Mice that overexpress human heat shock protein 27 have ... · phil inﬁltration in the huHSP27 OE mice relative to the HSP27 WTmice at 3h after renal IRI (Figure 6). At 24h after

Mice that overexpress human heat shock protein 27have increased renal injury following ischemiareperfusionSean W.C. Chen1, Minjae Kim1, Mihwa Kim1, Joseph H. Song1, Sang Won Park1, Dominic Wells2,Kevin Brown1, Jacqueline de Belleroche2, Vivette D. D’Agati3 and H. Thomas Lee1

1Department of Anesthesiology, Anesthesiology Research Laboratories, College of Physicians and Surgeons, Columbia University, NewYork, New York, USA; 2Department of Neuromuscular Diseases, Faculty of Medicine, Imperial College, London, UK and 3Department ofPathology, College of Physicians and Surgeons, Columbia University, New York, New York, USA

We previously showed that activation of the A1 adenosine

receptor protected the kidney against ischemia–reperfusion

injury by induction and phosphorylation of heat shock

protein 27 (HSP27). Here, we used mice that overexpress

human HSP27 (huHSP27) to determine if kidneys from these

mice were protected against injury. Proximal tubule cells

cultured from the transgenic mice had increased resistance

to peroxide-induced necrosis compared to cells from wild-

type mice. However, after renal ischemic injury, HSP27

transgenic mice had decreased renal function compared to

wild-type mice, along with increased renal expression of

mRNAs of pro-inflammatory cytokines (TNF-a, ICAM-1,

MCP-1) and increased plasma and kidney keratinocyte-

derived cytokine. Following ischemic injury, neutrophils

infiltrated the kidneys earlier in the transgenic mice. Flow

cytometric analysis of lymphocyte subsets showed that those

isolated from the kidneys of transgenic mice had increased

CD3þ , CD4þ , CD8þ , and NK1.1þ cells 3 h after injury. When

splenocytes or NK1.1þ cells were isolated from transgenic

mice and adoptively transferred into wild-type mice there

was increased renal injury. Further, depletion of lymphocytes

by splenectomy or neutralization of NK1.1þ cells resulted in

improved renal function in the transgenic mice following

reperfusion. Our study shows that induction of HSP27 in

renal tubular cells protects against necrosis in vitro, but its

systemic increase counteracts this protection by exacerbating

renal and systemic inflammation in vivo.

Kidney International (2009) 75, 499–510; doi:10.1038/ki.2008.572;

published online 19 November 2008

KEYWORDS: inflammation; keratinocyte-derived cytokine; lymphocyte;

natural killer cells; neutrophil

Acute renal failure (ARF) is a disease characterized by highpatient morbidity and mortality.1–3 Despite considerableresearch, there is no effective therapy for ARF.1 Ischemiareperfusion injury (IRI) is the major cause of ARF and occursfrequently due to the obligatory interruption of blood flowand undesirable hemodynamic changes during the peri-operative period.4–6

Our laboratory previously demonstrated that exogenousand endogenous A1 adenosine receptor (AR) activationprotected against renal IRI in mice7,8 as well as in rats.9,10

Mechanistically, we have previously shown that A1ARactivation phosphorylates heat shock protein 27 (HSP27) incultured renal proximal tubule cells and chronic activation oroverexpression (OE) of A1ARs leads to upregulation of totalHSP27.11 This finding was replicated in vivo, as mice treatedwith a selective A1AR agonist, 2-chloro-N6-cyclopentylade-nosine, before renal ischemia showed a reduction in renalcorticomedullary necrosis, apoptosis, and inflammation thatcorresponded with an increase in HSP27 expression.12

HSP27 is a member of family of chaperone proteins thatare upregulated in response to increases in temperature, aswell as a wide range of cellular stresses including hypoxia,ischemia, and exposure to toxic drugs.13–16 Increasedexpression of HSP27 serves to defend a cell against injuryor death by acting as chaperones facilitating properpolypeptide folding and aberrant protein removal.17–19

Furthermore, HSP27 is a potent antiapoptotic protein andis a key stabilizer of the actin cytoskeleton; both of thesecellular effects lead to increased resistance against celldeath.20–22 Not surprisingly, OE of HSP27 protected againstneuronal and cardiac injury.23–25

In this study, we tested the hypothesis that mice withglobal OE of HSP27 would show an increased resistanceagainst renal IRI. We determined in this study that althoughproximal tubules cultured from huHSP27 OE mice wereprotected against necrosis in vitro, huHSP27 OE mice in vivohad paradoxically worsened renal dysfunction compared toHSP27 wild-type (WT) mice. We subsequently tested thehypothesis that increased renal dysfunction in huHSP27 OE

http://www.kidney-international.org o r i g i n a l a r t i c l e

& 2009 International Society of Nephrology

Received 20 February 2008; revised 16 September 2008; accepted 23

September 2008; published online 19 November 2008

Correspondence: H. Thomas Lee, Department of Anesthesiology, Anesthe-

siology Research Laboratories, College of Physicians and Surgeons, Columbia

University, P&S Box 46 (PH-5), 630 West 168th Street, New York, New York

10032-3784, USA. E-mail: [email protected]

Kidney International (2009) 75, 499–510 499

http://dx.doi.org/10.1038/ki.2008.572

http://www.kidney-international.org

mailto:[email protected]

mice is due to the increased leukocyte activation andenhanced renal tubular inflammation after renal ischemiareperfusion (IR).

RESULTSHuman HSP27 mRNA and protein expression in huHSP27 OEmice

Figure 1 shows a selective human HSP27 transgene expres-sion in huHSP27 OE mice. huHSP27 OE and WT miceexpressed equivalent mRNA and protein expression ofmurine (endogenous) HSP27. However, only the huHSP27OE mice expressed human HSP27 mRNA and proteindistinguishable due to its larger size as a hemagglutinin-fused protein (Figure 1).

