NCC) Causes Severe Salt Wasting, Volume Depletion, and Renal Failure

Double knockout of pendrin and Na-Cl cotransporter (NCC) causes severe salt wasting, volume depletion, and renal failure

Manoocher Soleimania,b,c,1, Sharon Baronea,b,c, Jie Xua,b,c, Gary E. Shulld, Faraz Siddiquib, Kamyar Zahedia,b,c, and Hassane Amlalb,c

aResearch Services, Veterans Affairs Medical Center, Cincinnati, OH 45202; and Departments of bMedicine and dMolecular Genetics, Biochemistry, and Microbiology, and cCenter on Genetics of Transport and Epithelial Biology, University of Cincinnati, Cincinnati, OH 45267

Edited by Richard P. Lifton, Yale University School of Medicine, New Haven, CT, and approved July 2, 2012 (received for review February 15, 2012)

The Na-Cl cotransporter (NCC), which is the target of inhibition by with thiazide, can be an effective regimen for patients with fluid thiazides, is located in close proximity to the chloride-absorbing overload, such as congestive heart failure, nephrotic syndrome, transporter pendrin in the kidney distal nephron. Single deletion diuretic resistance, or advanced chronic kidney disease. of pendrin or NCC does not cause salt wasting or excessive diuresis under basal conditions, raising the possibility that these trans- Results porters are predominantly active during salt depletion or in re- Generation of Pendrin/NCC Double KO Mice. The double NCC/ sponse to excess aldosterone. We hypothesized that pendrin and pendrin KO mice were generated by crossing mice with single NCC compensate for loss of function of the other under basal con- deletion of pendrin or NCC with each other. Fig. 1 A and B ditions, thereby masking the role that each plays in salt absorption. demonstrates the generation of double NCC/pendrin KO mice, fi To test our hypothesis, we generated pendrin/NCC double knock- as veri ed by tail genotyping and Northern hybridization. Fig. out (KO) mice by crossing pendrin KO mice with NCC KO mice. 1C shows that 8-wk-old double KO mice are smaller than either single KOs or WT animals (Fig. 1C), with the average body Pendrin/NCC double KO mice displayed severe salt wasting and ∼ sharp increase in urine output under basal conditions. As a result, weight being 40% lower in age-matched double KO mice vs. WT or single KO mice (Fig. 1D)(P < 0.01; n = 5 in each group). animals developed profound volume depletion, renal failure, and Table 1 depicts body weights of WT and double KO mice at the metabolic alkalosis without hypokalemia, which were all corrected time of weaning (end of 3 wk) and at 8 wk of age. The double KO with salt replacement. We propose that the combined inhibition of mice were smaller at 3 wk and remained small at 8 wk of age. pendrin and NCC can provide a strong diuretic regimen without fl The failure to thrive was evident in both sexes. The double KO causing hypokalemia for patients with uid overload, including mice were born in the expected Mendelian distribution. Double patients with congestive heart failure, nephrotic syndrome, di- KO mice survive and live for at least 6 mo, with comparable uretic resistance, or generalized edema. survival rates to those in WT or single KO mice. (Longer-term survival analysis has not been performed.) diuretics | kidney tubules | nephrogenic diabetes insipidus Pendrin/NCC Double KO Mice Have Profound Renal Water and Salt he thiazide-sensitive Na-Cl cotransporter (NCC) (SLC12A3) Wasting. Animals were placed in metabolic cages, and, after ac- − − Tand the Cl /HCO3 exchanger pendrin (SLC26A4) are ex- clamation for 2 d, balanced studies (daily food intake, water in- pressed on apical membranes of distal cortical nephron segments take, urine volume, and body weight) were performed for 4 and mediate salt absorption, with pendrin working in tandem consecutive days. Results in Fig. 2 depict the collections on day 3, with the epithelial Na channel and NCC working by itself (1–6). which is representative of daily experiments, and demonstrate the Pendrin is expressed on the apical membrane of intercalated presence of profound polyuria (increased urine output), along cells in late distal convoluted tubule (DCT), connecting tubule with polydipsia (increased water intake), in double pendrin/NCC (CNT), and the cortical collecting duct (CCD) (7–9). The thia- KO mice but not in single KO mice (Fig. 2 A and B). Fig. 2C zide-sensitive NCC is primarily expressed on the apical mem- demonstrates that urine osmolality is reduced by ∼70% in double brane of DCT cells (10, 11). KO mice vs. the other genotypes. Fig. 2D shows sharp increases in Single deletion of pendrin or NCC does not cause salt wasting sodium and chloride excretion in double pendrin/NCC KO mice. or excessive diuresis under basal conditions (5, 12–15). Indeed, Double KO mice were eating normally; however, when adjusted even a mild degree of salt wasting has not been demonstrated in for body weights, they showed increased food intake vs. WT mice these two genetically engineered mouse models at steady state. (0.163 and 0.207 g food intake/g of body weight in WT and double Kidney functions, including sodium and chloride excretion, urine KO, respectively; P < 0.05, n = 5). Food consumption in single output, and blood urea nitrogen (BUN) levels in mutant mice KO mice (pendrin or NCC) was comparable to WT mice. are comparable to wild-type (WT) animals (12–15). Both pendrin KO and NCC KO mice, however, show signs of volume Pendrin/NCC Double KO Mice Have Severe Volume Depletion, Renal depletion or develop hypotension during salt restriction (12, 14). Failure, and Metabolic Alkalosis. To determine whether excessive These findings have led investigators to conclude that pendrin salt wasting causes volume depletion in double KO mice, we and NCC are predominantly active during salt depletion (and or in response to increased aldosterone levels), and their contribution to salt reabsorption at baseline conditions is small. Author contributions: M.S. and H.A. designed research; M.S., S.B., J.X., F.S., K.Z., and H.A. We hypothesized that NCC and pendrin may compensate for performed research; G.E.S. contributed new reagents/analytic tools; M.S. and H.A. ana- loss of the other under basal conditions, thereby masking the role lyzed data; and M.S. wrote the paper. that each plays in salt reabsorption. To test this hypothesis, we The authors declare no conflict of interest. generated double knockout of pendrin and NCC mice by crossing This article is a PNAS Direct Submission. animals with single deletion for NCC and pendrin. The double KO Freely available online through the PNAS open access option. mice show significant salt and fluid wasting, along with volume 1To whom correspondence should be addressed. E-mail: [email protected]. depletion and renal failure under baseline conditions. We propose This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. that pendrin could be a target for a diuretic that, in conjunction 1073/pnas.1202671109/-/DCSupplemental.

