Neutrophil peptidyl arginine deiminase-4 has a pivotal role in ischemia/reperfusion-induced acute kidney injury

Abstract

Ischemia/reperfusion is a common cause of acute kidney injury (AKI). However, mechanisms underlying the sudden loss in kidney function and tissue injury remain to be fully elucidated. Here, we investigated the role of peptidyl arginine deiminase-4 (PAD4), which converts arginine to citrulline and plays a role in epigenetic regulation and inflammation, in renal ischemia/reperfusion injury. PAD4 expression was highly induced in infiltrating leukocytes 24 hours following renal ischemia and reperfusion. This induction was accompanied by citrullination of histone H3 and formation of neutrophil extracellular traps in kidneys of wild-type mice. By contrast, PAD4-deficient mice did not form neutrophil extracellular traps, expressed lower levels of pro-inflammatory cytokines and were partially protected from renal ischemia/reperfusion-induced AKI. Furthermore, PAD4-deficient mice recovered kidney function 48 hours after ischemia/reperfusion, whereas kidney function in the wild-type mice progressively worsened. Administration of DNase I, which degrades neutrophil extracellular traps or the PAD-specific inhibitor YW3-56 before ischemia, partially prevented renal ischemia/reperfusion-induced AKI. Notably, transfer of neutrophils from wild-type, but not from PAD4-deficient mice, was sufficient to restore renal neutrophil extracellular trap formation and impair kidney function following renal ischemia/reperfusion. Thus, neutrophil PAD4 plays a pivotal role in renal ischemia/reperfusion-induced AKI.

Introduction

Acute kidney injury (AKI) results in high morbidity and mortality but has no effective treatment beyond supportive care.^{1, 2} AKI induced by ischemia/reperfusion (I/R), the most common cause of AKI,³ is characterized by injury to tubular epithelial cells^{4, 5} and vascular endothelial cells,^{6, 7} and robust inflammatory responses including leukocyte infiltration and upregulation of chemokines and cytokines in the kidney.^8–10 Although inflammatory responses to viral and bacterial infection are vital to host defense, tissue and wound repair, I/R-triggered “sterile” inflammation may lead to tissue injury.^{11, 12} A relatively recently described pathway of neutrophil-induced injury involves the formation of neutrophil extracellular traps (NETs) which involves the decondensation of chromatin and subsequent extravasation of DNA into the extracellular space.¹³ NET formation may aid clearance of bacteria during infection but has also been implicated in a growing list of autoimmune and inflammatory conditions,¹³ including ischemic injury to the heart,¹⁴ liver,¹⁵ muscle,¹⁶ brain^{17, 18} and kidney.^{19, 20}

The Peptidyl Arginine Deiminase (PAD) family is comprised of five closely related proteins (PADs 1 – 4 and PAD6) that post-translationally convert arginine to citrulline in proteins, thereby mediating signal transduction for diverse stimuli and affecting many biological processes.²¹ While PADs 1 – 3 and PAD6 are cytoplasmic enzymes,²¹ PAD4 occurs in both the cytoplasm and the nucleus^{22, 23} and is primarily expressed in leukocytes, particularly in granulocytes.²¹ The conversion of histone arginines to citrullines by PAD4 reduces the overall positive charges of histones and weakens histone-DNA binding, thereby disrupting the nuleosomes and triggering nuclear DNA release and formation of NETs.²¹ Thus, PAD4 may be involved in signal transduction leading to the inflammatory responses following I/R injury. Indeed, Rabadi et al²⁴ reported that renal tubular cell PAD4 is highly induced by I/R injury, and that PAD4-deficient mice are protected against I/R-induced kidney injury. Although implicated in renal I/R injury,^{20, 24} nothing is known regarding the specific role of neutrophil-PAD4 in this pathological context. Neutrophil PAD4 is especially relevant because it is essential to the formation of NETs, which evokes most aspects of inflammatory responses during autoimmunity and thrombosis.^{13, 25}

Here, we tested the hypothesis that neutrophil PAD4 and NET formation are mechanistically linked to renal I/R-induced AKI. We show in wild-type mice the induction of PAD4 after renal I/R. PAD4 promoted inflammation after renal I/R injury and was essential for NET formation. DNase I treatment prior to renal I/R suppressed NET formation and partially protected mice from I/R-induced renal injury. A PAD-specific inhibitor, YW3-56, potently suppressed I/R-induced kidney injury in mice. In PAD4-deficient mice, transfer of neutrophils derived from wild-type mice was sufficient to promote NET formation in the kidney and loss of kidney function after I/R. By contrast, PAD4-knockout mice receiving neutrophils from PAD4-deficient mice showed no detectable NETs in the kidney and were protected from I/R-induced renal injury.

