Tissue kallikrein permits early renal adaptation to potassium load

Abstract

Tissue kallikrein (TK) is a serine protease synthetized in renal tubular cells located upstream from the collecting duct where renal potassium balance is regulated. Because secretion of TK is promoted by K+ intake, we hypothesized that this enzyme might regulate plasma K+ concentration ([K+]). We showed in wild-type mice that renal K+ and TK excretion increase in parallel after a single meal, representing an acute K+ load, whereas aldosterone secretion is not modified. Using aldosterone synthase-deficient mice, we confirmed that the control of TK secretion is aldosterone-independent. Mice with TK gene disruption (TK-/-) were used to assess the impact of the enzyme on plasma [K+]. A single large feeding did not lead to any significant change in plasma [K+] in TK+/+, whereas TK-/- mice became hyperkalemic. We next examined the impact of TK disruption on K+ transport in isolated cortical collecting ducts (CCDs) microperfused in vitro. We found that CCDs isolated from TK-/- mice exhibit net transepithelial K+ absorption because of abnormal activation of the colonic H+,K+-ATPase in the intercalated cells. Finally, in CCDs isolated from TK-/- mice and microperfused in vitro, the addition of TK to the perfusate but not to the peritubular bath caused a 70% inhibition of H+,K+-ATPase activity. In conclusion, we have identified the serine protease TK as a unique kalliuretic factor that protects against hyperkalemia after a dietary K+ load.

Fig. 1.

Urinary excretion of K⁺ (A), aldosterone (B), kallikrein (C), and Na⁺ (D) in wild-type mice that have been taught to eat their daily ration over a 4-h period. n = 8 mice in each group. Values are means ± SE. Statistical significance is assessed by ANOVA and Newman-Keuls post hoc test. *P < 0.05 vs. the 10:00 am to 2:00 pm period. ^$P < 0.05 vs. the 6:00 pm to 10:00 am period.

Fig. 2.

Effect of aldosterone synthase deletion on urinary kallikrein excretion. Urinary kallikrein activity was measured on a standard (0.8% K⁺) rodent diet and after 24 or 48 h K⁺ loading by administration of a high (2% K⁺) diet. AS^+/+, wild-type mice; AS^−/−, aldosterone synthase-deficient mice. n = 5–7 mice per group. Values are means ± SE. Statistical significance is assessed by ANOVA followed by Bonferroni multiple comparison post hoc test when appropriate. *P < 0.05; **P < 0.01 vs. day 0, same genotype.

Fig. 3.

Effects of TK disruption on blood K⁺ concentration. (A) Blood K⁺ concentration on standard diet (0.8% K⁺) in five TK^+/+ and six TK^−/− mice, or after 24 h of feeding with a high (2%) K⁺ diet in eight TK^+/+ and six TK^−/− mice. (B) Blood K⁺ concentration in 7 TK^+/+ (filled bars) and 7 TK^−/− (open bars) littermate mice under fasting and postprandial states. Measurements were performed 1 h before and 5 h after the beginning of the feeding period in which the mice did ingest their daily food intake. Values are means ± SE. Statistical significance is assessed by ANOVA and, when significant, groups were compared by Newman-Keuls post hoc test. *P < 0.01 vs. TK^+/+ under postprandial state. ^$P < 0.001 vs. TK^−/− under fasting state.

Fig. 4.

