Research Introduction

9.17.13

• Altered metabolism in polycystic kidney

disease

• Telomerase activity in polycystic kidney disease cells

Autosomal dominant polycystic kidney disease • ADPKD is the most common inherited kidney disease.

Incidence 1 in 500 to 1 in 1000 worldwide. 600,000 patients in US

• Third most common single cause of ESRD world wide

and accounts for 5-10% of ESRD in US • ADPKD is caused by mutations of either PKD1 gene

(around 85% of cases) on chromosome 16 or the PKD2 gene (around 15% of cases) on chromosome 4

• There is evidence for a two hit mechanisms( germ line and somatic inactivation of two PKD alleles) explaining the focal development of renal and hepatic cysts

Polycystin 1 (PKD1) and Polycystin 2 (PKD2)

Intracellular

Cell Membrane

extracellular

Polycystins localize to cilia and are activated by mechanosensation

tubular lumen

Normal PKD1 or PKD2 Mutant

x

x x

PC1 PC2 PC1

PC2

Pathways up-regulated or down regulated in polycystic kidney disease

Altered Metabolism and Cancer

Altered Metabolism and Cancer

Resting cell Proliferating cells

Metabolism in proliferating cancer cells

Glucose Glutamine

Macro molecules

ATP

Proteins Lipids Nucleotides

Altered metabolism and Cancer

Glutamine metabolism and Cancer

Altered metabolism and Cancer

Do ADPKD cells behave like cancer cells?

Many of the signaling pathways activated in cancer cells are also active in ADPKD cells

Cells used for studies

1. NHK cells. Primary normal human kidney distal tubular epithelial cells isolated from nephrectomies.

2. ADPKD cells. Primary human ADPKD cells that are mutant for PKD1.

ADPKD cells Isolate cyst lining epithelial cells

PKD-/-

PKD+/-

Upregulation of PKM2 isoform in PKD cells

glutaminase

PKM2

actin

APKD NHK APKD NHK

PKM1

Glutaminase 1 is upregulated in ADPKD cells by cAMP

Forskolin/cAMP APKD APKD APKD NHK NHK NHK

Forskolin/cAMP

anti-glutaminase KGA anti-glutaminase GAC

glutaminase 1 KGA

glutaminase 1 GAC

Inhibition of glutaminase 1 blocks cyst formation by PDK-/- cells in cell culture

Inhibition of glutaminase 1 lowers ATP levels in ADPKD but not NHK cells

Alpha Ketoglutarate- most abundant metabolite in the urine of rat model of PKD

Nephrology 2012

Assess whether cyst formation in PKD-/- mice (knockout) is inhibited in by gls1 inhibitor

BPTES

Pkhd1-Cre; PKDfl/-

BPTES postnatal day 10-24

Vehicle control postnatal day 10-24

Sacrifice day 24

Treatment with BPTES slows cyst growth in PKD1 knockout model

Amino acids and glutaminolysis regulate mTORC1

Glutamine Glutamate Ketoglutarate GLS GDH

Leucine

mTORC1 Cell

proliferation

PS6K PS6

Cell 2012

Glutaminolysis regulates mTORC1

Cell 2012

Sirolimus and kidney growth in Autosomal Dominant Polycystic Kidney Disease

• 18 month randomized control trial

• 100 patients b/w the age group 18-40yrs- sirolimus (target dose 2 mg daily) or standard care

• Sirolimus level b/w 4 and 10 microgram/litre

• Estimated CrCl 70 ml/min • Serial MRI – to measure the

volume of polycystic kidney disease

• At randomization- median kidney volume was 907cm3 (sirolimus) and 1003 (control group)

• Primary outcome- kidney volume at 18 months

• Secondary outcomes –GFR and urine albumin excretion

Results: • Median increase in

sirolimus group was 99 cm3( interquartile range 43-173) Control group 97 cm3( interquartile range 37-181) at 18 months

• GFR did not differ between two groups

• Urinary albumin excretion rate was higher in sirolimus group

NEJM aug 2010

Everolimus in Patients with Autosomal Dominant Polycystic Kidney Disease

• 2 year, double blind trial • 433 patients with ADPKD

were assigned to receive either placebo or everolimus (2.5 mg bid)

• Estimated GFR of 30-89ml/min/1.73 m2

• Primary outcome was change in kidney volume at 12 and 24 months as measured on MRI

• Secondary outcome was renal function (GFR, serum creatinine, proteinuria, incidence of newly developed ESRD) at 24 months

Results: Total kidney volume increased between baseline and 1 year by 102 ml and 230 ml by 2 years in everolimus group Vs 157 ml by 1 year and 301 ml by 2 years in placebo group Mean decrement in estimated GFR after 24 months was 8.9 ml/min/1.73 m2 Vs 7.7 ml/min /1.73m2 in placebo group

NEJM august 2010

Rapamycin may not inhibit mTOR in ADPKD kidneys

Canaud et. al (2010) AJT

• Do polycystic kidney disease cells have up regulated telomerase activity?

TELOMERE and TELOMERASE

TELOMERES • Telomeres are DNA-protein structures that protect

the chromosome ends from degradation and fusion

• Telomere DNA sequence comprises of tandem repeats of the six nucleotide unit sequence TTAGGG. These sequences extend for thousands of bases at chromosome ends, averaging 10 kb in a newborn human’s cord blood.

• Telomere DNA is bound by a specialized group of protective proteins collectively called shelterin

TELOMERASE • Telomerase was first identified in ciliated protozoan “tetrahymena”

• Telomerase is a specialized DNA polymerase that synthesizes new

telomere sequences onto chromosome ends.

• Telomerase is composed of the core telomerase protein TERT, which contains the telomerase reverse transcriptase domain and and an essential RNA component called TERC which provides the template for telomeric sequence synthesis

• Telomerase activity is regulated during development and has a very low

level, almost undetectable in somatic cells. Telomerase is found in found in fetal tissue, germ cells

• Telomerase activity has been found to be up-regulated in most cancer cells thus allowing cancer cells to escape cellular senescence and becoming immortal.

Science 1994

Telomere length

293 T cells PKD

NHK

Thank You!