Results

PAD4 is upregulated in mouse kidneys after ischemia/reperfusion (I/R) injury

To establish the biological role for PAD4 in renal I/R injury, we first assessed the expression and distribution of PAD4 and one of its products, citrullinated histone H3 (Cit-H3) in the kidney after ischemia. Relative to sham controls, PAD4 mRNA levels measured by quantitative RT-PCR were three-fold higher 24 h after I/R injury (n = 4, p < 0.01; Figure 1a). Citrullination of Histone-H3 was not detectable in mice that underwent sham surgery and was markedly increased in mice 24 h after I/R (Figure 1b). Thus, renal I/R induces PAD4 expression and activity.

Figure 1. Expression of kidney PAD4 and citrullinated histone-H3 (Cit-H3) after ischemia and reperfusion (I/R) injury.

a) qPCR analysis of PAD4 mRNA level in mouse kidneys 24 h after renal I/R injury compared to sham operations (n = 4/group). b) Immunoblot analysis of Cit-H3 expression in wild-type (WT) mouse kidneys 24 h after I/R injury versus sham-operated control mice; actin was a loading control. Bottom panel: densitometric analysis of Cit-H3 band intensity (n = 3/group). c) and d) Immunofluorescence analysis of kidney sections from WT and PAD4^−/− (PAD4KO) mice following sham surgery (Sham) or after 28 min ischemia and 24 h reperfusion. c) PAD4 (green); Ly-6G (red); DAPI (blue). d) Cit-H3 (green); Ly-6G (red); DAPI (blue). Immunofluorescence signals double positive for PAD4 and Ly-6G (PAD4⁺Ly6G⁺), or Cit-H3 and Ly-6G (Cit-H3⁺Ly-6G⁺) were determined by averaging signal counts from 7 – 10 fields (20× objective) per kidney section per mouse. Data shown are mean ± SEM (n = 3/group). Data were compared one-way ANOVA using a significance level of p < 0.05; n. s. denotes not significant.

Although PAD4 is expressed mainly in hematopoietic cells,²¹ it has also been described in renal epithelial cells.²⁴ We performed immunolocalization of PAD4 and Cit-H3 in kidneys after ischemia to determine the sites of PAD4 expression and activity. Immunofluorescence microscopy showed few detectable PAD4 (Figure 1c) or Cit-H3 (Figure 1d) signals co-localizing with nuclear DNA in kidneys from wild-type (WT) and PAD4 knock-out (PAD4KO) mice following sham surgery. By contrast, 24 h after I/R injury, PAD4 (Figure 1c) and Cit-H3 (Figure 1d) were dramatically increased in the kidneys of WT mice, but were barely detectable in the PAD4KO mice. Double labeling of PAD4 or Cit-H3 with a neutrophil marker, Ly6G, revealed that the majority of the PAD4- and Cit-H3-expressing cells in the ischemic kidneys were neutrophils (Figures 1c – 1d). Although faint PAD4 signals were observed in renal epithelial cells from sham WT kidneys, they did not increase after I/R. Thus, renal I/R injury induces PAD4 expression and triggers Cit-H3 production mainly in infiltrating neutrophils.