Renal proximal tubules from huHSP27 OE mice show reducednecrosis after H2O2 injury

Treatment (6 h) with 1–5 mM hydrogen peroxide (H2O2)caused rapid necrosis of proximal tubule cells isolated andcultured from mice (Figure 2; N¼ 6 for each group). Renalproximal tubules cultured from the huHSP27 OE miceshowed increased resistance against H2O2-induced necrosiswith reduced lactate dehydrogenase released when comparedto the HSP27 WT mice proximal tubules (Figure 2).

huHSP27 OE mice showed increased renal injury after IR

After 30 min of renal ischemia and 3 h reperfusion, plasmacreatinine (Cr, mg/100 ml) rose significantly in both HSP27WT (Cr¼ 0.6±0.1, N¼ 6) and huHSP27 OE mice(Cr¼ 1.0±0.1, N¼ 6) compared with sham-operated miceat 3 h after surgery (Cr¼ 0.27±0.02, N¼ 6 for HSP27 WTsham-operated mice and Cr¼ 0.33±0.03, N¼ 5 for

huHSP27 OE sham-operated mice). Plasma Cr rose evenhigher at 24 h after renal ischemia in both HSP27 WT(Cr¼ 1.5±0.2, N¼ 8) and huHSP27 OE mice(Cr¼ 2.2±0.2, N¼ 8). However, huHSP27 OE mice showedsignificantly higher rise in plasma Cr at both 3 (Po0.05) and24 h (Po0.05) after reperfusion (Figure 3).

Because of the concerns for the potential geneticvariability associated with non-congenic strain of mice, webred our huHSP27 OE and HSP27 WT mice with C57BL/6mice for three generations. From the resulting male mice, wehave performed renal IR experiments and show that the

Mouse HSP27 protein

Human HSP27 protein

Mouse HSP27 mRNA

Human HSP27 mRNA

GAPDH mRNA

HSP27WT mice

HSP27OE mice

Figure 1 | Selective expression of human HSP27 mRNA andprotein in HSP27 OE mice. (a) Representative gel images ofsemiquantitative RT-PCR results of GAPDH, human HSP27, andmouse HSP27 mRNAs from HSP27 WT and huHSP27 OE mouserenal cortices. (b) Representative immunoblotting images forhuman HSP27 and mouse HSP27 protein expression in HSP27 WTand huHSP27 OE mouse renal cortices. Representative imagesfrom four experiments are shown.

0 1 2 3 4 50

10

20

30

40

HSP27 WT mice

HSP27 OE mice

*

*

*

mM [H2O2], 6 h treatment

% L

DH

rel

ease

d

Figure 2 | LDH release after vehicle or H2O2 treatment(0–5 mM) for 6 h in proximal tubule cells cultured from HSP27WT or huHSP27 OE mice. *Po0.05 vs LDH released fromproximal tubules isolated from HSP27 WT mice.

HSP27 W

T Sha

m

HSP27 W

T 3h

HSP27 W

T 24h

HSP27 O

E Sha

m

HSP27 O

E 3h

HSP27 O

E 24h

0.0

0.5

1.0

1.5

2.0

2.5

*

*#

*

*#

Pla

sma

Cr

(mg/

100

ml)

Figure 3 | Plasma creatinine (Cr in mg/100 ml) from sham-operated mice (Sham) or mice subjected to ischemia-reperfusion (IR). For the IR groups, plasma Cr were obtained at 3(IR 3 h) and 24 h (IR 24 h) after reperfusion. *Po0.05 vsappropriate sham. #Po0.05 vs HSP27 WT IR at appropriate time.Data are presented as mean±s.e.m.

500 Kidney International (2009) 75, 499–510

o r i g i n a l a r t i c l e SWC Chen et al.: HSP27 and renal ischemia reperfusion injury

huHSP27 OE mice backcrossed with C57BL/6 mice(Cr¼ 3.2±0.2 mg/100 ml, N¼ 7) show increased renalinjury after 30 min renal ischemia and 24 h reperfusioncompared to the HSP27 WT mice backcrossed with C57BL/6mice (Cr¼ 2.2±0.2 mg/100 ml, N¼ 6). Therefore, OE ofhuHSP27 in mice backcrossed with C57BL/6 mice continuedto result in increased renal injury after IR. We also note thatthe HSP27 WT mice backcrossed with C57BL/6 now showsimilar plasma Cr compared to the C57BL/6 mice subjectedto renal IR (2.2±0.3 mg/100 ml, N¼ 6). Moreover, theHSP27 WT and huHSP27 OE mice backcrossed withC57BL/6 mice show higher plasma Cr compared to theplasma Cr values of original HSP27 WT (Cr¼ 1.5±0.2 mg/100 ml, N¼ 8) and huHSP27 OE (Cr¼ 2.2±0.2, N¼ 8) micebefore backcrossing.

Renal tubular necrosis after IR

Renal ischemia (30 min) at 37 1C caused significant renaltubular necrosis at 3 and 24 h after IR in both HSP27 WT andOE mice (Figure 4) as evidenced by severe tubular necrosis,medullary congestion and hemorrhage, and development ofproteinaceous casts in all mouse kidney sections. Consistentwith plasma Cr, huHSP27 OE mice showed increased renaltubular necrosis 3 h after renal IR compared to HSP27 WTmice. However, HSP27 WT and huHSP27 OE mice showed asimilar degree of renal tubular necrosis after IR. TheJablonski scale renal injury score histology grading is usedto grade renal tubular necrosis after murine renal IRI.26

Thirty min of renal ischemia and 3 h of reperfusion resultedin greater renal injury score in huHSP27 OE mice (renalinjury score¼ 2.0±0.3, N¼ 4) when compared to HSP27WT mice (renal injury score¼ 0.88±0.3, N¼ 4, Po0.05).Twenty-four hour of reperfusion resulted in severe acute

tubular necrosis in both HSP27 WT mice (renal injuryscore¼ 3.0±0.0, N¼ 4) and huHSP27 OE mice (renal injuryscore¼ 2.8±0.4, N¼ 4).

huHSP27 OE mice show increased proinflammatory geneexpression in the kidney after renal IRI

We found significantly increased expression of intercellularadhesion molecule-1 (ICAM-1), monocyte chemoattractiveprotein-1 (MCP-1), and tumor necrosis factor-a (TNF-a)mRNA in huHSP27 OE mice compared to the HSP27 WTmice 3 h after renal ischemia. No significant differences in theexpression of keratinocyte-derived cytokine (KC) or macro-phage inflammatory protein-2 (MIP-2) mRNA were foundbetween HSP27 WT and OE mice (Figure 5).

huHSP27 OE mice show increased early renal neutrophilinfiltration after IR

Immunohistochemical assays showed an increase in neutro-phil infiltration in the huHSP27 OE mice relative to theHSP27 WT mice at 3 h after renal IRI (Figure 6). At 24 h afterrenal IR, polymorphonuclear infiltration was similar betweenthe two groups of mice. Manual counting of polymorpho-nuclear neutrophils in hematoxylin and eosin (H&E) slidesalso showed increased neutrophil infiltration in the huHSP27OE (0.72±0.20 neutrophils/� 400 field, N¼ 4) mice relativeto the HSP27 WT mice (0.05±0.02 neutrophils/� 400 field,N¼ 4, Po0.05) at 3 h after renal IRI.

