Tamm-Horsfall protein/uromodulin deficiency elicits tubular compensatory responses leading to hypertension and hyperuricemia

Abstract

Expression of Tamm-Horsfall protein (THP or uromodulin) is highly restricted to the kidney thick ascending limb (TAL) of loop of Henle. Despite the unique location and recent association of THP gene mutations with hereditary uromodulin-associated kidney disease and THP single nucleotide polymorphisms with chronic kidney disease and hypertension, the physiological function(s) of THP and its pathological involvement remain incompletely understood. By studying age-dependent changes of THP knockout (KO) mice, we show here that young KO mice had significant salt and water wasting but were partially responsive to furosemide, due to decreased luminal translocation of Na-K-Cl cotransporter 2 (NKCC2) in the TAL. Aged THP KO mice were, however, markedly oliguric and unresponsive to furosemide, and their NKCC2 was localized primarily in the cytoplasm as evidenced by lipid raft floatation assay, cell fractionation, and confocal and immunoelectron microscopy. These aged KO mice responded to metolazone and acetazolamide, known to target distal and proximal tubules, respectively. They also had marked upregulation of renin in juxtaglomerular apparatus and serum, and they were hypertensive. Finally, the aged THP KO mice had significant upregulation of Na-coupled urate transporters Slc5a8 and Slc22a12 as well as sodium-hydrogen exchanger 3 (NHE3) in the proximal tubule and elevated serum uric acid and allantoin. Collectively, our results suggest that THP deficiency can cause progressive disturbances in renal functions via initially NKCC2 dysfunction and later compensatory responses, resulting in prolonged activation of the renin-angiotensin-aldosterone axis and hyperuricemia.

Keywords: NKCC2; Tamm-Horsfall protein; hyperuricemia; lipid rafts; thick ascending limb; uromodulin.

Fig. 1.

Urine output, response to diuretics and localization of Na-K-Cl cotransporter 2 (NKCC2) in young Tamm-Horsfall protein (THP) knockout (KO) mice. A and B: 1-mo-old THP KO mice and age-matched wild-type (WT) mice were subject to 24-h urine measurement using single-mouse metabolic cages, after they were intraperitoneally administered with either normal saline as control or normal saline containing furosemide (FU; A) or acetazolamide (AZ; B); n = 8/diuretic/genotype for both (A and B). Note that, at the steady state, young THP KO mice excreted significantly more urine than age-matched WT mice (P < 0.01) in both cohorts (A and B); that young THP KO mice responded sensitively to furosemide by producing more urine than without furosemide treatment (P < 0.001), albeit less than the WT mice treated with furosemide (P < 0.05); and that young THP KO mice responded more robustly to acetazolamide than WT mice (P < 0.05). C: immunofluorescent staining of NKCC2 in the kidneys of 2 individual 1-mo-old WT and THP KO mice, representing 8 mice per genotype analyzed and showing that NKCC2 was predominantly located at the apical surface of thick ascending limb (TAL) in WT mice, but it was reduced at apical surface and increased in the cytoplasm in THP KO Mice. Bars equal = 30 μm.

Fig. 2.

Urine output, water consumption, and body weight of aged THP KO mice. A: 12-mo-old THP KO mice and age-matched WT control mice (n = 8 each genotype) were subjected to 24-h urine measurement using metabolic cages. B: the water supply of the same individual mice was measured at the beginning and end of the 24-h period, and the difference was calculated to determine the water consumption. C: the body weight was measured immediately before the 24-h urine collection. Each column represents the reading from the same mouse for the aforementioned 3 parameters aligned vertically, and the horizontal bars denote the averages. Note the significantly lower 24-h urine volume in aged THP KO mice, compared with the WT controls, and that this corresponded with reduced water intake and body weight.

Fig. 3.