K⁺ transepithelial absorption is due to increased expression and activity of colonic H⁺,K⁺-ATPase in CCDs isolated from TK^+/+ (filled bars) and TK^−/− mice (open bars). (A) CCDs isolated from TK^+/+ mice do not absorb Na⁺ (Bottom), do not generate a lumen-negative transepithelial voltage (Middle) and do not secrete K⁺ (Top) under basal conditions. CCDs isolated from TK^−/− mice absorb Na⁺ (Bottom) but do not generate a lumen-negative transepithelial voltage (Middle) and exhibited net K⁺ absorption (Top). The control group consists of littermate mice. Statistical significance is assessed by unpaired Student's t test. n = 7 in each group, *P < 0.05. (B) Intracellular pH recovery after an acute acid load in ICs of CCDs isolated from TK^+/+ and TK^−/− mice. Each trace represents the mean of pH_i changes recorded when NH₄Cl was added and removed in 8–9 CCDs (4–5 ICs were analyzed in each CCD). NH₄Cl removal leads to rapid intracellular acidification. (C) Average initial rates of pH_i recovery after intracellular acidification in ICs in the absence or presence of different inhibitors (30 μM Sch28080 or 1 mM oubain). Values on graph are means ± SE; n = 8 tubules for TK^+/+, n = 9 for TK^−/−, and n = 4 for TK^−/− + Sch28080 and TK^−/− + ouabain. Statistical significance is assessed by ANOVA and Newman-Keuls multiple comparison test. **P < 0.01 vs. TK^+/+. ^$P < 0.05. ^$$P < 0.01 vs. TK^−/− in the absence of inhibitor. The nadir pH_i achieved after washout of the NH₄Cl prepulse was identical in each group, 6.62 ± 0.05 for TK^+/+ (n = 8); 6.70 ± 0.11 for TK^−/− (n = 9); 6.72 ± 0.05 for TK^−/− + Sch28080 (n = 4) and 6.68 ± 0.09 for TK^−/− + ouabain (n = 4). Values are means ± SE and were not significantly different (one-way ANOVA). (D) Expression of the α2 subunit of H⁺,K⁺-ATPase and the α1 subunit of the H⁺,K⁺-ATPase in CNTs and CCDs. Results are expressed as arbitrary units (a.u.)/mm tubules. Data are means ± SE from four to six mice. Statistical significance was assessed by ANOVA followed by Bonferroni post hoc test: *P < 0.01 vs. TK^+/+.

Fig. 5.

Response to an acid load in TK^{-/ -} mice. Values in TK^+/+ (•) and TK^−/− (△) littermates are compared and represented as means ± SE. Blood pH (A) and HCO₃⁻ (B) were measured in mice challenged with an acid load consisting in 280 mM NH₄Cl in the drinking water. n = 5–7 mice per genotype. *P < 0.05 vs. TK^+/+.

Fig. 6.

Intracellular pH recovery after an acute acid load in ICs of CCDs isolated from TK^−/− mice. CCDs isolated from TK^−/− mice were perfused with either TK (10 μg/mL) or the vehicle alone for 30 min before recording. TK was able to inhibit the rate of recovery of intracellular pH after intracellular acidification of ICs by 70%. Values on graph are means ± SE, n = 9 tubules for TK^−/− with no TK, n = 5 for luminal TK, and n = 4 for peritubular TK. Statistical significance is assessed by ANOVA and Newman-Keuls multiple comparison test. *P < 0.05 vs. TK^−/− in the absence of TK. The nadir pH_i achieved after washout of the NH₄Cl prepulse was identical in each group, 6.70 ± 0.11 for no TK (n = 9); 6.61 ± 0.06 for luminal TK (n = 5); 6.60 ± 0.09 for peritubular TK (n = 4). Values are means ± SE and were not significantly different (one-way ANOVA).

Fig. 7.

Schematic model depicting how TK participate to the response against dietary K⁺ load. TK production by CNT cells is stimulated by dietary K⁺ loading. TK is then released into the urinary fluid and reaches the CCD. There, TK might favor K⁺ secretion by PCs through its stimulating action on ENaC mediated Na⁺ absorption, an effect that occurs through proteolytic processing of the γ-ENaC subunit (34). TK also inhibits K⁺ absorption by ICs by decreasing colonic H⁺/K⁺-ATPase expression and activity. It also up-regulates ENaC-independent electroneutral NaCl absorption presumably through a decrease in the local production of bradykinin (BK) (35).