Clinical and Experimental Pharmacology and Physiology (2014) 41, 911–920
doi: 10.1111/1440-1681.12298
ORIGINAL ARTICLE
Protection of Wistar-Furth rats against postischaemic acute renal injury: Role for nitric oxide and thromboxane? Viginie Voisin,* Anne-Emilie Decleves,* Virginie Hubert,* Vanessa Colombaro,* Laetitia Giordano,* Isabelle Habsch,* Nadine Bouby,† Denis Nonclercq‡ and Nathalie Caron* *Molecular Physiology Research Unit–Namur Research Institute for Life Sciences, Univerity of Namur, Namur, Belgium, † UMRS872 Team no. 2, Cordelier Research Center, Paris, France and ‡Department of Histology, University of Mons, Mons, Belgium
SUMMARY The Wistar-Furth (WF) rat strain is usually used in models of full major histocompatibility complex-mismatched kidney transplantation. Because these rats have been demonstrated to be resistant to several models of chronic kidney disease, the aim of the present study was to investigate their potential resistance to renal ischaemia–reperfusion (I/R) injury compared with another strain, namely Wistar-Hanover (WH) rats. Anaesthetized male WH and WF rats were submitted to I/R by occlusion of the left renal artery and contralateral nephrectomy. Urine, blood and tissue samples were collected at different time points after I/R to evaluate renal function, inflammation and tubular injury, along with determination of nitric oxide synthase (NOS) expression and thromboxane A2 (TxA2) production. Post-ischaemic renal function was better preserved in WF than WH rats, as evidenced by reduced levels of creatininaemia, urinary neutrophil gelatinase-associated lipocalin excretion and proteinuria. In addition, WF rats had less intrarenal inflammation than WH rats after I/R injury. These observations were associated with maintenance of neuronal NOS expression, along with lower induction of inducible NOS expression in WF versus WH rats. Moreover, WF rats excreted a significantly lower amount of TxB2. The results indicate that WF rats are more resistant to an I/R injury than WH rats in terms of renal function and inflammation. These observations are associated with differential regulation of intrarenal NOS expression, as well as a reduction in thromboxane production, which could contribute to a better outcome for the postischaemic kidney in WF rats. Key words: acute renal injury, nitric oxide synthase, renal ischaemia–reperfusion, thromboxane, Wistar-Furth rats.
Correspondence: Nathalie Caron, 61 rue de Bruxelles, B-5000, Namur, Belgium. Email: [email protected] Received 13 November 2013; revision 6 July 2014; accepted 7 August 2014. © 2014 Wiley Publishing Asia Pty Ltd
INTRODUCTION Acute kidney injury (AKI) is a very common clinical complication contributing to significant morbidity and mortality in the intensive care unit.1–3 Renal ischaemia–reperfusion (I/R) injury is a well-known experimental model used to investigate the pathogenesis of AKI, particularly because I/R insult is a prominent negative factor in the survival of renal allografts.4 Specifically, I/ R injury causes cellular hypoxia, increased oxidative stress, leucocyte infiltration and tubular and endothelial injuries. In particular, endothelial dysfunction is characterized by an imbalance in vasoactive factors in favour of the synthesis of endothelin, angiotensin II and thromboxane (Tx) A2, along with reduced nitric oxide (NO) production, thereby enhancing the degree of vasoconstriction.5,6 Furthermore, even a short episode of AKI can lead to long-term complications, including chronic kidney disease (CKD) or end-stage renal disease.7 These complications are usually associated with incomplete tubular repair, persistent tubulointerstitial inflammation, proliferation of fibroblasts and excessive accumulation of extracellular matrix.1 This highlights the urgent need to better characterize the pathogenesis of AKI to develop potent therapeutic approaches. One of the cellular responses to an ischaemic insult is decreased NO production, limiting the pool of vasodilator factors.5,8 Especially in the kidney, NO is involved in the regulation of vascular resistance and glomerular filtration rate (GFR). It also maintains renal structural integrity.9,10 However, NO has multiple actions: both beneficial and deleterious.9,11,12 Constitutive NO production by endothelial (e) NO synthase (NOS) induces vasodilation and reduces endothelial dysfunction,11 whereas neuronal (n) NOS-derived NO, produced in the macula densa in the kidney, plays a role in the regulation of renal haemodynamics by modulating tubuloglomerular feedback (TGF).10,13–15 In contrast, NO produced by inducible (i) NOS is primarily involved in inflammation or oxidative stress.16 Several studies have shown that inhibition of iNOS improves renal injury and reduces oxidative stress.17–19 Another potent mediator of renal function is TxA2. It has been shown that TxA2 controls afferent arteriole tone and is therefore involved in TGF.20,21 Hence, there is a balance between NO produced by nNOS and TxA2 produced by cyclo-oxygenase-(COX) 2. Indeed, COX2 can generate TxA2 and the free radical
V Voisin et al.
