Summary
Background and objectives Gitelman syndrome (GS) is a salt-wasting tubulopathy that results from the inactivation of the human thiazide–sensitive sodium chloride cotransporter located in the distal convoluted tubule. Tubular adaptation to renal sodium loss has been described and localized in the distal tubule in experimental models of GS but not in humans with GS.
Design, setting, participants, & measurements The tubular adaptation to renal sodium loss is described. Osmole-free water clearance and endogenous lithium clearance with furosemide infusion are used to compare 7 patients with genetically confirmed GS and 13 control participants.
Results Neither endogenous lithium clearance nor osmole-free water clearance disclosed enhanced proximal fluid reabsorption in patients with GS. These patients displayed significantly lower osmole-free water clearance factored by inulin clearance (7.1±1.9 versus 10.1±2.2; P<0.01) and significantly lower fractional sodium reabsorption in the diluting nephron (73.2%±7.1% versus 86.1%±4.7%; P<0.005), consistent with the inactivation of the thiazide-sensitive sodium chloride cotransporter. The furosemide-induced reduction rate of fractional sodium reabsorption in the diluting segment was higher in patients with GS (75.6%±6.1% versus 69.9%±3.2%; P<0.039), suggesting that sodium reabsorption would be enhanced in the cortical part of the thick ascending limb of the loop of Henle in patients with GS.
Conclusions These findings suggest that tubular adaptation to renal sodium loss in GS would be devoted to the cortical part of the thick ascending limb of the loop of Henle in humans.
Introduction
Before the molecular era, the term Bartter syndrome was used to designate congenital tubulopathies with impaired sodium reabsorption in the diluting segment, even though phenotypic heterogeneity was noted (1,2). In the past two decades, molecular genetic studies confirmed the distinction between Gitelman syndrome (GS) (3) and four genetic types of Bartter syndrome (4–7). The most prevalent forms diagnosed in adulthood are GS and type III Bartter syndrome. GS results from mutations in the SLC12A3 gene, which encodes the thiazide-sensitive sodium chloride cotransporter (NCC). Research efforts focusing on the renal phenotype of these salt-wasting tubulopathies have extensively used osmole-free water clearance and lithium clearance. Localization of sodium reabsorption defects and intrarenal compensatory mechanisms to renal sodium loss have been described. These efforts were limited by the uncertainty of the underlying molecular defect and the heterogeneity of the studied population, leading to paradoxical results (8). Furthermore, accumulating new information on renal sodium and lithium handling has disclosed pitfalls and led to more accurate use of clearance techniques.
To our knowledge, no functional studies based on clearance methods for genetically confirmed salt-wasting tubulopathy have been published. We used clearance studies to measure the capacity of the diluting nephron to reabsorb sodium and consequently to generate osmole-free water. This segment encompasses the cortical part of the thick ascending limb of the loop of Henle (cTAL), the distal convoluted tubule (DCT), the connecting tubule (CNT), and the cortical collecting duct (CCD). Sodium transport capacity of the diluting nephron can be impaired genetically in Bartter syndrome and GS or through pharmacologic interventions: Furosemide can inhibit the type 2 Na-K-Cl cotransporter (NKCC2) and mimic type I Bartter syndrome, hydrochlorothiazide can inhibit the NCC and mimic GS, and amiloride can inhibit the epithelial sodium channel (ENaC).
To identify an intrarenal compensatory mechanism for the chronic renal sodium loss in patients with GS, we performed clearance studies and used furosemide. We measured the capacity of the diluting nephron to reabsorb sodium before and after furosemide administration and compared patients with GS and healthy controls. We compared the results of osmole-free water clearance and endogenous lithium clearance and discuss the technical limitations of these methods.
Materials and Methods
This study was carried out in the Nephrology Department of the Strasbourg University Hospital, Strasbourg, France, in 8 patients with GS and 13 healthy controls who gave informed consent. The 8 patients were enrolled on the basis of a phenotype concordant with GS according to the following criteria: absence of arterial hypertension, hypokalemia of renal origin, hypomagnesemia, and hypocalciuria. For all patients, the diagnosis was genetically confirmed. The study was part of a standard renal evaluation for patients with Bartter syndrome or GS. Controls were healthy volunteers without signs of tubular injury (no glycosuria and normal bicarbonate and potassium serum levels), normal renal function, and normal BP.
Total DNA was extracted from blood peripheral leukocytes by standard procedures. Mutation analysis was performed by PCR amplification and direct sequencing of exons and flanking intronic sequences of the SLC12A3 gene, as described elsewhere (9), on an ABI Prism 3730XL DNA Analyzer Sequencer (Perkin Elmer–Applied Biosystems, Foster City, CA).
