μrnas: new frontiers in kideny disease

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μRNAs: New frontiers in kidney disease VISHAL S. VAIDYA, PH.D. ASSISTANT PROFESSOR OF MEDICINE & ENVIRONMENTAL HEALTH Brigham and Women’s Hospital Harvard Medical School Harvard School of Public Health

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Page 1: μRNAs: New frontiers in kideny disease

µRNAs: New frontiers in kidney disease

VISHAL S. VAIDYA, PH.D.ASSISTANT  PROFESSOR  OF  MEDICINE  &  ENVIRONMENTAL  HEALTH

Brigham and Women’s Hospital Harvard Medical School Harvard School of Public Health

Page 2: μRNAs: New frontiers in kideny disease

OUTLINE

Who we are and what do we do ?Who we are and what do we do ?

What’s special about µRNAsWhat’s special about µRNAs

Human profiling resultsHuman profiling results

Mechanistic studies with µRNAsMechanistic studies with µRNAs

Page 3: μRNAs: New frontiers in kideny disease

HOW CAN WE MODERNIZE TOXICOLOGY ?

Normal Increased risk Damage Kidney failureGFR Death

Translational Biomarkers Transform Kidney Safety Assessment

KIM

KIM’s Friend

New KIM

Page 4: μRNAs: New frontiers in kideny disease

HOW CAN WE MODERNIZE TOXICOLOGY ?

Normal Increased risk Damage Kidney failureGFR Death

Nat Biotech, 2010

AJP‐Renal, 2006JASN, 2007Clin Trans Sci, 2008

Kidney Intl, 2009JASN, 2013

Fibrinogen

Kidney Injury Molecule

A Panel of MicroRNAs

Blood, 2011

PLoS One, 2012

Am J Path, 2012

Tox Sci, 2012

Clin Chem, 2013

Translational Biomarkers Transform Kidney Safety Assessment

Paradigm shift in Predictive Kidney Toxicity Screening

Page 5: μRNAs: New frontiers in kideny disease

OUTLINE

Who we are and what do we do ?Who we are and what do we do ?

What’s special about µRNAsWhat’s special about µRNAs

Human profiling resultsHuman profiling results

Mechanistic studies with µRNAsMechanistic studies with µRNAs

Page 6: μRNAs: New frontiers in kideny disease

Weiland M et al, RNA Biol, 2012

HISTORY OF CIRCULATING NUCLEIC ACID DETECTION

Page 7: μRNAs: New frontiers in kideny disease

(1) Transcription of pri‐miRNAs from genes in the nucleus by RNA polymerase II 

(1) Processing of primary transcript into 70 nt long pre‐miRNA

(1) Export into cytoplasm in a Ran‐GTP dependent manner through Exportin 5 

(1) Cleavage of pre‐miRNA by RNase III enzyme Dicer, into a 22 nt double stranded RNA composed of the mature miRNA “guide” strand and the low abundance miRNA* “passenger” strand

(1) Incorporation of mature miRNA into RNA Induced Silencing Complex (RISC)

Saikumar, Ramachandran and Vaidya, 2013

Page 8: μRNAs: New frontiers in kideny disease

6. Packaging into multivesicular bodies (MVBs) that fuse with the plasma membrane and release as exosomesin a ceramide dependent pathway positively regulated by neutral sphingomyelinase 2 (nSMase2)

6. Encapsulation into high‐density lipoprotein (HDL) particles which is repressed by nSMase2 

6. Binding to RNA binding proteins, namely AGO‐2 and nucleophosmin 1 (NPM1) 

6. Incorporation into apoptotic bodies 

SECRETION OF MIRNAS IN TO EXTRACELLULAR SPACE

Page 9: μRNAs: New frontiers in kideny disease

NON‐IN

VASIVE M

ICRO‐

MARKERS

Saikumar, Ramachandran and Vaidya, 2013

Page 10: μRNAs: New frontiers in kideny disease

Sensitive: immediate release before mRNA transcription and protein translation.

Specific: tissue and disease specific regulation.

