METHODS

RESULTS

For clarity of presentation, we have gathered all of our TZ vs. no TZ comparisons in one table (Table 2), and all of our fasting vs. fed comparisons under no TZ and TZ conditions in a separate table (Table 4). Table 2 presents mean differences between thiazide and no thiazide, as this is the manner in which the computations were actually performed. Table 4 presents the actual mean values for every measurement along with an estimate of variance (SE).

Sodium and potassium.

Serum Li levels and filtered loads of Li both rose with TZ in fasting and fed states (Table 2) and Li clearance and FELi fell (Table 2 and Fig. 1, bottom left). Although TZ reduced serum Na levels, distal Na delivery and distal Na reabsorption (Table 2), urine Na excretion, filtered load (FLNa), and FENa were unaffected by the drug. Although reduced from pretreatment levels, Li clearance, FELi, distal Na delivery, and distal Na reabsorption during TZ treatment remained higher than in normal controls in the fed, but not the fasting state (Table 2, columns 4 and 5). Serum K fell, as expected. Mean values for all parameters for IH and N before and during TZ treatment are detailed in Table 4.

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Calcium.

Urine Ca excretion (Fig. 1, top left; Table 2) fell with TZ treatment in the fed state. The fraction of filtered Ca excreted (FECa) also fell both when fasting and with meals (Fig. 1, top right, Table 2). Filtered loads of Ca were unaltered even though serum Ca was increased by TZ in the fed state (Table 2). Because of stable filtered load and increased proximal tubule reabsorption, as evidenced by reduced FELi, distal delivery of Ca (Table 2 and Fig. 1, bottom right) fell in both the fasting and fed state, even though serum Ca rose in the fed state (Table 2). As expected, given a fall in distal delivery, distal Ca reabsorption fell (Table 2), but in the fed state the fraction of distal Ca excreted actually rose significantly (Table 2). Plasma PTH levels fell with TZ in the fasting and fed states (Table 2) and also fell with and without TZ in the fed compared with the fasting state (Table 4). Urine cAMP/g urine creatinine did not change significantly (Table 2). Filtered loads of cAMP could not be measured. Taken together, TZ appears to increase proximal tubule reabsorption and reduce delivery of Ca to more distal sites, which could contribute to a fall in urine Ca. But the drug did not bring values in our patients to those of our normal men in the fed state (Table 2). Distal Ca delivery and reabsorption, urine Ca excretion, and FECa all remained above normal.

Overnight and fasting, urine pH was lower with TZ than control (Fig. 2, top left), but differences did not achieve significance (Table 3). In the fed state, the fall in pH with TZ (Fig. 2, top middle) was significant (Table 3). Because of the fall in urine Ca and pH already noted, SS with respect to CaP fell markedly (Fig. 2, lower middle) in the fed state, to a statistically significant extent. The fall overnight is also dramatic but not significant, given only four patients (Fig. 2, lower left). In the fasted state, changes in CaP SS were minimal. CaOx SS also fell with TZ, but variably and without significance (Fig. 2, top and bottom right, Table 3). The fall was entirely due to the fall in urine Ca; urine volume was not affected by TZ (Table 3) nor was urine oxalate excretion (not shown).

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Table 3. Urine pH and supersaturation values

	Control			Thiazide
	Fast	Fed	ON	Fast	Fed	ON
Urine pH	6.56 ± 0.18	6.49 ± 0.09	6 ± 0.3	6.28 ± 0.18	5.9 ± 0.09*	5.47 ± 0.3
SS CAOX	6 ± 1	7.1 ± 0.6	15 ± 2	4 ± 1	6.3 ± 0.6	12 ± 2
SS CAP	1.3 ± 0.4	2.9 ± 0.2	2.5 ± 0.6	0.9 ± 0.4	1.1 ± 0.2*	1.1 ± 0.6
Urine volume, ml/h	153 ± 26	112 ± 14	49 ± 46	106 ± 28	120 ± 14	51 ± 45

Values are means ± SE during 3 periods of the day: fasting, fed, and overnight (ON); SS CaOx and SS CaP are supersaturations of calcium oxalate and calcium phosphate, respectively.

