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American Journal of Physiology - Regulatory, Integrative and Comparative Physiology logoLink to American Journal of Physiology - Regulatory, Integrative and Comparative Physiology
. 2015 May 6;309(1):R85–R92. doi: 10.1152/ajpregu.00071.2015

Sex differences in proximal and distal nephron function contribute to the mechanism of idiopathic hypercalcuria in calcium stone formers

Benjamin Ko 1,, Kristin Bergsland 1, Daniel L Gillen 2, Andrew P Evan 3, Daniel L Clark 3, Jaime Baylock 1, Fredric L Coe 1, Elaine M Worcester 1
PMCID: PMC4491535  PMID: 25947170

Abstract

Idiopathic hypercalciuria (IH) is a common familial trait among patients with calcium nephrolithiasis. Previously, we have demonstrated that hypercalciuria is primarily due to reduced renal proximal and distal tubule calcium reabsorption. Here, using measurements of the clearances of sodium, calcium, and endogenous lithium taken from the General Clinical Research Center, we test the hypothesis that patterns of segmental nephron tubule calcium reabsorption differ between the sexes in IH and normal subjects. When the sexes are compared, we reconfirm the reduced proximal and distal calcium reabsorption. In IH women, distal nephron calcium reabsorption is decreased compared to normal women. In IH men, proximal tubule calcium reabsorption falls significantly, with a more modest reduction in distal calcium reabsorption compared to normal men. Additionally, we demonstrate that male IH patients have lower systolic blood pressures than normal males. We conclude that women and men differ in the way they produce the hypercalciuria of IH, with females reducing distal reabsorption and males primarily reducing proximal tubule function.


idiopathic hypercalciuria (IH) is a common familial and presumably genetic trait among patients with calcium nephrolithiasis (52) to whose pathogenesis it appears to contribute by raising urine supersaturations (SS) with respect to stone-forming salts. The mechanism of hypercalciuria is not an increase of calcium filtered load but rather reduced renal tubule calcium reabsorption (54), which can be detected in the fasting state but differs most markedly from normal after eating (54).

The reduced tubule calcium reabsorption of IH cannot be ascribed to reduced serum parathyroid hormone (PTH) levels (51), or to insulin hormone (PTH) levels (51), but is, at least in part, related to an abnormally marked response to the slight rise of serum calcium with meals (51). The main factor that appears related to the fall in tubule calcium reabsorption seems to be food itself, suggesting an abnormally enhanced response of cell surface calcium receptor in kidney, bone, intestine, or elsewhere to such constituents of meals as aromatic amino acids, a conjecture that has as yet not been tested (11, 23, 41, 51).

The sites of reduced tubule calcium reabsorption are not fully known, but using endogenous lithium clearance measurements, we have found that proximal tubule (PT) reabsorption is lower in IH than in normal subjects, so that distal delivery of sodium, calcium, and magnesium is increased (53). Urine calcium excretion exceeds normal because distal calcium reabsorption does not increase, proportional to the increased delivery, whereas distal sodium reabsorption matches the increased delivery, so urine sodium excretion does not differ from normal.

Because many prior studies of tubule function have documented differences between men and women (4, 20, 39, 50), we have tested the hypothesis that patterns of segmental nephron tubule calcium reabsorption differ between the sexes in IH and normal (N). Our initial findings, presented here, support that conjecture. In particular, we have found that reduced PT reabsorption is more characteristic of men with IH, whereas, among women, IH arises mainly from reduced distal nephron calcium reabsorption.

METHODS

Patients and Normal Subjects

We studied 32 IH subjects (18 male) and 17 N subjects (6 male) Table 1. Some subjects have appeared in other studies from our laboratory (Table 1). The study was approved by the Institutional Review Board at the University of Chicago (protocols 12881A and 09-164B). Subjects were informed of the study and provided written informed consent to participate in the study.

Table 1.

Patients and normal subjects

Subject Number Type Age, yr Sex Weight, kg Height, cm
1 None*# 47 F 86 160
2 None*# 44 F 55 158
3 None*# 26 F 68 168
4 None*# 55 F 67 169
5 None*# 28 M 66 163
6 None*# 55 M 88 181
7 None*# 44 M 85 178
8 None 28 F 57 158
9 None* 24 F 71 178
10 None* 50 M 76 183
11 None* 29 F 62 161
12 None 38 F 78 150
13 None 25 F 65 164
14 None 26 M 93 172
15 None 38 M 141 174
16 None 29 F 53 156
17 None 48 F 95 165
    Mean 37 ± 3 77 ± 5 167 ± 2
18 IH*# 48 M 102 185
19 IH*# 64 M 93 173
20 IH# 66 M 88 185
21 IH# 55 M 87 168
22 IH# 58 F 58 158
23 IH*# 44 M 91 181
24 IH*# 45 F 57 164
25 IH*# 28 M 75 182
26 IH# 22 M 93 182
27 IH# 42 F 68 168
28 IH# 44 M 85 182
29 IH*# 29 M 89 185
30 IH 63 F 52 148
31 IH 66 M 105 169
32 IH# 59 M 85 177
33 IH# 40 F 82 164
34 IH*# 27 M 77 174
35 IH*# 52 F 61 158
36 IH 45 M 87 174
37 IH*# 56 M 95 178
38 IH 38 F 67 164
39 IH 56 F 91 168
40 IH 65 F 61 164
41 IH 46 M 57 177
42 IH 38 M 81 183
43 IH$ 52 F 61 169
44 IH$* 47 F 79 158
45 IH# 30 F 103 160
46 IH# 56 F 62 162
47 IH#¢ 30 M 75 179
48 IH# 41 M 80 168
49 IH 38 F 74 163
    Mean 47 ± 2+ 79 ± 3 171 ± 2

