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)
From Denby et al., 2011. Am J Pathol. 179:661-72.
From Denby et al., 2011. Am J Pathol. 179:661-72.
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
From Denby et al., 2014. J Am Soc Nephrol. 25:65-80.
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
From Denby et al., 2014. J Am Soc Nephrol. 25:65-80.
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?
From Denby et al., 2014. J Am Soc Nephrol. 25:65-80.
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
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