^*Significantly different from control, P < 0.05.

Table 4. Renal calcium handling with food intake and thiazide treatment

	IH No Thiazide		IH Thiazide		Normal Males
	Fast	Fed	Fast	Fed	Fast	Fed
SLi, μM	0.15 ± 0.01	0.11 ± 0.004*	0.20 ± 0.01	0.16 ± 0.004*	0.24 ± 0.03	0.19 ± 0.01
CCR, l/h	7.0 ± 0.6	7.6 ± 0.3	7.4 ± 0.6	7.4 ± 0.3	7.3 ± 0.5	7.7 ± 0.2
FLLi, μM/h	1.1 ± 0.1	0.9 ± 0.1	1.5 ± 0.1	1.2 ± 0.1*	1.7 ± 0.2	1.4 ± 0.1
U Li, μM/h	0.29 ± 0.04	0.22 ± 0.02	0.16 ± 0.04	0.21 ± 0.02	0.25 ± 0.05	0.22 ± 0.03
C Li, l/h	1.8 ± 0.2	2.2 ± 0.1	0.8 ± 0.2	1.3 ± 0.1*	0.89 ± 0.16	0.9 ± 0.07
FELi, %	26 ± 3	28 ± 1	11 ± 3	17 ± 1*	12 ± 2	12 ± 1
SNa, mM	139 ± 0.4	139 ± 0.2	137 ± 0.4	137 ± 0.2	139 ± 0.5	139 ± 0.2
FLNa, mM/h	973 ± 82	1051 ± 42	1017 ± 86	1007 ± 42	1018 ± 72	1074 ± 34
DDel Na, mM/h	253 ± 31	299 ± 16	106 ± 34	181 ± 16	124 ± 22	125 ± 10
ANaR, mM/h	241 ± 30	288 ± 16	97 ± 33	172 ± 16*	113 ± 23	114 ± 10
FEdDelNa, %	5.3 ± 0.7	3.8 ± 0.4	8.4 ± 0.8	4.9 ± 0.4*	20 ± 6	22 ± 3
UNa, mM/h	12 ± 2	11 ± 1	9 ± 2	8 ± 1	8 ± 1	9.5 ± 0.7
FENa, %	1.2 ± 0.1	1 ± 0.1	0.9 ± 0.2	0.8 ± 0.1	0.8 ± 0.1	0.9 ± 0.06
S K, mM	3.8 ± 0.1	4.0 ± 0.04	3.4 ± 0.1	3.5 ± 0.04	3.91 ± 0.07	3.88 ± 0.03
U K, mEq/h	3.9 ± 0.5	3.5 ± 0.2	2.6 ± 0.5	3.9 ± 0.2*	3.8 ± 0.5	3.1 ± 0.2
SCa, mM	2.32 ± 0.02	2.41 ± 0.008*	2.34 ± 0.02	2.44 ± 0.008*	2.34 ± 0.02	2.37 ± 0.006
FLCa, mM/h	9.1 ± 0.8	10.1 ± 0.4	9.5 ± 0.8	9.8 ± 0.4	9.5 ± 0.7	9.8 ± 0.3
DDel Ca, mM/h	2.3 ± 0.3	2.9 ± 0.2	1.0 ± 0.3	1.8 ± 0.2*	1.2 ± 0.2	1.2 ± 0.1
ACaR, mM/h	2.1 ± 0.3	2.3 ± 0.1	0.8 ± 0.3	1.3 ± 0.1	1.0 ± 0.2	0.9 ± 0.1
FEdDelCa, %	15 ± 3	20 ± 1	19 ± 3	29 ± 1*	40 ± 17	58 ± 8
UCa, mM/h	0.3 ± 0.05	0.53 ± 0.03*	0.19 ± 0.05	0.44 ± 0.03*	0.14 ± 0.03	0.28 ± 0.01*
FECa, %	3.2 ± 0.3	5.2 ± 0.2*	2 ± 0.3	4.4 ± 0.2*	1.5 ± 0.2	2.9 ± 0.1*
S Phos, mM	0.87 ± 0.06	0.95 ± 0.02	1.1 ± 0.06	1.1 ± 0.02	1.05 ± 0.04	1.15 ± 0.02*
S Mg, mM	2.64 ± 0.03	2.66 ± 0.01	2.54 ± 0.03	2.52 ± 0.01	2.61 ± 0.03	2.61 ± 0.01
P PTH, pg/ml	39 ± 2	32 ± 0.8*	33 ± 2	29 ± 0.8*	35 ± 2	27 ± 0.6*
UMg, mM/h	0.16 ± 0.03	0.28 ± 0.02	0.21 ± 0.03	0.32 ± 0.02	0.18 ± 0.02	0.31 ± 0.01*
FEMg, %	3.6 ± 0.6	5.8 ± 0.3*	5 ± 0.6	7.5 ± 0.3*	4.2 ± 0.5	6.4 ± 0.2*
FEP, %	12 ± 2	15 ± 1	14 ± 2	17 ± 1	11 ± 1	14 ± 0.6*
UcAMP/Cr, μM/g	4.3 ± 0.7	3.4 ± 0.4	2.1 ± 0.9	2.7 ± 0.4	ND	ND

