Whites Excrete a Water Load More Rapidly Than Blacks
A recent report demonstrated a racial difference in response to furosemide compatible with increased ion reabsorption in the thick ascending limb of the loop of Henle in blacks. Urinary dilution is another function of the loop-diuretic–sensitive Na,K,2Cl cotransporter in the thick ascending limb, and racial differences in urinary diluting capacity have not been reported previously. We assessed diluting segment (cortical thick ascending limb and distal convoluted tubule) function in black and white normotensives in 2 studies using a water-loading approach. In both studies, we found that whites excreted a water load more rapidly than blacks. In the first study, the final free water clearance rates (mean±SD) were 7.3±4.7 mL/min in whites (n=17, 7 females and 10 males) and 3.8±3.6 mL/min in blacks (n=14, 9 females and 5 males; P<0.03). In the second study, final free water clearance rates were 8.3±2.6 mL/min in whites (n=17, 8 females and 9 males) and 6.4±1.8 mL/min in blacks (n=11, 8 females and 3 males; P<0.01). We found no evidence of a racial difference in renal proximal tubular fluid reabsorption as assessed by renal endogenous lithium clearance or in plasma vasopressin level that could explain the difference in free water excretion. We conclude that our observations are most consistent with a lower capacity of ion reabsorption in the renal diluting segment in blacks. Slower excretion of an acute water load may have been an advantage during natural selection of humans living in arid, hot climates.
Chun et al1 recently demonstrated a racial difference in response to furosemide compatible with increased activity of the loop-diuretic–sensitive Na,K,2Cl (NKCC2) cotransporter in the thick ascending limb (TAL) of the loop of Henle in blacks compared with whites. In addition to helping to concentrate urine, the cortical TAL NKCC2 (in concert with the Na,Cl cotransporter in the water-impermeable segments of the distal convoluted tubule) generates free water by the net reabsorption of ions from tubular fluid, and a racial difference in NKCC2 activity could, therefore, affect the ability to dilute urine. Tubular diluting capacity can be characterized by measuring free water generation during water loading,2 and we report here the results of 2 experimental studies of renal responses to water loading in blacks and whites.
Both studies were approved by the University of Michigan Institutional Review Board, and all of the subjects gave written informed consent. For both studies, we recruited subjects by public advertisement from Ann Arbor, Michigan, and the surrounding area. Subjects were healthy normotensive black and white (non-Hispanic) men and women, aged 18 to 50 years. As per National Institutes of Health guidelines, race was self-determined. Normotensive status was established during a screening visit as a systolic blood pressure of <140 mm Hg and a diastolic blood pressure of <90 mm Hg for the average of 2 seated auscultatory blood pressure measurements performed by an experienced observer. A general health history was obtained and a physical examination performed to exclude other diseases including cancer, recent (within 6 months) stroke or myocardial infarction, diabetes mellitus, liver disease, chronic infections, and psychiatric disease of sufficient severity to interfere with a subject’s ability to adhere to the protocol. We screened for occult disease with a complete blood cell count, a thyroid-stimulating hormone level, a comprehensive automated biochemical profile, and a urinalysis; all of the values were required to be normal, including a serum creatinine (Cr) level of <1.3 mg/dL for women and <1.5 mg/dL for men and a serum potassium (K) level of >3.5 mmol/L. Pregnancy was excluded by a rapid urine pregnancy test. Drug therapies that could affect renal tubular Na handling (diuretics, nonsteroidal antiinflammatory drugs, caffeine, and theophylline) were not permitted.