Blood KC mRNA and protein expression and renal KC proteinexpression are increased in huHSP27 OE mice

Whole-blood KC mRNA was significantly upregulated in thehuHSP27 OE mice compared to the HSP27 WT mice atbaseline and at 24 h after renal IR (Figure 7). No other

HS

P27

WT

HS

P27

OE

3 h after IR 24 h after IRSham

Figure 4 | Representative of photomicrographs of four experiments (hematoxylin and eosin staining, original magnification� 200) of studies with HSP27 WT and huHSP27 OE mice. Pictures of outer medulla of the kidneys of sham-operated mice and micesubjected to renal ischemia-reperfusion (IR) injury 3 and 24 h prior are shown.


SWC Chen et al.: HSP27 and renal ischemia reperfusion injury o r i g i n a l a r t i c l e

proinflammatory mRNA expression (ICAM-1, TNF-a,MCP-1, MIP-2) was detectable in whole blood. With ELISA,MCP-1 and KC plasma levels were determined 24 h afterrenal IR. There were no differences in MCP-1 levels

(1519±421 pg/ml, N¼ 8 for HSP27 WT and 1544±465 pg/ml, N¼ 8 for huHSP27 OE mice), however, plasma KC levelsincreased significantly higher in the huHSP27 OE mice(36,535±4770 pg/ml, N¼ 5) compared to the HSP27 WT

GAPDH

Mouse HSP27

Human HSP27

ICAM1

KC

MCP1

MIP2

TNF-α

Sham 3 hafter IR

HSP27 WT mice HSP27 OE mice

Sham 3 hafter IR

TNFα

HSP27 W

T Sha

m

HSP27 W

T IR

HSP27 O

E Sha

m

HSP27 O

E IR

0

1

2

3

*

*#

TN

F-α

mR

NA

/GA

PD

H o

ver

PB

Sha

m (

rena

l cor

tical

mR

NA

)

ICAM1

HSP27 W

T Sha

m

HSP27 W

T IR

HSP27 O

E Sha

m

HSP27 O

E IR

0

1

2

3*#

TN

F-α

mR

NA

/GA

PD

H o

ver

PB

Sha

m (

rena

l cor

tical

mR

NA

)

KC

HSP27 W

T Sha

m

HSP27 W

T IR

HSP27 O

E Sha

m

HSP27 O

E IR

0

1

2

3

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**

KC

mR

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/GA

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H o

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enal

cor

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mR

NA

)

MCP-1

HSP27 W

TSham

HSP27 W

T IR

HSP27 O

E Sha

m

HSP27 O

E IR

0

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6

8

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*

*#

MC

P-1

mR

NA

/GA

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ver

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mR

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)

MIP-2

HSP27 W

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E IR

0

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*

MIP

-2 m

RN

A/G

AP

DH

ove

rP

B S

ham

(re

nal c

ortic

al m

RN

A)

Figure 5 | HSP27 OE mice show reduced proinflammatory mRNA expression after renal IR. (a) Representative gel images ofsemiquantitative RT-PCR of mouse and human HSP27 as well as proinflammatory markers ICAM-1, KC, MCP-1, MIP-2, and TNF-a from renalcortices of HSP27 WT and huHSP27 OE mice subjected to sham operation or to renal ischemia and 3 h reperfusion. (b) Densitometricquantifications of relative band intensities normalized to GAPDH from RT-PCR reactions for each indicated mRNA. *Po0.05 vs appropriatesham. #Po0.05 vs HSP27 WT IR. Error bars represent 1 s.e.m.



mice (14,466±6950 pg/ml, N¼ 5). Moreover, baseline KClevels were higher in the huHSP27 OE mice (538±105 pg/ml,N¼ 6) compared to the HSP27 WT mice (139±40 pg/ml,N¼ 6). Finally, baseline kidney KC protein expression wassignificantly higher in huHSP27 OE mice (18.4±2.4 pg/mgprotein, n¼ 4, Po0.01) compared to HSP27 WT mice(3.2±0.9 pg/mg protein, n¼ 4).

Renal apoptosis after IRI

We failed to detect significant terminal deoxynucleotidyltransferase biotin-dUTP nick-end labeling (TUNEL)-positivecells in kidney sections (corticomedullary junction) from

sham-operated mice (Figure 8). Mice subjected to 30 min ofrenal ischemia and 24 h of reperfusion demonstratedTUNEL-positive cells in the corticomedullary junction.However, there were no differences in TUNEL-positive cellsbetween huHSP27 OE mice and HSP27 WT mice (Figure 8).Finally, the degree of renal tubular apoptosis was alsoquantified by counting the number of apoptotic bodies inproximal tubules in the corticomedullary area of the kidney(expressed as apoptotic bodies per tubule) on the H&E-stained sections. A total of 25–50 tubules/field were countedfor each treatment group, and kidneys from four experimentswere examined. Renal IRI equivalently increased the numberof apoptotic bodies within the proximal tubules in bothHSP27 WT (0.22±0.1 apoptotic bodies/tubule, N¼ 4) andhuHSP27 OE mice (0.21±0.1 apoptotic bodies/tubule,N¼ 4).

FACS analysis of lymphocyte subtype

Utilizing flow cytometry, we identified subpopulations ofleukocytes that infiltrated the kidney following renal IR inHSP27 WT and OE mice. Flow cytometry staining of kidneyleukocytes showed increased renal trafficking of CD3, CD4,CD8, F4/80, Ly-6G, and NK1.1þ cells in both HSP27 WTand huHSP27 OE mice 3 h after renal IRI. However,significantly higher number of leukocytes infiltrated thekidneys in huHSP27 OE mice (Figure 9).

