Download - Jacqueline Ho Division of Pediatric Nephrology Children’s Hospital of Pittsburgh Oct. 9, 2014 SMALL RNAS AND SMALL KIDNEYS

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


FEWER DEVELOPING NEPHRONS (RENAL VESICLES) AT P0


FEWER DEVELOPING GLOMERULI AT P0


NO INCREASE IN APOPTOSIS IN NEPHRON PROGENITORS


DECREASED PROLIFERATION OF PROGENITORS


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


HISTOLOGICAL ABNORMALITIES AT 3 MO


PARTIAL FOOT PROCESS EFFACEMENT BY ELECTRON MICROSCOPY AT 3 MO


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


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

Download - Jacqueline Ho Division of Pediatric Nephrology Children’s Hospital of Pittsburgh Oct. 9, 2014 SMALL RNAS AND SMALL KIDNEYS

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