets H (n = 22) and L (n = 66) according to Mg retention (for calculation see Methods). Data are mean values (SE); +: p ≤0.05 vs. L; *: Limits of normalcy as observed in the authors’ laboratory or contained in literature [53]; n.d.: not determined. **: Urinary substance excretion rates are per mM creatinine; ***: substance concentrations are per l. For abbreviations see text.

L H L H Normal* L H Normal*

A. Urine Fasting1) Postprandial2) B. Fasting serum*** Volume; ml 214 (18) 188 (23) 260 (14) 167+ (19) >40 Creatinine; µM · l-1 96 (2) 94 (4) <97 pH 6.28 (0.10) 5.90+ (0.16) 5.81 (0.10) 5.41+ (0.13) 4.5 – 7.2 Lipids Total protein; mg · mM-1** 61 (20) 25 (5) 36 (8) 16+ (4) <6.0 Total-CH; mM · l-1 5.0 (0.13) 5.4 (0.21) <4.5 Alb 18 (8) 3 (1) 8 (4) 2+ (1) <1.7 HDL-CH; mM · l-1 1.1 (0.03) 1.0+ (0.08) >1.0 N-Alb-P 44 (12) 21 (4) 28 (5) 13+ (3) <4.0 LDL-CH; mM · l-1 3.5 (0.13) 3.7 (0.23) <4.9 MDA; nM · mM-1 112 (7) 97 (8) 129 (7) 104+ (12) n.d. HDL-CH/LDL-CH 0.36 (0.02) 0.30 (0.03) n.d. Ca; µM · mM-1 310 (0.02) 310 (0.04) 678 (28) 537+ (56) <339 VLDL-CH; mg · l-1 170 (10) 260+ (40) n.d. Pi (as phosphorus); mM · mM-1 1.35 (0.07) 1.28 (0.15) 0.89 (0.05) 0.95 (0.10) <1.8 TGL; mM · l-1 0.98 (0.06) 1.48+ (0.23) <1.8 Mean Ca/Pi; µM · µM-1 0.23 0.24 0.76 0.57 n.d. Total-CH + TGL; mM · l-1 6.0 (0.20) 6.9+ (0.45) <5.5 Na; mM · mM-1 11 (0.5) 9.8 (1.0) 9.8 (0.5) 7.9+ (1.0) <12 Lp(a); mg · l-1 53 (6) 90+ (35) n.d. Ox; µM · mM-1 18.7 (0.8) 17.3 (1.5) 18.5 (0.9) 15.9 (1.0) <25 Total Phospholipids (as phosphorus); mM · l-1 5.8 (0.1) 6.0 (0.3) <3.9 Cit; µM · mM-1 238 (12) 232 30) 220 (12) 208 (24) >178 Cit; µM · l-1 119 (3) 109+ (4) <160 UA; µM · mM-1 336 (13) 330 (34) 303 (7) 249+ (20) <269 UA; µM · l-1 343 (8) 368+ (12) <300 Undissociated UA; µM · l-1 554 (101) 826 (179) 810 (101) 1399+ (149) >600 C. General features RSP-CaOx 0.50 (0.07) 0.54 (0.14) 0.71 (0.04) 0.89+ (0.12) <1.1 Age; years 40.5 (1.5) 42.3 (2.4) >23 <62 RSP3)-Brushite -0.21 (0.08) -0.27 (0.14) 0.06 (0.08) -0.02 (0.11) <0.5 Body mass index; kg · (m2) -1 25.6 (0.4) 26.0 (0.5) >23 <32 RSP-UA -1.18 (0.20) -0.39+ (0.31) -0.34 (0.18) 0.59+ (0.22) <1.6 ASFP; score 37 (4) 42 (11) 1

1), 2): For total Mg excretion (µM per 2h and 3h, respectively) see Table 2, H and L. 3): Negative values indicate undersaturation, 0 = solubility, >1.0 homogeneous nucleation [45].

16 

 

Table 3 A: Ranked significant simple correlations of Mg retention (synonymous unrecovered Mg; see Table 1) as dependent variable in IRCU as a whole (n = 88) and several independent variables [fasting serum (S); postprandial urine (U)]. *: based on log10. For other details and abbreviations see section Material and methods.