Values are expressed as means ± SE;

^*Significant difference from fasting, P < 0.05. See Table 2 for abbreviation definitions. ND, not done.

The fall in urine pH seems to reflect an increase in net acid excretion (NAE, meq/h) during the fed period [TZ-control = 1.39 (0.11, 2.68) P = 0.028; the 95% confidence interval appears in parentheses]. The increase of NAE with TZ fasting was similar [1.56 (−1, 4) P, NS] but not significant. Both titratable acid (TA) and ammonia (NH₄) excretion rose in the fed state [0.29 (0.01, 0.56) P = 0.032 and 0.9 (0.44, 1.37) P < 0.001, TA and NH₄, respectively]. Changes in urine CO₂ were minimal [0.2 (−1.1, 0.69) P, NS]. Overnight changes in NAE and GI anion were well matched [1.15 (0.29, 2.00) P = 0.017 and −1.1 (−2.5, 0.3), P, NS, NAE, and GI anion, respectively]. The possible reason for increased NAE was a reduction of GI anion absorption (methods) [−1.91 (−3.39, −0.43) P = 0.005, fed; −1.73 (−4.68, 1.22) P, NS, fasting]. The components of GI anion absorption are simply the sum of urine nonmetabolite cations (Na + K + Ca + Mg) − anions (Cl + Phos), where all are expressed in milliequivalents per hour. None of these six constituents changed significantly, but the individual changes (−2.27, 0.38, 0.09, and −0.17 for the cations, respectively, and −0.42 and 0.36 for the anions) yield the overall change of −1.91. Given the loss of 1.91 meq/h of anion and increase of NAE of 1.39, one might presume a small increase in net systemic acid delivery of 0.52 meq/h for the fed state although given the variability involved net acid balance might well be neutral.

Magnesium and phosphorus.

TZ lowered serum Mg and raised fractional excretion of Mg in the fed period above both pretreatment values and values in normal males (Table 2). TZ lowered serum P levels below normal controls in the fed state but increased it above the low values of the patients in the control periods. It did not affect fractional excretion of P (Table 2) or urine P excretion (not shown).

DISCUSSION

In this study, as in our prior work, subjects with IH have increased Li clearance at baseline, compared with controls, reflecting decreased proximal tubule reabsorption of Na (41). Since Ca reabsorption at this site is passive and follows Na (16), the delivery of both Na and Ca out of the proximal tubule is increased in men with IH compared with N. TZ decreased Li clearance and distal delivery of Na and Ca out of proximal tubule in IH (Table 2), providing evidence that a significant mechanism for the hypocalciuric action of the drug is via increased proximal tubule reabsorption. Creatinine clearance, Ca filtered load, and urine Na excretion were not altered by chronic TZ treatment. A modest drop in intravascular volume has been shown to occur after initiation of TZ in other studies (7, 34), after which subjects come into balance at this new modestly decreased weight, and Na excretion returns to baseline; presumably volume depletion occurred in our subjects as well (Table 1).