Before the clearance study (at baseline), 24-hour urine was collected to determine urinary levels of sodium, potassium, chloride, urea, creatinine, and calcium. At the end of this collection period and before the water load, levels of sodium, potassium, chloride, calcium, magnesium, bicarbonate, creatinine, proteins, renin, and aldosterone were measured in plasma, and hematocrit was measured in blood. Heart rate and BP were recorded with an automatic oscillometer device (Centron) at baseline.
On the morning of the study, each participant received an oral water load of 10 mL/kg body weight. After a priming dose, a constant infusion of inulin (to estimate GFR) into a lower arm vein was started. Additional water was given to match the urinary output. After at least 1 hour of equilibration, when urine osmolality had reached a minimal value and when urinary flow rate was constant, three 30-minute urine collections were obtained. Then, 40 mg of furosemide was intravenously infused. After the infusion, three additional, 30-minute urine collections were made. Blood samples for clearance calculations were drawn at the beginning and at the end of each collection period via an intravenous canula placed on the lower arm, contralateral to the infusion arm. In each urine and plasma sample, osmolality and lithium, creatinine, chloride, sodium, and inulin levels were determined.
Osmolality was determined using freezing-point depression, and sodium, potassium, chloride, magnesium, calcium, bicarbonate, urea, and creatinine levels were determined using a standard automatized method. Endogenous lithium was determined with atomic absorption spectrophotometry, as described elsewhere (10). Inulin was hydrolyzed to fructose and then determined photometrically with indoleacetic acid (Technicon autoanalyzer, Domont, France). Immunoreactive renin was measured with a monoclonal antibody (Renin Irma Pasteur, Marnes-la-Coquette, France), and plasma aldosterone was measured by radioimmunoassay (SB-ALDO-2, CIS Bio International, Gif-sur-Yvette, France).
Clearances (mL/min) were calculated using the following standard formula:
where Us is the urine concentrations of s, Ps is the mean of serum value at the beginning and at the end of each urine collection, and V is the urine flow rate in mL/min. For calculation, two of the three 30-minute urine collections preceded and followed by a blood probe were used before and after furosemide administration. Osmole-free water clearance (CH2O) was calculated as follows:
CH2O during maximal water diuresis divided by inulin clearance (Cinulin) and multiplied by 100 was taken as an index of sodium reabsorption in the diluting segment, that is, distal to the point of isotonicity in the medullary TAL, and was written as follows:
Changes in fractional CH2O augmented by fractional chloride clearance (CCl) were taken as an index of changes in fractional sodium delivery to the diluting segment (FDDH2O) and were calculated as follows: 
Changes in the following equation:
were taken as an index of changes in sodium reabsorption in the diluting segment (FDRH2O) (11). FDRH2O reduction rate was calculated as follows: 
Endogenous lithium clearance was taken as an index of sodium delivery to the loop of Henle and was measured during maximal water diuresis. Fractional distal delivery of sodium was calculated as follows:
Fractional distal sodium reabsorption was calculated as follows (12):
Data are given as the mean ± SD. Paired observations are compared using U Mann–Whitney test for nonparametric data. The P value threshold for statistical significance is P<0.05.
Results
Patients with GS and controls had similar body mass index, heart rate, and systolic and diastolic BP. The patients in the control group were younger and predominantly female (Table 1).
Demographic and clinical features of patients with Gitelman syndrome and controls
Six different missense mutations and one splice mutation were detected in the eight patients with GS. Four of the patients were compound heterozygous and four were homozygous (Table 2). Patients 6-1 and 6-2 belonged to a consanguineous family. They had a known mutation in a donor splice site of exon 9. Patients 4-1 and 4-2 (brother and sister) were from a nonconsanguineous family.
Mutations in patients with Gitelman syndrome
Renal sodium loss in patients with GS resulted in the elevation of the serum aldosterone level (188±167 versus 57±22 pg/ml; P<0.005) and of the serum immunoreactive renin level (87.5±49.8 versus 5.8±3.6 pg/ml; P<0.005). It also resulted in a mild extracellular volume contraction, as attested by a higher hematocrit value and a higher plasma protein level (Table 3). Patients with GS displayed hypokalemic alkalosis with hypomagnesemia and a low ratio of urinary calcium to urinary creatinine (Tables 3 and 4). These features were all consistent with SLC12A3 mutations and were not found in the control group.
