micrornas in renal fibrosis and transplantation

Laura Denby BSc (Hons), PhD

Centre for Cardiovascular Science

University of Edinburgh

Denby Lab @dr_denby

MicroRNAs in renal fibrosis and transplantation

- mediators and therapeutic targets.

Non-coding RNA

DNA Sequencing revolution - Human Genome Project

- 22 287 known or predicted protein-coding gene loci.

- coding regions occupy 1.2% of the euchromatic genome.

- total fraction of bases occupied by known protein-coding transcripts is only about 2%.

- summation of the sequences covered by known genes, ‘mRNAs’ and spliced expressed

sequence tags (ESTs) indicated that (at least) 60 –70% of the mammalian genome is

transcribed on one or both strands.

The central tenet of molecular biology :-

RNA functions mainly as an informational

intermediate between a DNA sequence i.e.

gene and its encoded protein.

Gene mRNA Protein DNA

Transcription Translation

By brian0918 - Own

work, Public Domain,

https://commons.wikimedia.or

g/w/index.php?curid=404735

• Ribosomal RNA (rRNA)

• tRNA

• Long non-coding RNA (lncRNA)

• Small non-coding RNA

microRNA (miRNA)

snoRNA

siRNA

piRNA

• Circular RNA

Types of non coding RNA

Non-coding RNA

• miRNAs are small (<25nt) non-coding RNA molecules.

• 1st discovered in 1993 in C.elegans.

• 1st detected in humans 2000.

• Over a 1000 miRNA in humans.

• At least 60% of protein coding genes coding miRNA

complementary sites.

• Canonical and non-canonical biogenesis.

AGO

miRNA Biogenesis

Pri-miRNA transcript

- Transcribed by Pol II - Contain one or more stem loops

+ -

Ha and Kim, Nature Reviews 2014

Nucleus

AAA

Exportin 5

Cytoplasm

Pre-miRNA transcript

RNA induced silencing complex

AAA mRNA xxx

3 UTR

Translational repression / mRNA decay

mRNA cleavage

• Each pre-miRNA stem loop encodes 2

miRNA.

• Nomenclature agreement - 5p and 3p designation based on

position in stem loop.

• miRNA interact with mRNA with specific region called seed region.

• A single miRNA has the ability to hit multiple genes and influence many pathways.

• Frequently miRNA complementary sequences are found in functionally related but distinct mRNAs.

• Essential for normal kidney function.

• Phase I/II clinical trials underway with Anti-miRs for HCV, Alport’s Syndrome.

miR-200 family – TGFβ regulated

Seed Region – interacts with 3’UTR (5’ UTR and CDS)

miRNAs micromanagers of gene expression.

NEGATIVE regulators

5p

3p

Shi et al, 2008

miRNAs

Podocyte

Anti-Thy1.1 model – rat model of glomerulonephritis

Global

mesangial proliferation Segmental

mesangial proliferation

Normal glomeruli

Group

Glomerular

score Tubular score Total

Control 0.25 0.25 0.5

1 × ER4, 7 days 1 0.5 1.5

3 × ER4, 7 days 2 2 4

3 × ER4, 14

days

2.1 1.3 3.4

Histologic Evaluation of Kidney Damage in Anti-Thy1.1 Animals

25% of glomeruli had

a glomerular lesion.

Evidence of tubular

damage (5-25%)

Are miRNAs involved in glomerulonephritis??

ER-4

(2mg/kg)

From Denby et al., 2011. Am J Pathol. 179:661-72.

rno-miR-214

*p<0.05

3xGlomerulonephritis (D) vs Control (C) 7days

Immune-mediated glomerulonephritis

MiRNA Microarray

rno-miR-21

p=0.05

M

icro

RN

A e

xpre

ssio

n

(RQ

no

rma

lise

d to

U8

7)

1

2

3

4

5

6

7

*

*

Control

(PBS)

GN

(Anti-Thy1.1,

antibody ER-4)

miR-21

miR-214

Are miRNAs involved in glomerulonephritis??

