Activation of the alternative complement pathway and its relevance for sodium retention in experimental nephrotic syndrome

Abstract

The complement component C3, factor B (FB) and factor D (FD) belong to the alternative complement pathway and have been identified in urine samples from nephrotic mice. However, it is not yet known whether these factors are involved in mediating sodium retention in nephrotic syndrome (NS). Here we used a genetic mouse model of NS based on an inducible podocin deletion (Nphs2^Δipod). These mice were intercrossed with mice deficient for FB, FD or C3, yielding Nphs2^Δipod*Cfb^-/-, Nphs2^Δipod*Cfd^-/- or Nphs2^Δipod*C3^-/- mice, respectively. NS was induced after oral doxycycline treatment for 14 days. C3, FB and FD were detected in the nephrotic urine of Nphs2^Δipod mice as well as fragments of C3 and FB, indicating intrarenal activation of the alternative complement pathway. Lack of FB and FD had no impact on the activation of C3. Immunohistochemistry demonstrated positive C3 staining in protein casts and within the proximal tubule. Nephrotic mice of all genotypes experienced similar proteolytic activation of the epithelial sodium channel ENaC, developed sodium retention (urinary sodium concentration < 20 mM) and body weight gain. This was associated with a stimulation of proteolytic processing of epithelial sodium channel ENaC in all genotypes. In conclusion, components of the alternative complement pathway are detectable and activated in nephrotic syndrome. Mice with deletion of C3, FB or FD are not protected from proteolytic ENaC activation and sodium retention in NS.

Keywords: Alternative complement pathway; Edema; Epithelial sodium channel; Nephrotic syndrome; Sodium retention.

Fig. 1

Expression of FB, FD and C3 in the plasma of Nphs2^Δipod, Nphs2^Δipod*Cfb^-/-(FB^-/-), Nphs2^Δipod*Cfd^-/- (FD^-/-) and Nphs2^Δipod*C3^-/-(C3^-/-) mice before and after induction of experimental nephrotic syndrome. a, b Western blot for expression of C3 (green) and FB (red) under reducing a or non-reducing conditions b. c, d Western blot for expression of FD (red) under reducing c or non-reducing conditions d. e–i Densitometry of the obtained bands under reducing conditions. Note the specificity of the antibodies used as they lack any signal in the respective ko strain. ^# Significant difference (p < 0.05) between uninduced and nephrotic mice of the same genotype (unpaired t-test or Wilcoxon test). ^* Significant difference (p < 0.05) between genotypes and Nphs2^Δipod (ANOVA with Dunnetts multiple comparison test or Kruskal-Wallis with Dunn’s multiple comparison test)

Fig. 2

Induction of nephrotic syndrome in Nphs2^Δipod, Nphs2^Δipod*Cfb^-/-(FB^-/-), Nphs2^Δipod*Cfd^-/- (FD^-/-) and Nphs2^Δipod*C3^-/-(C3^-/-) mice. a–c Course of proteinuria after end of induction treatment at day 0. d maximal proteinuria normalized for urinary creatinine concentration after 8 days. e Western blot of urine samples after total protein staining. Note the albuminuria at 65 kDa after induction of nephrotic syndrome. f Densitometry of albumin abundance before and after induction of nephrotic syndrome. g Western blot of plasma samples after total protein staining. h Densitometry of albumin abundance before and after induction of nephrotic syndrome. Trajectories of Nphs2^Δipod mice are pooled and results with the respective knockout line were plotted separately for clarity. ^# Significant difference (p < 0.05) between uninduced and nephrotic mice of the same genotype (unpaired t-test or Wilcoxon test). ^* Significant difference (p < 0.05) between genotypes and Nphs2^Δipod (ANOVA with Dunnetts multiple comparison test or Kruskal-Wallis with Dunn’s multiple comparison test)

Fig. 3

Expression of FB, FD and C3 in the urine of Nphs2^Δipod, Nphs2^Δipod*Cfb^-/-(FB^-/-), Nphs2^Δipod*Cfd^-/- (FD^-/-) and Nphs2^Δipod*C3^-/-(C3^-/-) mice before and after induction of experimental nephrotic syndrome. a, b Western blot for expression of C3 (green) and FB (red) under reducing a or non-reducing conditions b. c, d Western blot for expression of FD (red) under reducing c or non-reducing conditions d. Note that the signal is weaker under non-reducing conditions, suggesting reduced recognition of FD by the antibody. e–i Densitometry of the obtained bands under reducing conditions. Note the specificity of the antibodies used as they lack any signal in the respective ko strain. ^# Significant difference (p < 0.05) between uninduced and nephrotic mice of the same genotype (unpaired t-test or Wilcoxon test). ^* Significant difference (p < 0.05) between genotypes and Nphs2^Δipod (ANOVA with Dunnetts multiple comparison test or Kruskal-Wallis with Dunn’s multiple comparison test)