Transfer of splenocytes or NK1.1þ cells from huHSP27 OEmice increases renal IRI in HSP27 WT mice

The adoptive transfer of splenocytes (Cr¼ 2.1±0.2, N¼ 6)or NK1.1± cells (Cr¼ 2.1±0.1, N¼ 6) isolated fromhuHSP27 OE mice increased renal injury at 24 h in HSP27WT mice (Figure 10). When the splenocytes (Cr¼ 1.2±0.2,

HS

P27

WT

HS

P27

OE

3 h after IR 24 h after IRSham

Figure 6 | Representative of photomicrographs of four experiments of immunohistochemistry for neutrophils (originalmagnification � 200) of the outer medulla of the kidneys of HSP27 WT and huHSP27 OE mice subjected to sham operation or torenal ischemia and 3 or 24 h reperfusion.

HSP27WT mice

HSP27OE mice

IRGAPDH

KC

GAPDH

KC

Sham

Sham

IR

Figure 7 | Representative gel images of semiquantitativeRT-PCR of whole-blood KC of HSP27 WT and huHSP27 OEmice subjected to sham operation or to renal ischemia and24 h reperfusion.



N¼ 5) or the NK1.1þ cells (Cr¼ 1.6±0.3, N¼ 6) fromHSP27 WT mice were given to the HSP27 WT mice, therewere no increases in renal injury (Figure 10).

Depletion of NK1.1þ cells or splenectomy unmasks theprotective phenotype of HSP27 after renal IR in mice

Figure 11 shows our ability to deplete NK1.1þ cellsselectively in mice. Mice were injected with either NK1.1antibody (PK136, 500 mg/mouse) or isotype control (mouseIgG2a). After 24 h, spleen and blood leukocytes were isolatedand subjected to fluorescence-activated cell sorting (FACS).Our PK136 antibody selectively depleted NK1.1þ cells in

blood (Figure 11, representative of three independentexperiments) and spleen (not shown) without effectingCD3þ T cells.

huHSP27 OE mice subjected to renal IRI after splenect-omy (Cr¼ 0.9±0.1, N¼ 8) or NK1.1þ cell depletion(Cr¼ 0.7±0.2, N¼ 6) were significantly protected againstrenal IRI compared to the HSP27 WT (Cr¼ 1.5±0.2, N¼ 8)or non-depleted huHSP27 OE mice at 24 h (Cr¼ 2.2±0.2,N¼ 8; Figure 12). However, HSP27 WT mice failed toimprove their renal function after splenectomy(Cr¼ 1.8±0.2, N¼ 7) or NK1.1þ cell depletion(Cr¼ 1.3±0.3, N¼ 6). Moreover, NK1.1þ neutralizingantibody treatment led to significant protection against renaltubular necrosis in huHSP27 OE mice (renal injuryscore¼ 1.3±0.4, N¼ 4; Figure 13a) compared to HSP27WT mice treated with NK1.1þ neutralizing antibody (renalinjury score¼ 2.3±0.4, N¼ 4). huHSP27 OE mice pre-

Table 1 | Primers used to amplify cDNAs encoding mouse or human HSP27, mouse GAPDH, and proinflammatory cytokinesbased on published GenBank sequences for mice

Primer Accession number Sequence (sense, antisense) Product size (bp) Cycle number Annealing T (1C)

Mouse HSP27 NM_024441 50-CCTAAGGTCTGGCATGGTA-30

50-AGGAAGCTCGTTGTTGAAGC-30373 26 66

Human HSP27 NM_001540 50-GTCCCTGGATGTCAACCAC-30

50-GACTGGGATGGTGATCTCG-30259 21 66

Mouse KC J04596 50-CAATGAGCTGCGCTGTCAGTG-30

50-CTTGGGGACACCTTTTAGCATC-30203 23 60

Mouse MIP-2 X53798 50-CCAAGGGTTGACTTCAAGAAC-30

50-AGCGAGGCACATCAGGTACG-30282 22 60

Mouse ICAM-1 X52264 50-TGTTTCCTGCCTCTGAAGC-30

50-CTTCGTTTGTGATCCTCCG-30409 21 60

Mouse TNF-a X02611 50-CCTCAGCCTCTTCTCCTTCCT-30

50-GGTGTGGGTGAGGAGCA-30290 24 65

Mouse MCP-1 NM_011333 50-ACCACAGTCCATGCCATCAC-30

50-CACCACCCTGTTGCTGTAGCC-30312 22 60

Mouse GAPDH M32599 50-ACCACAGTCCATGCCATCAC-30

50-CACCACCCTGTTGCTGTAGCC-30450 15 65

ICAM-1, intercellular adhesion molecule-1; KC, keratinocyte-derived chemokine; MCP-1, monocyte chemoattractant protein 1; MIP-2, macrophage inflammatory protein 2;TNF-a, tumor necrosis factor-a.Respective anticipated RT-PCR product size, PCR cycle number for linear amplification, and annealing temperatures used for each primer are also provided.

HSP27 WT Sham

HSP27 WT IR

HSP27 OE Sham

HSP27 OE IR

Figure 8 | Representative fluorescent photomicrographs ofkidney sections from identical fields illustrate apoptoticnuclei (TUNEL fluorescent stain). HSP27 WT and huHSP27 OEmice were subjected to sham operation or to renal ischemia and24 h reperfusion. Representative of six independent experiments.

CD3 CD4 CD8 NK1.1 F4/80 Ly-6G0

100

200

300

400

*

*

* *

*

*

Intr

aren

al le

ukoc

ytes

(%H

SP

27 W

T IR

)

Figure 9 | Kidney lymphocyte subtype (CD3, CD4, CD8, andNK1.1), macrophage (F4/80), and neutrophil (LY-6G) contentafter renal IR injury in HSP27 WT (white bars) and huHSP27OE mice (black bars). Kidney leukocyte subtype content in mice3 h after reperfusion was quantified using flow cytometry. Valuesare expressed as percent HSP27 WT mice subjected to IR. *Po0.05vs HSP27 WT IR. Error bars represent 1 s.e.m.



treated with NK1.1þ neutralizing antibody also showedreduced neutrophil infiltration compared to the HSP27 WTmice pretreated with NK1.1þ antibody (Figure 13b).Manual counting of polymorphonuclear cells in H&E slidesalso showed reduced neutrophil infiltration in the huHSP27OE mice treated with NK1.1þ antibody (0.87±1 neutro-phils/� 400 field, N¼ 4) relative to the HSP27 WT micetreated with NK1.1þ antibody (33±9 neutrophils/� 400field, N¼ 4, Po0.001) at 24 h after renal IRI. Similarprotection against renal tubular damage and neutrophilinfiltration was observed in splenectomized huHSP27 OEmice (data not shown).

