thengalreportermousedetects! the!response!ofthe!! … · supplementaryinformation!guide! ......

The Ngal Reporter Mouse Detects the Response of the Kidney to Injury in Real Time

Neal Paragas*1, Andong Qiu1*, Qingyin Zhang1, Benjamin Samstein1, Shi-Xian Deng1,

Kai M. Schmidt-Ott2, Melanie Viltard1, Wenqiang Yu1, Catherine S. Forster1, Gangli

Gong1, Yidong Liu1, Ritwij Kulkarni1, Kiyoshi Mori3, Avtandil Kalandadze1, Adam J.

Ratner1, Prasad Devarajan, Donald W. Landry1, Vivette D’Agati1, Chyuan-Sheng Lin1

and Jonathan Barasch†1.

1College of Physicians and Surgeons of Columbia University New York, N.Y. 10032 2Max-Delbruck Center for Molecular Medicine Berlin, Germany 3Kyoto University Graduate School of Medicine, Kyoto, Japan.

Nature Medicine: doi:10.1038/nm.2290

Supplementary Information Guide

A. Supplementary Methods

1. Western Blot

2. In situ hybridization and immunohistochemistry.

3. Kidney ischemia and cross transplantation

B. Supplementary Table 1: Sequence and primer information.

C. Supplementary Figures

1. Supplementary Fig. 1: Generation of the Ngal-Luc2/mC difusion reporter murine

model.

2. Supplementary Fig. 2: Functional verification of the Luc2/mC di-fusion reporter

gene.

3. Supplementary Fig. 3: PCR screening of the Ngal-targeted ES cell clones.

4. Supplementary Fig. 4: Ngal-Luc2/mC reported kidney damage in vivo after

ischemia reperfusion injury.

5. Supplementary Fig. 5: (a) Kidney Ngal-luc2 expression (left panel and outlined)

originates from injured kidney

6. Supplementary Fig. 6: uNgal derives from the kidney.

7. Supplementary Fig. 7: Neutrophils were not required for the expression of Ngal

in the kidney.

8. Supplementary Fig. 8: uNgal originates from the nephron.

9. Supplementary Fig. 9: Apoptotic S3 proximal tubule.

10. Supplementary Fig. 10: (a) Ngal-mCherry fluorescence was visible in the TAL

and CD.

11. Supplementary Fig. 11: Experimental NF-κB inhibitors

12. Supplementary Fig. 12: Model of sNgal reabsorption and uNgal excretion.


Supplementary Methods

Western Blot Urine and recombinant mouse Ngal standards were immunobloted using

polyclonal antibody to Ngal (R&D Systems, Minneapolis) and donkey anti-rabbit HRP-

labelled IgG antibodies (Jackson Immunoresearch).

In situ hybridization and immunohistochemistry. The paraffin sections were

dewaxed and then rehydrated by using Histoclear (Fisher Scientific) and a gradient of

ethanol, respectively, before in situ hybridization. Specific digoxigenin-labeled antisense

riboprobes were generated from mouse Ngal cDNA (Genbank accession number:

NM_008491) by using a Dig-labelling kit (Roche Applied Biosystems) and hybridized and

detected as previously described [(Li, J.Y., et al. Scara5 is a ferritin receptor mediating

non-transferrin iron delivery. Developmental cell 16, 35-46 (2009)). The hybridized

sections were counterstained with methyl green, dehydrated and mounted in Permount

(Fisher Scientific). Frozen and paraffin-embedded sections were used for

immunohistochemical analysis. Anti-mCherry (Clontech) and anti-v-ATPase B1/2 (Santa

Cruz Biotechnology) were used at a 1:50 dilution and antigen was localized by HRP-

DAB chromogen (R&D Systems) staining.

Kidney ischemia and cross transplantation The inferior vena cava (IVC) was

cannulated, and ice-cold heparinized saline (1mL, 10units/mL) administered via the IVC.

