Investigating the mechanism of renal cystogenesis in tuberous sclerosis and polycystic kidney disease

Tuberous sclerosis (TSC) is an autosomal dominant disorder caused by germline mutations in either TSC1 or TSC2 and characterised by the development of benign hamartomatous growths in multiple organs and tissues. Clinical trials are underway for the treatment of TSC-associated tumours using mammalian target of rapamycin (mTOR) inhibitors. Here, we show that many of the earliest renal lesions from Tsc1+/ and Tsc2+/ mice do not exhibit mTOR activation, suggesting that pharmacological targeting of an alternative pathway may be necessary to prevent tumour formation. Patients with TSC often develop renal cysts and those with inherited co- deletions of the autosomal dominant polycystic kidney disease (ADPKD) 1 gene (PKD1) develop severe, early onset, polycystic kidneys. Using mouse models, we crossed Tsc1+, and Tsc2+I mice with Pkd1+/ mice to generate double heterozygotes. We found that Tsc1+lPkd1+, and Tsc2+l Pkd1+, mice had significantly more renal lesions than their corresponding single heterozygote littermates indicating a genetic interaction between Tsd and Tsc2 with Pkd1. In agreement with our findings from Tsc1+/ and Tsc2+/ mice, we found that a large proportion of cysts from Tsc1+l Pkd1+, and Tsc2+l Pkd1+, mice failed to stain for pS6, suggesting that initiation of renal cystogenesis in these animals may occur independently of mTOR activation. We analysed primary cilia in phenotypically normal renal tubule epithelial cells by scanning electron microscopy (SEM) and found that those from Tsc1+, and Tsc2+I mice were significantly shorter than those from wild-type littermates (2.122pm and 2.016pm vs. 2.233pm, respectively, P<0.001). Primary cilia from epithelial cells lining renal cysts of Tsc1+' and Tsc2+I' mice were consistently longer (5.157pm and 5.091pm respectively). Interestingly, we found that Pkd1- deficiency coupled with either Tsd or 7sc2-deficiency altered the length of the primary cilia from both normal renal tubule cells (restored to 'wild-type' length) and epithelial cells lining cysts (Tsc1+tPkd1+, Mean 3.38pm and Tsc2+,Pkd1+l Mean 3.09pm). These novel data demonstrate that the Tsc and Pkd1 gene products help regulate primary cilia length which may prevent renal cystogenesis. Consistent with the observation that primary cilia modulate the planar cell polarity (POP) pathway, we found that many dividing pre-cystic renal tubule epithelial cells from Tsc1+/ , Tsc2+/ and Pkd1+/ mice were highly misorientated along the tubule axis. This could potentially lead to tubule dilation and subsequent cyst formation. We therefore propose that defects in cell polarity underlie both TSC and ADPKD-associated renal cystic disease and targeting of this pathway may be of key therapeutic benefit