Data reported here cover the first 90 minutes (three 30-minute periods) of a longer protocol designed to study the effects of dopaminergic control of renal sodium excretion; those data will be reported elsewhere. Subjects consuming their usual diet came to an outpatient research facility of the University of Michigan Medical Center during the morning. Subjects were asked to drink water before coming, because we wanted to ensure that they were well hydrated and could produce adequate urine volumes during the study. On arrival they were asked to drink 12 oz (360 mL) of water as rapidly as possible. An IV infusion of 5% glucose in water was begun and maintained throughout the study at a rate of 200 mL/h. Urine samples were collected every 30 minutes, and at 30 and 60 minutes, urine output was replaced orally milliliter for milliliter with water. Blood was obtained at baseline and at the end of each hour. Measurements of sodium (Na), K, Cr, and osmolality (Osm) were obtained for all of the urine and serum samples.
Inclusion and exclusion criteria and recruitment methods were the same as for protocol 1. On day 1, subjects who had previously agreed to participate in the study came to the outpatient research facility, where the details of the protocol were reviewed, a medical history was obtained, and a physical examination was performed. Before leaving the facility, subjects voided and were instructed to begin collecting all urine in 2 plastic jugs; “day” was the period from leaving the research facility until retiring to sleep, and “night” was the time from retiring through awakening, at which time subjects collected their first morning void, which completed the nighttime collection. The times of retiring and awakening were recorded. Subjects were instructed to collect any voids during the night in the night jug. Ambulatory blood pressure monitoring was performed during day 1 (Spacelabs Model 90207, Spacelabs, Inc).
At 9 am on day 2, subjects reported in the fasting state to the research facility and returned the urine collections. A catheter for blood sampling was placed in a forearm vein, and baseline blood samples were obtained. Subjects then voided and began the water-loading protocol by drinking 20 mL/kg of body weight of distilled water as rapidly as possible over ≤45 minutes. Hourly urine samples were collected for the following 2 hours by voiding. Urine volume was measured for both hourly collections, and the first hour’s volume was replaced orally milliliter for milliliter with distilled water. In addition to the baseline samples, blood was collected at the end of both urine collections. Serum analyses included Na, lithium (Li), Cr, and Osm. Plasma vasopressin was measured in the specimen obtained at the final collection. Urine Na and Li concentrations and Osm were measured in all of the urine specimens. Urinary clearances (ClX) were calculated by the standard formula: (urine X/serum X)×urine flow rate, where X can be Cr, Osm, or Li. Urinary free water clearance (CH2O) was calculated as urine flow rate−urinary Osm clearance. A concentration index (C.I.) was calculated as Urine Cr/Serum Cr.3
Serum and urine Na and K were measured by flame photometry and plasma Osm by freezing point depression. We measured endogenous serum and urine Li concentrations by mass spectrometry using a Finnigan Element inductively coupled high-resolution mass spectrometer in standard Meinhard nebulizer and Scott spray chamber configuration. Both Li-6 and Li-7 isotopes were analyzed in low-resolution mode, as well as Li-7 in medium resolution mode to confirm that isotopic interferences were negligible. The nominal instrument detection limit for Li is <0.002 μmol/L. This permits substantial dilution of the samples before analysis, which helps to minimize matrix effects. External calibrations are used for both urine and serum samples; the methods were validated by comparison with results obtained by standard additions. Serum and urine creatinine assays were performed using a modified Jaffe reaction. Plasma vasopressin was measured by radioimmunoassay.
Statistical comparisons between races were by unpaired t test. Statistical significance was accepted at the P<0.05 level. All of the data are expressed as means±SDs.
