Autosomal-dominant (ADPKD) is the most common life-threatening genetic disease. We have recently found that polycystin-1 (PC1), the protein affected in ADPKD, functions in the regulation of STAT6 activity by ciliary mechanotransduction in renal epithelial cells. PC1 undergoes flow-dependent proteolytic cleavage which releases its cytoplasmic tail from the membrane, followed by nuclear translocation. The PC1 tail binds to STAT6 and the transcriptional co-activator P100, and stimulates STAT6-dependent gene expression. STAT6 translocates from primary cilia to nuclei upon cessation of fluid flow. The nuclear PC1 tail is highly expressed in cyst-lining epithelial cells in ADPKD. Expression of the PC1 tail stimulates proliferation in MDCK cells and results in renal cyst-formation in zebrafish embryos. Furthermore, MDCK cells respond to interleukin-4 (IL4) and IL13 similar to immune cells in that they activate STAT6, and up-regulate the expression if IL4/13 receptor chains. These results strongly suggest that PC1 functions to silence STAT6 activity in the normal healthy kidney. Cessation of lumenal fluid flow, e.g. due to renal injury, triggers PC1 cleavage, STAT6 activation and a proliferative response. We hypothesize that lack of functional PC1 in ADPKD leads to constitutive STAT6 activity and an aberrant proliferative """"""""repair"""""""" response leading to cyst growth. Our preliminary results show that a clinically approved drug, known to inhibit STAT6, strongly inhibits renal growth and preserves renal function in a polycystic mouse model. We now propose to study in detail the mechanism of the regulation of STAT6 activity by PC1 and to test our hypotheses regarding the role of this novel signaling pathway in renal injury repair and ADPKD.
In Aim 1, we will characterize the regulation of STAT6 in MDCK cells expressing a STAT6-responsive GFP reporter. The effects of the state of differentiation, PC1 tail expression, apical fluid flow and IL4/13 will be investigated. We will test whether STAT6 is activated in a scratch-wounding model. We will investigate the expression and localization/secretion of IL4/13 and IL4/13 receptor chains in response to STAT6 activation. Analysis of ADPKD tissue and polycystic mouse models will reveal whether IL4/13 receptors are up-regulated in cysts and whether they secrete IL4/13 into the lumen. Finally, we will test our hypothesis that the IL4/13/STAT6/PC1 pathway is activated in a mouse model of renal ischemia/reperfusion injury.
In Aim 2, we will test whether crossing of STAT6 null mice with polycystic mouse models will result in suppression of renal cystic disease. Furthermore, we will investigate the amelioration of renal cystic disease by treatment with the STAT6 inhibitor and delineate the mechanism of action.
In Aim 3, we will generate a transgenic mouse line over-expressing the soluble PC1 tail in a kidney-specific and doxycycline-inducible manner. Based on our results in MDCK cells and zebrafish, we anticipate that these animals will develop renal cystic disease and will mimic human ADPKD most closely mechanistically. Project Narrative ADPKD is the most common life-threatening genetic disease and affects over 600,000 patients in the US. Due to the need for kidney transplantation or life-long dialysis in most patients, the personal burden on patients and the burden on the health care system are enormous. Currently, there is no available treatment to prevent or slow the disease onset. Our work has led to the identification of two signaling mechanisms that are aberrantly regulated in ADPKD and involve the kinase mTOR and the transcription factor STAT6, respectively. We could validate both of these pathways as feasible drug targets. Based on animal experiments, two clinically approved drugs (the mTOR inhibitor rapamycin and the STAT6 inhibitor leflunomide) appear to be highly promising. In the case of mTOR inhibitors, this work has already led to at least three clinical trials that are in early stages. The present proposal is aimed at understanding the molecular mechanisms underlying one of the two pathways (STAT6) that we have discovered and to further test the feasibility of targeting this pathway with an existing, already FDA-approved drug. Therefore, this proposal is designed to yield new mechanistic insights and also to provide information that will be directly relevant for the guidance of future clinical trials. Leflunomide is currently used for long-term treatment of rheumatoid arthritis. Our results using animal models make us cautiously optimistic that leflunomide - or related compounds - have promise to become the first available treatment to slow the disease progression in ADPKD. Even a delay of the onset of end-stage renal disease by a number of years would be a tremendous advance and would dramatically improve the quality of life of a large number of patients. In addition to the immediate clinical relevance, our proposed work will also shed light on our unexpected discovery that renal tubular epithelial cells appear to possess a STAT6-dependent signaling pathway similar to the one previously described in immune cells. Compared to the wealth of knowledge in the immune system, little is known about the function of this pathway in renal tubular epithelial cells, or anywhere else outside of the immune system, despite the fact that STAT6 is widely expressed. We hypothesize that this signaling pathway regulates epithelial, proliferative repair responses after renal injury. If correct, then this would impact on most forms of acute and chronic renal damage.
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