in vivo microscopy - hÔpital necker · in vivo microscopy janos peti-peterdi professor department...

In vivo microscopy

Janos Peti-Peterdi Professor

Department of Physiology and Biophysics, Zilkha Neurogenetic Institute

Keck School of Medicine

University of Southern California

1

Outline Visualization of

renal/glomerular

structure/function

Pathology/podocyt

e injury

• Quantitative

imaging of organ

functions (GFR, RBF,

RAS elements)

•Serial MPM imaging

of the same glom.

over time in vivo

•New mouse models

with fluorescent cell

lineage tags

•Tracking cell fate,

function, migration

•Podocyte shedding

•Leaky GFB, GFR↓

•Cell-to cell

propagation of

injury, calcium

signaling

Tissue remodeling,

regeneration

•The highly dynamic

glomerular

environment

•New concept on

endogenous nephron

repair master

program

•Role of body salt

and fluid

conservation, classic

salt sensing

mechanisms

Imaging approach using in vivo multiphoton microscopy

Dunn et al AJP Cell Physiol. 283: C905-16, 2002.

Kang et al AJP Renal Physiol. 291: F495-502, 2006.

Look inside the intact, living kidney

Observe the function of cells, vascular/tubular structures

photon 1 photon 2

focal plane Low Energy

Ar HeNe MP

IR

680-1300nm

Abdominal Imaging Window

Oscillations in glomerular/tubular function

Time (s) 0 50 100 150 200 250 300 Flu

ore

scen

ce

in

ten

sit

y

Glom

PT

CCD

Frequency: 0.12 Hz (myogenic)

0.023 Hz (TGF)

Period: 6-10 s (myogenic)

20-50 s (TGF)

• The mechanisms of glomerular injury/dysfunction,

albuminuria are incompletely understood due to technical

limitations in studying the GFB in its native environment in

vivo.

• Multiphoton microscopy is able to visualize the intact GFB

with high temporal and spatial resolution in the intact living

kidney.

Glomerular structure and function

Urinary pole

Vascular pole

albumin

podocytes

70kDa dextran

quinacrine

Lucifer yellow

ROI1

ROI2

C

Time (s) 2 4 6 8 10 12 14

LY

in

ten

sit

y X

10

3

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

ROI1

ROI2

Dt

A G

PT

D S

NG

FR

(n

l/m

in)

ROI2 distance (µm)

B

G

0

20

40

60

0 100 200 300 400 500

Quantitative imaging of kidney functions - SNGFR

Kang et al AJP Renal Physiol 291: F495-502, 2006

70kDa dextran

quinacrine

Lucifer yellow

SNGFR

Control STZ-

diabetes STZ+ARB

SN

GF

R (

nL

/min

)

0

10

20

30

40

50

60

70

80

*

ns

Control

STZ-diabetes

Renal blood flow - RBC velocity

70kDa dextran

quinacrine

B

Tim

e (

s)

0

2.0

0 20 X(µm)

Dx Dt

C D A

PT DT

DT

Renal blood flow - RBC velocity

Kang et al AJP Renal Physiol 291: F495-502, 2006

Multiphoton imaging allows high sensitivity measurement of glomerular permeability

©2012 by American Society of Nephrology

Nakano D et al. JASN 2012;23:1847-1856

500 kDa dextran inulin

Glomerular albumin permeability

Progressive glomerular pathology

in a mouse model of FSGS (NEP25 mice) Day 1 Day 3

Day 4 Day 5

Podocyte-GFP

Alexa594-albumin

Prorenin receptor

A

AA

G

B

CCD G

The two sites of (patho)physiological renin expression

in the renal cortex

Classic vascular site - JGA CNT - CCD

Renin

Alexa594-albumin

JGA

AA

AA

CCD

CCD

Renin content in

both the JGA and

CCD increased in

untreated

diabetes. However,

olmesartan

treatment

increased JGA

renin, but

decreased CCD

renin.