13368–13373 | PNAS | August 14, 2012 | vol. 109 | no. 33 www.pnas.org/cgi/doi/10.1073/pnas.1202671109 Downloaded by guest on September 30, 2021 examined the kidney expression of renin in WT and mutant mice. As shown in Fig. 3A (Upper gels), mRNA expression and protein abundance of renin robustly increased in kidneys of double KO mice relative to single KO or WT mice, with enhanced mRNA expression and protein abundance by ∼7-fold (P < 0.0001 vs. WT; n = 4) and >10-fold (P < 0.001 vs. WT; n = 5), respectively. These results indicate that the double KO mice are in a state of severe volume depletion. To determine whether salt wasting (Fig. 2) and the subsequent volume depletion (Fig. 3A) impair the kidney function, BUN levels were measured in WT and mutant mice. Results demonstrated a sharp increase in BUN levels in double KO mice relative to other genotypes, consistent with significant prerenal failure (Fig. 3B), which signifies impaired kidney function subsequent to decreased perfusion as a result of volume depletion. Double KO animals have normal serum sodium and potassium concentrations (Table 2). To ascertain the impact of severe volume depletion and the subsequent renal failure on acid base balance, systemic acid base parameters in various genotypes were examined (Experi- mental Procedures). As indicated, double pendrin/NCC KO mice have profound metabolic alkalosis vs. WT mice, as determined by elevated serum bicarbonate and arterial pH (Fig. 3C; P < 0.01 vs. WT mice). Mice with NCC deletion have normal serum bicarbonate levels, and mice with pendrin deletion demonstrate mild elevation in serum bicarbonate (12, 15). Blood pressure measurement by the computerized tail cuff method indicated that double KO mice are hypotensive (Fig. 3D; P < 0.01 vs. WT). MEDICAL SCIENCES Effect of Salt Replacement on Volume Depletion, Renal Failure, and Metabolic Alkalosis in Pendrin/NCC Double KO Mice. To ascertain the role of volume depletion in the pathogenesis of renal failure and metabolic alkalosis, double pendrin/NCC KO and WT mice were placed on high-salt (7%) diet for 7 d. Fig. 4A (Left and Right) demonstrates that the mRNA expression levels of renin decreased by ∼90% in kidneys of double KO mice on high-salt intake relative to normal diet. The above results demonstrate that salt replacement significantly improves the vascular volume depletion in double KO mice. Fig. 4B shows that BUN levels, although mildly higher in double KO vs. WT mice, are significantly decreased in double KO mice on high-salt diet relative to normal diet (comparison vs. Fig. 3B). Fig. 4C indicates significant improvement in the severity of metabolic alkalosis, as verified by a sharp reduction in serum bicarbonate levels in double KO mice on high-salt vs. normal-salt diet (P < 0.001; n = 4).

