Autosomal dominant polycystic kidney disease is caused by mutations in the Pkd1 (85%) or Pkd2 (15%) genes. PKD morbidity is associated with primarily renal, but also liver, pancreatic, and vascular abnormalities such as intracranial aneurysms and aortic artery weakness. There are no effective therapies at present. Primary cilia house important regulators of cell growth and division. Defects in genes regulating cilia formation and function result in serious lifelong and adult-onset diseases. Here we focus on polycystins, the genes mutant in polycystic kidney diseases that are among the most common inherited disorders. The organizing principal of this proposal is that ciliary polycystin channels modify ciliary calcium levels that in turn modify ciliary signaling pathways regulating growth and cell division, and ultimately cyst formation. We measured ciliary calcium concentration and identified ion channel currents (Icilium) in single cilia. We found that calcium changes are largel confined to the cilium. We directly measured primary cilia currents under whole-cilium voltage clamp and found that the basal current was mediated by PC1-L1/PC2-L1 heteromers. These channels rapidly and dramatically alter cilia calcium levels. We were able to conduct these experiments by generating fluorescent indicators for calcium measurements that specifically target cilia. We showed that ciliary calcium levels regulate Smoothened-activated Gli1 transcription and ciliary tip accumulation of Gli2. We are crossing Pkd-mutant mice with these indicator mice. We focus on the function of the polycystin proteins themselves rather than downstream signal transduction pathways. Here we propose to measure ion channel currents in primary cilia from freshly isolated kidney collecting duct tubules, and tubular cells in intact tubules. The overall goal is to determine whether PC1 and PC2, the proteins mutant in polycystic kidney disease, also function as an ion channel complex in cilia. Our hypothesis is that PC-L1 proteins regulate cilia calcium to high levels and as cells mature and differentiate, they express PC proteins and reduce cilia calcium. In the experiments proposed, we will first identify the type of channel, PC or PC-L1, that is present in embryonic, neonatal, young adult, and mature adult mice. This requires careful evaluation of PC and PC-L1 biophysical and pharmacological properties. We will then determine and compare the domains of PC and PC-L1 that enable them to regulate calcium in cilia in distinct ways. Finally, we will determine how PC1, the more commonly affected protein in polycystic kidney disease, regulates the pore of the PC2-forming channel.

Public Health Relevance

Autosomal dominant polycystic kidney disease (ADPKD) is an inherited disease in which a person has one mutated copy and one normal copy of either Pkd1 or Pkd2 genes (PC1 or PC2 proteins). Gradual growth of existing cysts and new cyst formation over decades results in a decline in renal function, with 50% of patients developing end stage renal disease (ESRD) by age 55. The disease can begin before birth or in later life. Currently there are no effective medicines. Most patients require dialysis or renal transplantation, and ADPKD accounts for approximately 4-8% of all patients with ESRD. Cysts can also form in liver, pancreas, seminal vesicles and arachnoid membrane. Many patients have cardiovascular abnormalities such as aortic root dilation, arterial aneurysms, heart valve abnormalities and intracranial aneurysms. Although the genes associated with ADPKD are known, the underlying mechanisms for the disease are not. In fact, we do not currently understand the normal `job' of either PC1 or PC2 proteins. Recent evidence suggests that polycystic kidney diseases are associated with the malfunction of primary cilia (ciliopathies). Other kidney cystic diseases are caused by mutations in known ciliary proteins, including. nephronophthisis. The primary cilium is a single antenna-like structure that extends from the apical side of polarized cells, such as cells of the kidney medulla collecting ducts. While it is known that PC1 and PC2 localize to the primary cilia, we do not understand what they do in this organelle. We recently measured polycystin-mediated ionic currents in single cilia from immortalized cell lines and embryonic fibroblasts. Although we had assumed they would be from PC1 and PC2 proteins, they were not. Instead, relatives of these proteins, called PC1-L1 and PC2-L1, mediated current flow. Among several observations, we found that primary cilia maintain a high resting calcium level (? 600 nM). We also observed that high Ca2+ in the cilia compartment inactivates PC-L1, which suggests that Ca2+ flowing through the channel will turn-off the current when Ca2+ accumulates. Here we propose to measure ion channel currents in primary cilia from freshly isolated kidney collecting duct tubules, and tubular cells in intact tubules. We use mice since many mouse genetic model mimic the human disease. The overall goal is to determine whether PC1 and PC2, the proteins mutant in polycystic kidney disease, also function as an ion channel complex in cilia. Our hypothesis is that PC-L1 proteins regulate cilia calcium to high levels and as cells mature and differentiate, they express PC proteins and reduce cilia calcium. This is important because the cilium is a sequestering compartment for developmental pathways, such as the primarily growth and cell division signal transduction system, Sonic hedgehog. In the experiments proposed, we will first identify the type of channel, PC or PC-L1, that is present in embryonic, neonatal, young adult, and mature adult mice. This will require careful evaluation of PC and PC-L1 biophysical and pharmacological properties. We will then determine and compare the domains of PC and PC- L1 that enable them to regulate calcium in cilia in distinct ways. Finally, we will determine how PC1, the more commonly affected protein in polycystic kidney disease, regulates the pore of the PC2-forming channel. Through these studies, we aim to understand the basic function of the polycystins and why mutations in these proteins cause life-threatening polycystic kidney disease. This is a necessary step in designing drugs or therapies to treat the disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
High Priority, Short Term Project Award (R56)
Project #
1R56DK103739-01A1
Application #
9108684
Study Section
Special Emphasis Panel (KMBD)
Program Officer
Rasooly, Rebekah S
Project Start
2015-09-01
Project End
2016-08-31
Budget Start
2015-09-01
Budget End
2016-08-31
Support Year
1
Fiscal Year
2015
Total Cost
$132,688
Indirect Cost
$57,688
Name
Children's Hospital Boston
Department
Type
DUNS #
076593722
City
Boston
State
MA
Country
United States
Zip Code
02115