DISCUSSION

The major findings of this study are that (1) isolatedproximal tubules from huHSP27 OE mice were protectedagainst H2O2-induced necrosis compared to the proximaltubules isolated from HSP27 WT mice; (2) in contrast,huHSP27 OE mice showed worse renal function, increasedrenal tubular inflammation (early leukocyte influx and renalproinflammatory mRNA expression), and exacerbated earlyrenal tubular necrosis after renal IRI in vivo; (3) huHSP27 OEmice showed increased plasma KC levels at baseline and 24 hafter renal IRI; (4) huHSP27 OE mice had increased renal KC

HSP27 W

T IR

HSP27 O

E IR

HSP27 W

T WT sp

lenoc

yte IR

HSP27 W

T OE sp

lenoc

yte IR

HSP27 W

T WTNK1.

1 IR

HSP27W

T OE N

K1.1

IR0.0

0.5

1.0

1.5

2.0

2.5

**

*

Pla

sma

Cr

(mg/

100

ml)

Figure 10 | Plasma creatinine (Cr in mg/100 ml) from HSP27WT and huHSP27 OE mice subjected to 30 min renal ischemiaand 24 h reperfusion. Some HSP27 WT mice were injectedwith huHSP27 OE splenocytes or NK1.1þ cells before renalischemia. *Po0.05 vs HSP27 WT 24 h IR. Data are presented asmean±s.e.m.

NK1.1

CD

3

NK ABIsotype AB

CD

3

CD45

Isotype AB NK AB

25.1% 25.8%

0.9%6.9%

(cals) (cals)02

03

01

04

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03

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04

(cals) 02

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(cals) 02

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100 101 102 103 104 100 101 102 103 104

100 101 102 103 104 100 101 102 103 104

Figure 11 | Representative FACS analysis of CD3þ lymphocytes and NK1.1þ lymphocytes in blood of HSP27 WT mice 24 h aftertreatment with either NK1.1þ neutralizing antibody (PK136) or with isotype control.



protein expression; (5) adoptive transfer of NK1. 1þ cells orsplenocytes isolated from huHSP27 OE mice exacerbatedrenal injury in HSP27 WT mice; and (6) depletion ofNK1.1þ cells or splenectomy unmasked the protectiveeffects of HSP27 OE against renal IRI in mice.

Renal IRI is the leading cause of ARF in hospitalizedpatients.1,27 Moreover, the mortality and morbidity ofperioperative ARF are very high and remain virtuallyunchanged for the past 40 years.3,28 We have demonstratedin our earlier studies that activation of renal A1ARs producedrenal protection in vivo as well as in vitro.7,8,11,29 Themechanisms of renal protection with A1AR activation aremediated at least in part by phosphorylation as well asupregulation of HSP27 protein expression.11,12 Transientactivation of renal tubular A1ARs led to phosphorylation ofHSP27 whereas OE or chronic stimulation of A1ARs led toupregulation of HSP27. To the best of our knowledge,modulation of HSP27 expression on renal function after IRIin vivo has never been studied previously.

HSP27 is generally regarded as a cytoprotective proteinand is well known to attenuate apoptosis, stabilize cytoske-letal architecture, and decrease necrotic cell death.19,22,30 It iswell established that HSP27 phosphorylation or upregulationimproves cell survival against stress or injury.14,30 Theseprinciples as well as our previous findings led us tohypothesize that huHSP27 OE mice would show improvedrenal tubular survival against necrotic injury in vitro andbetter renal function after IRI in vivo. As we expected, theproximal tubule cells cultured from huHSP27 OE miceshowed increased resistance against H2O2-induced necrosiscompared to the cells cultured from HSP27 WT mice.However, we unexpectedly demonstrated that the huHSP27OE mice showed enhanced renal IRI with a heightenedinflammatory response (earlier intrarenal leukocyte (forexample, neutrophils, lymphocytes) infiltration, increasedrenal tubular proinflammatory mRNA expression, and higherplasma and kidney KC levels) after renal IRI. We also showenhanced renal necrosis early (3 h after reperfusion) after IR.Therefore, although HSP27 expression in renal proximaltubules is associated with increased resistance against necrosisin vitro, systemic HSP27 OE led to increased systemic andrenal tubular inflammation in these mice, leading toworsened renal injury after IR in vivo.

Although HSP27 expression is usually associated withincreased tolerance against cell death and injury, HSP27 mayalso orchestrate physiological events that increase immunecell-mediated cytotoxicity. For instance, HSP27 phosphor-ylation enhances neutrophil chemotaxis and function.31

Furthermore, a cytoplasmic OE of HSP25, a murine formof HSP27, increases natural killer (NK) cell-mediated lysis ina murine melanoma model.32 In addition, OE of HSP27 in abreast cancer cell line increases T-cell-mediated cytolysis.33 InHeLa cells, HSP27 is required for proinflammatory cellsignaling and the expression of proinflammatory genes.34 Inthis study, we also showed increased inflammatory changes inhuHSP27 OE mice leading to worse renal dysfunction after invivo renal IRI. Our study also suggests that exogenousproinflammatory signaling distant to the organ or site ofinjury is important in regulating injury as increasedinflammatory response overrides the protective effects ofrenal tubule HSP27 overexpression. We propose that

HSP27 W

T 24

h IR

HSP27 O

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Figure 12 | Plasma creatinine (Cr in mg/100 ml) from HSP27WT and huHSP27 OE mice subjected to 30 min renal ischemiaand 24 h reperfusion. Some HSP27 WT and OE mice wereinjected with NK1.1þ neutralizing antibody (NK AB) or weresplenectomized before renal ischemia. *Po0.05 vs appropriateHSP27 WT 24 h IR. #Po0.05 vs huHSP27 OE 24 h IR. Data arepresented as mean±s.e.m.