Pendrin/NCC Double KO Mice Have Impaired Kidney Response to Exogenous Vasopressin. The presence of low urine osmolality, despite severe volume depletion, suggests either the impairment in arginine vasopressin (AVP) secretion or dysregulation in the collecting duct aquaporin 2 (AQP-2) and/or thick-limb Na-K-2Cl cotransporter (NKCC2). Our results demonstrate the down- regulation of AQP2 in medullary collecting duct in pendrin/NCC double KO mice (Fig. S1 A and B). The expression levels of AVP mRNA in the brain were enhanced in double KO mice (Fig. S1D), consistent with increased circulating AVP levels. NCC double KO mice failed to increase their urine osmolality significantly 24 h after 1-desamino-8-D-AVP (dDAVP) (an AVP analog) injection, consistent with the impaired response to AVP in double KO mice (Fig. S1E). The expression of NKCC2, the apical Na-K-2Cl cotransporter in the thick limb, increased (Fig. S1C), presumably to minimize the salt wasting. In the last series of experiments, the expression of sodium- − − dependent Cl /HCO3 exchanger (NDCBE) (Slc4a8) and so- Fig. 1. Generation and examination of NCC/pendrin double KO mice. (A) Generation of double pendrin/NCC KO mice. Tail genotyping was used to verify the generation of double KO mice. (B) Northern hybridization show- mice are small and have growth retardation. (D) Body weights of WT and ing the generation of double Pendrin/NCC KO mice. Pendrin/NCC double KO mutant animals. Double pendrin/NCC KO mice fail to thrive and are ∼40% mice lack the expression of pendrin and NCC in their kidneys. (C) Double smaller than either single KOs or WT animals at 8 wk after birth. *Signiﬁcant pendrin/NCC KO mice display growth retardation. Double pendrin/NCC KO difference (see Results for more information).

Soleimani et al. PNAS | August 14, 2012 | vol. 109 | no. 33 | 13369 Downloaded by guest on September 30, 2021 Table 1. Body weights of WT and double KO mice at 3 and 8 wk of age 3wk 8wk

WT body weight, g 12.2 ± 1.2 22.6 ± 2 dKO body weight, g 7.2 ± 1.00 14.6 ± 2 P <0.05 <0.05

The double KO (dKO) mice were signiﬁcantly smaller at 3 and 8 wk of age (n = 6 in each group). Both male and female mice are included in calculations.