Dynamic range: ~ 50,000 copies of miRNA/cell. Translational potential: highly conserved with a high degree of inter- and intra-species

homology. Stability: more resistant to degradation than mRNA and proteins; and able to be detected in

formalin fixed paraffin embedded tissues (although DNA is more stable, it is less tissue specific). Accessible in plethora of biological fluids allowing noninvasive detection.

Quantitative by PCR amplification.

WHAT MAKES MIRNASGOOD BIOMARKERS ?

Plasma miRNAS:• Harsh boiling conditions (>80oC)• Low or high pH• > 5 freeze thaw cycles• Storage at room temperature for > 1

week

Urinary miRNAS:• No degradation for up to

1 day at room temperature

• > 4 freeze thaw cycles

STABILITY

Page 11: μRNAs: New frontiers in kideny disease

10 20 30 40 50 60 70 80 90T G T A C C A C C T T G T C G G G T A G C T T A T C A G A C T G A T G T T G A C T G T T G A A T C T C A T G G C A A C A G C A G T C G A T G G G C T G T C T G A C A T T T T G G T A T C- - - - - - - - - - T G T C G G G T A G C T T A T C A G A C T G A T G T T G A C T G T T G A A T C T C A T G G C A A C A C C A G T C G A T G G G C T G T C T G A C A - - - - - - - - - -- - - - - - - - - - - - - - - - - T A G C T T A T C A G A C T G A T G T T G A C T G T T G A A T C T C A T G G C A A C A G C A G T C G A T G G G C T G T C - - - - - - - - - - - - - - -- - - - - - - - - - T G T C G G G T A G C T T A T C A G A C T G A T G T T G A C T G T T G A A T C T C A T G G C A A C A G C A G T C G A T G G G C T G T C T G A C A - - - - - - - - - -T G T A C C A C C T T G T C G G G T A G C T T A T C A G A C T G A T G T T G A C T G T T G A A T C T C A T G G C A A C A G C A G T C G A T G G G C T G T C T G A C A T T T T G G T A T CT G T A C C A C C T T G T C G G A T A G C T T A T C A G A C T G A T G T T G A C T G T T G A A T C T C A T G G C A A C A G C A G T C G A T G G G C T G T C T G A C A T T T T G G T A T C- - - - - - - - - - T G T C G G G T A G C T T A T C A G A C T G A T G T T G A C T G T T G A A T C T C A T G G C A A C A C C A G T C G A T G G G C T G T C T G A C A - - - - - - - - - -

10 20 30 40 50 60 70 80 90 100- - - - - - - - - - - - - - - - - - GAAGT T AGGCT GAGGGGCAGAGAGCGAGACT T T T CT AT T T TCCAAAAGCT CGGT CT GAGGCCCCT CAGT CT T GCT T CC- - - - - - - - - - - - -- - - - - - - - - - - - AT AAAGGAAGT T AGGCT GAGGGGCAGAGAGCGAGACT T T T CT AT T T TCCAAAAGCT CGGT CT GAGGCCCCT CAGT CT T GCT T CCT ACCCCGCG- - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - T GAGGGGCAGAGAGCGAGACT T T T CT AT T T TCCAAAAGCT CGGT CT GAGGCCCCT CAGT - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - AT AAAGGAAGT T AGGCT GAGGGGCAGAGAGCGAGACT T T T CT AT T T TCCAAAAGCT CGGT CT GAGGCCCCT CAGT CT T GCT T CCT ACCCCGCGC- - -- - - - - - - - - - - - - - - - - - GAAGT T AGGCT GAGGGGCAGAGAGCGAGACT T T T CT AT T T TCCAAAAGCT CGGT CT GAGGCCCCT CAGT CT T GCT T CCT - - - - - - - - - - - -ACT T GT GAGGAAAT AAAGGAAGT T AGGCT GAGGGGCAGAGAGCGAGACT T T T CT AT T T TCCAAAAGCT CGGT CT GAGGCCCCT CAGT CT T GCT T CCT ACCCCGCGCTT G- - - - - - - - - - - - AT AAAGGAAGT T AGGCT GAGGGGCAGAGAGCGAGACT T T T CT AT T T TCCAAAAGCT CGGT CT GAGGCCCCT CAGT CT T GCT T CCT AACCCGCGC- - -