Values are expressed as means ± SE. M, male, F, female; IH, idiopathic hypercalciuria. $Subject previously reported by Worcester et al. (54).

*

Subject previously reported by Worcester et al. (53).

Subject previously reported by Bergsland et al. (4b).

#

Subject previously reported by Bergsland et al. (4a).

Subject previously reported by Worcester et al. (51).

¢

Subject previously reported by Worcester et al. (51).

+

P < 0.05, vs. none.

Protocol

All subjects were studied in the General Clinical Research Center (GCRC) at the University of Chicago, as previously described (51). Briefly, the study began at 6 AM. Two 1-h fasting urine samples were collected between 6 and 8 AM, after which subjects ate breakfast. Hourly urine collections continued during the day for a total of 14 clearance periods. A 15th collection was made overnight from 8 PM until 6 AM the following morning. Matching blood samples are collected hourly and every half hour during the 2 h following meals.

Every patient collected and brought in a 24-h urine sample that began the day before the study and ended at 6 AM on the day of the study. For this collection, they had been instructed to adhere to the study diet. This collection is reported separately.

All subjects ate a diet consisting of three isocaloric meals composed of common foods. The 1,800-kcal base diet provided 1,160 mg of calcium, and 1,240 phosphorus daily, as determined by laboratory analysis of the three meals (Covance Laboratories, Madison WI). The base diet also provided 2,141 mg sodium, 2,427 mg potassium, and 2,018 mg of magnesium daily. Subjects were stratified to one of three caloric levels (1,800, 2,100, or 2,400 kcal/day), according to an estimate of individual energy needs using the Schofield equation (43), and the diet was planned by a nutritionist in the GCRC using Nutritionist 4, version 4.1 software (N-Squared Computing, San Bruno, CA). Blood pressures were taken hourly (Datascope Trio or Philips Sure Signs VS3).

Laboratory Methods

Serum ultrafiltrate and urine calcium, magnesium, sodium, potassium, creatinine, and urine volume were measured, as described elsewhere (51). Lithium was measured in deproteinated serum and urine by atomic emission spectroscopy (Instrument Laboratory model 951 AA/AE) with an air-acetylene-N2O flame at a wavelength of 670.8 nm and bandwidth of 0.5 nm with background correction, as previously described (53).

Calculations

All excretion rates, clearances, and deliveries are expressed as corrected per 1.73 m2.

Distal delivery.

For Ca, Mg, and Na, distal delivery out of proximal tubule is

DDel[x]=UF[x]gFEpt (1)

where DDel is distal delivery, UF is ultrafiltrate concentration of Ca, Mg, or Na, g is glomerular filtration rate, x is a given solute, and FEpt is the fraction of filtrate delivered into PT that is excreted out of PT downstream to more distal segments. We have used endogenous creatinine clearance to estimate g. For these calculations, we have relied on CrCl, assuming that in subjects with generally normal renal function, secretion artifacts will be minimal. We have tested this assumption in our first publication of this protocol and found the creatinine and iothalamate markers of glomerular filtration rate (GFR) gave indistinguishable results (54).

It is established that endogenous lithium is freely filtered and reabsorbed almost all in PT (15). Some reabsorption may occur in the thick ascending limbs, especially with extracellular volume contraction (3) and perhaps also in the distal tubule (2, 3), but even so, the majority of reabsorption is in PT (2, 3). Therefore,

DDel[x]=UF[x]gFELi (2)

and since FELi is simply ULi·V/UFLi·g, urine excretion divided by filtered load, or more simply clearance of lithium (CLi) divided by g,

DDel[x]=UF[x]CLi. (3)

Given uncertainty about contributions of post PT Li reabsorption, we performed our studies on well-hydrated subjects eating identical diets that contain adequate Na (∼100 mEq/day).