PAD4 mediates kidney I/R injury

To determine whether PAD4 is involved in renal I/R injury, we assessed kidney function 24 h and 48 h after I/R in WT and PAD4KO mice. As expected, WT mice developed severe kidney dysfunction as reflected by elevated plasma creatinine (Figure 2a) and BUN (Figure 2b) concentrations. In contrast, BUN (p < 0.001) and creatinine (p < 0.001) levels were significantly lower in PAD4KO mice compared with WT 24 h post I/R. Indeed, 48 h after I/R injury, levels of both creatinine and BUN returned to the normal ranges in the PAD4KO mice, but continued to climb in the WT mice (Figure 2a – 2b). Likewise, mRNA levels of kidney injury molecule-1 (KIM-1), an established biomarker for renal proximal tubule injury,²⁶ were barely detectable in the kidneys of both WT and PAD4KO mice that underwent sham surgery but increased dramatically 24 h after renal I/R injury in WT mice (Figure 2c). The induction of KIM-1 expression after I/R was lower in PAD4KO mice (p < 0.001, Figure 2c). Finally, histological evidence of injury was assessed in kidneys from WT and PAD4KO mice subjected to I/R (Figure 2d). Kidneys from sham-operated mice of either genotype displayed normal morphology, with well-preserved brush border membranes and no loss of tubular epithelial cells. Kidneys from WT mice subjected to I/R showed marked tubular injury as reflected by loss of brush border, cast formation and extensive loss of tubular epithelial cells, tubular dilation, and intra-tubular debris (Figure 2d). Kidneys from PAD4KO mice subjected to I/R also showed evidence of injury, but less than those seen in WT mice (Figure 2d). Semi-quantitative assessment of kidney tissue injury yielded tubular necrosis scores of 0.07 ± 0.01 and 0.09 ± 0.04, respectively, in WT and PADK4KO mice that received sham surgery, and 3.4 ± 0.06 and 2.1 ± 0.4 (P < 0.05) in WT and PAD4KO mice 24 h after I/R injury (Figure 2e). These data support the conclusion that PAD4 mediates renal I/R-induced injury.

Figure 2. PAD4 mediates kidney I/R injury.

Plasma creatinine (a) and BUN (b) concentrations were measured before, 24 h-, and 48 h- after renal I/R injury in WT and PAD4^−/− (KO) mice. c) qPCR analysis of KIM1 expression in WT and PAD4^−/− mice 24 h after renal I/R injury compared to WT mice following sham surgery (n = 6). d) Periodic acid-Schiff (PAS)-staining of kidney sections from WT and PAD4^−/− mice 24 h post I/R or sham surgery. e) Renal tissue injury score was used to grade renal tubular necrosis in PAS-stained kidney sections from mice following a sham surgery or 28-min ischemia and 24-h reperfusion (scale = 0 – 4, n = 3). ***p < 0.001, **p < 0.01.

PAD4 promotes inflammation after renal I/R injury

Infiltration of inflammatory cells into the renal parenchyma occurs early in the course of renal I/R injury.^{10, 27} Neutrophils are key effectors of the inflammatory cascade in a variety of kidney injury models, including I/R.^{10, 27} We therefore assessed the role of PAD4 in renal I/R-triggered inflammatory responses and neutrophil infiltration. As shown in Figure 3a, neutrophil elastase (NE)-positive neutrophils were hardly detectable in the kidneys of WT and PAD4KO mice subjected to sham surgery. I/R induced significant neutrophil infiltration in kidneys of WT mice and, to a lesser extent, in PAD4KO mice 24h after I/R (p < 0.01; Figure 3a). To test whether PAD4 deficiency affects neutrophil mobility, we performed a Boyden trans-well migration assay using 10% FBS as a chemo-attractant. Supplemental Figure 1 shows that neutrophil migration rates were similar in WT and PAD4KO mice. In addition, no difference in total number of white blood cells in the peripheral blood was observed (Supplemental Figure 2a). Next, we tested whether PAD4 deficiency reduced the circulating platelet number, which could impair neutrophil infiltration following I/R injury.¹⁹ Supplemental Figure 2b shows that total platelet count was similar in WT and PAD4KO mice.

Figure 3. PAD4 is implicated in inflammatory responses in the kidney.

a) Quantification of neutrophil elastase (NE) signals in kidney sections from WT and PAD4^−/− mice following a sham surgery or 28-min ischemia and 24-h reperfusion (n = 3 – 4). b) qPCR analyses of pro- and anti-inflammatory cytokine expression.

To identify the role of PAD4 in pro- and anti-inflammatory cytokine production, we performed quantitative RT-PCR analyses 24 h after renal I/R injury. Basal expressions of TNF-α, IL-6, IL-8, and IL-10 were not affected by PAD4-deficiency (Figure 3b). The expression of TNF-α, IL-6 and IL-8 increased in WT mice 24 h after I/R injury but these increases were significantly blunted in PAD4-deficient mice (Figure 3b). In contrast, the expression of the anti-inflammatory cytokine, IL-10, was significantly enhanced in the absence of PAD4 (Figure 3b). These data support the hypothesis that PAD4 mediates renal I/R-induced upregulation of pro-inflammatory cytokines, and that endogenous PAD4 activity may also suppress the expression of anti-inflammatory cytokines.