RNA

N=3/gp 3 injections

1 week apart Sacrificed at 7 days post last injection

ER-4 (2mg/kg)



6

SHRSP WKY

Malignant

hypertension -

Gross vascular lesions

(onion skin pattern)

Hypertension induced nephropathy

40

30

20

10

0

Kidney Pathology Total Score

SHRSP WKY

(26.00, 31.00) 40

30

20

10

0

SHRSP WKY

Mic

roR

NA

expre

ssio

n

(RQ

norm

alis

ed to U

87)

WKY SHRSP

2

4

6

**

**

miR-21

miR-214

UUO SHAM

- is the final common pathway for most forms of progressive renal disease.

- involves glomerular sclerosis and/or tubulointerstitial fibrosis.

- tubulointerstitial fibrosis is best histological predictor of progression.

Unilateral ureter obstruction (UUO) model of fibrosis

MiR

NA

expre

ssio

n

(RQ

norm

alis

ed to U

87)

SHAM

UUO

* *

0

1

2

3

4

5

6

7

21 214 200b 200c 192 24 329 26b 30c

***

***

* ** * **

145

miRNA *p<0.05, **p<0.01, ***p<0.001 From Denby et al., 2011. Am J Pathol. 179:661-72.

Renal Fibrosis

0

0.5

1

1.5

2

2.5

3

3.5

4

Wildtype miR21 -/- miR214-/-

SHAM UUO

% F

ibro

sis

sco

re

*

***

NS

#

#

*p<0.05, p<0.001 vs SHAM; #p<0.001 vs WT UUO

WT

UUO

miR21-/-

UUO

miR-214-/-

UUO

92% reduction

From Denby et al., 2014. J Am Soc Nephrol. 25:65-80.

miR-21 and miR-214 are mediators of renal fibrosis.

Global

miR-214 -/-

miR-21 -/-

Anti-miR-214-3p as a potential therapeutic for renal fibrosis.

SHAM UUO UUO

+

Control Anti-miR

UUO

+

Anti-miR214

% F

ibro

sis

***

#

#

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

SHAM UUO Control Anti-miR Anti-miR214

86% reduction

SC

5mg/kg


0

2000

4000

6000

8000

10000

12000

14000

16000

Kidney Spleen Heart Liver Lung

pm

ol/g t

issue

Day -8 Day -1

Day 0

Surgery

UUO

or

sham

Day

7

Day -15

Anti-miR/

Control Anti-miR/

PBS

Anti-miR/

Control Anti-miR/

PBS

Anti-miR/

Control Anti-miR/

PBS

Sacrifice

-RNA

-Tissue for FFPE

-Protein

-Blood

SC SC SC

~5X

Favourable kidney biodistribution

Mechanism of action of miR-214

751 genes

upregulated

1160 genes

downregulated

DAVID Functional Annotation

Enriched Functional Annotations

Extracellular Matrix p-value 1E-13

Including

Collagen type 3, α 1

Collagen type 4, α 1

Collagen type 7, α1

MMP2

MMP14

TIMP2

Alpha SMA

1911 differentially expressed genes

analysed by Rank Products

>5000 differentially expressed genes

analysed by LIMMA

~20%

3’UTR predicted targets of miR-214

KIDNEY

Illumina microarray

UUO kidney (n=4)

Control Anti-miR treated vs Anti-miR-214 treated

• miRNA targets can be organ/cell type/injury specific!

• Algorithms give false positives – regulation of biogenesis and expression exist!

UUO kidney – Control anti-miR

UUO kidney – Anti-miR-214

Vs KIDNEY

Illumina microarray


CKD patient 1 CKD patient 2 CKD patient 3

*

scramble

H&E

miR-214

monocytes

Chronic kidney

disease

TGF-β

Smad3 ?

•Tubulointerstitial fibrosis

•Immune mediated glomerulosclerosis

•Hypertension induced damage

•Ischaemia/reperfusion injury

miR-214

Renal damage

miR-21 miR-214

?

monocytes

Chronic kidney

disease

TGF-βTGF-β

Smad3 ?Smad3 ?

•Tubulointerstitial fibrosis

•Immune mediated glomerulosclerosis

•Hypertension induced damage

•Ischaemia/reperfusion injury

miR-214

Renal damage

miR-21 miR-214

?

Renal damage

miR-21 miR-214

?

Li, L.-M., et al., The Journal of Immunology, 2011. 186(4): p. 2552-2560.