B: Multivariate regression analysis with Mg retention (see A, n = 88) as dependent, sum of fasting serum total CH and TGL, postprandial urinary volume and Ca excretion as independent variables: R2 = 0.399, p <0.00001, as adjusted for deleted independent variables. *: partial regression coefficient; %: contribution to variation within the model.

A r p = r p =

Age 0.222 0.046 U-Na -0.236 0.034 S-VLDL-CH 0.263 0.018 U-Total protein* -0.268 0.016 U-pH 0.299 0.007 U-Ca -0.374 0.001 Body mass index 0.300 0.007 U-Volume -0.477 0.0001 S-Total CH + S-TGL 0.355 0.001

B Beta* % Standard error p =

S-Total CH + S-TGL 0.219 4.8 0.086 0.013202 U-Volume -0.347 12.0 0.087 0.000133 U-Ca -0.413 17.1 0.085 0.000006

17 

Table 4: Data of rats fed normal (Controls) or Mg-deficient (MgD) diet. Mean values (SE); n: number of animals, except [ ].

Controls MgD Controls MgD

n=12 n=11 p n=12 n=11 p

A. Daily urinex Continuation: Volume; ml 6.4 (0.6) 9.6 (3.5) 0.08 Lipids Total protein; µg 5 (1) 13 (3) 0.02 Total CH; mM · l-1 1.4 (0.13) 2.0 (0.18) 0.03 Alb 4 (1) 9 (3) 0.16 HDL-CH; mM · l-1 0.73 (0.05) 0.65 (0.08) 0.53 N-Alb-P 1 (2) 5 (3) 0.32 LDL-CH; mM · l-1 0.55 (0.16) 1.1 (0.16) 0.03 MDA; nM 17 (2) 11 (2) 0.05 Free fatty acids; mM · l-1 0.89 (0.03) 0.71 (0.07) 0.01 Mg; µM 11 (1.3) 3.2 (0.8) <0.001 Total phospholipids (as phosphorus); mM · l-1 2.2 (0.13) 1.2 (0.23) <0.001 Ca; µM 15 (1.8) 26 (7.0) 0.13 C. Tissue++ Pi (as phosphorus); µM 742 (32) 548 (64) 0.02 Kidney Ox; µM 3.89 (042) 1.84 (0.37) [8] 0.003 Ox; nM · g-1 242 (28) [8] 210 (44) [7] 0.54 Cit; µM 69 (7) 100 (18) 0.10 Cit; nM · g-1 602 (154) [6] 5185 (1048) [8] 0.003 RSP CaOx 1.17 (0.03) [9] 1.02 (0.08) [10] 0.10 Aorta RSP CaPi (as brushite) 1.12 (0.06) [9] 1.14 (0.15) [10] 0.92 Mg nd [5] nd [4] pH 6.35 (0.05) 6.68 (0.24) 0.17 Ca; nM · mg-1 · mm-1 284 (16) [5] 308 (21) [4] 0.40 B. Fasting serum Pi; µM · mg-1 · mm-1 2.1 (0.11) [5] 1.9 (0.2) [4] 0.41 Total Mg; mM · l-1 0.76 (0.04) 0.30 (0.02) <0.001 Mean Ca/Pi; nM · nM-1 0.14 0.19 ndt Ionized Mg; mM · l-1 0.16 (0.004) 0.13 (0.004) <0.001 D. General features Total protein; g · l-1 55 (1) 45 (1) <0.001 Initial body weight; g 214 (3) 216 (3) 0.55 Total Ca; mM · l-1 2.5 (0.03) 2.4 (0.08) 0.30 Body weight gain; g 336 (18) 75 (11) <0.001 Total Ca+; mM · l-1 2.5 (0.03) 3.0 (0.1) <0.001 Final body weight; g 580 (19) 291 (11) <0.001 Pi (as phosphorus); mM · l-1 1.7 (0.13) 1.8 (0.16) 0.96 Food intake; g 10.1 (1.0) 8.0 (1.0) 0.15 Rat PTH; ng · l-1 49 (10) 34 (4) 0.17 Food efficiency; g · g-1 38.1 (3.5) 5.9 (1) <0.001 1,25(OH)2 D; ng · l-1 13 (1) 12 (2) 0.68 Creatinine; µM · l-1 65 (3) 49 (3) <0.001

x: Mean excretion rates per 100 g final body weight are (Controls, followed by MgD): Volume 1.1, 3.3; Total protein 0.9, 4.5; Alb 0.7, 3.1; N-Alb-P 0.2, 1.7; MDA 2.9,