PAD4 is essential for NET Formation after I/R injury

Whereas neutrophil extracellular traps (NETs) mediate certain forms of tissue injury,^{28, 29} their role in renal I/R injury is not well-defined. To determine whether infiltrating neutrophils form NETs after I/R-induced injury, we examined kidney sections by confocal microscopy using DAPI and anti-NE antibody to test for co-localization of DNA and neutrophil granule proteins. Among intact neutrophils, NE staining revealed granular cytoplasmic patterns surrounding clearly defined multi-lobulated nuclei (Figure 4a, top panels). In WT mice 24 h after I/R injury, DAPI staining revealed occasional sheets of extracellular DNA co-localizing with diffuse NE signals signifying NET formation (Figure 4a, bottom panels). We next determined whether PAD4 activity is required for renal I/R-induced NETs formation. First, we confirmed that PAD4 activity is essential for histone citrullination, a prerequisite for NET formation; Figure 4b shows that I/R-induced histone citrullination was completely abolished by PAD4-deficiency, demonstrating that PAD4 activity is essential for histone citrullination in response to I/R. Quantification of NETs showed that kidneys of WT and PADKO mice that underwent sham surgery contained no detectable NETs, while those of WT mice 24 h after I/R injury contained NETs, albeit involving only a small fraction of the total neutrophils (Figure 4c). Kidney sections from PAD4KO mice contained fewer NETs compared to WT mice (P < 0.001), corroborating the Cit-H3 immunoblot data (Figure 4b). Taken together, these findings are consistent with previous evidence supporting the hypothesis that PAD4 is essential for efficient NET formation.³⁰ More broadly, we conclude that NETs are formed in the kidney after I/R in a PAD4-dependent fashion.

Figure 4. Neutrophil extracellular traps (NETs) in mouse kidney following I/R injury.

a) Representative confocal images. Top panel: intact neutrophils in kidney sections of mice following sham surgery. Bottom panel: NETs formation in kidney sections of mice following 28-min ischemia and 24-h reperfusion. NETs are defined by co-localization of NE (green) and diffuse DAPI staining patterns. b) Immunoblot analysis of Cit-H3 expression. c) Quantification of NETs in kidney sections from WT and PAD4^−/− mice following sham surgery or I/R injury (n = 3 – 4).

NETs contribute to renal I/R injury

Concentrations of circulating cell-free DNA (cfDNA) are a potential marker of NET formation³¹ and may be mechanistically linked to inflammation through activation of TLR receptors.^32–35 Twenty-four hours after I/R, circulating cfDNA concentrations increased in WT mice versus WT mice that underwent sham surgery (p < 0.0002; Figure 5a). By contrast, PAD4KO-deficiency blunted, but did not completely prevent, the renal I/R triggered increase in circulating cfDNA (p < 0.005; Figure 5a). Thus, PAD4 at least partially mediates renal I/R induced cfDNA release.

Figure 5. PAD4 and NETs contribute to elevated levels of circulating cell-free DNA (cfDNA) and renal injury after kidney I/R.

a) Circulating cfDNA concentrations were determined in WT and PAD4^−/− mice 24 h post I/R or sham surgery (n = 3). b) Circulating cfDNA concentrations in WT mice treated with saline or DNase I (n = 7). c) Quantification of NETs (n = 3 – 5); d) and f) plasma creatinine (n = 7 – 12); e) and g) plasma BUN concentrations (n = 7 – 12).

Our findings indicate that NETs are formed in the kidney after I/R. Since NETs contain extracellular DNA, we tested the therapeutic potential of DNase I administration for renal I/R-induced NET formation, cfDNA release and kidney dysfunction. Intravenous injection of DNase I suppressed the increase in plasma cfDNA 24 h after renal I/R injury (p < 0.05, Figure 5b) with a concomitant reduction of kidney NETs compared to I/R WT mice that received saline (p < 0.001, Figure 5c). DNase I injection also reduced plasma creatinine (p < 0.05, Figure 5d) and BUN levels (p < 0.01, Figure 5e) compared to saline treated mice. These results further support the conclusion that NET formation and release of extracellular DNA are pathogenic components of renal I/R.

Conversely, to determine whether inhibition of PAD4 activity is sufficient to prevent I/R-induced NET formation and renal injury, we treated WT mice with either vehicle or YW3-56, a PAD-specific inhibitor,³⁶ 30 min prior to renal I/R. Twenty-four hours after I/R injury, plasma concentrations of creatinine (p < 0.01, Figure 5f), and BUN (p < 0.001, Figure 5g) were both markedly lower in mice that received YW3-56 compared to those which received vehicle, indicating that PAD4 inhibition protects mice from I/R-induced kidney injury.

