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

2. 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

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

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).

5. MIR-17~92 CLUSTER

6. 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.

7. NEPHRON PROGENITORS IN KIDNEY DEVELOPMENT S SP D2D1 Self-renewal: Maintenance of Stem cell pool Multipotent: Generation of differentiated progeny Nephron progenitor

8. 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?

9. ABLATION OF MIR-17~92 IN NEPHRON PROGENITORS Six2-Cre Mouse miR-17~92 Floxed mouse miR-17~92 miR-17~92 is removedmIR-17~92 function is untouched Six2

10. LOSS OF MIR-17~92 IN NEPHRON PROGENITORS RESULTS IN RENAL HYPOPLASIA AT P0 ControlHetMutant Marrone AK et al (2014). J Am Soc Neph 2014, published online February 7; doi10.1681/ASN.201304039.

11. 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.

12. 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.

13. FEWER DEVELOPING GLOMERULI AT P0 Marrone AK et al (2014). J Am Soc Neph 2014, published online February 7; doi10.1681/ASN.201304039.

14. 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.

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

16. MIR-17~92 LOSS IN NEPHRON PROGENITORS Developmental defects: (1) Fewer developing nephrons formed (2) Heterozygous renal hypoplasia (3) Decreased proliferation in nephron progenitors

17. 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.

18. HISTOLOGICAL ABNORMALITIES AT 3 MO Marrone AK et al (2014). J Am Soc Neph 2014, published online February 7; doi10.1681/ASN.201304039.

19. 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.

20. 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

21. HOW DOES MIR-17~92 REGULATE NEPHRON NUMBER AND PATTERN? N-myc and/or C-myc (ChIP) Candidate downstream targets: Bim, PTEN, p21 RNA sequencing approach to identify novel targets. Regulation of: -apoptosis/cell death -nephron differentiation -renal size Feingold syndrome patients: Renal US Urine protein to creatinine

22. HIGH THROUGHPUT RNA SEQUENCING OF CONTROL AND MUTANT KIDNEYS April Marrone and Dennis Kostka

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

24. CANDIDATE MIR-17~92 TARGETS Olive et al (2010). Int J Bioch & Cell Biol, 42(8): 1348-5. X

25. INCREASED P21 EXPRESSION IN MUTANTS Marrone AK et al (2014). J Am Soc Neph 2014, published online February 7; doi10.1681/ASN.201304039.

26. MIR- 17~92 IN KIDNEY DEVELOPMENT AND DISEASE miR-17~9 2 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?

27. ACKNOWLEDGMENTS University 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

28. Thanks