Targeted rescue of a polycystic kidney disease mutation by lysosomal inhibition

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic cause of end-stage renal disease. The molecular pathogenesis of ADPKD is not completely known, and there is no approved therapy. To date, there is limited knowledge concerning the molecular consequences of specific disease-causing mutations. Here we show that the ADPKD missense variant TRPP2(D511V) greatly reduces TRPP2 protein stability, and that TRPP2(D511V) function can be rescued in vivo by small molecules targeting the TRPP2 degradation pathway. Expression of the TRPP2(D511V) protein was significantly reduced compared to wild-type TRPP2. Inhibition of lysosomal degradation of TRPP2(D511V) by the US Food and Drug Administration (FDA)-approved drug chloroquine strongly increased TRPP2 protein levels in vitro. The validation of these results in vivo requires appropriate animal models. However, there are currently no mouse models harboring human PKD2 missense mutations, and screening for chemical rescue of patient mutations in rodent models is time-consuming and expensive. Therefore, we developed a Drosophila melanogaster model expressing the ortholog of TRPP2(D511V) to test chemical rescue of mutant TRPP2 in vivo. Notably, chloroquine was sufficient to improve the phenotype of flies expressing mutant TRPP2. Thus, this proof-of-concept study highlights the potential of directed therapeutic approaches for ADPKD, and provides a rapid-throughput experimental model to screen PKD2 patient mutations and small molecules in vivo.

Keywords: PKD2; chloroquine; lysosome; polycystic kidney disease.

FIGURE 1. The ADPKD missense variant TRPP2^D511V reduces TRPP2 protein abundance

a) The human TRPP2^D511V mutation is localized in the highly conserved third transmembrane segment (TRPP2^506–527) of TRPP2. b) Western blot analysis of wild-type TRPP2 shows two distinct bands. Compared to wild-type, TRPP2^D511V shows reduced protein levels in HeLa cells. c) This effect is not cell-type-specific, because a similar reduction is observed in HEK293T cells. d) Group data from b and c show a significant reduction of TRPP2^D511V protein levels by 85% compared to wild-type TRPP2 in HeLa cells (n=5; p=2·10⁻⁶), and by 63% in HEK293T cells (n=5; p=3·10⁻⁴).

FIGURE 2. TRPP2^D511V is temperature-sensitive

a) mRNA of transiently transfected HeLa cells was isolated. TRPP2 wild-type and TRPP2^D511V mRNA abundance is similar as assessed by qPCR. b) Comparison of TRPP2 wild-type and TRPP2^D511V expression at 27°C in HeLa cells by Western blot. c) Group data from b.

FIGURE 3. TRPP2^D511V is degraded in lysosomes

a) Western blot analysis shows that TRPP2^D511V is more abundant in cells treated with 200 µM chloroquine (CQ) compared with control cells. b) MG-132 (210 µM) does not affect TRPP2^D511V protein levels. c) Group data from a and b show that chloroquine significantly increases abundance of TRPP2^D511V (mean=1,461%; n=4; p=0.003), whereas MG132 shows no effect (mean=108%; n=4).

FIGURE 4. Chloroquine rescues mutant D. melanogaster TRPP2 (Amo) function in vivo

a) Wild-type (w¹¹¹⁸) flies have a large number of progeny (mean=251; n=8), while amo mutant males (amo^−/−) are infertile (mean number of progeny=2.4; n=8; p=9·10⁻¹¹). A third chromosomal amo transgene in the amo^−/− mutant background can rescue the fertility phenotype (amo^−/−;amo) (mean number of progeny=236; n=8). b) Introduction of an amo^D627V transgene on the third chromosome fails to rescue male infertility of amo^−/− males. Feeding of 3 mM chloroquine to amo^−/−;amo^D627V males, however, significantly increases fertility compared to untreated flies of the same genotype (mean number of progeny=20; n=10; p=0.007). Lower concentrations of chloroquine (0.2 mM) showed no effect, whereas dietary chloroquine at or above 10 mM caused significant toxicity. Please note that the amo^D627V transgene causes a small but significant increase in male fertility compared to amo^−/− males suggesting residual function of the amo^D627V variant (mean number of progeny: 2.4 versus 7.2, respectively; n=10; p=0.002). In contrast, amo^−/− males fed chloroquine have progeny similar to untreated controls (mean number of progeny=1.3; n=10). c)–e) Localization of Amo in mature sperm in wild-type (w¹¹¹⁸), amo^−/−;amo^D627V, and chloroquine-treated amo^−/−;amo^D627V flies (anti-Amo: green; scale bar 5 µm; n=8). Chloroquine treatment results in an increase of flagellar Amo expression, without enrichment at the tip of the sperm tail. Previous studies have shown that Amo localization in amo^−/−;amo flies is similar to wild-type. f) Western blot of Amo protein from male flies of the indicated genotypes. Amo was immuno-precipitated from wild-type (w¹¹¹⁸), amo^−/−;amo^D627V, and chloroquine-treated flies (3 mM). Amo expression was reduced in amo^−/−;amo^D627V flies compared to wild-type (w¹¹¹⁸)(−76.1%; standard deviation = 12.8; n=2). Chloroquine-treatment of amo^−/−;amo^D627V flies increased Amo protein levels (mean=+40.3%; standard deviation=16.7; n=2). Equal numbers of flies were used for all experimental conditions. Tubulin served as a loading control.