Characterisation of the roles of the inositol polyphosphate 5-phosphatase, INPP5E, in polycystic kidney disease pathogenesis

2017-02-21T22:56:05Z (GMT) by Hakim, Sandra
INPP5E is an inositol polyphosphate 5-phosphatase that dephosphorylates PI(3,4,5)P₃ and PI(4,5)P₂ at the D-5 position of the inositol ring. INPP5E mutations have been identified in the human ciliopathy syndromes, Joubert syndrome and related disorders (JSRD) and mental retardation, truncal obesity, retinal dystrophy and micropenis (MORM). Ciliopathy syndromes are autosomal recessive congenital disorders characterised by multi-organ dysfunction that arise due to defective cilia function. Germline inactivation of Inpp5e in mice is embryonically lethal and produces severe developmental defects that resemble human ciliopathy syndromes, including polydactyly, exencephaly and polycystic kidneys. Although the features of Inpp5e-null mice resemble human ciliopathy syndromes, whether cilia defects contribute to the pathogenesis of all of the phenotypes in these mice is not well established. Polycystic kidney disease (PKD) results from the development of multiple large fluid filled kidney cysts arising from the tubular epithelial cells that comprise the nephrons. PKD disrupts normal kidney architecture and function and represents a significant clinical burden, resulting in end stage renal failure (ESRF) requiring costly interventions such as peritoneal dialysis and kidney transplantation. There are multiple genetic causes of PKD that have been identified in humans, although the disease mechanisms underlying PKD in many cases are poorly understood. The molecular basis of PKD arising from Inpp5e inactivation in mice is not well characterised and forms the basis of the studies for this PhD. This thesis reports the generation of Inpp5e-null mice (Inpp5e-/-) which display a ciliopathy phenotype with polycystic kidneys, consistent with that reported previously by others, although with additional features including oedema and lung hypoplasia. The Inpp5e-/- embryos reported in this thesis display PKD that is characterised by cilia depletion, however, further characterisation of Inpp5e-/- embryos was limited due to lethality between E15.5 - E18.5. In order to examine PKD postnatally and to specifically investigate the roles Inpp5e plays in renal epithelium, renal epithelial cell-restricted inactivation of Inpp5e was carried out using Ksp1.3-Cre transgenic mouse crosses (Inpp5efl/fl;Ksp-Cre). Inpp5efl/fl;Ksp-Cre mice developed severe and aggressive PKD postnatally that was also characterised by cilia depletion. By crossing Inpp5efl/fl;Ksp-Cre mice with a transgenic Cre reporter strain, lacZ/EGFP (Z/EG), analysis of pre-cystic tubules with Inpp5e inactivation was enabled, and revealed that cilia loss did not occur prior to cyst development. Therefore, loss of cilia may represent a late event in cyst development and may not contribute to cyst development in Inpp5efl/fl;Ksp-Cre mice. INPP5E is a potent PI(3,4,5)P₃ 5-phosphatase that acts downstream of phosphoinositide 3-kinase (PI3K) to inhibit Akt phosphorylation and downstream mammalian target of rapamycin complex 1 (mTORC1) signalling in cultured cells, although whether it performs this role in vivo is not reported. mTORC1 signalling is amplified in other models of PKD and may contribute to PKD pathogenesis. Strikingly, immunohistochemical and immunoblot analysis revealed that activation of Akt/mTORC1 signalling occurs in Inpp5efl/fl;Ksp-Cre renal epithelium, and furthermore, progressively increases with cyst development. Cilia depletion is also proposed to contribute to mTORC1 activation. To examine the contribution of cilia depletion to mTORC1 activation in cystic Inpp5efl/fl;Ksp-Cre mouse kidneys, two approaches were employed. Adenosine monophosphate (AMP) – activated protein kinase (APMK) is phosphorylated and activated to repress mTORC1 in response to flow induced cilia bending, and this mechanism is disrupted in cultured cells with defective cilia formation. Immunoblot analysis showed that there was no change in AMPK phosphorylation in cystic Inpp5efl/fl;Ksp-Cre mouse kidneys. Additionally, immunohistochemical analysis of mTORC1 activation and cilia number in cystic versus non-cystic Inpp5efl/fl;Ksp-Cre;Z/EG mouse kidneys was performed, revealing that cells harbouring mTORC1 activation do not display increased cilia loss. Collectively, these studies suggest that cilia depletion does not drive activation of mTORC1 in renal epithelial cells harbouring Inpp5e depletion. Finally, treatment of Inpp5efl/fl;Ksp-Cre mice from P8-P20 with an allosteric mTORC1 inhibitor attenuated cyst development and improved renal function. Therefore, Inpp5e maintains basal PI3K/Akt/mTORC1 activity in renal epithelial cells, and inactivation of Inpp5e leads to aberrant hyperactivation of this signalling axis to generate PKD. This thesis identifies INPP5E as a critical repressor of PI3K/Akt/mTORC1 signalling in renal epithelial cells, a function required for the maintenance of normal kidney function and morphology to prevent development of PKD. In addition, these studies highlight that PI3K/Akt signalling represents an input that activates mTORC1 in PKD pathogenesis. These findings may have implications for future studies investigating novel therapeutic interventions for PKD.