H&E

PMN IHC

HSP27 WT + NK antibody HSP27 OE + NK antibody

Figure 13 | NK1.1þ neutralizing antibody decreases renalnecrosis and PMN infiltration. (a) Representativephotomicrographs (hematoxylin and eosin staining, originalmagnification � 200) from studies with HSP27 WT and huHSP27OE mice injected with NK1.1þ neutralizing antibody. Pictures ofouter medulla of the kidneys of sham-operated mice and micesubjected to renal ischemia-reperfusion (IR) injury 24 h prior areshown. (b) Representative photomicrographs ofimmunohistochemistry for neutrophils (original magnification� 200) of the outer medulla of the kidneys of HSP27 WT andhuHSP27 OE mice injected with NK1.1þ neutralizing antibodyand subjected to renal ischemia and 24 h reperfusion.



although the antiapoptotic and anti-necrotic effects of HSP27on the renal tubules promote direct cellular protection, theenhanced systemic inflammation of huHSP27 OE micepromotes the exacerbation of immune-mediated renal tubuleinjury after IR.

With regard to the previous studies involving huHSP27 OEmice, Hollander et al.23 demonstrated that OE of HSP27protected against cardiac IRI evidenced by reduced release of Crkinase, lipid peroxidation, and myocyte protein oxidation.Similar results were obtained by Efthymiou et al.35 utilizing thesame strain of huHSP27 OE transgenic mice utilized in thisstudy. In these previous studies, an isolated Langendorff-perfused heart IR model was utilized. Therefore, the effects ofcirculating inflammatory cells in cardiac IRI could not be tested.It remains to be determined whether the cardiac protectionpersists in intact, whole animal models of cardiac IRI.

Neutrophils, macrophages, and lymphocytes have impor-tant functions in initiating and propagating inflammationafter renal IR.4,36,37 In this study, we showed that huHSP27OE mice had increased early (3 h) neutrophil infiltration afterrenal IR. The appearance of neutrophils as well as macro-phages mediates antigen-independent mechanisms of tissuedamage due to innate immunity activation.38–41 Neutrophilinfiltration begins early, within hours after the initiation ofreperfusion and peaks at B24 h after reperfusion. However,although renal neutrophil infiltration after IRI is a well-known phenomenon, the direct role of neutrophils inpromoting the pathogenesis of renal IRI has been contro-versial. Some studies report a beneficial effect of neutrophilneutralization/blockade whereas other studies showed noeffects.36,38,40,42,43 Our study showed that the huHSP27 OEmice had an early increase in neutrophil infiltration (3 h) aswell as worsened renal injury after IR show. Therefore, weconclude that neutrophil infiltration after renal IR doesdirectly contribute to increased renal injury after IR and thatneutrophil-mediated renal injury may outweigh the cyto-protective effects of renal tubular HSP27 OE.

We detected increased T lymphocyte as well as NK1.1þcell infiltration in huHSP27 OE mice compared to HSP27WT mice 3 h after renal IR. The pathophysiological role ofNK1.1þ cells and T lymphocytes in initiating liver andcardiac IRI has been described.37,44,45 In the kidney, NK1.1þcells as well as T cells have important functions in thepathogenesis of IRI.44,46,47 When the mice were splenecto-mized (to deplete circulating lymphocytes) or treated with aneutralizing NK1.1þ antibody, less renal injury occurred inthe huHSP27 OE mice. In fact, after splenectomy andNK1.1þ cell depletion, huHSP27 OE mice had significantlybetter renal function compared to the splenectomized orNK1.1þ cell-depleted HSP27 WT mice. These findingsfurther support the hypothesis that huHSP27 OE mice haveexacerbated inflammatory response after renal IR anddepletion of key cellular components of the inflammatoryresponse uncovers the protective phenotype of systemicHSP27 OE. Surprisingly, depletion of NK1.1þ cells orsplenectomy failed to protect HSP27 WT mice against renal

IRI. This finding contrasts with the study by Li et al.48 whereNK1.1 cell depletion using the same antibody as in this studyprovided profound protection against renal IR. Our studysuggests that the NK1.1 cells are important in generatingrenal injury only in huHSP27 OE mice. The reason for thisdiscrepancy is not clear. Strain differences may explain thisdiscrepancy as Li et al. used a C57BL/6 strain whereas weused mice with a C57BL/10 and CBA/Ca background.

The role of increased inflammation after renal IR causingworsened renal dysfunction in huHSP27 OE mice is furthercorroborated by our adoptive cellular transfer studies. HSP27WT mice infused with splenocytes or NK1.1þ cells isolatedfrom huHSP27 OE (but not from HSP27 WT) mice showedincreased renal injury. These data further support ourconclusion that the increased renal dysfunction in huHSP27OE mice after IR is due to the enhanced inflammatory andimmune-mediated renal injury.

One of the limitations of this study is that we do notconclusively identify the specific leukocyte subtype(s) respon-sible for the exacerbated renal injury in huHSP27 mice afterrenal IR. We implicate one of the cell types involved as NK1.1cells as we show increased renal injury in HSP27 WT infusedwith huHSP27 OE NK1.1 cells. However, we cannot rule out theheightened proinflammatory role of other leukocytes such asmacrophages and T cells in huHSP27 OE mice as these celltypes have also been shown to be important in renal IRI.Identifying the specific role of T lymphocytes and/or macro-phages in creating the enhanced proinflammatory phenotype inhuHSP27 remains to be investigated.

We also demonstrated that huHSP27 OE mice hadincreased expression of whole-blood KC mRNA and proteinexpression at baseline and after renal IR. Moreover, wedemonstrate increased renal KC protein expression inhuHSP27 mice. It is unclear as to why overexpression ofsystemic HSP27 would lead to upregulation of kidney andblood KC expression in mice. Nevertheless, we propose thatincreased renal KC production may account for thedifferences in renal neutrophil infiltration after IR. Althoughthe rodents lack a homologue of human IL-8, KC is one ofthe two murine IL-8-like CXC cytokines that acts throughCXCR2 and functions as granulocyte chemoattractant similarto the function of IL-8.41,49 KC is a well known and majorchemoattractant for neutrophils that subsequently causetissue and cellular damage after injury and inflammation.41

In a rat model of cold ischemia followed by kidneytransplantation, inhibition of the chemokine receptor CXCR2reduced kidney graft failure due to ischemia/reperfusion.50

KC is also important in initiating injury in other diseasesassociated with increased inflammation. Frink et al.51

demonstrated that administration of the anti-KC antibodybefore trauma and hemorrhage in mice prevented increasesin KC plasma levels, which was accompanied by ameliorationof neutrophil infiltration and edema formation in lung andliver after trauma and hemorrhage.