Renin content in the diabetic kidney

Un-

treated

ARB

JGA CCD renin

Kang JJ et al, Hypertension 51: 1597-604, 2008

Renin

Alexa594-albumin

Lasagni L and Romagnani P. J Am Soc Nephrol 2010.

Tracking the fate of podocytes in vivo using serial

multiphoton imaging in mice

with fluorescent lineage tags

t=0 h t=24 h t=48 h

Cell tracking by serial MP imaging of same glomerulus

every 24 hours

Hackl et al. Nat. Med. 2013

MPM imaging in vivo reveals signs of podocyte migration

in the intact kidney after UUO

Glo

meru

li w

ith

pa

rieta

l G

FP

(%

)

Days after UUO Ctrl UUO

0

20

40

60

80

100

Glo

meru

li w

ith

pa

rieta

l G

FP

(%

)

0

20

40

60

80

100

*

a b c

d e f

g h i 0 20 40 60 80 100 120 140 160


GFP+ cells in the parietal layer after UUO


Identification and tracking of single podocytes in the new

multi-color Pod-Confetti mouse model using in vivo MPM

imaging

Cell 2010

Pod-Confetti after UUO


Zariwala et al, J. Neuroscience (2012)

Podocin GCaMP3

New mouse models with cell-specific expression

of the genetically encoded calcium indicator GCaMP3

Mice expressing GCaMP3

in:

• Podocytes

X

Burford JL et al, J Clin Invest, 2014

Podocyte calcium waves visualized in vivo in the new Pod-

GCaMP3 mouse model using MPM imaging

0s 20s 40s

0

1

2

3

Ctrl ANGII injury

ΔF

/F0

*

*


Podocyte calcium waves visualized in vivo in the new Pod-

GCaMP3 mouse model using MPM imaging


0

200

400

600

Ctrl Suramin

0

400

800

1200

Ctrl/WT Suramin P2Y2-/-

0

4

8

12

Ctrl/WT Suramin P2Y2-/-

*

* * * *

F b

as

eli

ne

(U

)

ΔF

(U

)

Velo

cit

y (

µm

/s)

The effects of pharmacological (suramin) or genetic (P2Y2-/- mice)

P2Y2 blockade on the propagation of podocyte [Ca2+]i wave

induced by podocyte injury


Tracking TRPC6-mediated podocyte [Ca2+]i and glomerulopathy

development in

vivo

in the

same glomerulus

GC

aM

P3 F

(×

10

3)

Pod-GCaMP3

/WT

Pod-GCaMP3

/TRPC6KO

Pod-

GCaMP3

/TRPC6TG

0

10

20

30

‡

‡

* †

40 Baseline ANG II

B

*

Baselin

e

14

days a

fter

AN

G I

I in

fusio

n

Pod-GCaMP3

/TRPC6-TG Pod-GCaMP3

/TRPC6-KO

Pod-GCaMP3

/WT A

Intravital imaging of podocyte cell functions

Highly sensitive foot process and podocyte [Ca2+]i imaging

in iPod-Confetti and Pod-GCaMP5/Tomato mice

Fixed tissue SEM In vivo MPM in mouse

Intravital imaging of podocyte

cell functions

Highly sensitive podocyte

[Ca2+]i imaging

in Pod-GCaMP5/Tomato mice

x y

z

Tomato

Ca2+

Ca2+

Albumin

Experimental approach to study renal stem cells:

(1) Transgenic mouse models, genetic cell fate mapping

mesenchymal progenitor cell lineages

NG2-CreERT2-Tomato; Ren1d-Confetti; Foxd1-CreERT2-Confetti

(2) Serial intravital imaging of same glomerulus over days,

tracking cell fate Urinary pole

Vascular pole

A

C

B

• Migration pattern

• Dynamics

• Function

• Heterogeneity

Progenitor cells

G

G

Visual clue: Progenitor cells home to the glomerular entrance

NG2-CreERT2-Tomato mice Low salt diet + ACEi

500 kDa dextran-Alexa 488

Macula densa cells may control the homing of

mesenchymal progenitor cells

to the glomerular vascular entrance

TAL

DT ? NO

PGE2

Macula Densa (MD) cells: chief medical doctor of nephron?