Baseline plasmatic levels of main variables and hematocrit in patients with Gitelman syndrome and controls
Baseline daily urinary output of main variables in patients with Gitelman syndrome and controls
Water load was performed for the clearance study. One patient with GS was excluded because of digestive intolerance to the water load (n=7 available for study measurements). Before furosemide administration, patients with GS displayed a higher osmolar clearance and a higher chloride clearance secondary to SLC12A3 mutations (Table 5). After furosemide injection, urinary output, osmolar clearance, sodium clearance, and chloride clearance were enhanced in patients with GS and controls, which attested to the normal function of the loop of Henle in both groups (Table 5). Lithium clearance did not differ between groups (Table 5). Osmole-free water clearance was lower in patients with GS patients, before and after furosemide injection (Table 6), disclosing a sodium reabsorption defect in the diluting nephron, as expected with SLC12A3 mutations.
Effect of furosemide on urinary output on solute and osmolar clearances
Osmole-free water and endogenous lithium clearance before and after furosemide administration
According to osmole-free water clearance and to endogenous lithium clearance, FDD did not differ between groups (Table 6). According to the endogenous lithium clearance, there was no difference in fractional distal sodium reabsorption between groups. However, according to the osmole-free water clearance, FDRH2O was significantly lower in patients with GS (Table 6), as expected with SCL12A3 mutations. The administration of furosemide modified FDDH2O and FDRH2O in both groups, consistent with the pharmacologic effect of furosemide on sodium and chloride reabsorption in the cTAL (Table 6). Furosemide administration disclosed an increased reduction rate of FDRH2O in patients with GS (Table 6), suggesting that the sodium transport capacity of the cTAL was enhanced in those with GS. This enhancement could be viewed as a compensation process for the chronic renal sodium loss resulting from the SLC12A3 mutations.
Discussion
Clearance studies provided insight into the sodium transport capacity of the diluting nephron. The main result of our study was a higher furosemide-induced FDRH2O reduction rate in patients with GS than in healthy controls, a finding that indicates an enhanced sodium transport capacity of the cTAL in patients with GS. This capacity can be interpreted as an adaptive process.
Concerning the genotype of our patients (Table 2), we detected seven different mutations in this group of eight patients with GS. Four patients were homozygous and four were compound heterozygous. Patients 6-1 and 6-2 were homozygous for a known splice mutation, particularly prevalent among gypsies from Europe (13). The diversity of mutations in the SLC12A3 gene supports a high prevalence of heterozygosity in the general population. In our patients, no severe phenotype was found (14) and there was a majority of missense mutations, as described in the literature (3,9,15–17).
Before the molecular era, osmole-free water clearance was used to assess the site of action of diuretics in humans (18) and to localize the tubular defect of sodium reabsorption in Bartter syndrome (19,20). Chaimovitz and colleagues (19) described the case of a 5-year-old boy with Bartter syndrome. This child had failure to thrive, hypokalemic alkalosis, extracellular fluid volume contraction, and hyposthenuria. Osmole-free water clearance and FDRH2O were low, and hydrochlorothiazide enhanced natriuresis. These results suggested that the tubular defect concerned the TAL. Sutton and colleagues (20) reported the cases of five young patients (mean age, 28 years) with Bartter syndrome. They had low FDRH2O and severely increased natriuresis after furosemide infusion, but not after chlorothiazide administration. These results suggested that the tubular defect was located in the DCT, and today those patients would probably be considered to have GS or type III Bartter syndrome.
The osmole-free water clearance and the FDRH2O were impaired in our sample of patients with GS. This is in line with the findings of many studies from the premolecular era in which a suppressed sodium reabsorption in the diluting nephron was found in Bartter syndrome (21) and agrees with the expected effect of SLC12A3 mutations. In light of many experimental data in transgenic mice, we postulated that chronic renal sodium loss could be compensated for by an enhanced sodium reabsorption capacity involving segments from the diluting nephron. Cancelling cTAL sodium reabsorption with furosemide and comparing FDRH2O before and after furosemide administration, we sought to determine whether SLC12A3 mutations would affect the activity of one (or many) of these segments. The possible effect of furosemide on the proximal tubule did not differ between patients with GS and controls because FDDH2O was not statistically different between groups after furosemide injection. We found a higher furosemide-induced FDRH2O reduction rate in patients with GS than in controls, suggesting an increased sodium reabsorption rate in the cTAL of patients with GS. Of note, in a sample of patients with Bartter syndrome who did not undergo genotyping, Sutton and colleagues had already noticed that furosemide administration resulted in a higher sodium–creatinine urinary ratio, which was consistent with an enhanced sodium transport in the loop of Henle (20).