Absolute reabsorption and fractional excretion of distally delivered Ca, Mg, or Na. By mass balance,

U[x]V=DDel[x]R[x] (4)

where U is urinary excretion, V is urine volume, R is absolute reabsorption of x, by distal nephron segments that for Ca and Mg would include thick ascending limb of the loop of Henle and distal convoluted tubule (DCT), and for Na also include the collecting ducts. The fraction of DDel[x] excreted, FED[x] is urine excretion/distal delivery,

FED[x]=(U[x]V)/DDel[x] (5)

and therefore, looked at another way, urine excretion is the simple product of distal delivery and fraction of distal delivery excreted:

U[x]V=DDel[x]FED[x]. (6)

Statistical Analyses

Patient characteristics were summarized via the mean and standard deviation for continuous covariates and frequency and percentage for categorical covariates. To account for within-subject correlation resulting from repeated measurements on the same subject throughout the day, generalized estimating equations (13) utilizing an identity link function and independence working covariance structures were used to estimate and test mean laboratory values between IH and N, and between fasting and fed periods. To correct inference for within-subjects correlation, robust variance estimates clustering on “patient” were used (57). Multiplicative interactions were used to estimate fasting state and sex-specific mean differences between IH and N. Simultaneous tests of regression model coefficients were conducted using multivariate Wald tests. No adjustment for multiple comparisons was made. All statistical calculations were performed using R (21) and Systat 13 software (Systat Software, Chicago, IL).

RESULTS

Men and Women Combined Fasting and Postprandial Measurements

Determinants of distal delivery.

Fractional lithium excretion (FELi) did not differ between IH and N in the fasting or fed condition (Table 2) but did increase significantly with meals among IH subjects. Ultrafiltrate (UF) Ca rose with meals equally in IH and N; UF Mg and serum Na did not change. Creatinine clearance (CCr) did not differ with meals or between groups.

Table 2.

Generalized estimating equations results for filtered loads, distal deliveries, distal handling, and excretions

Fast
Fed
Overnight
24 Hour
N IH N IH N IH N IH
FELi 14.8 ± 3.1 18.4 ± 1.5 17.6 ± 1.9 20.9 ± 1.2 11 ± 2 9 ± 2 14 ± 2 18 ± 1
UF [Ca], mmol 1.31 ± 0.01 1.31 ± 0.01 1.34 ± 0.01 1.35 ± 0.01 1.29 ± 0.02 1.31 ± 0.02 1.28 ± 0.01 1.32 ± 0.01
CCr, l/h 8.3 ± 0.8 6.9 ± 0.4 7.9 ± 0.6 7.2 ± 0.3 6.5 ± 0.6 6.6 ± 0.5 8.4 ± 0.7 6.5 ± 0.5
FLCa, mmol/h 10.8 ± 1.1 9.0 ± 0.5 10.5 ± 0.8 9.7 ± 0.4 8.4 ± 0.7 8.7 ± 0.7 10.7 ± 0.9 8.5 ± 0.6
DDELCa, mmol/h 1.28 ± 0.2 1.63 ± 0.2 1.66 ± 0.1 2.00 ± 0.1 0.8 ± 0.1 0.8 ± 0.1 1.4 ± 0.2 1.5 ± 0.1
ACaR 1.13 ± 0.2 1.39 ± 0.2 1.38 ± 0.1 1.52 ± 0.1 0.6 ± 0.1 0.46 ± 0.09 1.28 ± 0.2 1.2 ± 0.1
FEDCa, % 14.0 ± 1.6 18.0 ± 1.8 20.0 ± 1.9 28.6 ± 1.7* 27 ± 4 41 ± 4* 16 ± 2 21 ± 2
UCa, mmol/h 0.15 ± 0.02 0.25 ± 0.02* 0.29 ± 0.03 0.49 ± 0.03* 0.14 ± 0.04 0.28 ± 0.01* 0.17 ± 0.02 0.28 ± 0.02*
ADJ UCa, mmol/h 0.17 ± 0.02 0.26 ± 0.02* 0.29 ± 0.03 0.48 ± 0.03*
FECa, % 1.4 ± 0.2 2.7 ± 0.2* 2.9 ± 0.2 5.2 ± 0.03* 2 ± 0.3 3.5 ± 0.3* 1.8 ± 0.2 3.4 ± 0.2*
SNa 137.9 ± 0.4 138.6 ± 0.3 137.8 ± 0.4 138.2 ± 0.3 138 ± 0.4 139 ± 0.4 137 ± 0.3 139 ± 0.2*
FLNa 1141 ± 114 959 ± 52 1084 ± 86 996 ± 44 900 ± 80 919 ± 74 1152 ± 98 900 ± 74
DDELNa 135.4 ± 19.6 173.7 ± 16.6 172.2 ± 11.9 206.0 ± 15.1 84 ± 11 80 ± 10 150 ± 21 157 ± 16
ANaR 128.4 ± 19.4 166.7 ± 15.9 163.2 ± 12.4 197.4 ± 14.6 80 ± 11 77 ± 10 148 ± 22 151 ± 16
FEDNA 5.9 ± 1.3 4.2 ± 0.5 6.3 ± 1.1 4.5 ± 0.3 5.8 ± 0.9 4.3 ± 0.8 5.2 ± 0.6 4.1 ± 0.4
UNaHR 7.0 ± 1.4 6.9 ± 1.0 8.9 ± 1.1 8.6 ± 0.7 3.8 ± 0.5 3.1 ± 0.4 6.4 ± 0.7 5.9 ± 0.5
FENa 0.65 ± 0.12 0.70 ± 0.10 0.84 ± 0.10 0.84 ± 0.05 0.47 ± 0.07 0.35 ± 0.06 0.61 ± 0.06 0.66 ± 0.04
FLMg 4.9 ± 0.5 4.0 ± 0.2 4.6 ± 0.4 4.3 ± 0.2 3.8 ± 0.4 3.9 ± 0.3 4.9 ± 0.4 3.8 ± 0.3*
DDELMg 0.59 ± 0.09 0.74 ± 0.08 0.74 ± 0.05 0.90 ± 0.08 0.37 ± 0.05 0.34 ± 0.04 0.64 ± 0.09 0.66 ± 0.07
AMgR 0.42 ± 0.08 0.56 ± 0.07 0.49 ± 0.06 0.62 ± 0.08 0.21 ± 0.04 0.16 ± 0.04 0.47 ± 0.08 0.47 ± 0.46
FEDMg 37.2 ± 3.4 27.6 ± 2.8* 40.8 ± 2.8 39.1 ± 2.4 52 ± 6 54 ± 6 34 ± 4 31 ± 3
UMgHR 0.17 ± 0.02 0.17 ± 0.02 0.25 ± 0.01 0.29 ± 0.02 0.15 ± 0.02 0.11 ± 0.004 0.18 ± 0.02 0.19 ± 0.01
ADJ UMgHR 0.18 ± 0.02 0.18 ± 0.02 0.26 ± 0.01 0.28 ± 0.02
UKHR 3.1 ± 0.4 3.3 ± 0.4 3.2 ± 0.2 3.01 ± 0.2 1.6 ± 0.2 1.6 ± 0.2 2.7 ± 0.3 2.4 ± 0.2