Neutrophil PAD4 is essential for renal R/I injury

PAD4 is expressed at high levels in neutrophils.²¹ However, other cells, such as monocytes,³⁷ macrophages,³⁷ and renal epithelial cells²⁴ have been reported to express PAD4. To determine if neutrophil PAD4 mediates renal I/R-induced kidney injury, we performed neutrophil transfer experiments. Specifically, we isolated neutrophils from either WT or PAD4KO mice and transferred them to PAD4KO mice 30 min prior to performing renal I/R. Immunofluorescence microscopy of tissue obtained 24 h after renal I/R showed that the number of infiltrating neutrophils in the kidneys from PAD4KO mice receiving WT or PAD4-deficient neutrophils was similar (Figure 6a). However, large numbers of NETs were present in the kidneys of PAD4KO mice that received WT neutrophils whereas PAD4KO mice receiving PAD4-deficient neutrophils did not show detectable NETs in the kidney (p < 0.001, Figure 6b). Notably, PAD4-deficient mice receiving WT neutrophils developed substantially more severe renal dysfunction 24 h after renal I/R, as evidenced by increased creatinine (1.83 ± 0.17 vs. 0.44 ± 0.04, p < 0.0001, Figure 6c) and BUN (136 ± 8 vs. 63 ± 10, p < 0.001, Figure 6d) levels than did PAD4-deficient mice receiving PAD4-deficient neutrophils (Figures 6c – 6d). These results strongly support an important role for neutrophil-PAD4 in renal I/R injury and NET formation.

Figure 6. Neutrophil transfer reveals pivotal role for neutrophil PAD4 in renal NETs formation and I/R-induced injury.

WT (n = 8) or PAD4^−/− (n = 6) neutrophils were injected into PAD4^−/− mice 30 min before ischemic surgery. WT (n = 3) and PAD4^−/− mice (n = 3) also underwent ischemic surgery as bench marks. a) Quantification of neutrophil elastase-positive cells in kidney sections. B) Quantification of NETs. C) Plasma concentrations of creatinine. D) Plasma BUN concentrations.

Discussion

Although neutrophils have been implicated in kidney I/R injury,³⁸ the mechanisms whereby neutrophils contribute to injury remain unclear. The release of nuclear constituents, such as DNA, histones and high mobility group box-1 (HMGB1), triggers inflammatory pathways to exacerbate tissue ischemia and injury. While neutrophil extracellular traps (NETs), formed by the decondensation and extrusion of nuclear DNA,^{13, 25} may protect against bacterial infections, recent work indicates that NETs may cause tissue injury during sterile inflammation, such as I/R injury.^{11, 12, 29} Citrullination of histone proteins by peptidyl arginine deiminases (PADs), which alters histone charges and promotes chromatin decondensation, is a critical step in NET formation. PAD4, which is highly expressed in neutrophils and contains a nuclear localization signal, is indispensable for NET formation in neutrophils.²¹ Although first described in neutrophils, it is now recognized that extracellular trap formation (ETosis) occurs in other cell types.^{39, 40} Our studies reported here were designed to uncover the role of NETs and PAD4 in kidney I/R injury. Several noteworthy findings were obtained.

First, our studies showed that PAD4 expression is increased in kidneys after ischemia and that PAD4 contributes to I/R injury. Studies from the Lee laboratory using chemical PAD4 inhibitors⁴¹ and more recently a different PAD4 deficient mouse,²⁴ and the Anders lab²⁰ also concluded that PAD4 contributes to renal IRI. There are some differences, however, between our results and those previously reported. In the Lee studies, PAD4 was observed mainly in renal epithelial cells, leading them to conclude that epithelial PAD4 mediates injury.²⁴ While we observed faint signals in epithelial cells, the majority of signals were from neutrophils. Moreover, transfer of PAD4-expressing neutrophils to PAD4-deficient mice restored susceptibility to I/R injury. The latter result strongly supports an important role for neutrophil PAD4 in I/R injury but cannot exclude a role for PAD4 expressed in other cell types. Studies using tissue- or cell type-specific deletion of PAD4 may further define the role of PAD4 in other PAD4-expressing cell types, including macrophages,³⁷ dendritic cells⁴² and epithelial cells.²⁴