Is miR-214-3p a translatable target?


miRNAs in biopsy proven IgAN

Hennino et al, Scientific Reports, 2016

miR-214-3p and miR-21-

5p

are overexpressed in renal

tissue of patients with

moderate-severe fibrosis.

For example:

miR-214-3p

- with moderate fibrosis

(T1: median RQ = 2.20

[1.67–3.07])

- with severe fibrosis

(T2: median RQ = 2.48

[2.03–5.93])

miRNAs in transplantation

Is there a role of miR-214 in chronic allograft damage (CAD) ? CAD - significant cause of graft failure following renal transplantation. - characterised by interstitial fibrosis and tubular atrophy (IFTA). - leads to the failure of up to 5% grafts annually. - there are no specific therapies available.

BM12 C57Bl/6

CAD 12 wks

Murine Model 12 weeks Allograft

PSR

miR-214 expression

Isograft

miR-214 -/-

Victoria Banwell

??Potential Non-invasive biomarker

of CAD in combination with

imaging.

miRNA biomarkers

seNSOR Non-invasive biomarkers of renal disease

AIM:

- predicts functional outcome in IgAN patients (including in a validation cohort). n=100 historical with followup; n=400 validation cohort

- is limited to IgAN or is present in other renal diseases. n=300 generic age, sex and eGFR matched CKD patients with IgAN pts n=200.

- reflects the severity of renal pathological changes. n=75 newly diagnosed IgAN

510 CKD patients 54 native biopsies

? Treatment effect- disease modifiable biomarker would be useful.

- Identified a miRNA signature in IgAN patients that correlated with fibrosis on biopsy and eGFR.

0 1 2 3 5

0

1

2

3

4HO-1 mRNA

Day

RQ

HO

-1

Controls

Low HO-1

High HO-1

1b

0 1 2 3 5

0.0

0.5

1.0

1.5

2.0

RQ

miR

-A

Controls

Low HO-1

High HO-1

*

**

**

miR-A

**

*

*

*

***

**

**

a

-2 0

0

20

40

60

80

HO-1 Protein

D H

O - 1

P B

M C

p r o

t e i n

D 0

- D 1

***

***

n.s.

HA +

Surgery HA

miRNAs in transplantation

Rel

ativ

e ex

pre

ssio

n o

f H

O-1

mR

NA

Rel

ativ

e ex

pre

ssio

n o

f m

iRN

A A

PBMCs Katie Connor

Kim1

Col1a1

Egf

Up in UUO Down in UUO

miRNA as mediators of repair?

miR-214

miR-21

Reversible Unilateral ureteric obstruction model (rUUO)

RNA Seq

Picture – Spike Clay; https://www.jove.com/video/52559/a-murine-model-irreversible-reversible-unilateral-ureteric

miRNA as mediators of repair?

Cell type ?

CD68

sh

am

C

D3

1

uu

o C

D3

1

ruu

o C

D3

1

sh

am

F4

/80

uu

o F

4/8

0

ruu

o F

4/8

0

sh

am

LT

L

uu

o L

TL

ruu

o L

TL

sh

am

PD

GF

Rb

uu

o P

DG

FR

b

ruu

o P

DG

FR

b

0

5

10

15sham CD31

uuo CD31

ruuo CD31

sham F4/80

uuo F4/80

ruuo F4/80

sham LTL

uuo LTL

ruuo LTL

sham PDGFRb

uuo PDGFRb

ruuo PDGFRb

2 p

ow

er-

delt

a C

T

Kidney FACS - Endothelial cells - PDGFBR+ - Proximal Tubules - Gli +ve - Myeloid cells e.g. Macrophages

KIM-1 Col 1a Col 3a

Team Denby

Vasudev Menon

Lorna Marson

Prof S Wigmore

Bryan Conway

Carolynn Cairns

Prof Jeremy Hughes

David Ferenbach

Jon Manning

Patrick Mark

Prof E van Rooji

Prof E Olson

Emily McQuarrie

Prof Jon Barratt

Kate Stevens

Alison Taylor

Prof Alan Jardine

Prof Andy Baker

Liz, Hayley, Deborah, Rowan – Clinical Research Nurses

Renal and Transplant Patients of Glasgow and Edinburgh.

Acknowledgements

micrornas in renal fibrosis and transplantation

Documents