dium channel (ENaC) subunits were examined in kidneys of four WT, single KO, and double KO mice. Fig. S2A is a Northern hybridization and shows that the expression of NDCBE increased significantly in kidneys of pendrin KO mice and NCC KO mice but not in double KO mice. Examination of ENaC subunits indicated a consistent increase in the expression levels of α, β, and γ subunits only in NCC KO mice. In kidneys of double KO mice, the expression of ENaC α and β subunits achieved statistically significant up-regulation vs. WT animals (P < 0.05; n = 4) (Fig. S2B). Western blots showed significant up- regulation of ENaC β and γ subunits in kidneys of double KO mice vs. WT animals (Fig. S2C; n = 4 in each group; P < 0.05 for each subunit). Antibodies against NDCBE (Experimental Proce- dures) did not yield any specific bands on Western blots. Discussion Thiazides are the specific inhibitors of NCC in the distal tubule and the most widely used diuretic in the world, in large part because they are considered mild agents and do not cause severe salt wasting (16, 17). The DCT is responsible for the reabsorption of 7–10% of filtered salt, and published reports indicate that majority of this process is mediated via NCC (18, 19). The absorption of the remaining filtered sodium in DCT is estimated to occur via sodium channel, with the residual component being + + absorbed via the Na /H exchanger (20, 21). The mild salt- wasting effect of thiazides is disproportionate to the magnitude of NCC inhibition, suggesting that other salt-absorbing trans- porters in the proximity of NCC might be compensating and, therefore, blunting the diuretic effect of thiazides. However, despite the presence of several sodium-absorbing molecules (above), the identity of chloride-absorbing transporter(s) in the distal nephron, which might become active in the setting of NCC inactivation, remains speculative. Pendrin, which was first identified by linkage analysis in − − patients with Pendred syndrome (22), can function in Cl /HCO3 exchange mode (4) and works in conjunction with the sodium channel in the kidney (6). Mice with the genetic deletion of pendrin, and human beings with inactivating mutation of pendrin, do not display any evidence of salt wasting under baseline conditions (5, 15). Similar to pendrin KO mice, NCC-null mice do not demonstrate any noticeable salt wasting under baseline conditions (12). Both mutant mouse models, pendrin and NCC KO mice, show signs of volume depletion and salt wasting or develop hypotension in response to salt restriction (12, 14). Both NCC and pendrin are regulated by excess aldosterone (6, 12, 14, 23– 26). These findings have led investigators to conclude that pendrin and NCC are predominantly active during salt depletion and/or in response to aldosterone, and their contribution to salt reabsorption at baseline conditions is insubstantial. Two lines of evidence support an important role for pendrin in Fig. 2. Fluid balance and salt excretion in WT and mutant animals. (A) Urine compensatory salt absorption in response to NCC inactivation. output in WT and mutant animals. Double pendrin/NCC KO mice display First, pendrin expression increases significantly in kidneys of profound polyuria (increased urine output). (B) Water intake in WT and mutant animals. Double pendrin/NCC KO mice have significant polydipsia NCC KO mice (25, 27). Second, treatment with hydrochloro- fl (increased water intake). (C) Urine osmolality in WT and mutant animals. thiazide causes increased uid loss in pendrin KO mice relative Double pendrin/NCC KO mice have low urine osmolality relative to WT and to WT littermates (27). It should be noted, however, that neither single KO mice. (D) Salt excretion in WT and mutant animals. Double pen- NCC KO mice nor pendrin KO mice display any evidence of salt drin/NCC KO mice display a sharp increase in sodium and chloride wasting wasting, volume depletion, or renal failure under baseline con- compared with other genotypes. *Significant difference (see Results for ditions (12–15). more information).

13370 | www.pnas.org/cgi/doi/10.1073/pnas.1202671109 Soleimani et al. Downloaded by guest on September 30, 2021 Fig. 3. Double pendrin/NCC KO mice have severe volume depletion and display renal failure and metabolic alkalosis. (A) mRNA expression and protein abundance of renin in kidneys of WT and mutant animals. The mRNA expression levels of renin (Left and Center) increased by ∼700% in double

pendrin/NCC KO mice relative to WT or single KO MEDICAL SCIENCES mice (P < 0.0001). The renin protein (Right)was induced in kidneys of double KO mice (P < 0.00001). (B) BUN levels in WT and mutant animals. Double pendrin/NCC KO mice demonstrate significant elevation in BUN level relative to other genotypes, consistent with prerenal failure. (C) Acid–base parameters in WT and mutant mice. Double pendrin KO/NCC KO mice have profound metabolic alkalosis vs. WT mice. Mice with NCC or pendrin deletion demonstrate normal or borderline elevation in their serum bicarbonate. (D) Blood pressure measurement in WT and mutant animals. Pendrin/NCC double KO mice show significant hypotension (P < 0.01 vs. WT). *Significant difference (see Results for more information).