Second, our studies indicate that NETs are formed in the kidney after ischemia-reperfusion and contribute to renal dysfunction. Specifically, NETs were observed in WT kidneys after ischemia but were absent in kidneys from PAD4-deficient mice. This finding is consistent with prior studies, which found that PAD4 is necessary for NET formation.⁴³ In addition, PAD4-deficient mice also had lower kidney expression of pro-inflammatory chemokines and cytokines (Figure 3b), which could reduce inflammatory cell recruitment, thereby reducing further tissue damage and NETs formation. The apparent vicious cycle between NETs formation and inflammation complicates the definition of the proximate effects of PAD4. Circulating cell-free DNA concentration also increased after I/R but was lower in the absence of PAD4. This result suggests that NET formation contributes to cell-free DNA release after ischemia-reperfusion. Other sources of DNA, such as necrosis, are also possible contributors.¹⁹ Indeed, while we observed a marked reduction in renal NET formation in PAD4KO mice following I/R injury (Figure 4c), there was only a moderate reduction in circulating cfDNA (Figure 5a), supporting the notion that NETs formed in the kidney in response to I/R injury contributes to circulating cfDNA but is not the only source for cfDNA in the circulation. The findings that treatment with DNase I, which degrades NETs, or a PAD-specific inhibitor YW3-56, reduced both the frequency of NETs in the kidneys and also improved kidney function, support the hypothesis that PAD4 has a pivotal role in NETs formation and renal I/R injury.

Third, our studies uncover PAD4 and DNase I as potential therapeutic targets for preventing kidney ischemic injury. Small molecule inhibitors of PAD4 are under development, and we show that the PAD-specific inhibitor YW3-56 reduced IR injury. YW4-03, another PAD-specific inhibitor, was also shown to reduce hepatic I/R injury.¹⁵ Likewise, Ham et al⁴¹ and Nakazawa et al²⁰ showed reduced kidney I/R injury using a non-selective PAD inhibitor, Cl-amidine. The finding that DNase I reduced I/R injury confirms recent studies.^{24, 44} Treatment with DNase I also reduced the number of renal NETs. While tempting to speculate that DNase I reduces kidney injury by degrading NETs, other mechanisms, such as reduced toll-like receptor (TLR) activation by circulating cell-free DNA, are possible. In contrast to the protective actions of DNase I in renal I/R injury, DNase I is thought to mediate cisplatin-induced AKI wherein DNase I-deficient mice are relatively protected.⁴⁵ The mechanisms underlying the differential effects of DNase I in I/R versus cisplatin nephrotoxicity are not known and require further study.

Although our studies strongly support the view that neutrophil PAD4 and NET formation are critical mediators of renal I/R injury, the mechanism whereby PAD4 and NETs are induced after I/R, and the mechanism whereby PAD4 and NETs lead to tissue injury remain to be elucidated. Jansen et al recently showed that renal I/R induces necrosis of renal tubular epithelial cells and triggers DNA release, thereby activating platelets and inducing the formation of platelet-granulocyte-DNA complexes.¹⁹ Likewise, platelet-induced formation of NETs has been noted in sepsis, acute lung injury and arterial and venous thrombosis.⁴⁶ Although we did not measure platelet aggregation, the number of circulating platelets was similar in WT and PAD4KO mice (Supplemental Figure 2b). In vitro, the release of histones and HMGB1 from injured hepatocytes¹⁵ and renal epithelial cells²⁰ can also trigger NET formation through the activation of TLR on neutrophils. Conversely, NET constituents may be directly toxic to tubular epithelial cells,²⁰ creating an amplifying loop of injury wherein epithelial injury promotes NET formation that leads to further epithelial injury. We also found that IL-10 levels were increased in the absence of PAD4. Since IL-10 reduces ischemic injury,⁴⁷ the protective actions of PAD4 deficiency/inhibition may be related to increased IL-10 production. The significance and source of IL-10 in PAD4-mediated injury requires further study.

In summary, we provide evidence that NETs are formed within the kidney after I/R and contribute to I/R-induced injury, and that PAD4 in neutrophils is essential for kidney NET formation and ischemic injury. Accordingly, maneuvers that prevent the formation of NETs, such as PAD4 inhibition, or increase degradation of NETs may be of value in preventing or treating IRI.