Other investigators have studied adaptation to renal sodium loss in mouse models of GS and have reported contradictory findings. In the homozygous NCC-deficient mice, micropuncture experiments showed that fluid reabsorption was enhanced in the proximal tubule. No morphologic or functional modifications of the TAL were found, but the CNT profile revealed a marked epithelial hypertrophy that was accompanied by an increased apical abundance of all three ENaC subunits; this finding suggested that the sodium transport rate would be increased downstream of the DCT (22). In the homozygous NCC Ser707× knockin mouse, protein levels of the sodium-hydrogen exchanger 3 and of the NKCC2, which are the main sodium transporters of the proximal tubule and of the TAL, respectively, were not altered, and the β-ENaC subunit was upregulated, consistent with an enhanced sodium reabsorption rate downstream of the DCT (23). Sterile 20/SPS1-related proline/alanine–rich kinase (SPAK)–regulated NCC and SPAK-null mice also exhibited a GS phenotype. In this GS mouse model, expression of NKCC2 and its phosphorylated active form were upregulated, suggesting that the TAL would be the site of an adaptive process resulting in the enhancement of its sodium reabsorption capacity. The segments located downstream of the DCT were not described in this model (24).
Our results did not exclude the possibility of an increased sodium transport in the CNT and CCD of patients with GS. Actually, the increased delivery of sodium to these segments and the aldosteronism secondary to NCC dysfunction probably account for potassium and bicarbonate imbalance in humans. However, our results suggested that the blockade of the mineralocorticoid receptor or of ENaC, which are commonly used to limit life-threatening hypokalemia in patients with GS, might have a limited effect on extracellular fluid volume.
Regarding proximal tubule sodium reabsorption, we did not find any statistically significant difference with osmole-free water clearance or endogenous lithium clearance between patients with GS and healthy controls. An enhancement of sodium reabsorption was expected in the proximal tubule of patients with GS because they displayed contraction in extracellular fluid volume. Our findings might be due to the potential confounding effect of the water load on the proximal tubule. The water load is mandatory to depress the antidiuretic hormone secretion and to maintain an adequate urine flow rate, enabling reproducible urine collection to be obtained during the clearance study. It has been shown that an acute water load could modify proximal tubular sodium handling in healthy volunteers (25). The endogenous lithium clearance method did not disclose a sodium transport defect in the diluting nephron of patients with genetically confirmed GS. This might be explained by the sodium or potassium depletion in patients with GS. Briefly, for lithium clearance to be a quantitative measure of end-proximal fluid delivery, two conditions must be met: The fractional reabsorption of lithium in the proximal tubule should be the same as that of water, and no net lithium reabsorption or secretion should occur beyond the proximal tubule. Physiologic studies demonstrated that these conditions were not met but that somehow the “excess” of lithium delivered to the loop of Henle would match the “unwanted” amount of lithium reabsorbed in the distal tubule, resulting in a null net balance. However, in case of sodium or potassium depletion, distal lithium reabsorption increased dramatically (26,27). These latter conditions were both present in GS and might explain the observed discrepancy between the results of osmole-free water clearance and of the endogenous lithium clearance regarding sodium handling in the diluting nephron.
Taking our results as a whole, we propose that the chronic renal sodium loss secondary to SLC12A3 mutations in patients with GS could be compensated for by the enhancement of sodium reabsorption upstream of the DCT, namely in the cTAL, and that pharmacologic blockade of sodium transport in segments located downstream of the DCT would not significantly increase the extracellular fluid volume contraction in patients with GS.
Disclosures
None.
Acknowledgments
We are indebted to Dr. Anne Blanchard and Prof. Pascal Houillier for helpful discussions, and we are grateful to the nurses and clinical staff of the Department of Nephrology of the Strasbourg University Hospital.
We thank the Institut National de la Santé et de la Recherche Médicale for providing G.F. with a research contract (Contrat d'Interface pour Cliniciens) for the period 2010–2015.
Footnotes
Published online ahead of print. Publication date available at www.cjasn.org.
See related editorial, “Adaptation in Gitelman Syndrome: “We Just Want to Pump You Up”,” on pages 379–382.
Access to UpToDate on-line is available for additional clinical information at www.cjasn.org.
- Received January 31, 2011.
- Accepted December 11, 2011.
- Copyright © 2012 by the American Society of Nephrology
References
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