The most salient feature of the present studies is the profound expression levels in the kidney and a sharp increase in BUN fluid and salt wasting that are observed subsequent to the com- levels in mutant mice (Results). Interestingly, the serum potas- bined deletion of pendrin and NCC, strongly suggesting that sium levels in double KO mice remained normal (Results), which each transporter masks the absence of the other under baseline is intriguing by and in itself, given the fact that similar magni- conditions. The fluid and electrolyte loss resulted in massive tudes of volume depletion, salt wasting, and renin/aldosterone volume depletion with subsequent prerenal failure and metabolic activation in subjects treated with loop diuretics (such as furo- alkalosis in double KO mice. This picture is completely distinct semide) is usually associated with hypokalemia, subsequent to from that in mice with single deletion of pendrin or NCC, which potassium secretion into the lumen in exchange for sodium ab- do not display any significant electrolyte or fluid loss under sorption via the sodium channel in the CNTs and collecting baseline conditions. This report highlights the essential role of ducts. One plausible explanation could be that pendrin works in pendrin in salt and fluid reabsorption in the distal nephron in the conjunction with the sodium channel (ENaC) to absorb NaCl, setting of NCC inactivation. and the inhibition of pendrin would blunt the activity of ENaC, + The double knockout of NCC and pendrin caused significant therefore impairing the secretion of potassium via K channels volume depletion, as verified by a robust increase in renin in exchange for sodium absorption through ENaC. A recent study showed that hydrochlorothiazide treatment resulted in severe volume depletion, along with metabolic alka- Table 2. Serum sodium and potassium concentrations in WT losis and decreased kidney function, in a patient with Pendred and double KO mice syndrome resulting from an inactivating mutation in Pendred WT double KO Syndrome (PDS) gene (28). This presentation is extremely unusual for thiazides and closely mimics the clinical picture in our Na+, mEq/L 136 ± 1.2 138 ± 1.5 + ± ± double KO mice and supports the conclusion that a functional K , mEq/L 4.6 0.3 4.2 0.4 pendrin blunts the diuretic effect of NCC inhibition subsequent to Serum sodium and potassium concentrations were not different between treatment with thiazides by compensatory absorption of salt in the WT and double KO mice. distal nephron.

Soleimani et al. PNAS | August 14, 2012 | vol. 109 | no. 33 | 13371 Downloaded by guest on September 30, 2021 Fig. 5. Proposed schematic diagram depicting the synergistic diuretic effects of thiazides and inhibitors of pendrin in the distal nephron. The combined inhibition of NCC and pendrin (Lower) can cause signiﬁcant salt and water wasting vs. control (Upper).

The pendrin/NCC double KO mice have low urine osmolality despite severe volume depletion (Figs. 2 and 3), along with the down-regulation of AQP2 and impaired response to exogenous vasopressin analog (dDAVP) (Fig. S1), consistent with the generation of nephrogenic diabetes insipidus, which is extremely unusual in humans or animals with massive volume depletion and elevation of vasopressin and activation of renin–angiotensin–aldosterone axis. The impaired urine-concentrating ability indicates that factors unknown at the present blunt the action of endogenous vasopressin and, as a result, down-regulate AQP2 expression and activity in pendrin/NCC double KO mice. Salt replacement corrected almost all physiologic abnormalities observed in pendrin/NCC double KO mice, as verified by >85% correction in the severity of volume depletion (improvement in renin expression levels), renal failure (reduction in BUN levels), and metabolic alkalosis (decrease in serum bicarbonate concen- tration). These results strongly indicate that salt and water wasting with the subsequent volume depletion are the central events in pendrin/NCC double KO mice, and their correction with salt replacement minimizes the severity of the volume depletion. A recent publication indicated that pendrin could work in tandem with NDCBE in B-intercalated cells to absorb sodium and chloride (29). Such an interconnecting mechanism in the intercalated cells can provide an ENaC-independent salt-reab- Fig. 4. Effect of increased dietary salt intake on vascular volume, kidney sorption pathway in the collecting duct, which is electroneutral + function, and acid–base parameters in double pendrin/NCC KO mice. (A) and not associated with a net K transport. It is plausible that the Expression of renin in kidneys of WT and mutant animals. The mRNA ex- interruption of this pathway plays an important role in the pre- pression levels of renin (Upper and Lower) were decreased by 90% in kid- vention of hypokalemia in NCC/pendrin double KO mice (Table neys of double pendrin/NCC KO mice on high-salt relative to normal-salt diet. (B) BUN levels in WT and mutant animals. In salt-replete animals, the increase in BUN levels in pendrin/NCC double KO mice is significantly blunted relative to baseline conditions. (C) Acid–base status in WT and mutant mice. pendrin KO/NCC KO mice on high-salt vs. normal diet. *Significant difference The magnitude of metabolic alkalosis was significantly improved in double (see Results for more information).

