Kidney Disease Research

Transcription

1 Thomas Weimbs Assistant Professor, Department of Molecular, Cellular & Developmental Biology Kidney Disease Research

2 The mtor pathway is regulated by polycystin 1, and its inhibition reverses renal cystogenesis in polycystic kidney disease From the lab of Thomas Weimbs (and collaborators) Department of Molecular, Cellular, and Developmental Biology University of California Santa Barbara

3 Autosomal dominant polycystic kidney disease (ADPKD) the most common life threatening genetic disease affects >600,000 Americans and 12.5 million people worldwide affects more people than cystic fibrosis, muscular dystrophy, hemophilia, Down syndrome and sickle cell anemia combined. frequency: 1 in 200 1,000 births. one of the leading causes of kidney failure in the US. two genes are responsible for ADPKD: PKD1 gene encoding for polycystin 1 1 (~85% of cases) PKD2 gene encoding for polycystin 2 2 (~15% of cases) the functions of polycystins 1 1 and 2 2 are unknown.

4 Clinical Features of ADPKD Epithelial lined renal cysts Also hepatic and pancreatic cysts Intracranial aneurysms Hypertension Renal failure in 50% of patients by age 50 Treatment: dialysis or kidney transplantation No available treatment directed at the disease process Prevention: none

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6 Model of cyst formation Characteristic features of cystic epithelial cells: Increased proliferation Increased apoptosis Increased fluid secretion

7 Polycystin 1 Tuberin mtor STAT6 P100

8 Expression Constructs

9 Expression of NTM PC1 in MDCK cells causes G1 cell cycle arrest and apoptosis NTM PC1

10 Expression of NTM PC1 in MDCK cells causes G1 cell cycle arrest and apoptosis NTM PC1

11 Expression of NTM PC1 in MDCK cells causes G1 cell cycle arrest and apoptosis NTM PC1

12 Expression of NTM PC1 in MDCK cells causes G1 cell cycle arrest and apoptosis NTM PC1

13 NTM PC1 targets to the Golgi apparatus NTM PC1

14 Tuberous Sclerosis Complex (TSC) Affects 50,000 Americans and 1 million individuals worldwide. Clinical: benign tumors in the skin, kidney, brain, heart, eyes, lungs, teeth and oral cavity, and other organs. Two genes have been identified: TSC1 (hamartin) and TSC2 (tuberin). TSC patients also have kidney cysts. The TSC2 and PKD1 genes are immediately adjacent on chromosome 16 (only 60 nucleotides away). If both, the TSC2 and PKD1 genes are affected, patients have severe, earlyonset polycystic kidney disease. Tuberin was reported to affect intracellular trafficking of polycystin 1: Kleymenova, E., O. Ibraghimov Beskrovnaya, H. Kugoh, J. Everitt, H. Xu, K. Kiguchi, G. Landes, P. Harris, and C. Walker (2001) Mol Cell. 7: Hypothesis: tuberin and polycystin 1 may be functionally linked.

15 NTM PC1 forces re localization of tuberin to the Golgi apparatus NTM PC1

16 NTM PC1 forces re localization of tuberin to the Golgi apparatus NTM PC1

17 NTM PC1 forces re localization of tuberin to the Golgi apparatus NTM PC1

18 Tuberin co immunoprecipitates with PC1 tail FLM PC1 CTM PC1

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20 NTM PC1 forces re localization of mtor to the Golgi apparatus

21 mtor is activated in ADPKD cysts ADPKD Normal Phospho mtor (Ser 2448)

22 mtor is activated in ADPKD cysts ADPKD Normal Phospho S6K (Thr 389)

23 mtor is activated in PKD mouse models Pkd1 cond/cond : TgN(balancer1)2Cgn MAL transgenic orpk rescue Phospho S6 (Ser 235/236)

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25 The mtor inhibitor rapamycin reduces cysts in the orpk rescue rescue mouse model Rapamycin treated mutant mouse Vehicle treated control mouse Vehicle treated mutant mouse Rapamycin treatment: Daily I.P. From P150 > P178 (n=4 each)

26 Mouse MRI Scanning

27 The mtor inhibitor rapamycin causes regression of existing cysts (n=6 each)

28 Rapamycin induces apoptosis and lumenal shedding in cysts

29 Vehicle treated control mouse Rapamycin treated mutant mouse Vehicle treated mutant mouse Rapamycin reduces cysts in the bpk mouse model Rapamycin treatment: Daily I.P. From P7 > P21

30 Rapamycin reduces cysts in the bpk mouse model

31 Rapamycin improves kidney function in the bpk mouse model

32 Rapamycin treatment reduces kidney volumes in ADPKD patients

33 Working Model

34 Ac kn ow le dg em en ts Current lab members (UCSB): * Jonathan Shillingford * Seng Hui Low Shivakumar Vasanth Sambuddho Mukherjee Nikunj Sharma Pallavi Bhattaram Josie Widjaja Recent past lab members (Cleveland Clinic): Collaborators * Noel Murcia, Nicole Brown, Chris Flask, Tomoko Obara, Michelle Kane (Case Western Reserve University) * Gerd Walz, Albrecht Kramer Zucker (University of Freiburg, Germany) * Gregory Germino, Klaus Piontek (Johns Hopkins University) * Michael Kinter, Andrew Novick, David Goldfarb (Cleveland Clinic) * Claire Larson * Ryan Hedgepeth Michelle Koo Connie Marcks Tammera Davis

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