We have previously demonstrated that A1AR agonists orOE of A1ARs produced renal protection in vivo as well as



protection against renal tubule cell death in vitro throughincreases in renal tubular HSP27 expression.7,8,11,29 There-fore, unlike HSP27, systemic A1AR activation produces bothin vivo and in vitro renal protection. We have performed pilotstudies and determined that A1AR activation failed toupregulate HSP27 in two leukocyte cell lines (Jurkatlymphocyte cell line, RAW 263.7 macrophage cell line, datanot shown). Therefore, we speculate that a selectiveupregulation of renal HSP27 with A1AR activation leads torenal protection whereas global HSP27 OE leads to increasedrenal injury after IR due to increased leukocyte activation andKC overproduction.

Although significant efforts had been made to maintain aconstant hybrid background, because of the concerns for thepotential genetic variability associated with non-congenicstrain of mice, we bred our huHSP27 OE and HSP27 WTmice with C57BL/6 mice for three generations. We showedOE of huHSP27 in mice backcrossed with C57BL/6 micecontinued to result in increased renal injury after IR.Therefore, although strain differences do have an importantfunction in renal IR injury, global OE of human HSP27protein led to increased renal injury after IR before and afterbackcrossing with the C57BL/6 strain.

In summary, we demonstrate in this study that global OEof HSP27 does not confer protection after renal IRI. Incontrast to what we hypothesized and what others havereported in cardiac and neuronal injury studies, OE of HSP27in vivo led to increased renal injury after IR. HSP27 doesserve to protect the renal proximal tubules from necrosis invitro but its protective effects are counteracted by increasedimmune-mediated renal tubular injury after IR. Futurestudies will compare the direct cytotoxicity profiles ofNK1.1þ cells and lymphocytes between HSP27 WT andhuHSP27 OE mice.

MATERIALS AND METHODSMiceHeterozygous breeder pairs of mice overexpressing (OE) humanHSP27 were generated as described previously.52 huHSP27 OEtransgenic mice overexpress human HSP27 tagged with hemagglu-tinin to track the expression of the transgene. The generation andinitial characterization of the huHSP27 OE and WT mice with aC57BL/10 and CBA/Ca background has been described previously.52

Heterozygous HSP27 transgenic mice were bred and resulting malelittermates (huHSP27 OE or HSP27 WT mice) were used in thesubsequent studies. We performed tail DNA PCR genotyping as wellas human HSP27 RT–PCR from the RNAs extracted on every mousestudied using primers that distinguish human from mouse HSP27.In preliminary studies, we also confirmed human HSP27 proteinoverexpression with immunoblotting for mouse and human form ofHSP27 (Santa Cruz Biotechnologies, CA) as described pre-viously.11,53 We also purchased C57BL/6 mice (Harlan Laboratories,Indianapolis, IN) to breed with huHSP27 OE and HSP27 WT mice.

Primary culture of renal proximal tubulesProximal tubules from male HSP27 WT and huHSP27 OE micewere enriched using the methods of Vinay et al.54 and cultured in

50:50 mix of Dulbecco’s modified Eagle’s medium high glucose andF12 plus 5% fetal bovine serum (FBS).

Induction of necrosis in cultured mouse renal proximaltubulesAfter 24 h deprivation of FBS, primary cultures of renal proximaltubules isolated from HSP27 WT and huHSP27 OE mice weretreated with 1–5 mM H2O2 for 0–6 h to induce necrosis. Wedetermined in preliminary studies that the ability of H2O2 to killrenal tubule cells is dose and time related. Our preliminary studiesalso showed that high-dose (1–5 mM) treatment with H2O2 for 1–6 hcauses negligible apoptosis (data not shown).

Renal ischemia-reperfusion injuryAfter Columbia University IACUC approval, adult (25–30 g)male huHSP27 OE mice and their littermate control HSP27 WTmice were anesthetized with an intraperitoneal injection ofpentobarbital (50 mg/kg or to effect), and placed supine on aheating pad under a warming light to maintain body temperature at37 1C. Additional pentobarbital was given as needed based on themouse’s response to a tail pinch. Bilateral flank incisions were madeand following right nephrectomy, a microaneurysm clip occludedthe left renal pedicle (artery and vein) for 30 min. Ischemia (30 min)was chosen as previous studies have shown that this length ofischemic injury minimizes mortality whereas maximizing reprodu-cible IRI mice.7,8 After 30 min, the microaneurysm clip wasremoved, 0.5 cc of saline was given intraperitoneally, the woundsclosed in two layers and animals were allowed to awaken fromanesthesia. The sham mice were subjected to anesthesia and rightnephrectomy only.

Adoptive transfer of splenocytes or NK1.1þ cellshuHSP27 OE or WT spleens were crushed, splenocytes were passedthrough a nylon cell strainer (BD Biosciences, San Jose, CA) andcollected in phosphate-buffered saline. Red blood cells were lyzed andsingle-cell splenocyte suspensions from huHSP27 OE or WT micewere transferred intravenously (6� 106 to 1� 107 splenocytes pertransfer, 200ml) to the HSP27 WT mice. NK1.1þ lymphocytes wereisolated utilizing SpinSep murine NK cell enrichment kit (StemCellTechnologies, Vancouver, Canada, BC), resulting in 490% pureNK1.1þ cells. To confirm NK1.1þ phenotype and purity of isolatedcells, column-purified cells were stained for 30 min with fluoresceinisothiocyanate-conjugated anti-mouse NK1.1 (PK136; eBioscience,San Diego, CA) and analyzed with Quanta FACS (Beckman Coulter,Fullerton, CA). Purified NK1.1þ cells isolated from HSP27 WT orhuHSP27 OE mice were injected intravenously (1–1.2� 106 NK1.1þcells per transfer, 200ml) to HSP27 WT mice. The recipient HSP27 WTmice were subjected to renal IR 24 h later.

NK1.1þ cell depletion and splenectomyNK cells were depleted by injecting each mouse with 500 mg rabbitanti-mouse NK1.1þ antibody (eBioscience) intravenously 24 hbefore renal ischemia. Splenectomized mice had their spleensremoved 15 min before renal ischemia.