Baseline characteristics, including serum Osm, did not differ between the races (Table 1), except for a higher diastolic blood pressure in blacks. Urine flow rate was higher for all 3 of the periods in whites (period 1 [0 to 30 minutes]: 11.9±7.9 versus 4.8±3.5 mL/min, P<0.01; period 2 [30 to 60 minutes]: 10.3±6.1 versus 4.8±2.7 mL/min, P<0.01; period 3 [60 to 90 minutes]: 9.6±4.2 versus 6.9±3.7 mL/min, P<0.05; white versus black for all of the comparisons). CH2O was higher in whites during periods 2 and 3 (Figure 1). Because flow rate differed during the first 30 minutes and water was replaced orally milliliter for milliliter at the 30- and 60-minute time points, the total amount of water consumed during the study differed between the races (1144±395 versus 763±163 mL, white versus black; P=0.002). The total urine volume excreted during the 90 minutes after water loading was greater in whites than in blacks (994±419 versus 490±241 mL, white versus black; P=0.001), as was the average percentage of the water load excreted (81.1±22.5 versus 60.8±21.4%, white versus black; P=0.015). Urine osmality (UOsm) declined in both races and was similar during all of the collection periods (Figure 1). Urinary excretion of Na during the 90 minutes of the study was greater, although not significantly so, in whites (39.4±20.1 versus 20.1±11.7 mmol, white versus black; P=0.07); there was no difference during any of the individual periods.
There were no significant differences between the white and black groups for urine volume, flow rate, Na or Osm excretion, or CI during the day or night (Table 3). Total Osm excretion (day plus night) was also similar between the groups: 746±274 versus 707±366 mOsm, white versus black; P=0.77). K excretion was lower in blacks during the daytime but not at night (Table 3).
Urinary endogenous lithium clearance did not differ between the races (day: 19.9±8.0 versus 18.3±10.9 mL/min, white versus black [n=10], P=0.53; night: 16.0±5.8 versus 16.7±11.2 mL/min, white versus black [n=10], P=0.92). Because of sample mishandling, 1 black subject did not have urinary Li values determined for the day or night urines.
During water loading, the amount of water consumed did not differ between the groups (2091±410 versus 2156±566 mL, white versus black; P=0.77). The total urine volume excreted during the 120 minutes of water loading was greater, although not significantly so, in whites compared with blacks (1131±471 versus 930±256 mL, white versus black; P=0.15). Urine flow rate did not differ between the groups during the first hour (8.8±4.2 versus 8.3±2.6 mL/min, white versus black; P=0.55); flow rate increased more in whites than blacks and was significantly greater during the second hour (11.4±3.1 versus 8.8±2.8 mL/min, white versus black; P<0.01). CH2O also increased significantly in both groups from the first to the second hour of water loading: during the second hour, CH2O was significantly greater in whites (Figure 2), whereas lithium clearance was similar (26.9±9.6 versus 22.3±12.2 mL/min, white versus black; P=0.32). UOsm during the first and second hourly collections did not differ by race (Figure 2). Plasma vasopressin was below the lower limit of the assay (<0.5 pg/mL) in all of the subjects at the end of 2 hours, and serum Na, K, Cr, and Osm did not differ between the groups at the end of either hour 1 or 2 (data not shown).
The observations in our 2 studies are consistent in showing that whites excrete a water load more rapidly than blacks. The racial difference observed in the first study could have been affected by the differences in water intake resulting from the subjects’ ad libitum and unmeasured water consumption before their presentation to the research facility and by the procedure of replacing urine volume milliliter for milliliter at the end of each period. However, in the second study, subjects presented in the fasting state, and the amount of water ingested was very similar between the groups; the lower urine flow rate and lesser CH2O in blacks therefore appear to be attributed to a difference in renal water handling. We acknowledge that because the water loads were delivered orally in that study, it is possible that there is a racial difference in the gastrointestinal absorption of water or its distribution in the body,4 but we have no way of assessing those possibilities and contend that our observations most likely result from a difference in renal water handling.