3.8; Mg 1.9, 1.1; Ca 2.6, 8.9; Pi 128, 189; Ox 0.7, 0.6; Cit 12, 34;. +: Corrected for serum mean total protein of Controls; ++: data are per unit dry tissue weight and length, respectively; nd: not detectable; ndt: not determined;

18

Legends to figures in text

Figure 1: Composite view of fasting (A), post-test meal load (B) and daily (C) urinary (U) Mg

excretion of individuals in the subsets H and L, and IRCU as a whole (ALL). Horizontal

thick lines in A, B and C indicate mean values (●: normocalciuria; x: idiopathic

hypercalciuria); n: number of patients. Horizontal dotted lines in C: a is the mean value

reported for non-stone-forming human males in southern [7] and northern [46] Germany

and observed in the authors’ laboratory (unpublished); b is the mean value as

extrapolated from Mg intake (assuming 35% intestinal absorption) by working healthy

normals in Sweden [47]; c and d are the mean values (derived as in b from

recommended dietary Mg intake per day) in Germany (14.6 mM) [48] and France (18.8

mM) [49], respectively; e and f are the mean values of IRCU patients reported for

western Germany [50] and former Yugoslavia [51], respectively; g denotes urinary Mg

per day as derived from mean daily Mg intake of males in USA (4.5 mM) [52].

Figure 2: Mg in fasting, postprandial and daily urine (U) of IRCU patients with normocalciuria

(light columns) and idiopathic hypercalciuria (hatched columns). Mean values (SE); n:

number of patients.

Figure 3: Mean percentage unrecovered Mg, Ca, Na (synonymous retention; for calculation see

Methods section) in 3h postprandial urine collected after intake of two types of TM,

differing with respect to content of Mg and Na. A: Type 1 TM, VLMg–NNa; B: Type 2

TM, LMg–LNa. For more details of composition of TMs see Methods section. :

IRCU; : controls; n: number of individuals studied; *: p = 0.003 vs. Mg in A; **,

***: p = 0.005, p = 0.003 vs. Na in A (all according to Wilcoxon test).

Figure 4: Minerals in the three renal anatomic regions (A) and femur (B) of rats fed normal

or Mg-deficient diet. Mean values, SE. *: p <0.01 vs. normal diet; n: number of

rats.

Figure 5: Tentative view of events in pathophysiology of IRCU. Thin and thick arrows indicate

moderate and strong increase ( , ) and decrease ( , ), respectively, or no change

(↔); ?: uncertain or unknown. *: transient (recurrent) episodes, or permanent; **: in the

acute setting; ***: epithelial-interstitial transition, fibrosis (upon repeat episodes). For

other abbreviations see Text.

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24

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30

APPENDIX

Supportive online material (AF 1 – 3): Additional comments with illustrations and references from

previous reports are given, all with permission of the publishers; for more details see the respective

original work [ ].

AF 1

Comments to "IRCU and Low Mg status have alteration of blood lipids, ROS and blood vasculature in

common?"

In IRCU and experimental animals there is impressive indication from available literature of 1) renal

disease progression in response to low density lipoprotein and Lp(a) [76, 77], 2) intimate relationship

between IRCU and the risk of myocardial infarction [92i], the morphological state of large blood

vessels [93i, 94i, 95i], 3) correlation of serum ionized Mg with blood lipids [96i, 97i], 4) a stimulating

role of low dietary Mg as modulator of blood lipids, atherogenesis [98i], ROS production [99i], and of

Mg deficit in alteration of vascular dynamics [100i, 101i], 5) lipoprotein oxidation as fundamental

event in vascular responses [102i], especially intima-media thickness and blood pressure [103i], 6)

endothelial dysfunction in association with high normal blood CH levels [104i], 7) endothelial

adaption to hypoxic metabolism [105i] that is introduced by ischemic injury [84] and oxidative stress

[87], factors associated with renal lipid accumulation, 8) Mg deficit and ROS release during hypoxia-

reoxygenation, affecting heart activity [106i, 107i] and renal microvasculature [108i]. Although from

these reports alone a concept unifying IRCU pathophysiology and initial Mg status related oxidative

damage to blood vasculature and heart cannot be derived, an early metabolic step pointing into this

direction is seen in oxidant/antioxidant imbalance (co-incidence of high urinary MDA and low plasma

UA (Table 2, A and B)). IRCU exhibits a slight rise of plasma Ca/Pi in presence of low urinary nitrate

[30], an indicator of low production of nitric oxide (NO), the major endothelium-derived vasorelaxant

[109i], and IRCU shares low NO with non-stone forming chronic renal diseases of humans [110i].