The kidneys were removed from the retroperitoneum with the aorta and the vena cava

en bloc, and the ureters were removed with a large patch of bladder. The recipient’s left

kidney was then removed following ligation of the vessels and cauterization of the ureter,

and its abdominal IVC and aorta exposed from the renal vessels to the iliac bifurcation.

The donor aorta and IVC were anastomosed to the recipient’s aorta and IVC using 10-0

suture, and the kidney graft reperfused. The donor ureter was inserted into the recipient

bladder and fixed by suturing and the abdominal incision was then closed. Four days

later, the previous incision was reopened and the recipient’s right kidney was removed,

and the abdominal incision was closed again. Both closure sutures are absorbable so

that suture removal was not necessary. Each mouse was given a sub-cutaneous

injection of Lactated Ringers (1-2 cc) that was pre-warmed to body temperature, and

was placed on warming water-blanket. Supplementary oxygen was administered during

and immediately post-operatively to minimize hypoxia.


Supplementary Table 1: Sequence and primer information.


Supplementary Figures

Supplementary Fig. 1: Generation of the Ngal-Luc2/mC difusion reporter murine model.

(a) The Ngal targeting BAC DNA was constructed by knockin of the Luc2/mC gene with

an LNL cassette (light blue box) at the translational start codon of the Ngal gene (Ngal

exon represented by grey box and UTR represented by dark blue). A DTA-Ampr

cassette, a negative selection marker, was placed 2kb downstream of the LNL cassette

in place of a 1kb DNA fragment. (b) The Ngal-targeting construct was electroporated into

KV1 ES cells, and homologous recombination led to the Ngal-targeted ES cells. (c) The

Ngal-targeted ES cells responded to 1mM cyanide and the TLR agonist, lipid A (4µg/ml)

with Luciferase luminescence. (d) PCR genotying of germline-transmitted F1 pups with

primers F1, R1 and R2 identified both the targeted and the wild type alleles at 476bp and

345bp, respectively. (e) The correct integration of the Luc2/mC gene in Ngal-targeted F1

mice was verified by long distance PCR (6819bp) with primers F2 and R3.

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Supplementary Fig. 2: Functional verification of the Luc2/mC di-fusion reporter gene.

The Luc2/mC gene was cloned into pCI-neo, and transiently expressed in HeLa cells.

After 24 hours, luminescence and fluorescence were imaged in a Zenogen Bioimager.

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Supplementary Fig. 3: PCR screening of the Ngal-targeted ES cell clones. (a) Ngal

targeting construct and location of screening primers. (b, c) PCR screen for Ngal

targeted ES clones using primers F3 and R3 (b) and F1 and R3 (c).

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Supplementary Fig. 4: Ngal-Luc2/mC reported kidney damage in vivo after ischemia

reperfusion injury. (a) Heterozygous Ngal-Luc2/mC male mouse received 30 min of

unilateral ischemia (left kidney) and then was visualized at 0, 6, 12 and 24 hours after

reperfusion in a Zenogen Bioimager to detect Ngal-Luc2/mC activity. Peak expression of

Ngal-Luc2/mC occurred at 12 hours after reperfusion. Ngal-Luc2/mC was tonically

expressed in the testis which could occupy inguinal or pelvic sites. (b) Immunoblot of

uNgal at time-points in parallel with the assays of kidney Ngal-Luc2/mC activity. (c)

Heterozygous Ngal-Luc2/mC male mouse received 30 min of unilateral I/R (right kidney),

and was then visualized at 12 hours after reperfusion in a Zenogen Bioimager to detect

Ngal-Luc2/mC activity.

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Supplementary Fig 5: (a) Kidney Ngal-luc2 expression (left panel and outlined)

originates from injured kidneys after 30 min of bilateral I/R (right panel and outlined). (b)

Ngal expression in the lung was induced by lipid A inhalation. Ngal-Luc2 was induced

within 6 hours after aspiration of 10 µg of lipid A.