The recent report from Chun et al,1 demonstrating that there is a racial difference in urinary responses to furosemide administration concluded that TAL NKCC2 activity may be higher in blacks: our demonstration of lower free water clearance in blacks is more consistent with decreased TAL NKCC2 activity. It is important to stress that quite different functions were assessed in the 2 studies, neither of which provides an unequivocal assessment of TAL function. We used a water-loading protocol to assess TAL function, but because free water is generated by solute reabsorption in both the water-impermeable cortical segment of the TAL and in the distal-convoluted tubule, we cannot distinguish between those segments.2 However, based on the observed effects of furosemide and thiazides on free water clearance in humans5 and the lack of a significant defect in concentrating ability in human genetic disorders with markedly impaired Na,Cl cotransporter activity,6 the TAL is thought to be quantitatively much more important than the distal-convoluted tubule. Nonetheless, clinical observations of thiazide-induced hyponatremia suggest that, particularly in some elderly individuals, distal-convoluted tubule function can be a quantitatively important determinant of water balance.7
In addition to ion reabsorption in the TAL, vasopressin-mediated water reabsorption in the collecting tubule is an important determinant of CH2O. Vigorous water loading is expected to largely eliminate, or at least greatly attenuate, the action of vasopressin, although, because the vasopressin assay is not sensitive enough to ensure that suppression of vasopressin is complete, we cannot exclude an effect at the collecting duct. In addition, it is possible that nonvasopressin-dependent back diffusion of water in the collecting duct is greater in blacks than whites. However, the similarity of the minimum urine osmolarities observed in blacks and whites in our study argues against an important contribution of water reabsorption in the collecting duct.
Urinary free water clearance is influenced by differences in tubular flow rate and the delivery of Na and K from the proximal tubule8: we addressed the possibility of a racial difference in proximal tubular ion reabsorption by measuring urinary ClLi.9 Our observation that ClLi did not differ between the races before or during water loading suggests that a difference in the delivery of fluid from the proximal tubule does not account for the racial difference in urinary free water clearance. Because average minimum UOsm is similar in blacks and whites after water loading, and in both groups is far below that of the plasma, the difference in CH2O between the groups reflects the rate at which the diluting segment can generate free water and implies that the determinants of minimum UOsm, including the osmolality of the medullary interstitium and the efficacy of the countercurrent mechanism, as well as the permeabilities of the TAL and collecting duct, are intact.
We did not observe significant differences in urine volume or UOsm between whites and blacks during the day or night preceding our second water-loading study. This is in contrast to the observations of Bankir et al,3 who observed a difference in urine flow rate and CI during the day, although not at night, and of Chun et al,1 who observed a racial difference in urine volume and Osm in a 12-hour overnight collection. In part, the differing observations might be accounted for by differences between the protocols followed by those investigators and ourselves, because they hospitalized subjects and imposed a controlled diet, whereas our subjects were studied as outpatients, and no dietary or fluid restrictions were imposed. We also note, however, that in our study, both UOsm and CI did trend in the direction of greater concentration in blacks, particularly during the daytime, and our smaller sample size may have been insufficient to permit detection of a statistically significant difference.
We agree with Bankir et al3 that natural selection could have played a role in shaping racial differences in renal water handling and suggest that, in addition to enhanced urine concentrating ability, slower excretion of a water load could have conferred a selective advantage during the evolution of humans in the hot, arid climate of east Africa. Much of the interest in adaptations to such environments has focused on sodium homeostasis, but it is likely that obligatory water requirements are much more stringent than those for sodium. For instance, whereas average sodium excretion varied by some 200-fold between the populations with the lowest and highest excretions in INTERSALT, urine volume varied by only ≈3-fold, and even that might overestimate the magnitude of variation in water intake, because there is no way to estimate insensible losses in that study.10 Because water is available only intermittently to hunter-gatherers, slowing excretion of an acute water load could help to optimize water retention and improve reproductive success. Such a phenotype would be particularly advantageous if coupled with an ability to increase maximum urinary concentrating ability.
The contents of this article are solely the responsibility of the authors and do not represent the official views of National Center for Research Resources or the National Institutes of Health.
Sources of Funding
This work was supported in part by grant M01-RR000042 from the National Center for Research Resources of the National Institutes of Health and by the Faculty Group Practice of the University of Michigan.
- Received August 21, 2008.
- Revision received September 16, 2008.
- Accepted January 13, 2009.
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