Assessment of renal function after ischemia-reperfusioninjuryRenal function was assessed by measurement of plasma Cr 3 or 24 hafter renal ischemia by a colorimetric method based on the Jaffereaction.55



Histological examinations to detect necrosis and apoptosisHistological examinations to detect necrosis and apoptosis wereperformed as described previously.7,8 Morphological assessment wasperformed by an experienced renal pathologist (VDD), who wasunaware of the treatment each animal had received. An establishedgrading scale (renal injury scores of 0–4) for assessment of necroticinjury to the proximal tubules was used for the histopathologicalassessment of IR-induced damage (3 and 24 h after IR), as outlinedby Jablonski et al.26 Renal tubular apoptosis was assessed bycounting the number of apoptotic bodies in proximal tubules in theouter stripe of the corticomedullary junction (expressed as meannumber of apoptotic bodies per tubule). This area is the mostseverely injured area after renal IRI. At least 25–30 tubules werecounted per field and six fields were examined per slide.

Assessment of renal apoptosis with TUNEL StainingWe used TUNEL staining to detect DNA fragmentation in apoptosis24 h after renal injury. In situ labeling of fragmented DNA wasperformed with TUNEL staining (green fluorescence) with acommercially available in situ cell death detection kit (Roche,Indianapolis, IN), according to the manufacturer’s instructions.

Assessment of renal inflammationRenal inflammation after IRI was determined with (1) detection ofneutrophil infiltration with immunohistochemistry 3 and 24 h afterrenal IR; (2) measurements of mRNA encoding markers ofinflammation (Table 1),8 including KC, ICAM-1, MCP-1, MIP-2,and TNF-a; and (3) immunophenotyping of T lymphocyte (CD3þ ,CD4þ , CD8þ ), NK1.1þ lymphocyte (PK136), macrophage (F4/80), and neutrophil (LY-6G) influx into the kidneys after IR withflow cytometry.

Neutrophil immunohistochemistryNeutrophil immunohistochemistry was performed as describedpreviously7,8 with a monoclonal antibody raised against Ly-6G(551459; BD Biosciences). A primary antibody that recognized IgG2b

(MCA691XZ; Serotec, Raleigh, NC) was used as a negative isotypecontrol in all experiments. Intrarenal neutrophil infiltration was alsoquantified by manual counting of polymorphonuclear cells in H&Eslides by a renal pathologist who was blinded to the treatmentgroups. Neutrophils were quantified in 10 randomly chosenmicroscope fields (magnification � 400) in the corticomedullaryjunction, and results were expressed as neutrophils counted per� 400 HPF.

Semiquantitative reverse transcription-PCR assaySemiquantitative reverse transcription-PCR assay for proinflamma-tory mRNAs was performed as described previously.11,56,57

Isolation of lymphocytes from mouse kidney and flowcytometry analyses of leukocyte subtypesFlow cytometric analysis was performed with a Quanta SC flowcytometer (Beckman Coulter, Miami, FL) to analyze the whole-kidney leukocyte content as described previously.58 These studiesassessed kidney content of T cell (CD3, CD4, and CD8), NK cells(PK136), macrophages (F4/80), and neutrophils (Ly-6G) inhuHSP27 OE or WT mice subjected to 30 min renal ischemia and3 h reperfusion. In brief, kidney was extracted, minced, thoroughlycrushed, and then passed through a 50-mm mesh prewetted withphosphate-buffered saline containing 10% FBS. The kidney cell

suspension was diluted with phosphate-buffered saline containing10% FBS and carefully layered on an equal volume of Histopaque-1083 (Sigma, St Louis, MO) to isolate mononuclear cells. Sampleswere centrifuged at 400 g for 30 min at room temperature. The pelletfrom this spin was processed to detect neutrophils. The mono-nuclear cell interface containing lymphocytes and macrophages wascollected, washed twice with phosphate-buffered saline with 10%FBS, and pelleted. The mononuclear cells recovered from the mousekidney (105 cells in a volume of 100 ml) were preincubated with ananti-mouse CD16/32 (2.4G2) antibody for 10 min to blocknonspecific antibody binding. Samples were then incubated withdifferent combinations of antibodies (for example,CD45þCD3þ 7AAD, CD3þCD4þ 7AAD) for 20 min at roomtemperature. Flow cytometry was conducted under conditions toeliminate the interference of nonspecific staining by dead and non-leukocyte (for example, renal) cells. For discrimination of viable anddead cells, samples were stained with 7-AAD for 5 min at roomtemperature. Anti-mouse CD45 was used to determine the totalleukocyte cell number. Kidney leukocyte infiltrations were expressedas percent of HSP27 WT mice subjected to renal IR.

Antibodies for FACSPurified rat IgG2a (clone YTH71.3) was purchased from Serotec(Oxford, UK). Anti-CD3e (clone 145–2C11), anti-F4/80 (cloneBM8), anti-Ly-6G (Gr-1), anti-CD45 (clone 30-F11) were purchasedfrom eBioscience. Anti-NK1.1 (LY-55; clone PK136), anti-CD4/L3T4 (clone GK1.5), and anti-CD8a/Lyt-2 (clone 53–6.7) wereobtained from Beckman Coulter.

Measurement of plasma cytokines by ELISAMurine plasma MCP-1 (eBioscience) and KC (R&D Systems,Minneapolis, MN) concentrations were measured using commerciallyavailable ELISA kits according to the manufacturer’s instructions fromsham-operated mice and mice subjected to renal IR 24 h before.

Statistical analysisThe data were analyzed with t-test when means between two groupswere compared or with one-way (for example, plasma Cr) ANOVAplus Tukey’s post hoc multiple comparison test to compare meanvalues across multiple treatment groups. The ordinal values of theJablonski scale were analyzed by the Kruskal–Wallis nonparametrictest with Dunn’s post-test comparison among groups. In all cases,Po0.05 was taken to indicate significance. All data are expressed asmean±s.e.m.

ReagentsUnless otherwise specified, all chemicals were obtained from Sigma.

DISCLOSUREAll the authors declared no competing interests.

ACKNOWLEDGMENTSThis work was supported by National Institutes of Health grant RO1DK-58547.

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Documents

Mice that overexpress human heat shock protein 27 have ... · phil inﬁltration in the huHSP27 OE mice relative to the HSP27 WTmice at 3h after renal IRI (Figure 6). At 24h after