The effect of TZ on distal Ca delivery and fractional urine Ca excretion is present in both fasting and fed states; however, patients with IH still exhibit sensitivity to nutrient ingestion. TZ tends to normalize proximal tubule Na and Ca handling in IH, so that, at least in the fasting period, delivery of Na and Ca out of proximal tubule does not differ between control men and men with IH on TZ, nor does urine Ca excretion. During the fed period, absolute and fractional Ca excretion increases in both normal men and men with IH. However, in IH, unlike normal men, distal delivery of Na and Ca rises after feeding, even during TZ administration and exceeds that of normal men eating the same food. Thus, TZ may ameliorate, but not totally correct, the abnormalities of Na and Ca handling found in IH, which are expressed in decreased proximal tubule solute reabsorption, particularly after meals. A further implication of these data is that the increase in Ca excretion with meals in normal men is due to changes in Ca handling beyond the proximal tubule; in IH, the augmented delivery of Ca out of the proximal tubule may amplify this distal event. Alternatively, there may be additional abnormalities in distal Ca handling in IH, which cannot be detected in this study.

During the fed period, much of the increased Na and Ca delivered out of proximal tubule is eventually reabsorbed in the distal nephron, such that distal Na and Ca reabsorption in IH exceeds that of normal subjects (41). The site of this reabsorption is not known, however, and for Ca, it may be located in either ascending limb or distal tubule. Our current data do not allow us to discriminate between these sites at present. However, probably because of decreased delivery, absolute distal Ca reabsorption in IH is significantly lower on TZ than at baseline, suggesting that there may be less Ca furnished to sites in the inner medulla, where Randall's plaque is formed. Future studies may be able to confirm whether TZ administration is able to decrease plaque formation in patients with IH.

We recognize that our calculations rely on the assumption that calcium reabsorption follows sodium reabsorption in proximal tubule. Whether or not this relationship is disrupted, in part, by active calcium transport in the S3 segment (31), as has been demonstrated in perfused rabbit S3 segments, cannot be determined in humans at the present time. Likewise, under conditions of diuretic-induced volume contraction, lithium reabsorption in the distal nephron via ENaC-mediated transport may render interpretation of lithium clearance less certain as a marker of proximal tubule reabsorption (18, 35). We calculated the urine Na/K molarity ratio as a gauge of such an effect and found the ratio fell with TZ in the fed period [−1.18 (−2.32, −0.05) P = 0.037]. Given this finding, we must accept the possibility of an overestimate of the reduction of Li clearance with TZ; we cannot quantify its magnitude further.

Our data confirm those of other investigators, who have found a decrease in urine SS with respect to Ca salts in patients with IH treated with TZ. Woelfel et al. (39) measured SS only for CaOx, and they found that it decreased significantly in four of seven patients treated with TZ for 3–4 mo. Borghi et al. (4) assessed SS for both CaOx and CaP at regular intervals during their placebo-controlled trial of indapamide, and they found significant decreases in both at 6 mo in the indapamide arms, which declined further by 12 mo. However, unlike other studies, we found a decrease in urine pH in IH patients while on chlorthalidone, which is significant during the fed period, and this has consequences for urine SS. Nicar et al. (26) found a slight fall in pH (5.97 ± 0.49 vs. 5.84 ± 0.49) with thiazide, but 24-h urine samples were used in a diet-controlled, but outpatient, setting; perhaps with a CRC design, this fall might have achieved significance. Although there is a trend to decreased urine SS with respect to both CaOx and CaP, only the drop in CaP SS during the fed period is significant among our patients. The fact that there is only one overnight urine sample per patient in each study phase limits the ability for changes to reach significance at that time period. Although decreases in both types of SS have importance for prevention of Ca stones, CaP may be of particular importance, as even for stones composed primarily of CaOx, the initiating phase is CaP (15).

