Jacqueline Ho

Division of Pediatric Nephrology

Children’s Hospital of Pittsburgh

Oct. 9, 2014

SMALL RNAS AND SMALL KIDNEYS

MIRNAS: A NOVEL REGULATORY MECHANISM FOR GENE EXPRESSION

• ~500-600 miRNAs in humans

• Up to ½ of all mRNA transcripts are miRNA targets

• Regulate a variety of processes:

• Differentiation

• Proliferation

• Apoptosis

• Cell signaling

• Stem cells and miRNAs

MIRNA BIOGENESIS

Ho J and Kreidberg JA (2012); JASN 23(3): 400-4.

MIRNAS AND ACUTE KIDNEY INJURY• Analysis of differential miRNA expression profiles in acute kidney injury:

• Mouse models: cisplatin, ischemia-reperfusion injury (Lee et al (2014), Kidney Int, epub ahead of print doi: 10.1038/ki.2014.117); (Bellinger et al (2014), PLoSOne, Apr 2; 9(4): e93297)

• Patients with AKI (Ramachandran et al (2013), Clin Chem, 59(12):1742-52)

• Possible biomarkers?

• MicroRNAs as drug targets or drugs themselves

• Eg. miR-24 antagonism was protective against IRI (Lorenzen et al (2014), JASN, epub ahead of print, pii: ASN.2013121329).

• Eg. miR-126 (overexpression was protective post IRI) (Bijkerk et al (2013), JASN, epub ehead of print, doi: 10.1681/ASN.201 3060640).

MIR-17~92 CLUSTER

MIR-17~92 CLUSTER (ONCOMIR-1)

• Genomic amplification and elevated expression in human B-cell lymphomas

• Increased expression of miR-17 and miR-106a following IRI (Kaucsar et al (2013), Nucleic Acid Ther; 23(5): 344-54)

• Deletion of miR-17~92 results in several developmental defects:

• Lung hypoplasia, ventricular septal defect, impaired B-cell development

• (Ventura et al (2008), Cell, 132: 875-886)

DePontual et al (2011). Nature Genetics, 43(10): 1026-1030.

NEPHRON PROGENITORS IN KIDNEY DEVELOPMENT

S

S P

D2D1

Self-renewal:Maintenance of Stem cell pool

Multipotent:Generation of differentiated

progeny

Nephron progenitor

HYPOTHESIS

• miR-17~92 was expressed in the developing kidney in nephron progenitors

• Linked to a human syndrome associated with renal anomalies

• miR-17~92 is required for normal kidney development and function

• Nephron number?

• Nephron pattern?

• Impact on kidney function?

ABLATION OF MIR-17~92 IN NEPHRON PROGENITORS

Six2-Cre Mouse miR-17~92 Floxed mouse

miR-17~92

miR-17~92

miR-17~92 is removed mIR-17~92 function is untouched

Six2

LOSS OF MIR-17~92 IN NEPHRON PROGENITORS RESULTS IN RENAL HYPOPLASIA AT P0

Control Het Mutant

Marrone AK et al (2014). J Am Soc Neph 2014, published online February 7; doi10.1681/ASN.201304039.

PRESERVED EXPRESSION OF NEPHRON PROGENITOR MARKERS AT PO

Marrone AK et al (2014). J Am Soc Neph 2014, published online February 7; doi10.1681/ASN.201304039.

FEWER DEVELOPING NEPHRONS (RENAL VESICLES) AT P0

Marrone AK et al (2014). J Am Soc Neph 2014, published online February 7; doi10.1681/ASN.201304039.

FEWER DEVELOPING GLOMERULI AT P0

Marrone AK et al (2014). J Am Soc Neph 2014, published online February 7; doi10.1681/ASN.201304039.

NO INCREASE IN APOPTOSIS IN NEPHRON PROGENITORS

Marrone AK et al (2014). J Am Soc Neph 2014, published online February 7; doi10.1681/ASN.201304039.

DECREASED PROLIFERATION OF PROGENITORS

Marrone AK et al (2014). J Am Soc Neph 2014, published online February 7; doi10.1681/ASN.201304039.

MIR-17~92 LOSS IN NEPHRON PROGENITORS

• Developmental defects:

• (1) Fewer developing nephrons formed

• (2) Heterozygous renal hypoplasia

• (3) Decreased proliferation in nephron progenitors

LONG-TERM CONSEQUENCES : ALBUMINURIA AT 6 WEEKS AND 3 MONTHS

Marrone AK et al (2014). J Am Soc Neph 2014, published online February 7; doi10.1681/ASN.201304039.

HISTOLOGICAL ABNORMALITIES AT 3 MO

Marrone AK et al (2014). J Am Soc Neph 2014, published online February 7; doi10.1681/ASN.201304039.

PARTIAL FOOT PROCESS EFFACEMENT BY ELECTRON MICROSCOPY AT 3 MO

Marrone AK et al (2014). J Am Soc Neph 2014, published online February 7; doi10.1681/ASN.201304039.

MIR-17~92 LOSS IN NEPHRON PROGENITORS

• Evidence for glomerular disease:

• (1) Albuminuria (with heterozygous and homozygous loss of miR-17~92)

• (2) Focal glomerulosclerosis

• (3) Partial foot process effacement

• (4) Decreased renal function

HOW DOES MIR-17~92 REGULATE NEPHRON NUMBER AND PATTERN?

N-myc and/or C-myc (ChIP)

Candidate downstream targets: Bim, PTEN, p21RNA sequencing approach to identify novel targets.

Regulation of:-apoptosis/cell death-nephron differentiation-renal size

Feingold syndrome patients:Renal USUrine protein to creatinine

HIGH THROUGHPUT RNA SEQUENCING OF CONTROL AND MUTANT KIDNEYS

April Marrone and Dennis Kostka

VALIDATION OF MOST UPREGULATED TRANSCRIPTS PREDICTED TO BE MIR-17~92 TARGETS

CANDIDATE MIR-17~92 TARGETS

Olive et al (2010). Int J Bioch & Cell Biol, 42(8): 1348-5.

X

INCREASED P21 EXPRESSION IN MUTANTS

Marrone AK et al (2014). J Am Soc Neph 2014, published online February 7; doi10.1681/ASN.201304039.

MIR-17~92 IN KIDNEY DEVELOPMENT AND DISEASE

• miR-17~92 is required in nephron progenitors for normal renal development:

• Nephron number and pattern

• Ablation of miR-17~92 in nephron progenitors and their derivatives results in kidney disease:

• Renal disease and renal hypodysplasia

• Outstanding questions:

• Which miR (or miRs) in the cluster are responsible for this phenotype?

• Is there an intrinsic defect in nephron progenitors? Eg. specification or self-renewal?

• What are the downstream targets of miR-17~92 in the kidney?

ACKNOWLEDGMENTSUniversity of Pittsburgh/CHP

• Yu Leng Phua

• April Marrone

• Jessica Chu

• Andrew Bodnar

Collaborators:

• Carl Bates

• Sunder Sims-Lucas

• Donna Stolz

• Sheldon Bastacky

• Dennis Kostka

Collaborators:

• Cliff Tabin

• Andrew McMahon

Funding:

• NIDDK- R00DK087922

• Pittsburgh Center for Kidney Research Pilot Project – NIDDK P30 DK079307

• March of Dimes Basil O’Connor Starter Scholar Research Grant

• Norman S. Coplon Extramural Grant

Thanks