Values are expressed as means ± SE. Bold indicates significant difference from fast, same group. IH, intermittent hypoxia; N, normoxia; FELi, urine excretion divided by filtered load; UF, ultrafiltrate; [Ca], calcium concentration; CCr, calcium creatine; FLCa, filtered load of calcium; DDELCa, distal delivery of calcium; ACaR, absolute calcium reabsorption; FEDCa, fraction of distal calcium delivery excreted; UCa, urine calcium excretion; ADJ UCa, urine calcium excretion adjusted for DDELCa; FECa, fractional calcium excretion; SNa, serum sodium; FLNa, filtered load of sodium; DDELNa, distal delivery of sodium; ANaR, absolute sodium reabsorption; FEDNA, fraction of distal calcium delivery excreted; UNaHR, hourly urine sodium excretion; FENa, fractional sodium excretion; FLMg, filtered load of magnesium; DDELMg, distal delivery of magnesium; AMgR, absolute magnesium reabsorption; FEDMg, fraction of distal magnesium delivery excreted; UMgHR, hourly urine magnesium excretion; ADJ UMgHR, hourly urine magnesium excretion adjusted for DDELMg; UKHR, hourly urine potassium excretion. Similar values are shown for Mg and Na. CCr and all excretions and deliveries expressed/1.73 M2.

*

Significantly different from c, same meal period.

Calcium.

Fasting, urine calcium of IH exceeded N, but distal delivery of calcium (DDELCa) did not differ significantly (Table 2) between IH and N. With meals, urine Ca rose in both groups and remained higher in IH vs. N (Table 2). DDELCa also rose in both groups but did not differ significantly between IH and N. Because urine calcium differences between IH and N exceeded differences in DDELCa, our results imply additional differences between IH and N in distal nephron calcium reabsorption.

One way to assess the magnitude of this distal effect is to compare differences between IH and N, fasting and fed. In subjects in the fasting condition, DDELCa and urine calcium of IH exceeded N by 0.35 and 0.1 mmol/h, respectively. Postprandially, the corresponding differences were 0.34 and 0.2 mmol/h, respectively. Since urine calcium differences doubled, whereas delivery differences were the same, the increased urine calcium must arise from a distal effect of about 0.1 mmol/h in reabsorption.

Another way is to use the fraction of distal calcium excreted, FEDCa, which is simply the ratio of urine calcium to DDELCa. FEDCa was higher in IH vs. N, fasting and fed (Table 2). The difference in FEDCa between IH and N became significant only in the postprandial period, partly because the discrepancies between differences of delivery and urine excretion were larger, and also perhaps because there were more observations. In the fasting condition, the FEDCa difference was 4%, while in the fed condition, it was 8.6%, reflecting the larger difference in urine calcium.