Methods

Acute Ischemia/Reperfusion Injury in Mice

PAD4-deficient mice on a C57BL/6J background were provided by Dr. Yanming Wang (Penn State University, University Park, PA).⁴⁸ All protocols concerning the use of laboratory animals were approved by the Institutional Animal Care and Use Committee at University of Texas Health Science Center at San Antonio and Penn State Hershey College of Medicine. Acute ischemic kidney injury was induced as previously described.⁴⁹ Briefly, wild-type and PAD4-deficient mice (7 – 8 weeks old, males) were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight) and placed on a heating pad to maintain core body temperature at 37°C. Both kidney pedicles were identified through two small paramedial dorsal incisions and clamped for 28 min. Control animals underwent the same surgical procedure without having their kidney pedicles clamped. Blood samples were collected before clamping and at indicated times post-clamping and reperfusion.

Histology and Immunofluorescence Microscopy

Kidneys were fixed in 10% neutral-buffered formalin overnight, dehydrated, and embedded in paraffin. Tubular injury was assessed in Periodic acid-Schiff (PAS)-stained sections using a semi-quantitative scale as described previously⁵⁰. For immunofluorescence, mounted sections (5 µm) were dewaxed, rehydrated, and pretreated with antigen retrieval solution (Tris-EDTA-Tween-20, pH 9.0) in a decloaking chamber (Biocare Medical, Concord, CA) following manufacturer’s instructions. Sections were blocked (60 min) in phosphate buffered saline (PBS) supplemented with 10% goat serum and 5% bovine serum albumin, incubated with antibodies against PAD4, Cit-H3, Ly-6G, and neutrophil elastase (NE; Abcam) for 16 h (4°C). Sections were washed in PBS, and incubated with Alexa Fluor 488- or Alexa Fluor 568-conjugated goat anti-rabbit or goat anti-rat IgG (Invitrogen) for 50 min at room temperature (RT), followed by incubation with DAPI (1 µg/mL, 5 min at RT), and 0.05% Sudan Black prepared in 70% ethanol (Sigma, St Louis, MO) for 10 min at RT, rinsed with deionized water, and cover-slipped in VectaShield antifade mounting media (Vector Labs, Burlingame, CA). Fluorescence images were acquired using an Olympus FV-1000 laser scanning confocal microscope; 405-, and 488-nm lasers were used to excite DAPI, and Alexa 488 conjugates, respectively. Single plane 512×512×12 bit images were captured in a frame-sequential manner using a 60× oil objective.

Analysis and Quantification of Leukocytes and NETs

Images used for quantitative analyses were captured using an Olympus Provis AX70 microscope equipped with a 20× objective and an Olympus DP70 camera. The number of NE-, PAD4-, and/or Cit-H3- positive cells was determined by averaging the number of positive cells in each 20× field per kidney section. Neutrophils and NETs were determined by overlaying DAPI staining with those for NE, PAD4, or Cit-H3. Intact cells were classified as neutrophils only if they were NE positive and contained well-defined nuclei. NETs formation was defined by the colocalization of diffused, spread-out DAPI-positive nuclear materials, and NE or Cit-H3 signals. For quantification, signals from 7 – 10 different fields per section were counted and averaged; 3 – 7 kidneys were examined per experimental condition.

Neutrophil Transfer

Neutrophils were isolated from bone marrow of donor mice as described in Supplemental Method, and were counted using a hemocytometer and 3×10⁶ cells were injected into the tail vein 30 minutes prior to I/R surgery.

Assays of Renal Function

Plasma BUN levels were measured using a QuantiChrom Urea assay kit according to the manufacturer’s instructions (BioAssay Systems, Hayward, CA). Plasma creatinine concentrations were determined using an enzymatic creatinine assay kit (Diazyme Labs, San Diego, CA), which is more resistant to interference than the conventional Jaffe methods⁵¹.

DNase I and PAD4 Inhibitor Treatment

Wild-type mice (7 – 8 weeks old males) were injected intraperitoneally (i.p.) with 5 mg/kg of DNase I or saline, 15 min before and once again 8 h after I/R surgery. The PAD-specific inhibitor YW3-56 (10 mg/kg) or 10% DMSO in saline (vehicle control) was injected i.p. 2 h prior to I/R surgery.

Statistical Analyses

Statistical differences among groups were analyzed using Student’s t-test (two groups), and one-way ANOVA (three and more groups) using Prism software. p < 0.05 was considered statistically significant.

Additional methods are presented in the online Supplemental Materials.