Response to diuretics in aged THP KO mice. A–C: 12-mo-old THP KO mice and age-matched WT mice were subject to 24-h urine measurement, after they were intraperitoneally administered with normal-saline as control or normal saline containing furosemide (FU; A); metolazone (MET; B); or acetazolamide (AZ; C). n = 8/group. Note that, at the steady state, aged THP KO mice excreted considerably less urine than age-matched WT mice (P < 0.001) in all 3 cohorts (A–C), that aged THP KO mice were completely unresponsive to furosemide (A), and that aged THP KO mice responded moderately to metolazone (B) and robustly to acetazolamide (C). NS, no statistical significance.

Fig. 4.

Effects of THP loss on the apical translocation of NKCC2. A: lipid raft flotation assay of the partitioning of NKCC2 in the lipid rafts. Triton X-100-insoluble protein extracts from 12-mo-old THP KO mice and age-matched WT mice were centrifuged on a stepwise sucrose density gradient (5–40% in 5% increments), and equal volumes from different fractions (1–9: lighter density fractions to the left and heavier density fractions to the right) were resolved on an SDS-PAGE and immunoblotted with anti-NKCC2 and reblotted with anti-flotillin, a lipid raft marker, and anti-THP. The results are representative of 3 similar experiments. Note the lack of partitioning of NKCC2 in the lighter fractions (6, 7) in THP KO mice, as opposed to the situation in WT mice and the coexistence of NKCC2 and THP in the same fractions in WT mice. Short bars on the left denote positions of molecular mass standards (from top to bottom): 170 and 135 kDa; 55 and 40 kDa; 135 and 100 kDa; 170 and 135-kDa; 55 and 40 kDa; 135 and 100 kDa. B, top: cellular fractionation and Western blotting. Renal cells from aged WT and THP KO mice (n = 3 per genotype) were fractionated into plasma membrane-enriched and cytosol-enriched fractions. The resultant fractions were dissolved in SDS-PAGE loading buffer and analyzed by Western blotting using anti-NKCC2 and anti-p-NKCC2 antibodies. Anti-flotillin and anti-β-actin served as loading controls for membrane and cytosol, respectively. Short bars on the right denote positions of molecular mass standards (from top to bottom): 170 and 135 kDa; 170 and 135 kDa; 55 and 40 kDa. B, bottom: densitometry of the Western blotting results shown is top, expressed as ratio to flotillin or -β−actin. Note the remarked reduction of NKCC2 and p-NKCC2 in the membrane fraction and the significant increase of NKCC2 in the cytosol. NS, no statistical significance. C: confocal immunofluorescent microcopy showing colocalization of NKCC2 with membrane marker flotillin at the apical surface of TAL of aged WT mice and, in contrast, the reduced apical labeling and increased cytoplasmic labeling of NKCC2 in the TAL of aged THP KO mice. All panels are of the same magnification; bar = 30 μm: ×400. D: immunoelectron microscopy of NKCC2 of 12-mo old THP KO mice and age-matched WT mice showing apical association of NKCC2 in WT mice and the cytoplasmic staining of NKCC2 in THP KO mice. Please refer to materials and methods for the operationally defined “Apical membrane” and “Cytoplasm”, and procedures of enumeration and tabulation of the gold particles in these two compartments. Bar = 200 nm. Bottom: average number of gold particles and SD per counted area in the apical membrane and cytoplasm.

Fig. 5.

Triple immunofluorescent detection and localization of renin (color purple), NKCC2 (green), and THP (red) in young (1-mo-old) and aged (12-mo-old) THP KO mice and their age-matched WT controls. G, glomerulus; JGA, juxtaglomerular apparatus; TAL, thick ascending limb; MD, macula densa. Note: 1) in young WT mice, NKCC2 and THP colocalized at the apical surface of TAL; MD was positive for NKCC2 but lacked THP; 2) in young THP KO mice, MD did not have apparent upregulation of NKCC2 and JGA was negative for renin; 3) in aged WT mice, NKCC2 and THP colocalized at the apical surface of TAL and MD was positive for NKCC2 but lacked THP; and 4) in aged THP KO mice, NKCC2 remained at the apical surface in MD, along with marked upregulation of renin in JGA, but it was primarily cytoplasmic in TAL cells. All the panels are of the same magnification, and bar = 30 μm.