The fall of urine pH with TZ clearly reflects an increase of NAE, and this can be ascribed to a fall in GI organic anion absorption as calculated from the difference between the sum of the major nonmetabolizable cations and anions (29). The numerically main reason for the fall in GI anion absorption, as calculated, is a fall in urine Na not balanced by a corresponding fall in urine chloride. Given the fixed diets used with TZ and control periods, we have to presume these changes could reflect effects of TZ on GI, as well as renal transport. As this is simply an observation during the course of our experiment, the present study cannot elucidate mechanisms further.

Several metabolic changes that were expected were noted during TZ treatment, such as modest decreases in serum Na, Mg, and K, and a rise in serum Ca that occurred during the fed period. Prior studies in which PTH was evaluated during TZ treatment of IH have shown conflicting results. In one study, no change was found in PTH in IH patients during TZ treatment compared with baseline (37). However, in accordance with two other studies, we found a small, but significant, decrease in PTH related to TZ treatment (8, 9). In our study, PTH was measured at multiple time points throughout the day, which may facilitate the detection of small, but potentially significant, changes in PTH related to TZ treatment. The drop may be the result of the modest rise in serum Ca that occurs during TZ treatment, although this rise is seen predominantly in the fed period, while PTH is significantly lower in both the fasting and fed states. The effect on distal Ca reabsorption from the decrease in PTH is unclear. Of note, vitamin D tended to fall in all four patients, but the difference was not significant (Table 1).

Urine Mg does not increase during chronic TZ therapy, but because serum Mg and the filtered load of Mg decrease, the fractional excretion of Mg rose with TZ treatment and presumably led to the fall in serum Mg that we observed. This could reflect decreased reabsorption in either the thick ascending limb or in the distal nephron. Nijenhuis et al. (28) found that mice exposed to TZ had a decrease in the expression of the magnesium channel TRPM6 in the distal tubule. This could be one potential explanation for the relative decrease in magnesium reabsorption seen in our subjects.

Our previous studies using endogenous Li clearance have shown that many patients with IH have decreased proximal tubule Na reabsorption and deliver increased Ca into the distal nephron where reabsorption, although increased, is inadequate to prevent hypercalciuria (41, 42). This explanation for IH would also provide a mechanism for the initiation and growth of Randall's plaque, via increased delivery of Ca to the thick ascending limb and resultant loading of the medullary interstitium with the augmented Ca reabsorption that would result (10). TZs should be an ideal treatment for this abnormality, by potentially normalizing Ca reabsorption in the proximal tubule. Since overgrowth on exposed plaque surface is the major way by which idiopathic CaOx stones form (15), decreasing plaque formation may be a second mechanism by which TZs can prevent recurrent stones, in addition to lowering urine Ca.

Our study suggests that a main hypocalciuric effect of chronic TZ treatment in IH is increased proximal tubule reabsorption of Na and Ca, with consequent decrease in urine Ca excretion and urinary SS with respect to CaOx and CaP. This may have the additional effect of decreasing formation of Randall's plaque, which might provide additional protection from stone recurrence.

GRANTS

This publication was made possible by National Institutes of Health (NIH) grant P01 DK56788 and by grant UL1 RR024999 to the University of Chicago GCRC from the National Center for Research Resources (NCRR), a component of the NIH, and NIH Roadmap for Medical Research. Its contents are solely the responsibility of the authors and do not necessarily represent the official view of NCRR or NIH.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Author contributions: K.J.B. performed experiments; K.J.B. and F.L.C. analyzed data; K.J.B. prepared figures; K.J.B. and E.M.W. drafted manuscript; K.J.B., E.M.W., and F.L.C. edited and revised manuscript; K.J.B. and F.L.C. approved final version of manuscript; E.M.W. and F.L.C. conception and design of research; E.M.W. and F.L.C. interpreted results of experiments.