Surprizingly, opposite development of urinary Ca/Pi and the ratio urinary Ca/Pi to plasma Ca/Pi

appears as similar exponential function in stone-free as well as –bearing IRCU patients (Fig. 1i, and

), whereas urinary Mg relative to Ca/Pi (in urine and plasma) correlates linearly (Fig. 1i, ) [30];

these findings suggest that factor(s) other than Mg in urine, Ca and Pi in urine and blood plasma must

come into play, allowing for stone formation.

AF 2

Comments to "IRCU initiation as sequel of a cascade of cellular events?"

Impairment of NO’s activity, turned on by dominance of degradation and consumption relative to

production of this vascular dilation effector, has been accused as underlying its association with high

P-UA in cardiovascular disease [111i]. In IRCU P-UA not only is elevated but also directly correlated

to plasma total antioxidant capacity (Fig. 2i, and ), suggesting that the lower the latter, the higher

31

are systemic ROS excess and oxidative damage to proteins [112i], UA synthesizing xanthine oxidase

(XO) included (note that in mice XO gene ablation induces renal interstitial fibrosis and tubular

accumulation of UA precursor oxypurine hypoxanthine (HX) and lipids [113i]), and loss of function

of mineralization-inhibiting osteopontin [112i, 114i]). Together these factors may become devastating

for extra- and intracellular homeostasis of fluid, Ca/Pi in plasma and red blood cells (Fig. 2i, - );

provided intracellular total Na (not intracellular total Mg) is low due to inappropriate renal regulation

of Na and fluid, Ca/Pi rises in plasma and red blood cells, a phenomenon almost completely separating

IRCU from healthy control individuals (Fig. 2i, ). Additional support for deranged cell metabolism

related early events in IRCU etiology comes from unpublished data of patients showing low (not high)

Mg retention after intake of TM type 1 (Fig. 3i, ): Fasting P-UA is decreased, whereas urinary HX

excretion (a documented marker of tissue hypoxia [115i, 116i]) is increased, urinary nitrate and blood

pressure are unchanged, urinary volume and pH are elevated as is ASFP, the crude marker of activity

of stone forming processes in IRCU [38] (Fig. 3i, - ). This scenario, although of uncertain cause

to date, may reflect that during borderline or low Mg status high renal cellular Ca content is induced

via impairment of Na/Ca-exchange [117i] and generalized modulation of cell membrane Na-H-

exchanger protein [118i, 119i], in agreement with postprandially high renal Na reclaim of IRCU vs.

healthy controls (Fig. 3), whereas the nature of still higher postprandial urine pH remains unexplained

(Table 2, A). Consequently, cell swelling and subsequent lowering of intracellular Na concentration

would emerge as epiphenomena, becoming partially reversed during night hours as reflected by mean

higher natriuresis and higher urine volume (due to cell shrinkage) during fasting hours at morning

(Table 2, A). In earlier work into IRCU etiology we have alluded to altered urinary volume, Na and Ca

homeostasis during a daily cycle and possible modulation by a natriuretic peptide hormone [120i].

AF 3

Comments to "NC and Ca stones – Endpoints of a vasculopathy?"