Sodium.

Fasting, urine sodium excretion and DDELNa did not differ between IH and N (Table 2). With meals, urine sodium and DDELNa increased in both groups but did not differ between IH and N. In other words, unlike the case for calcium, distal reabsorption estimated by FEDNa rose to match delivery equally in IH and N.

Magnesium.

In the fasting condition, urine magnesium excretions of IH and N were identical, and DDELMg did not differ (Table 2). With meals, urine magnesium rose in both groups and remained equal. DDELMg rose similarly with meals in IH and N; the increase became significant only in IH. However, differences between fasting and postprandial were nearly identical in magnitude for IH and N (0.15 and 0.16, respectively). In this sense, sodium and magnesium handling appears similar. Differences and changes in FEDMg add no additional information.

Men and Women Analyzed Separately in the Fasting and Fed Condition

Determinants of distal delivery.

FELi was higher in IH vs. N males, for fasting and postprandial conditions (Table 3). FELi rose with meals in IH females and N males. CCr was higher in male IH than female IH fasting and postprandial despite correction to 1.73 m2 BSA (methods). During postprandial, but not fasting, conditions, filtered load of calcium among IH males exceeded N males and female IH subjects. Filtered load of sodium by IH males exceeded corresponding fasting and postprandial females. Altogether, IH males stand apart from the other groups with respect to FELi, CCr, FLCa, and FLNa.

Table 3.

Generalized estimating equations results for deliveries and distal Fe of Ca and Na in male and female exposed to intermittent hypoxia and normoxia in fasted and fed groups

Fast
Fed
Female
Male
Female
Male
N IH N IH N IH N IH
DDELCa, mmol/h 1.29 ± 0.25 1.33 ± 0.17 1.24 ± 0.23 1.87 ± 0.22* 1.72 ± 0.15 1.66 ± 0.19 1.56 ± 0.14 2.26 ± 0.18*#
FEDCa, % 12.5 ± 1.4 17.2 ± 2.5 16.7 ± 3.3 18.7 ± 2.5 18.5 ± 1.8 29.5 ± 3.2* 22.9 ± 4.3 28.0 ± 1.8
UCa, mmol/h 0.13 ± 0.02 0.19 ± 0.02 0.18 ± 0.03 0.29 ± 0.02*# 0.27 ± 0.03 0.40 ± 0.02* 0.32 ± 0.05 0.56 ± 0.03*#
ADJ UCa, mmol/h 0.15 ± 0.02 0.19 ± 0.02 0.20 ± 0.04 0.29 ± 0.02*# 0.27 ± 0.03 0.39 ± 0.02* 0.32 ± 0.06 0.53 ± 0.03*#
DDELNa, mmol/h 138 ± 27.5 141.5 ± 18.9 130.5 ± 22.9 199.7 ± 23.5* 176.9 ± 16.1 167.1 ± 19.1 163.1 ± 13.9 234.4 ± 19.2*#
FEDNa, % 3.6 ± 0.69 3.4 ± 0.57 10.3 ± 2.6# 4.9 ± 0.8* 5.0 ± 0.61 4.2 ± 0.51 8.9 ± 2.7 4.8 ± 0.3
UNa, mmol/h 4.6 ± 1.2 4.2 ± 0.7 11.5 ± 2.5# 9.1 ± 1.4# 7.6 ± 1.0 6.3 ± 0.9 11.3 ± 2.1 10.2 ± 0.8#
FECa, % 1.45 ± 0.23 2.53 ± 0.30* 1.39 ± 0.07 1.88 ± 0.17* 2.94 ± 0.30 5.2 ± 0.48* 2.56 ± 0.20 5.18 ± 0.29*
DDELMg, mmol/h 0.57 ± 0.01 0.58 ± 0.01 0.58 ± 0.02 0.86 ± 0.09 0.75 ± 0.05 0.68 ± 0.05 0.71 ± 0.08 1.06 ± 0.04*#
FEDMg, % 38 ± 3 23 ± 3 38 ± 4 31 ± 3 39 ± 2 38 ± 2 44 ± 2 40 ± 1
UMg 0.15 ± 0.02 0.12 ± 0.02 0.2 ± 0.02 0.22 ± 0.02# 0.24 ± 0.01 0.21 ± 0.01 0.28 ± 0.01 0.34 ± 0.01*#
FELi, % 16.7 ± 4.3 16.9 ± 1.7 11.3 ± 3.1 19.6 ± 2.2* 19.6 ± 2.3 20.6 ± 1.7 13.8 ± 2.3 21.1 ± 1.6*
CCr, l/h 7.52 ± 0.92 6.11 ± 0.60 9.75 ± 1.50 7.58 ± 0.40# 7.09 ± 0.57 5.99 ± 0.47 9.38 ± 1.20 8.08 ± 0.25#
FLCa, mmol/h 9.91 ± 1.24 8.00 ± 0.79 12.6 ± 1.83 9.87 ± 0.54 9.57 ± 0.78 8.17 ± 0.63 12.4 ± 1.58 10.8 ± 0.33*#
FLNa, mmol/h 1034 ± 123 847 ± 84 1342 ± 204 1050 ± 55# 978 ± 76 826 ± 65 1288 ± 163 1119 ± 35#

Values are expressed as means ± SE. Bolded values indicate significant difference from fast, same sex, and code.