10 20 30 40 50 60 70 80 90 100 110- - - - - - - - - - - - - - GGT T GGGT A GGGGCCCT CGT CT T A CCCA GCA GT GT T T GGGT GCT GGT T GGGA GT CT CT A A T A CT GCCGGGT A A T GA T GGA GGCCCCT GT C- - - - - -A CCCA A GGT GGGCGGGCT GGGCGGGGGCCCT CGT CT T A CCCA GCA GT GT T T GGGT GC- GGT T GGGA GT CT CT A A T A CT GCCGGGT A A T GA T GGA GGCCCCT GT CCCT GT G- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - CGT CT T A CCCA GCA GT GT T T GGGT GCT GGT T GGGA GT CT CT A A T A CT GCCGGGT A A T GA T GGA - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - CGT CT T A CCCA GCA GT GT T T GGGT GCT GGT T GGGA GT CT CT A A T A CT GCCGGGT A A T GA T GGA GG- - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - CCCT CGT CT T A CCCA GCA GT GT T T GGGT GCT GGT T GGGA GT CT CT A A T A CT GCCGGGT A A T GA T GGA GG- - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - CCCT CGT CT T A CCCA GCA GT GT T T GGGT GCT GGT T GGGA GT CT CT A A T A CT GCCGGGT A A T GA T GGA GG- - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - CCCT CGT CT T A CCCA GCA GT GT T T GGGT GC- GGT T GGGA GT CT CT A A T A CT GCCGGGT A A T GA T GGA GG- - - - - - - - - - - - - -

HamsterChimpanzee

DogCowRat

MouseHuman

miR-21 5p miR-21 3p

miR-200c 5p

miR-423 5p

miR-200c 3p

miR-423 3p

HamsterChimpanzee

DogCowRat

MouseHuman

HamsterChimpanzee

DogCowRat

MouseHuman

WHAT MAKES MIRNASGOOD BIOMARKERS ?

SPECIES CONSERVATION

Page 12: μRNAs: New frontiers in kideny disease

µRNAs as mechanistic biomarkers of kidney disease

Exosomal and circulating miRNAs have been shown to be mechanistic biomarkers in various cancers, organ damage and disease states.

MiR‐210: plasma and urinary biomarker for acute and chronic forms of injury and acute allograft rejection as well as ESRD. Modulates cellular response and mitochondrial metabolism in hypoxic conditions.

MiR‐155: a key regulator of the immune response and its expression is decreased in the in urine and plasma in both acute and chronic kidney damage as well as in ESRD.

MiR‐21: silences metabolic pathways; its expression significantly increases in kidney and urine following damage.

Saikumar, Ramachandran and Vaidya, 2013

Page 13: μRNAs: New frontiers in kideny disease

OUTLINE

Who we are and what do we do ?Who we are and what do we do ?

What’s special about µRNAsWhat’s special about µRNAs

Human profiling resultsHuman profiling results

Mechanistic studies with µRNAsMechanistic studies with µRNAs

Page 14: μRNAs: New frontiers in kideny disease

HEALTHY AKIExpressed [E] 345 281Borderline-expressed [BE] 275 223Non-expressed [NE] 1287 1402E = 19-30 (Ct); BE = 30-32 (Ct); NE = >32 (Ct)

RNA ISOLATION

HEALTHY AKI

58

39

28

5

E in both248

E in Healthy E in AKI

BE in Healthy BE in AKI

NE in Healthy NE in AKI

PooledURINES

PooledURINES

REVERSE TRANSCRIPTION

PRE-AMPLIFICATION

MIRNA PROFILING (1809 miRNAS) 378 miRNAs selected

Ramachandran et al., Clin Chem, 2013

Page 15: μRNAs: New frontiers in kideny disease

378

miR

NA

Pro

filin

g

8 excluded

54 excluded

Fold Change< 5-fold

299 excluded

-5 51.5

Melt Curve Failures

Std Dev > 1.5p > 0.01

miR-502-5p

Fold change over healthy93 60

miR-4640-5pmiR-21-5p

miR-4698

miR-4650-3p

miR-200b-3p

let-7d-5p

miR-23a-3p

miR-3679-5p

miR-4724-5p

miR-4301let-7b-5p

let-7c

miR-191-5p

miR-373-5pmiR-1301

miR-320b

miR-3620-3p

378 79 25 17

HEALTHY AKI

Page 16: μRNAs: New frontiers in kideny disease

Expression levels of miR‐21, 200c, 423 and 4640 are significantly different in patients AKI (n=117) as compared to 

healthy volunteers (n=97)

Page 17: μRNAs: New frontiers in kideny disease

The combined cross‐validated area under the receiver operator curve for miR‐21, ‐200c, ‐423 and ‐4640 was computed to be 

0.91.