Progressive narrowing of vascular intima-media thickness due to lipids and Ca that invade these

tissues, subsequent hypoxia and formation of vascular "plaques" are pathologies common to athero-

and arteriosclerosis of humans [91, 92, 121i] and rodents fed inadequate dietary Mg [122i];

noteworthy, in human arteries plaques are reduced by Ca entry blocking agents [121i] and in rabbits

prevented by dietary Mg [123i]. Thus, if not abated by treatment, tissue hypoxia emerges as driving

force for ROS damage to mitochondria [124i], a risk factor of reduction of tubular electrolyte transport

energy and efficiency, tubulo-interstitial production of Ca binding collagen I (indicator of

fibrogenesis) and inflammation [125i], oxidative damage to DNA and lipids [126i], intra-cytoplasmic

CaPi precipitation, segregation within vesicles, extrusion of these into interstitium of calcifying

matrices [127i]. In interstitium crystal ghosts, an organic phase able to react with Ca and Mg [128i,

129i], are present during physiologic (bone) and dystrophic (intrarenal) calcification and potentially

32

able to serve as template for deposition of ACP and scaffold for seizure of Ca ions to form HAP,

although Ca needs dissipation from blood vessel walls (to halt calcification). Extracellular matrix

calcification, occurring in soft tissues such as blood vessels, is prevalent in patients with chronic

kidney disease [130i], and in IRCU association of calcified collagen fibers and cell membrane vesicles

with Randall plaques has been described [131i]. Thus, if one accepts that in IRCU transient or

permanent hypoxia of renal tissue (see above) coincides with increasing renal Mg retention, viz. low

Mg status and decreasing urine volume and Ca (Table 3, B), then these factors together with

increasing blood lipids (Tables 2 and 3, B) likely act in concert to allow for extratubular but still

intrarenal CaPi precipitation, growth of this mineral, and formation of plaques [13]. For several

aspects of such a scenario a mosaic of data is available from our experiment in rats fed a diet rich in

CH and lipids but normal Mg content [20]: Compared with rats fed normal chow, this diet not only

induces cortico-medullary NC and CaPi (brushite; Ca/Pi 1.0) stones, but also increase of intrarenal Ca

and, stronger, Mg, accompanied by decrease and increase of Pi in cortex and medulla, respectively; in

glomerula-bearing cortex Ca/Pi is 4-fold increased, and in total kidney mean Ca/Mg is higher than in

controls (Fig. 4i; A, - ). There is in addition impressive destruction of microarchitecture of

tubule-lining cells (Fig. 4i; B, ), intracellular accumulation and broad dissemination of Mg (Fig. 4i;

B, ) and Ca (not shown), in sharp contrast with the normal situation (Fig. 4i; B, and ) [132i]. At

the level of mouse kidney cells (from interstitium, endothelium, glomeruli, tubuli) adaptation to

hypoxia occurs via activation of target genes and expression of proteins [133i]. In cerebral tissue

vitally threatened by ischemia-induced Ca overload intracellular Mg accumulation occurs via divalent

cation transport channel protein, a member of the TRPM membrane protein family [134i]. A similar

structure-function relationship, serving to balance intracellular Ca and Mg, has been observed in

kidney diseases [135i]. Co-existence of rat renal cortical-medullary-papillary increase of Mg vis-à-vis

Ca (on a molar basis) with calcifications and stones [Fig. 4i; 20], and rise of Mg in renal medulla of rat

with isolated NC in present work [Fig. 4] are unprecedented findings. However, existence and details

of this complex interplay of Ca and Mg [136i] in CaPi calcification and stone-bearing human kidney

await demonstration.

In the 1970s the "Vascular response to injury" hypothesis has been inaugurated to understand

similarities between atherosclerosis and the response of arteries to experimental injury [137i]; later it

was recognized that ROS and tissue injury can be partners in biology of disease (ROS are released

during both hypoxia and hyperoxia [124i]), leading to direct probing of ROSs roles in this setting

[138i] and in mouse kidney developing interstitial fibrosis after ischemia-reperfusion [139i]. Further

support for a central causative role of transient tissue ischemia-reperfusion in both extra-renal vascular

and intra-renal parenchymal calcifications, almost ideally fulfilling the above theory [137i], comes

from data of our experiment with highly inbred Lewis rats fed normal diet but carrying an infrarenal

aortic graft: The graft-bearing animals develop, besides histologically detectable NC [140i],

calcifications within media of the graft and a high positive correlation of plasma ionized Mg with P-

33

UA [141i] (note that in injured vascular endothelium ROS-producing XO is ubiquitous [142i]), but no

change of blood pressure, urinary Mg and total protein [140i]; however, there is imbalance of

endothelium derived endothelin (vasoconstrictor) and NO (vasodilator), and rise of renal cortical Ca/Pi

(Fig. 4i; C, - ). In studies using a superior technology increasing extracellular Mg has a protective

effect against post-ischemic damage of rat kidney, including maintenance of low intracellular Na,

changes probably related to beneficial role of Mg in recovery of renal ATP synthesis during post-

ischemic reperfusion [143i]. Although the significance of findings in rodents as a basis for Ca stone

formation in IRCU is uncertain to date, Mg supplementation of daily food not only is effective in stone

metaphylaxis of IRCU [14] but also in prevention of NC [140i] and vascular calcifications [141i].

