Zymogen-locked mutant prostasin (Prss8) leads to incomplete proteolytic activation of the epithelial sodium channel (ENaC) and severely compromises triamterene tolerance in mice

Abstract

Aim: The serine protease prostasin (Prss8) is expressed in the distal tubule and stimulates proteolytic activation of the epithelial sodium channel (ENaC) in co-expression experiments in vitro. The aim of this study was to explore the role of prostasin in proteolytic ENaC activation in the kidney in vivo.

Methods: We used genetically modified knockin mice carrying a Prss8 mutation abolishing proteolytic activity (Prss8-S238A) or a mutation leading to a zymogen-locked state (Prss8-R44Q). Mice were challenged with low sodium diet and diuretics. Regulation of ENaC activity by Prss8-S238A and Prss8-R44Q was studied in vitro using the Xenopus laevis oocyte expression system.

Results: Co-expression of murine ENaC with Prss8-wt or Prss8-S238A in oocytes caused maximal proteolytic ENaC activation, whereas ENaC was activated only partially in oocytes co-expressing Prss8-R44Q. This was paralleled by a reduced proteolytic activity at the cell surface of Prss8-R44Q expressing oocytes. Sodium conservation under low sodium diet was preserved in Prss8-S238A and Prss8-R44Q mice but with higher plasma aldosterone concentrations in Prss8-R44Q mice. Treatment with the ENaC inhibitor triamterene over four days was tolerated in Prss8-wt and Prss8-S238A mice, whereas Prss8-R44Q mice developed salt wasting and severe weight loss associated with hyperkalemia and acidosis consistent with impaired ENaC function and renal failure.

Conclusion: Unlike proteolytically inactive Prss8-S238A, zymogen-locked Prss8-R44Q produces incomplete proteolytic ENaC activation in vitro and causes a severe renal phenotype in mice treated with the ENaC inhibitor triamterene. This indicates that Prss8 plays a role in proteolytic ENaC activation and renal function independent of its proteolytic activity.

Keywords: ENaC; Prostasin; Prss8; Prss8-R44Q; Prss8-S238A; epithelial sodium channel.

Figure 1:. Co-expression of zymogen-locked Prss8-R44Q stimulates ENaC currents to a lesser extent than wild-type Prss8 or proteolytically inactive Prss8-S238A

A-D Representative whole-cell current traces recorded in an oocyte expressing murine ENaC alone (ENaC; A) or in combination with murine wild-type Prss8 (ENaC + Prss8-WT; B), zymogen-locked Prss8-R44Q (ENaC + Prss8-R44Q; C), or catalytically inactive Prss8-S238A (ENaC + Prss8-S238A; D). Presence of amiloride (Ami, 2 μM) or chymotrypsin (Chym, 2 μg/ml) in the bath solution is indicated by corresponding black or grey bars, respectively. E Summary of data obtained in similar experiments as in A-D. Individual and mean ± SEM values of ΔI_ami before (−) and after (+) application of chymotrypsin. Measurements performed in the same oocyte are connected by a line (n=32 from 4 different batches of oocytes). ‡, p<0.001; †, p<0.01; n.s. not significant; Student’s t test. F Summary of the data shown in E normalized as relative stimulatory effect of chymotrypsin on ΔI_ami, i.e. the ratio of ΔI_ami after chymotrypsin to ΔI_ami before chymotrypsin. Thus, a ratio of one (dotted line) indicates the absence of a stimulatory effect of chymotrypsin on ENaC. Individual and mean ± SEM values are shown. ‡, p<0.001; n.s. not significant; one-way ANOVA with Bonferroni post hoc test.

Figure 2:. Analysis of protease activity at the cell surface of oocytes expressing wild-type Prss8, zymogen-locked Prss8-R44Q or catalytically inactive Prss8-S238A.

Proteolytic activity of prostasin (A, B) or of trypsin-like proteases (C, D) at the cell surface of oocytes detected using Ac-KHYR-AMC or Boc-QAR-AMC fluorogenic substrate, respectively. Progress curves of proteolytic activity (mean ± SEM) are shown for oocytes expressing ENaC alone or co-expressing ENaC with Prss8-wt, Prss8-R44Q or Prss8-S238A. In A and C, representative recordings of absolute fluorescent signal values (RFU=relative fluorescent unit) obtained in oocytes from one batch are shown (A: n=8; C: n=10). In B and D, RFU values obtained in different batches of oocytes (B: n=40, N=5; D: n=78, N=9) were normalized to account for the batch to batch variability of the absolute RFU values and to correct for the signal decline due to photobleaching. In each individual recording RFU values were normalized to the initial RFU value at the beginning of the measurement. In addition, for each time point the average normalized RFU values measured in oocytes expressing ENaC alone were subtracted from the corresponding individual normalized RFU values. ‡, p<0.001; †, p<0.01; n.s., not significant; one-way ANOVA with Bonferroni post hoc test (at the time point 180 min).