13372 | www.pnas.org/cgi/doi/10.1073/pnas.1202671109 Soleimani et al. Downloaded by guest on September 30, 2021 2). These observations predict that crossing the NDCBE KO Tail DNA Genotyping for NCC KO, pendrin KO, and Pendrin/NCC Double KO mice (29) with the NCC KO mice may result in severe salt Mice. The details of tail DNA genotyping are included in SI Experimental wasting with normal serum potassium in NCC/NDCBE double Procedures. KO mice, a phenotype similar to that observed in NCC/pendrin double KO mice. Antibodies, Western Blotting, and Immunofluorescence Labeling. Details of Whether specific inhibitors of pendrin can adversely affect the antibodies, Western blots, and immunofluorescence labeling are included in function of inner ear and thyroid, two organs with abundant SI Experimental Procedures. expression of pendrin (22, 30), remains speculative. Pendrin is essential for the embryonic development of structures involved RNA Isolation and Northern Hybridization. Details regarding RNA isolation, in normal hearing (cochlea, the vestibular labyrinth, and the hybridization, and gene-specific PCR fragments are included in SI endolymphatic sac of the inner ear) (22, 30), and its mutation in Experimental Procedures. humans or in its deletion in mice causes structural abnormalities in the above organs during their embryonic stage, which results Blood Composition and Urine Electrolytes Analysis. Details of blood compo- in deafness (30). It is, therefore, highly plausible that the in- sition and urine electrolyte analysis are included in SI Experimental hibition of pendrin after the normal development of inner-ear Procedures. structures is completed will not interfere with normal hearing. The impact of pendrin inhibitors on thyroid function remains Blood Pressure Monitoring. Details of blood pressure measurements are in- speculative at the present. cluded in SI Experimental Procedures. In conclusion, we propose that the combined inhibition of pendrin and NCC can provide a strong diuretic regimen without Vasopressin Analog Injection. Animals (WT and double KO mice) were placed causing hypokalemia for patients with fluid overload (schematic in metabolic cages, and their urine output and water intake, along with food diagram in Fig. 5 Upper and Lower), such as those with conges- intake, were measured before and 24 h after s.c. injection with the vaso- tive heart failure, nephrotic syndrome, diuretic resistance, or pressin 2 (V2)-receptor–specific vasopressin analog dDAVP at 5 μg/100 g of generalized edema. Whether the combination of thiazides and body weight. mineralocorticoid antagonists can be as effective as the combination of thiazides and pendrin inhibitors remains speculative. Statistical Analysis. The results for chloride excretion, sodium excretion, urine Furthermore, whether alterations in the milieu of the CCD volume, urine osmolarity, and blood pressure are presented as means ± SE. subsequent to pendrin inhibition (such as acidic luminal pH) are Statistical significance between WT and double KO mice was determined by important determinants of the salt wasting phenotype in pendrin/ Student’s unpaired t test or ANOVA, and P < 0.05 was considered significant. NCC double KO mice (and by inference pendrin inhibitor/thia- Unless indicated, at least four separate animals from each group (WT, pen- MEDICAL SCIENCES zide combination) is currently unknown. drin KO, NCC KO, or double KO) were used for determination of expression fl Experimental Procedures levels (Northern, Western, or immuno uorescence) and balanced studies. Animal Models. Details of generation of Slc26a4 (pendrin) and the thiazide- ACKNOWLEDGMENTS. This work was supported by a Merit Review award sensitive cotransporter (NCC)-null mice have been reported by our group (12, from the Department of Veterans Affairs, funds from the Center on Genetics 15). Double pendrin/NCC KO mice were generated by crossing pendrin KO of Transport and Epithelial Biology at the University of Cincinnati, National mice with NCC KO mice. The WT, single KO, and double KO mice have a Institutes of Health Award R56DK62809, and grants from US Renal Care and mixed BALB/c and C57BL/6J background. Dialysis Clinic, Inc. (DCI) (to M.S.).

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