Involvement of ceramide biosynthesis in increased extracellular vesicle release in Pkd1 knock out cells

Abstract

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is an inherited disorder characterized by the development of renal cysts, which frequently leads to renal failure. Hypertension and other cardiovascular symptoms contribute to the high morbidity and mortality of the disease. ADPKD is caused by mutations in the PKD1 gene or, less frequently, in the PKD2 gene. The disease onset and progression are highly variable between patients, whereby the underlying mechanisms are not fully elucidated. Recently, a role of extracellular vesicles (EVs) in the progression of ADPKD has been postulated. However, the mechanisms stimulating EV release in ADPKD have not been addressed and the participation of the distal nephron segments is still uninvestigated. Here, we studied the effect of Pkd1 deficiency on EV release in wild type and Pkd1^-/- mDCT15 and mIMCD3 cells as models of the distal convoluted tubule (DCT) and inner medullary collecting duct (IMCD), respectively. By using nanoparticle tracking analysis, we observed a significant increase in EV release in Pkd1^-/- mDCT15 and mIMCD3 cells, with respect to the wild type cells. The molecular mechanisms leading to the changes in EV release were further investigated in mDCT15 cells through RNA sequencing and qPCR studies. Specifically, we assessed the relevance of purinergic signaling and ceramide biosynthesis enzymes. Pkd1^-/- mDCT15 cells showed a clear upregulation of P2rx7 expression compared to wild type cells. Depletion of extracellular ATP by apyrase (ecto-nucleotidase) inhibited EV release only in wild type cells, suggesting an exacerbated signaling of the extracellular ATP/P2X7 pathway in Pkd1^-/- cells. In addition, we identified a significant up-regulation of the ceramide biosynthesis enzymes CerS6 and Smpd3 in Pkd1^-/- cells. Altogether, our findings suggest the involvement of the DCT in the EV-mediated ADPKD progression and points to the induction of ceramide biosynthesis as an underlying molecular mechanism. Further studies should be performed to investigate whether CerS6 and Smpd3 can be used as biomarkers of ADPKD onset, progression or severity.

Keywords: ADPKD; Autosomal Dominant Polycistic Kidney Disease; exosomes; extracellular ATP; extracellular vesicles; purinergic signaling.

Figure 1

Characterization of EVs obtained from culture medium of mDCT15 and mIMCD3 cells. EVs from culture medium of mDCT15 wild type (A) and Pkd1^-/- (B) cells and from mIMCD3 wild type (C) and Pkd1^-/- (D) cells were characterized by NTA and transmission electron microscopy (E). Scale bar: 50 nm.

Figure 2

Increase in EV release in Pkd1^-/- models in vitro and in vivo. The concentration of EVs was analyzed by NTA in preparations obtained by ultracentrifugation from mDCT15 (A) and mIMCD3 cells (B) and by precipitation with the Total Exosome Isolation Reagent (from cell culture medium) from mDCT15 (C) and mIMCD3 cells (D). The particle concentration was normalized to the total cell count. Data are shown as mean ± SEM (n = 3). Urinary EVs in samples from Pkd1^-/- and wild type (WT) mice were isolated with the Total Exosome Isolation Reagent (from urine) and analyzed by NTA. Data are shown as mean ± SEM (n = 8 for WT mice and n = 5 for Pkd1^-/- mice) (E). * indicates statistical difference from WT (p < 0.05).

Figure 3

Role of the extracellular ATP and purinergic signaling in EV release in wild type and Pkd1^-/- mDCT15 cells. Expression of P2rx7 at the mRNA level was analyzed in wild type and Pkd1^-/- mDCT15 cells and normalized to the expression of Gapdh, used as housekeeping gene (A). Data (mean ± SEM) are expressed as fold change of the gene expression in wild type (WT) cells. * indicates statistical difference from WT (p < 0.05, n = 3). Extracellular ATP levels were analyzed in WT (B) and Pkd1^-/- mDCT15 cells (C) exposed to vehicle (control) or apyrase (6 U/ml, 24 hours). Data (mean ± SEM) are expressed as fold change of extracellular ATP levels in control cells. EV release was quantified in suspensions obtained from wild type (D) and Pkd1^-/^- mDCT15 cells (E) treated with vehicle (control) or with 6 U/ml apyrase by NTA. Data are shown as mean ± SEM. * indicates statistical difference from control (p < 0.05, n = 4).

Figure 4

Principal component analysis of the RNAseq data transcripts of wild type and Pkd1^-/- mDCT15 cells. The plot depicts a clear separation between the Pkd1^-/- mDCT15 cell samples (red circles) and the wild type mDCT15 cell samples (blue circles) (n = 3/group).

Figure 5

Effect of Pkd1 knockout on the expression of ceramide biosynthesis enzymes. The expression of the CerS6 (A) and Smpd3 (B) in wild type (WT) and Pkd1^-/- mDCT15 cells was analyzed by qPCR and normalized to the expression of Gapdh, used as housekeeping gene. Data (mean ± SEM) are expressed as fold change of the expression in the wild type cells. * indicates statistical difference from WT (p < 0.05, n = 3).

Figure 6

Proposed role of polycystin-1 deficiency in EV release in the distal convoluted tubule. Loss of Pkd1^-/- in mDCT15 cells is associated with an increase in the EV release and an increase in the expression of P2xr7, Smpd3 and CerS6, genes encoding for proteins involved in ATP signaling and ceramide synthesis, respectively. These findings suggest a link between the loss of Pkd1 expression, ceramide biosynthesis and EV release. The latter can stimulate cysts development in the surrounding tissue contributing to ADPKD disease progression. ADPKD, Autosomal dominant polycystic kidney disease; CD, collecting duct; CerS6, Ceramide Synthase 6; DCT, distal convoluted tubule; EV, extracellular vesicles; LOH, Loop of Henle; PC1, polycystin 1; PT, proximal tubule; P2rx7, Purinergic Receptor P2X 7 (gene); P2X7, Purinergic Receptor P2X 7 (protein); Smpd3, Sphingomyelin Phosphodiesterase 3.