*

Significant difference from N same sex and food status.

#

Significant difference from female same code and food status. Constituents of calculated distal delivery and fractional excretion are shown in the last four rows for completeness.

Calcium

Fasting.

Neither urine calcium nor DDELCa of N men and women differ; likewise, for IH and N women (Table 3). Among men, both DDELCa and urine calcium of IH exceeded N. Because the relative differences in DDELCa and urine calcium between male IH and N are similar in magnitude, as reflected by nearly identical values for FEDCa, the higher urine calcium in IH men can be ascribed to increased distal calcium delivery.

Fasting to postprandial.

Urine calcium rose for different reasons in women and men. DDELCa did not change in women, so the increase is due to decreased distal reabsorption, as indexed by increase of FEDCa. In men the increase of urine calcium was caused by both increased delivery and reduced distal reabsorption because DDELCa and FEDCa both rose.

Postprandial.

Among females, urine calcium excretion of IH exceeded N, even though values for DDELCa were nearly identical. The higher urine calcium excretion of fed IH females can, therefore, only have arisen from differences in distal nephron function, as reflected in their increased FEDCa.

Among males, urine calcium excretion and DDELCa of IH both exceeded N; however, even though values for FEDCa did not differ significantly between IH and N males, there is some hint of a food-related distal effect. Fasting values for DDELCa of IH vs. N males differed by 0.63 and urine calcium by 0.11 mmol/h, whereas corresponding fed values were 0.7 and 0.23. In other words, the difference of DDELCa was nearly the same in fasting and fed conditions, but the difference of urine calcium was twice as high postprandially as it was during fasting.

Sodium

Fasting.

Differences in DDELNa are not reflected uniformly in urine sodium excretion. Sodium excretion was higher in men than women, but their DDELNa values did not differ (Table 3). On the other hand, DDELNa was higher in IH males vs. N males, yet urine Na values did not differ. FEDNa accurately reflects these nonuniformities, being higher in N men than women, and lower in male IH than male N.

Fasting to postprandial.

Urine Na rose in females but not in males, DDELNa rose in males, but not in females. Changes in DDELNa and urine Na excretion, therefore, dissociated completely in both sexes. As one reflection of this dissociation, FEDNa increased among N females.

Postprandial.

Urine Na and DDELNa of IH males exceeded IH females (Table 3). DDELNa of IH males exceeded N males, even though urine Na excretions did not differ. FEDNa did not differ among the four groups. However, it is worth pointing out that given the much higher value of DDELNa among male IH, the fact that urine sodium excretions of male IH and N are not different underscores the remarkable balancing of distal reabsorption to distal delivery.

Magnesium

Fasting.

Urine Mg excretion of male IH exceeded female IH (Table 3). There were no significant differences in DDELMg or FEDMg. DDELMg and FEDMg of males with IH exceeded females with IH though without statistical significance.

Fasting to postprandial.

Urine Mg increased in all four groups, but the increase was not significant among N males. There were no significant changes in DDELMg. FEDMg increased significantly only in IH females. The increases of urine Mg cannot be specifically traced to either delivery or distal FE, except among female IH, in whom delivery did not change, whereas urine excretion nearly doubled.

Postprandial.

Urine Mg excretion and DDELMg were both higher in male vs. female IH. This strongly supports a role for increased delivery in the males producing higher excretion.

Blood Pressures

Unadjusted.

Given the differences in sodium handling between IH and N and between males and females, we measured systolic and diastolic blood pressure throughout the protocol (methods). Unadjusted (Table 4), systolic pressures of females were less than males in corresponding subject types. Systolic pressures of male IH were lower than male N. Diastolic pressures of female IH were less than male IH (Table 4).

Table 4.

Blood pressure by sex and patient type fasting and fed

Unadjusted
Adjusted
Female
Male
Female
Male
N IH N IH N IH N IH
Systolic 117 ± 4.6 110 ± 4.6 129 ± 3.5 120 ± 1.7* 118 ± 4.3 112 ± 4.4 126 ± 3.4 120 ± 1.8*
Diastolic 68 ± 3.3 63 ± 2.3 75 ± 4.1 70 ± 1.6 68 ± 3.1 64 ± 2.1 72 ± 3.9 70 ± 1.6

Values are expressed as means ± SE. Bolded values indicate significant difference from female, same patient type.

*

Significant difference from control, same sex. Adjusted means represent mean blood pressure among a subpopulation with mean DDELNa (187.7) and FEDNa (5.1).