• Strategic anatomical localization at the glomerular entrance

• Upside-down cells, organelle-rich basal region facing the glomerulus

• Very thin, fragmented basement membrane

MD

Brenner and Rector’s The Kidney

The nephron: evolutionary and developmental links

Romagnani, P. et al. (2013) Renal progenitors: an evolutionary conserved strategy for kidney regeneration Nat. Rev. Nephrol. doi:10.1038/nrneph.2012.290

• Migration from sea to land

• Successful adaptive

mechanisms in nephron

structure and function to

conserve body salt and fluids

• Strategic localization of

macula densa cells during

nephron development

• Elongation of loop of Henle

after birth

• Hypothesis: Master program exists for nephron repair

• We need to learn from evolution, physiology, adaptation

to environment

• Loss of body fluid and salt, the most robust and

organ-specific stimulus

• Apply and augment strategy in disease

"We can't solve problems by using the

same kind of thinking we used when we

created them." - Albert Einstein

•Nephrology research focuses on injured kidney – a failure

(in repair)

•Physiology research focuses on healthy organ,

adaptive changes in kidney structure and function – a success

(in remodeling)

Physiology-based concept

The condition associated with the

loss of body fluid and salt may be the

most appropriate and effective stimulus

for nephron-level kidney repair

Baseline Day 3 Day 5

Day 7 Day 10 Day 13

Tracking the same glomerular area over two weeks LS+ACEI:

NG2-Tomato cells home to the glomerular vascular pole

Progenitor cells 500 kDa dextran-Alexa 488

G

Serial multiphoton imaging of cells of the NG2 lineage

in NG2-CreERT2-Tomato mice

Progenitor cells

Robust increase in renal cortical NG2+ cell density

in response to 5 days low salt+ACEI diet

Ctrl Low salt+ACEI

G G

G

G

PTs 0

10

20

30

40

50

60

70

80

Ctrl LS+ACEI

# N

G2+

cell

s p

er

fie

ld

* C

Similar results after 10 days

furosemide treatment G

NG2-Confetti

AA2

G1

G2

AA1

H NG2-Confetti

AA2

G1

G2 AA1

G3 AA3

I

AA

ɑ-SMA NG2-Tomato A renin NG2-Tomato

20%

Ng2+ 80%

Ng2-

DAPI B renin NG2-Tomato

77%

Ng2+

23%

Ng2-

DAPI C

G

AA G

AA

G

D E F G PDGFRβ

NG2-Tomato

G

Claudin-1 NG2-Tomato

G

Podocin NG2-Tomato

G

Villin NG2-Tomato

PTs

Tracking the fate of mesenchymal progenitor (NG2+) cells

Low salt+ACEi treated mice, selective COX-2 inhibition with SC58236

COX-2 and nNOS inhibition (MD cell markers) block

NG2+ cell homing to the glomerular vascular pole

Control LS+ACEi

NG2 cell

DAPI

LS+ACEi+COX

2i LS+ACEi

+7-NI

*

# #

*p<0.05 vs. control #p<0.05 vs. LS+ACEi

Low salt+ACEi treated mice, selective nNOS inhibition (7-NI)

0

2

4

6

8

10

12

14

16

18

LS+

ACEi

LS+ACEi+

7-NI

Ctrl Ctrl+

7-NI N

um

ber

of

Ki6

7+

cell

s p

er

JG

A a

rea

*

§

Ki67

DAPI

Control

Ctrl+7-NI

LS+ACEi

LS+ACEi

+7-NI

nNOS inhibition (MD cell marker) blocks

NG2+ cell proliferation at the glomerular vascular pole

Baseline Day 3 Day 6

Day 9 Day 12

Tracking the same glomerular area over two weeks NT IgG:

Ren1d-Confetti cells migrate along the PEC layer into PT

G

G

MD→

MD cell isolation and bioinformatics workflow

GFP mouse model

nNOS/CreERT2-mTmG/fl

Digestion and harvest Isolation

Transcriptome and bioinformatics Confirmation

Macula densa-specific expression of novel angiogenic

secreted proteins in the human kidney

MD→

←MD

Pappa-2 CCN1/Cyr61

Pregnancy-associated plasma protein A2

• Cleaves IGFBP-5

• Local regulator of IGF bioavailability

• Tissue hypertrophy

Cyr61 (Cystein-rich protein 61)

• αVβ5 Integrin binding

• NF-kB signaling

• Angiogenesis

CYR61 Expression in Ju CKD 2 Discovery Cohort: Minimal Change Disease vs. Healthy Living Donor

Under-expression Gene Rank: 39 (in top 1%) P-value: 5.55E-7

Reporter: 3491 t-Test: -5.818

Fold Change: -2.145

Legend

0. No value (42) 1. Healthy Living Donor (31) 2. Arterial Hypertension (20) 3. Diabetic Nephropathy (17) 4. Focal Segmental Glomerulosclerosis (17) 5. IgA Nephropathy (25) 6. Lupus Nephritis (32) 7. Membranous Glomerulonephropathy (18) 8. Minimal Change Disease (14) 9. Thin Basement Membrane Disease (6) 10. Vasculitis (21)

Ju CKD 2

Sci Transl Med 2015/12/01 243 samples

mRNA 17,379 measured genes

Affymetrix Human Genome U133 Plus 2.0 Array (altCDF v10)

© 2015 The Regents of The University of Michigan. Images from Nephroseq may be used in publications with proper citation. The citation is as follows: Nephroseq (The

Regents of The University of Michigan, Ann Arbor, MI) was used for analysis and visualization. For further information, refer to the terms of use. Nephroseq Source: https://www.nephroseq.org/resource/main.html#a:1N10406;cv:detail;d:1N156636767;dso:geneUnderex;dt:predefinedClass;ec:[1N2];epv:1N1.1N3;et:under;f:195810567;g:3491;gt:boxplot;p:1N200014695;pg:1;pvf:9809,1N9,1N800098054;scr:datasets;ss:all;th:g100.0,p0.050,fc0.0;v:17

Macula densa cells may function as inducers and master

regulators of mesenchymal progenitor cell-mediated

vascular, glomerular, and tubular remodeling

TAL

DT

NO

PGE2

Pappa2

CCN1

Dysfunctional macula densa cells as

the root cause of glomerular disease?

Future goal

• Fine-tuning of the MD-targeting regenerative approach

• Identification and activation of the critical molecular players

THANK YOU

Peti-Peterdi lab

Anne Riquier-Brison

Kengo Kidokoro

Toshiki Doi

Ju-Young Moon

Georgina Gyarmati

Jasmine Castillejos

Donna Ralph

Urvi Shroff

Jim Burford

Haykanush Gevorgyan

Lisa Lam

Karie Villanueva

Sarah Vargas

Ildiko Toma

Arnold Sipos

McMahon lab

Sanjeev Kumar

Jing Liu

Institute of Urology

Inderbir Gill

Andre Abreu

Nariman Ahmadi

AHA ADA

UKRO Amgen

U Regensburg, Germany

Ina Schieβl

Hayo Castrop

U Southern Denmark, DK

Per Svenningsen

Waitemata DHB/U Bristol

Andy Salmon

U Washington

Stuart Shankland

Jeff Pippin

Kagawa U, JPN

Daisuke Nakano

Akira Nishiyama

U Cologne, Germany

Thomas Benzing

Bernhard Schermer

Matthias Hackl

Agnes Prokai

Mario Negri, Bergamo, IT

Paola Rizzo

Ariela Benigni

Giuseppe Remuzzi

DK64324, DK10944

S10RR024754

SE/UAB/USC

Laszlo Rosivall

Darwin Bell

Alicia McDonough

in vivo microscopy - hÔpital necker · in vivo microscopy janos peti-peterdi professor department...

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