Figure 3.. Renal expression and urinary excretion of prostasin in Prss8-wt, Prss8-S238A and Prss8-R44Q mutant mice

A Western blot appearance of wild-type and mutant prostasin in kidney tissue as well as a recombinant truncated murine prostasin (amino acids 30-289, predicted mass 28 kDa) with or without deglycosylation by PNGaseF. Note that deglycosylation leads to single clear bands, compatible with glycosylation of prostasin in vivo. B Expression of prostasin as analyzed by Western blot from kidney lysates treated with PNGaseF for deglycosylation. Representative lanes with n=3 for each genotype. C Densitometric analysis of the obtained bands from two Western blots with n=5-6 for each genotype. D Urinary excretion of prostasin measured with an ELISA in 24 h urine samples.

Figure 4:. Sodium conservation is not impaired in Prss8-wt, Prss8-S238A and Prss8-R44Q mutant mice

(A, B) 24 h urinary sodium and potassium excretion under a control, low sodium (LS/NK) or low sodium + high potassium diet (LS/HK) on the 5^th day. (C) Change in body weight calculated as the difference between baseline and value on the 5^th day. (D) Time course of plasma aldosterone concentration after switch to a low sodium diet with normal potassium content (LS/NK, solid lines) or a low sodium with a high potassium content (LS/HK, dashed lines). # indicates significant difference between low sodium and control diet, * indicates significant difference between the genotypes

Figure 5:. Effect of diuretics in Prss8-S238A and Prss8-R44Q mutant mice

A Natriuretic response expressed as urinary Na/K ratio to the acute administration of vehicle (V, injectable water, 5μL g⁻¹) or the ENaC inhibitor triamterene (T, 10 μg g⁻¹). Urine was collected for 6 hours. B, C Effect of a four day treatment with the ENaC inhibitor triamterene on urine output and urinary sodium/potassium ratio. D, E Course of body weight as well as fluid and food intake during triamterene treatment. F Kaplan-Meier curve for the probability to reaching the end point weight loss greater than 25% of the baseline value which prompted termination of the experiment and euthanasia in that particular mice. G-I Effect of a four day treatment with the NKCC2 inhibitor furosemide or the NCC inhibitor HCT on urine output, natriuresis and body weight. abbreviations: C control, V vehicle, T triamterene, Furo furosemide, HCT hydrochlorothiazide # indicates significant difference between control and diuretic treatment, * indicates significant difference between the genotypes

Figure 6:. Tissue expression of ENaC subunits in kidneys from Prss8-wt, Prss8-S238A and Prss8-R44Q mutant mice under control and triamterene treatment

Immunohistochemical staining of fixed kidney tissue. In all genotypes, there is increased abundance of the α-subunit and apical staining for all subunits after a two day triamterene treatment.

Figure 7.. Protein expression of ENaC subunits in kidneys from Prss8-wt, Prss8-S238A and Prss8-R44Q mutant mice under control and triamterene treatment

A Representative Western blot for α-, β-ENaC from native kidney lysates and γ-ENaC from deglycosylated kidney lysates. Full length α-, β-, and γ-ENaC migrate at 90, 88 and 72 kDa, respectively. Furin-cleaved α- and γ-ENaC migrate at 25 and 61 kDa, respectively. Fully cleaved γ-ENaC migrates at 54 kDa. B-G Densitometric analysis of the obtained bands after normalization for total protein content of each lane. N=5-6 for each genotype and treatment obtained from 2 gels of which one is shown. # indicates significant difference between control and triamterene treatment, * indicates significant difference between the genotypes

Figure 8:. Model of the proteolytic ENaC activation by prostasin in the kidney

A Cleavage of Prss8 at R44 during intracellular maturation induces a conformational change to a two-chain form that enables the recruitment of another serine protease (depicted in red) to the cell surface and facilitates proteolytic activation of ENaC. This scaffold function of prostasin is independent of its proteolytic activity of prostasin as the effect of proteolytically inactive Prss8-S238A on ENaC is similar to that of Prss8-wt. The exact identity of the recruited serine protease is unknown. B The scaffold function is impaired in zymogen-locked prostasin (Prss8-R44Q) and there is no recruitment of another serine protease. This is most likely to due to a different conformation of single-chain prostasin with the intact activation bond.