Adjusted.

In a stepwise general linear model with systolic blood pressure as dependent and age, sex, fed vs. fast (food), subject type (code), DDELNa, and FEDNa as covariates, code, sex, DDELNa and FEDNa entered significantly. F values were 22, 40, 4.6, and 42, respectively, meaning that DDELNa had marginal effects. With adjustment (Table 4), systolic pressures of male IH remained lower than their same sex N, and male IH diastolic pressure remained higher than female IH. In other words, adjustment removed the sex difference in systolic pressure within subject type but did not remove the systolic pressure difference between normal and IH men or diastolic pressure between IH men and women.

Overnight Values

FELi, CCR, and, therefore, DDELCa, DDELNa, and DDELMg no longer differed between IH and N (Table 2). Urine Ca of IH exceeded N, however, as does FECa (Table 2). The higher urine calcium excretion of IH vs. N is due to a higher value of FEDCa, as filtered load and DDELCa are not different. All values for Na and Mg are the same in IH and N (Table 2). Given only one overnight point for each subject, numbers were too small to permit separate analysis by sex.

Twenty-Four-Hour Urine Collections

Apart from increased urine Ca excretion and FECa, 24-h samples reveal no useful distinctions between IH and N (Table 2). Presumably, this is because they combine the GCRC data period from 6 AM through 8 PM with the overnight period, in which deliveries no longer differ. Therefore, it is the 14-h urine from 6 AM to 8 PM, which should best disclose PT and distal abnormalities. We did not collect such split 24-h samples. Numbers of samples were too small to support analysis by sex.

DISCUSSION

Mechanism of Hypercalciuria

Sex differences in calcium handling.

Our central new finding here is that women and men differ in the way they produce the hypercalciuria of IH. Among women, IH arises mainly, if not completely, from a fall in distal nephron calcium reabsorption, whereas among men, hypercalciuria arises to a significant extent from an increase of distal calcium delivery with an additional element of reduced distal calcium reabsorption.

In addition, we have found evidence of differences in blood pressure, which were detectable in the past (53) and now can be clearly demonstrated, and delimited to differences between IH and N males.

Sex differences in Mg handling.

A sex difference is present, albeit in a muted form, for magnesium. Females do not display differences in delivery or excretion of Mg between IH and N, so their hypercalciuria occurs in the absence of a corresponding magnesuria. Postprandially, males do have a difference in delivery of Mg between IH and N, and IH males display a corresponding magnesuria. This is consistent with a higher delivery of Mg not fully compensated for in distal segments.

Lack of differences in Na handling.

Differences of calcium and magnesium handling are not reflected in renal handling of sodium, in which changes in distal delivery are equally balanced by changes in distal reabsorption, so no selective imbalances are documented.

In other words, IH arises from an imbalance between distal delivery and distal reabsorption of calcium in males, and from a reduction of distal calcium reabsorption in females. This imbalance is present but minimal for Mg, and absent altogether for sodium.

DCT is the likely site of Ca-Na-Mg dissociation in IH.

Although our studies cannot possibly locate which distal segments are most important in this difference of compensation, we favor the idea it may be the DCT, including the connecting tubule. Because these segments have separate channels for calcium (TRPV5) (26) and magnesium (TRPM6) transport (48), the DCT can dissociate calcium from magnesium handling. Such dissociation is seen in Gitelman's syndrome (24), voltage-gated potassium channel (KCNAl) mutations (17), epidermal growth factor mutations (18), and cyclin M2 mutations (46), all of which are hypomagnesuric conditions in which urinary calcium is either reduced or unaffected.

By contrast, the thick ascending limb is an unlikely site for calcium and magnesium dissociation, as it lacks appropriate transporters to create the observed differences in calcium and magnesium handling. Our study cannot carry this issue further.

Sex Heterogeneity in Other Studies

Humans.

This finding of sex heterogeneity in tubule function is not surprising, as sex differences in kidney function have been observed both in humans and animals (7, 9, 25, 42). In humans, sex hormone receptors are present throughout the kidney (10, 14, 47, 49), and kidney mass is known to vary with sex (27). Hemodynamically, men and women are known to respond differently to ANG II infusions, with only men preserving GFR with ANG II administration (29). Few human studies directly compare tubule function in men to women, but heterogeneity in tubule function has been demonstrated. For example, the organic anion transporters (OAT1-4) display a sex difference in activity, evidenced by different renal clearances of various medications (20, 39, 50).

Previous reports using lithium clearance have clearly demonstrated a matching of sodium delivery and distal reabsorption, a finding recapitulated in our study, and our values for FELi were comparable with previously published results (6, 12, 15, 32). Although direct comparisons between sexes using endogenous lithium clearances have not been rigorously examined previous to our study, a pattern of differential tubular response to salt loading has been seen between sexes (3335, 56). These studies demonstrated that men, and women in the follicular phase of their menstrual cycle, decrease sodium reabsorption both proximally and distally in response to a salt load, whereas women in the luteal phase only vary by their distal response (3335, 56). Although our study did not specifically determine hormonal levels or menstrual phase, fasting female mean lithium clearances were consistent with those previously reported for those in the luteal phase (3335).

