Abstract
ABSTRACTIn autosomal dominant polycystic kidney disease (ADPKD), cyst inflation and continuous enlargement are associated with marked transepithelial ion and fluid secretion into the cyst lumen via cystic fibrosis transmembrane conductance regulator (CFTR). Indeed, the inhibition or degradation of CFTR prevents the fluid accumulation within cysts. The in vivo mechanisms by which the lack of Polycystin-2 leads to CFTR stimulation are an outstanding challenge in ADPKD research and may bring important biomarkers for the disease. However, hampering their study, the available ADPKD in vitro cellular models lack the three-dimensional architecture of renal cysts and the ADPKD mouse models offer limited access for live-imaging experiments in embryonic kidneys. Here, we tested the zebrafish Kupffer's vesicle (KV) as an alternative model-organ. KV is a fluid-filled vesicular organ, lined by epithelial cells that express both CFTR and Polycystin-2 endogenously, being each of them easily knocked-down. Our data on the intracellular distribution of Polycystin-2 support its involvement in the KV fluid-flow induced Ca2+-signalling. Mirroring kidney cysts, the KV lumen inflation is dependent on CFTR activity and, as we clearly show, the knockdown of Polycystin-2 results in larger KV lumens through overstimulation of CFTR. In conclusion, we propose the zebrafish KV as a model organ to study the renal cyst inflation. Favouring its use, KV volume can be easily determined by in vivo imaging offering a live readout for screening compounds and genes that may prevent cyst enlargement through CFTR inhibition.
Highlights
The major clinical manifestation of autosomal dominant polycystic kidney disease (ADPKD) is the development of massive fluid-filled kidney cysts that destroy the renal parenchyma
While not affecting cell proliferation which is a marked difference to the renal cyst formation, we demonstrate that pkd2-knockdown causes a significant increase in the CFTR-mediated fluid-secretion into the Kupffer’s vesicle (KV) lumen mirroring the cyst inflation process
Numerous studies using cellular (Li et al, 2004, 2012; Reif et al, 2011; Yuajit et al, 2013) or mouse models for ADPKD (Blazer-Yost et al, 2010; Yang et al, 2008; Yuajit et al, 2014) have proved the key role of CFTR in the cyst inflation process. This can be slowed down by the use of CFTR-interfering molecules, such as: steviol and its derivatives that inhibit CFTR and promote its proteasomemediated degradation (Yuajit et al, 2013, 2014); CFTRinh-172 (Li et al, 2004), the CFTR blocker used in our experiments; and pioglitazone, an agonist of the peroxisome proliferator-activated receptor γ which reduces the CFTR mRNA levels (Blazer-Yost et al, 2010)
Summary
The major clinical manifestation of autosomal dominant polycystic kidney disease (ADPKD) is the development of massive fluid-filled kidney cysts that destroy the renal parenchyma. This can be explained by the maternal contribution of pkd mRNA previously detected in the early stages of the cup mutants’ embryonic life (Schottenfeld et al, 2007) Corroborating this data, our whole-mount immunostaining experiments showed that, in a less extent than in their normal tail siblings (Fig. S1D-G), at 36 hpf Polycystin-2 is still present near the basal body and along the pronephric cilia from curly up tail cup−/− mutants (Fig. S1H-J). It was only at the 3 s.s. that the rate of inflation of the KVs from pkd2-morphants increased and became significantly different from WT embryos (Fig. 4A,D,H) This suggests that the overstimulation of CFTR upon the knockdown of Polycystin-2 occurs early in the KV morphogenesis, but not immediately after its formation
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