Autophagy is a process that takes place in all eukaryotic cells that keeps cells alive under stressful conditions. In autophagy there is the sequestration of damaged organelles into double-membraned autophagosomes that subsequently fuse with lysosomes where their cargoes are delivered for degradation and recycling. In the healthy kidney, autophagy plays an important role in the homeostasis and viability of renal tubular epithelial cells. There is indirect evidence that PKD is a case of suppressed autophagy: 1) Many of the agents that protect against PKD are autophagy inducers. 2) Preliminary/published data demonstrate decreased autophagic flux in Pkd1 -/- mouse kidneys and PC1 -/- cells. 3) There is cross-talk between autophagy and apoptosis, a key process in cyst growth. The overall hypothesis is that autophagic flux (autophagosome-lysosome fusion and degradation) is decreased early in the polycystic kidney and that interventions that block autophagy will worsen PKD and conversely autophagy inducers like caloric restriction, 2-deoxy-glucose or trehalose will increase autophagy, decrease apoptosis and improve PKD in an autophagy?dependent manner. The presence of autophagy (autophagic flux, presence of autophagosomes and autolysosomes), mitophagy (autophagosomes containing mitochondria) and the effect of autophagy inhibition (using genetic techniques), autophagy induction (using autophagy-inducers that are known to protect against PKD) on cyst growth and apoptosis will be determined in Pkd1 -/- mice and PKD1 -/- cells. An improved understanding of the mechanism by which induction of autophagy can prevent PKD may lead to the identification of new targets for both diagnosis and therapy of PKD.
In Aim 1, the effect of autophagy inhibition or induction in will be tested Pkd1 -/- mouse kidneys. Autophagic flux and apoptosis will be determined during a time course in Pkd1 -/- mouse kidneys. It will be determined whether autophagy knockout in kidney- specific double knockout ATG7, Pkd1 -/- mice worsens apoptosis, proliferation, PKD and kidney function. The effect of autophagy inducers (caloric restriction, 2-deoxy-glucose or trehalose) on autophagy, apoptosis and proliferation, PKD and renal function will be determined in Pkd1 -/- mice and ATG7, Pkd1 double knockout mice. The effect of a novel autophagy inducer (trehalose) on PKD will be determined.
In Aim 2, pathways of autophagy in PKD1 -/- tubular cells will be determined. These experiments will use more sophisticated techniques of detection of autophagic flux and mitophagy and pharmacological and genetic techniques of autophagy induction and inhibition that cannot be performed in vivo. The effect of autophagy induction by mechanistically distinct methods (glucose deprivation, trehalose, Beclin1 peptide) and autophagy inhibition (3 methyladenine, chloroquine, expression of dominant negative ATG4) on autophagy pathways and flux, mitophagy, apoptosis, proliferation, cyst size/number will be determined. Our results will provide a definitive test of the hypothesis that PKD represents a case of decreased autophagy. Our results will offer mechanistic insights into the mechanism of action of caloric restriction or chemical caloric restriction that protect against PKD and that are also autophagy inducers. Our results will provide therapeutic insights into the potential future use of autophagy inducers in PKD.
Autosomal dominant polycystic kidney disease (ADPKD) is the most common life threatening hereditary disease in the USA. As complications of ADPKD like hypertension, chronic kidney disease, need for kidney transplantation are common and the number of veterans with ADPKD is large, the health care burden of ADPKD patients for the Department of Veteran's Affairs is large. The overall hypothesis is that autophagy, that plays an important role in the homeostasis and viability of normal tubular epithelial cells, is decreased early in the polycystic kidney and that decreased autophagy drives apoptosis and proliferation of tubular cells to form cysts. An improved understanding of the mechanism by which induction of autophagy can prevent PKD may lead to the identification of new targets for both diagnosis and therapy of PKD. A therapy that slows cyst formation and growth may potentially reduce the huge healthcare burden that ADPKD patients present to the Department of Veteran's Affairs.