Therefore, one so far neglected anti-calcification mechanisms of Mg may be sought in achievement of

higher solubility of CaPi, viz. soft tissue Mg/Ca ratios >0.5 - 1 (Fig. 4i, D) [23]. Corroborating this

view would be that in primarily mineralizing tissue such as bone Mg/Ca <0.5 and a rise of Ca/Pi

develop in response to food Mg deficit (Fig. 4, B).

34

Legends to figures (marked "i")

1i: , : Relationship of urinary Ca/Pi divided by plasma Ca/Pi (UM · PM-1), and urinary

Ca/Pi (UM); note that UM and PM are synonymous with UCa · UPi-1, and PCa · PPi

-1,

respectively. ●, ○: stone-free and –bearing male IRCU patients. : Correlations of UM ·

PM-1 and UM · PM

-1 divided by UMg in fasting urine of stone-free (SF) and -bearing (SB)

patients, respectively. Dotted vertical lines in - span the range of Log UM · PM-1,

within which the vast majority of patients clusters. Stippled horizontal lines in , : at

molarity of UM ≤1.0 only nanosized and poorly crystallized ACP is present. Redrawn

from [30].

2i: : Plasma UA (P-UA) of younger male healthy controls, younger (≤37 years) and older

(>37 years) IRCU patients. : Correlation of P-UA and plasma total antioxidants (P-

TAS) of stone-free (▲) and –bearing (■) patients, respectively. : Plasma and red blood

cell (RBC) Ca/Pi; : (Ca/Pi)/Mg; : (Ca/Pi)/Na. Light and hatched columns are from

healthy controls and IRCU, respectively. : Correlation of (Ca/Pi)/Na in plasma and

RBC from (▲) IRCU patients and (●) age-matched healthy controls (note that only one

patient overlaps). ●: Mean value and SE (bars) of 14 controls. Redrawn from [29].

3i: ( - ): Unpublished own data illustrating in IRCU the yet hypothetical link between

Mg retention ( ), ASFP ( ) and several associated variables ( - ); note that along

increase of Mg retention ASFP rises too. Light and hatched columns: low and high Mg

retention, respectively. Mean values, SE (bars); ***: p <0.005; **: p <0.01; *: p <0.05; a: p = 0.08; b: p = 0.15; n: number of animals.

4i: A: Renal cortico-medullary-papillary gradients of Mg, Ca, Pi, ratios Ca/Mg and Ca/Pi

(dimension is µM per g dry weight) of rats fed a CH- and fat-rich diet ( - ); redrawn

from [20]). Light columns: normal diet; hatched columns: experimental diet. Mean

values, SE (bars). *: p ≤0.05; n: number of animals.

B: Electron microscopy with energy-dispersive elemental X-ray analysis of intracellular

Mg ( , ) and the corresponding areas of renal tubular cells ( , ) from the cortico-

medullary region of rats fed the CH- and fat-rich diet; note that intracellular Ca is

similarly distributed (not shown), and that ionized and bound (chelates, complexes)

elements are not discernible. Left ( , ) and right ( , ) panels are from rat on normal

and experimental diet, respectively. ––––– : bar 5 µm. Modified from [132i].

C: Urinary (U) excretion of endothelin and nitrate per day (mean of two collection days)

and 100 g body weight, and ratio of these vasoeffectors ( , , ). ( ): Ca/Pi ratio in

renal cortical dry tissue; - - - - : Lower limit of CaPi precipitation in vitro [58]. Light

35

columns: controls, undergoing sham operation; hatched columns: rats bearing an aortic

graft. For details see [140i].

D: X-ray diffraction pattern of Ca and phosphorus (P) peaks at increasing ambient Mg

molarity; note that Ca and P peaks are not detectable at Mg/Ca = 1. Modified from [23].

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