Fig. 6.

Measurement of serum renin, angiotensin II and aldosterone as well as sodium, chloride, and potassium in THP KO mice. Note that in A the significantly higher levels of renin and aldosterone in 5-mo-old THP KO mice (n = 4) than age-matched WT mice (n = 4). Angiotensin II was also higher although it was less statistically significant. Note that in B serum renin, angiotensin II, and aldosterone were all significantly higher in 12-mo old THP KO mice (n = 4) than in age-matched WT mice (n = 4). Also, note in C the significantly increased sodium and chloride and decreased potassium concentrations in 12-mo-old THP KO mice compared with age-matched WT mice.

Fig. 7.

Blood pressure measurement in aged THP KO mice. THP KO mice and WT mice (both 12-mo old; n = 8) were subjected to measurement of systolic (A) and diastolic (B) blood pressure using a noninvasive tail cuff system (see materials and methods for details). Note that THP KO mice had higher blood pressure than WT controls.

Fig. 8.

Renal expression and localization of urate transporters in aged THP KO mice. Total RNAs extracted from 12-mo-old THP KO mice (n = 4) and age-matched WT mice (n = 4) were subjected to quantitative real-time PCR using oligonucleotide primers specific for Slc5a8 (A), Slc5a12 (B), and Slc22a12 (URAT1; C). Values were calculated using ΔΔCt formula and expressed as ratios against simultaneously amplified β-actin. Note the increased expression of all three urate transporters, with the differences of Slc5a8 and Slc22a12 between KO and WT mice reaching statistical significance. D: Western blotting detection (left) and densitometry (right) of Slc5a8 and Slc22a12 showing significantly their increased expression on the protein level in aged THP KO mice. Each lane was from an individual mouse. E: representative images of immunofluorescent staining of Slc5a8 and Slc22a12 (URAT1) in 12-mo-old WT and THP KO mouse kidneys. Note the markedly increased expression of both urate transporters at the luminal surface of some of the proximal tubules of the KO mice. Arrows denote the proximal tubules with urate transporter overexpression; whereas arrowheads denote those without significant overexpression. Bar = 30 μm.

Fig. 9.

Expression of sodium-hydrogen antiporter 3 (NHE3) in aged THP KO mice. Western blotting (A) and immunofluorescence staining (B) using an anti-NHE3 antibody showing elevated expression of NHE3 in 12-mo old THP KO mice, compared with age-matched WT mice. A, left: each lane is from an individual mouse. A, right: densitometry result from left. B: representative images from 5 mice examined per genotype; bars = 30 μm.

Fig. 10.

Measurement of serum levels of uric acid and allantoin in THP KO mice. Note the lack of significant difference in serum uric acid and allantoin levels between 5-mo old THP KO and WT mice (A and B; n = 10/genotype). Also, note the significantly higher levels of uric acid and its metabolic product allantoin in 12-mo-old THP KO mice (n = 8) than in age-matched WT mice (C and D; n = 8/genotype).

Fig. 11.

Schematic diagram depicting the age-dependent physiological responses during THP deficiency. A: under normal conditions when THP is sufficient, THP helps anchor NKCC2 to the apical surface of TAL where NKCC2 transports physiological amounts of Na, Cl, and K (K not drawn for brevity). Salt sensing at MD, renin synthesis at JGA, and angiotensin synthesis and uric acid reabsorption at PT are all kept at physiological levels. B: when THP is deficient in young animals, NKCC2 becomes partially cytoplasmic, hence partially dysfunctional. This results in salt wasting and increased urine output. Since NKCC2 is only partially dysfunctional, the physiological disturbances as well as the compensatory responses including the activation of renin-angiotensin axis are minimum. C: when THP is deficient in aged animals, NKCC2 is completely dysfunctional at TAL. Lower filtered load plus higher reabsorption of NaCl at PT led to robust response at MD and marked upregulation of renin and angiotensin. The salt wasting is compensated in part by increased reabsorption of NaCl at PT and functionally linked increased reabsorption of uric acid, leading to hyperuricemia.