Fig. 4

Tissue expression of C3 in Nphs2^Δipod, Nphs2^Δipod*Cfb^-/-, Nphs2^Δipod*Cfd^-/- and Nphs2^Δipod*C3^-/- mice before and after induction of nephrotic syndrome. Representative staining of kidney sections stained for C3 at 20- (upper panel, scale 20µm) and 63-fold (lower panel, scale 5µm) magnification. The antibody was the same as used for Western blot. No signal is obtained in Nphs2^Δipod*C3^-/- mice. * marks the C3-positive tubular protein casts

Fig. 5

Amiloride-sensitive natriuresis in Nphs2^Δipod, Nphs2^Δipod*Cfb^-/-, Nphs2^Δipod*Cfd^-/- and Nphs2^Δipod*C3^-/- mice before and after induction of nephrotic syndrome. a Natriuretic response to the acute administration of the ENaC inhibitor amiloride (A, 10 µg/g) or vehicle injection (V, injectable water, 5µl/g). b Fold-increase of the natriuretic response after amiloride administration before (uninduced, U) and after (nephrotic, N) induction of nephrotic syndrome. ^# Significant difference (p < 0.05) between uninduced and nephrotic mice of the same genotype (unpaired t-test or Wilcoxon test). ^* Significant difference (p < 0.05) between genotypes and Nphs2^Δipod (ANOVA with Dunnetts multiple comparison test or Kruskal-Wallis with Dunn’s multiple comparison test)

Fig. 6

Sodium retention in Nphs2^Δipod, Nphs2^Δipod*Cfb^-/-(FB^-/-), Nphs2^Δipod*Cfd^-/- (FD^-/-) and Nphs2^Δipod*C3^-/-(C3^-/-) mice after induction of nephrotic syndrome. Course of sodium intake a–c, urinary sodium excretion in spot urine samples e–g and body weight i–k after induction of nephrotic syndrome. d arithmetic mean of sodium intake. h, l minimal urinary sodium excretion h and maximal body weight gain l, both reflecting maximal ENaC activation. ^# Significant difference (p < 0.05) between uninduced and nephrotic mice of the same genotype (unpaired t-test or Wilcoxon test) ^* Significant difference (p < 0.05) between genotypes and Nphs2^Δipod (ANOVA with Dunnetts multiple comparison test or Kruskal-Wallis with Dunn’s multiple comparison test)

Fig. 7

Tissue expression of γ-ENaC in Nphs2^Δipod, Nphs2^Δipod*Cfb^-/-, Nphs2^Δipod*Cfd^-/- and Nphs2^Δipod*C3^-/- mice before and after induction of nephrotic syndrome. Representative staining of kidney sections stained for γ-ENaC at 20- (upper panel, scale 20µm) and 63-fold (lower panel, scale 5µm) magnification. The antibody was the same as used for Western blot

Fig. 8

Expression of ENaC subunits and proteolytic processing in kidney lysates from Nphs2^Δipod, Nphs2^Δipod*Cfb^-/-(FB^-/-), Nphs2^Δipod*Cfd^-/- (FD^-/-) and Nphs2^Δipod*C3^-/-(C3^-/-) mice before and after induction of nephrotic syndrome. a Representative Western blots showing the expression of α-, β- and γ-ENaC in a plasma membrane preparation of kidney cortex lysates before (healthy) and after induction (nephrotic) of nephrotic syndrome. Note that the samples were deglycosylated before analyzing expression of γ-ENaC and its cleavage products [25]. The white line is only for optical discrimination, it is one blot each, no vertical cutting. b–g Densitometry of the obtained bands normalized for total protein content of each lane. ^# Significant difference (p < 0.05) between uninduced and nephrotic mice of the same genotype (unpaired t-test or Wilcoxon test) ^* Significant difference (p < 0.05) between genotypes and Nphs2^Δipod (ANOVA with Dunnetts multiple comparison test or Kruskal-Wallis with Dunn’s multiple comparison test)