Page 18: μRNAs: New frontiers in kideny disease

WHICH NORMALIZER TO USE ?

Page 19: μRNAs: New frontiers in kideny disease

WHAT IS THE TEMPORAL VARIABILITY OF µRNAs

Ramachandran et al., Clin Chem, 2013

Page 20: μRNAs: New frontiers in kideny disease

DRUG‐INDUCED KIDNEY TOXICITY

• All 70 patients had high ALT (>150 IU/L) and received NAC treatment

• Expression levels of miR‐21, 200c and 423 are significantly higher in patients with  Acetaminophen‐induced kidney toxicity.

Collaboration with Dr. Dan Antoine, Center for Drug Safety Science, UK n =65 n = 27 n = 43

Control vs. SCr > 1

miR-21 0.87miR-200c 0.82miR-423 0.83

Page 21: μRNAs: New frontiers in kideny disease

LONGITUDINAL STUDY

Urinary levels of miRNA‐21 and miRNA‐423 are significantly higher in patients admitted to the ICU who develop AKI  which progresses to Renal Replacement Therapy (RRT) or death as compared to those who do develop AKI but do not require RRT as well as those who do not develop AKI. 

Collaboration with Dr. Jay Koyner, Univ of Chicago 

Page 22: μRNAs: New frontiers in kideny disease

Can we perform small RNA sequencing in the urine ?

Healthy Prostate Cancer

miR‐451

miR‐31

miR‐185miR‐874

Collaboration with Dr. Martin Sanda, Emory Univ 

Page 23: μRNAs: New frontiers in kideny disease

OUTLINE

Who we are and what do we do ?Who we are and what do we do ?

What’s special about µRNAsWhat’s special about µRNAs

Human profiling resultsHuman profiling results

Mechanistic studies with µRNAsMechanistic studies with µRNAs

Page 24: μRNAs: New frontiers in kideny disease

0 24 48 72 96 1200

200

400

600

800

1000

Kim

-1 (f

old

chan

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Seru

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reat

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g/dL

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iew

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hours post Cisplatin injection  hours post Cisplatin injection 

+/+

‐/‐

+/+

‐/‐

MIR‐155 KO ARE SUSCEPTIBLE TO KIDNEY TOXICITY

Page 25: μRNAs: New frontiers in kideny disease

• We conducted a screen of the entire miRNomein human urine and have identified miR‐21, miR‐200c, miR‐423, and miR‐4640 as sensitive (and noninvasive) biomarkers of kidney damage

• miR‐155 plays a protective role in cisplatin‐induced kidney toxicity

SUMMARY

CONCLUSION

QUESTIONS

• Will there be a unique and distinct panel of miRNA for subtype of kidney damage?

• How will a panel of miRNAs and proteins perform as biomarkers for early detection?

• Can we reduce kidney damage by targeting candidate miRNAs?

• Extracellular RNAs have an immense potential as biomarkers

• Understanding the biology of extracellular RNAs is likely to yield in newer insights in kidney health and disease

Page 26: μRNAs: New frontiers in kideny disease

VAIDYALAB.ORG

Melanie Adler Toxicologist

Susanne RammToxicologist

Systems Tox

Ajay AmrendraMolecular Biologist

Shreyas JadhavRNA Biologist

Mechanisms

Florin CraciunMouse Genetics

Biomarkers

Outstanding New Environmental Scientist Award

Innovation in Regulatory Science Award

Kathryn PellegriniImmunologist

Oana NicoaraPediatric Nephrologist

µRNAs

Poornima RaoChemical Engg

CollaboratorsSushrut Waikar, BWH

Vanesa Bijol, BWH

Jay Koyner, U.Chicago

Donna Mendrick, US-FDA