Animals

Studies in animals have confirmed human findings regarding renal mass (22), sex hormone receptor abundance (5, 31), and sex variability in OAT function (44). Endothelin 1 (ET-1) levels vary between males and females, and ET-1 has also been associated with modulation of tubule function in animals (19, 36, 58). Other sex differences in both proximal and distal tubule transporter expression and function have been described as well (1, 37, 38, 42). Interestingly, male rats have increased thick ascending limb expression of the Na+K+2Cl cotransporter (NKCC2) (8, 30), while female rats have been reported to have increased abundance of NCC (40). This suggests that the DCT exerts a greater influence with regard to sodium and calcium handling in females compared to males, providing a potential mechanism for the greater distal dissociation of sodium and calcium in female IH compared to male IH.

Blood Pressure Differences Between IH and N

Previously, we have demonstrated lower blood pressures in IH vs. N when adjusted for distal sodium delivery (53). Here, we found lower unadjusted systolic pressures in male IH vs. N, but differences among women were not significant. After adjustment for distal sodium delivery and fractional distal sodium excretion, systolic blood pressures remained lower in IH men than in normal men, and no differences were found among women. We have no mechanism to account for this blood pressure difference and no data bearing upon its effects; however, the higher blood pressure in men could possibly reduce proximal reabsorption vs. women via effects in membrane channel insertion (55).

Apart from our studies of IH, the role of altered proximal vs. distal sodium handling on blood pressure is evolving, but evidence favors an association between hypertension and proximal tubule function. Studies in nonhypercalciuric patients have associated salt-sensitive hypertension with low FELi (12, 45). This phenomenon cannot be further explored within the present study but requires new experiments focused on blood pressure regulation. We recognize that our studies do not correspond with large-scale epidemiological studies (16, 28), which show higher blood pressures among stone formers. Data from our study relied upon detailed study of individuals on fixed diets, which does not correspond with general surveys of the IH population.

Twenty-Four-Hour Samples Do not Differentiate IH From N in Their Proximal and Distal Reabsorptions

At least in the modest sample of subjects from whom we gathered 24-h samples with lithium measurements, mean values for DDELCa and FEDCa were not different between IH and N, meaning that combining overnight and fed periods blurs distinctions that we clearly demonstrated in the CRC. Possibly, the use of only one fasting serum was part of that blurring.

Nocturnal Hypercalciuria Reflects Reduced Distal Ca Reabsorption

Overnight, urine calcium molarity and SS are higher in IH than N. Surprisingly, the difference between IH and N arises completely from reduced distal calcium reabsorption in IH vs. N (Table 2). It is perhaps overnight when sodium and calcium handling appear most dramatically different in that FEDNa values for IH and N are identical. Likewise, FEDMg is identical between IH and N. These dissociations again support a main role for DCT in permitting overnight hypercalciuria while abolishing any differences in Mg and Na excretions. We did not compare men to women, as numbers of samples would be too low to draw conclusions.

IH phenotype Is Heterogeneous with Regard to Proximal and Distal Abnormalities

Overall, IH arises from a combination of nephron abnormalities. Females mostly create hypercalciuria by reducing distal reabsorption and often do not exhibit increased distal delivery. Males, by contrast, exhibit reduced proximal tubule function, leading to increased distal delivery of calcium and sodium. Although the distal nephron enhances both sodium and calcium reabsorption, hypercalciuria in males seems to arise from a combination of increased distal delivery and reduced distal reabsorption. Consuming meals enhance all aspects of tubule function, causing calcium levels to rise in the urine, but the sex differences do not appear to vary with food.

Perspectives and Significance

These findings lead to a new and more refined understanding of differential tubule transport defects in IH, but they are also a gross oversimplification. This general schema varies among patients and potentially indicates genetic and/or hormonal variation beyond the scope of this work. Further study into genetic effects will require, at a minimum, family studies. The sex differences in tubule function imply a role for hormonal regulation of calcium transport beyond calcitriol and PTH, by estrogen, progesterone, and testosterone. An investigation into this will require careful studies to examine the interplay between these hormones and tubule function.

GRANTS

This publication was made possible by National Institutes of Health (NIH) Grants P01-DK-56788 and UL1-RR-024999 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.

DISCLOSURES

F. L. Coe and E. M. Worcester are consultants for Litholink, Labcorp.

AUTHOR CONTRIBUTIONS

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

ACKNOWLEDGMENTS

Contents of this article are solely the responsibility of the authors and do not necessarily represent the official view of the National Center for Research Resources or the National Institutes of Health.

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