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Supplementary Fig. 6: uNgal derives from the kidney. Cross-transplantation of kidneys

between Ngal-/- and Ngal +/+ mice was followed by ischemia-reperfusion injury for 10 min.

(a) Ngal mRNA was markedly upregulated in Ngal+/+ kidneys placed in Ngal+/+

(228.1±18.8 fold) or in Ngal-/- hosts (184.6±56.7 fold), but not in Ngal-/- kidneys located in

the Ngal+/+ hosts (6.3±0.86 fold). Liver Ngal was not elevated 24 hours after ischemia-

reperfusion (n=15). Kim1 was significantly induced in ischemic wild-type to wild-type,

wild-type to knockout, and knockout to wild-type kidneys (7.9±0.4 fold, 7.8±3.8 fold and

10.2±4.8 fold, respectively) 24 hours after 10 min of ischemia. (b) Average urinalysis of

uNgal collected from transplants at 0, 12, and 24 hours after ischemia reperfusion injury,

revealing that uNgal increased when a Ngal+/+ kidney was placed in a Ngal+/+ (WT->WT;

n=6) or in a Ngal-/- host (Wt->KO; n=3), while there was a smaller change in uNGAL

when a Ngal-/- kidney was placed in a Ngal+/+ host (KO->WT; n=2).

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Supplementary Fig 7: Neutrophils were not required for the expression of Ngal in the

kidney. (a, b) FACS analysis of blood cells from RB6-8C5-treated mice showed that the

peripheral neutrophil population was ablated (a; region R1), whereas this population was

visible in control IgG2B-treated mice (b; region R1). (c, d) Ngal mRNA was expressed

by nephrons, 24 hours after 30 minutes of ischemia, as shown by in situ hybridization,

with (c) or without (d) the ablation of neutrophils.

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Supplementary Fig 8: Ngal is expressed by the nephron. (a, b) Ngal mRNA is

expressed by nephrons after 30 min of ischemia. Prominent expression was found in the

TAL and CD. (c) Ngal-mC protein was faithfully expressed in the same cells as the Ngal

mRNA. Serial sections showed that Ngal mRNA (in situ hybridization; Left panel) was

expressed in dilated tubules containing casts (H&E; middle panel), and colocalized with

mC reporter protein (immunohistochemical staining with an anti-mC antibody and DAB

chromogen visualization; right panel). (d) Unilateral ureteral obstruction (UUO) model

induced Ngal mRNA, which was visualized by in situ hybridization.


Supplementary Fig 9: Apoptotic S3 proximal tubule cells (top; boxed area) were

detected by Tunel assay. They do not overlap with Ngal expressing medullary rays and

CD cells (bottom; open arrowheads).

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Supplementary Fig 10: (a) Ngal-mCherry fluorescence was visible in the TAL (closed

arrowheads), but not in the adjacent thin Limbs of Henle (open arrowheads). (b) Ngal-

mCherry fluorescence was visible in the alternating epithelial cells of the CD revealing

specific expression of Ngal mRNA in α-IC (closed arrowheads).

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Supplementary Fig 11: Experimental NF-κB inhibitors

InhibitorA:3-methyl-5H-Quino[8,7-c][1,2]benzothiazine-6,6-dioxide (Analogue 30)26

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InhibitorC:1-(5-methoxy-2-(thiophen-2-yl)quinazolin-4-ylamino)-3-methyl-1H-pyrrole-

2,5-dione (Analogue 30)25.

InhibitorD:5-bromo-N-(2-methylquinolin-8-yl)thiophene-3-sulfonamidepyrrole-2,5-dione

(Analogue 27)26.

MG132:(S)-4-methyl-N-((S)-4-methyl-1-((S)-4-methyl-1-oxopentan-2-ylamino)-1-

oxopentan-2-yl)-2-(3-phenylpropanamido)pentanamide24.

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Supplementary Figure 12: Model of sNgal reabsorption and uNGAL excretion in two

compartments of the kidney.


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