RESULTS
(a) 700
Creatininaemia (µmol/L)
superoxide, leading to a simultaneous reduction in NO bioavailability.20 This may impair the TGF response. The aim of the present study was to investigate the potential resistance of Wistar-Furth (WF) rats to I/R injury and to determine whether this could be linked to NO production. Indeed, several studies have reported that WF rats are resistant to the development of CKD in experimental rodent models, such as mineralocorticoid hypertension-induced renal injury,22,23 the 5/6 nephrectomy model,24 puromycin-aminonucleoside (PAN)induced CKD25 and DOCA salt-induced CKD.26 In these studies, it was suggested that NO production, and particularly nNOS expression, were maintained after renal injury in WF rats, which could explain their resistance to CKD. Interestingly, another study reported that WF rats were protected against renal microcirculation injury after chronic NOS inhibition.26 The findings of that study suggest that in addition to NO other mechanisms may be involved in the protection of WF rats against progression to CKD.26 Thereby, demonstrating reduced susceptibility to AKI in WF rats could be of particular interest to investigate therapeutic approaches. Moreover, because this rat strain is often used in rat models of full major histocompatibility complex-mismatched kidney transplantation,27,28 this potential resistance to AKI should be taken into consideration when interpreting the results of such studies. In the present study, we sought to determine whether WF rats were resistant to AKI in a model of renal I/R. The renal response to I/R injury in WF rats was then compared with that in another rat strain, namely Wistar-Hanover (WH) rats. To further characterize the potential resistance, we also evaluated renal NOS expression and TxA2 production after I/R.
*
600 500 400
†
300 200 100
0 0 (b)
NGAL (×105 U/24 h)
912
12
48
168
*
10 8 6
*
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48
168
Time after I/R (h) Fig. 1 Evaluation of renal function. Temporal evolution of (a) creatininaemia and (b) 24 h urinary neutrophil gelatinase-associated lipocalin (NGAL) excretion in Wistar-Hanover (■, WH) and Wistar-Furth (□, WF) rats under control conditions (Time 0) and at different time points after ischaemia–reperfusion (I/R). Results are the mean SEM (n = 6–9 per group). *P < 0.05 compared with Time 0; †P < 0.05 compared with WH rats (two-way ANOVA (a) or two-way ANOVA for repeated measurements (b)).
General observations The mortality rate in WF and WH rats after I/R was 1.4% and 3.9%, respectively. The weight of the right kidney was similar in all groups, with a mean ( SEM) of 1.01 0.03 g. In control WH and WF rats, left kidney weight did not differ significantly from that of the right kidney. However, after I/R, the weight of the left kidney in WH rats increased progressively from 0.93 0.05 g on Day 0 to 2.16 0.07 g on Day 7 after I/R (P < 0.05). The progressive increase in postischaemic left kidney weight in WF rats was less pronounced, with an increase from 0.93 0.05 g on Day 0 to 1.76 0.06 g on Day 7 after I/R (P < 0.05); the difference between the WH and WF groups was significant on Day 7 (P < 0.05). Renal function and renal injury after I/R To determine whether WF rats may be protected against renal ischaemic insult, we measured levels of two markers of AKI,5,29 namely plasma creatinine and urinary neutrophil gelatinase-associated lipocalin (NGAL). As shown in Fig. 1, under control conditions, creatininaemia and NGAL excretion were similar in WH and WF rats. However, 48 h after I/R, creatininaemia (Fig. 1a) had increased nearly fivefold in WH rats (P < 0.001), but thereafter returned to baseline levels by 72 h. In WF rats, despite a slight (non-significant) increase in creatin-
inaemia at 48 h after I/R, there were no significant changes in creatininaemia throughout the observation period. There was a 130-fold increase in NGAL excretion in WH rats (Fig. 1b) 48 h after I/R (P < 0.05), but NGAL excretion had returned to baseline levels 72 h after I/R. In WF rats, there was also a significant increase in NGAL excretion 48 h after I/R, but this increase corresponded only to a 30-fold change (P < 0.05) compared with baseline, and NGAL excretion had returned to baseline levels by 72 h after I/R. Proteinuria was also determined in WH and WF rats (Fig. 2a). Under control conditions, levels were similar in both strains. A significant increase in proteinuria was observed 48 h after I/R in both strains (P < 0.05), but the magnitude of the increase was less in WF than WH rats (P < 0.05). To further characterize renal function, and particularly tubular function, we evaluated renal sodium handling by measuring fractional excretion of sodium (FENa), which enables determination of the ability of renal tubules to reabsorb sodium properly (Fig. 2b). As indicated in Fig. 2b, FENa under control conditions was similar in WH and WF rats (1.17 0.07% vs 1.49 0.06%, respectively; NS). Forty-eight hours after I/R, FENa was increased significantly in both strains, but the increase was 2.5-fold higher in WH than WF rats (9.20 2.09% vs
© 2014 Wiley Publishing Asia Pty Ltd
Ischaemic kidney injury in Wistar-Furth rats
Proteinuria (mg/24 h)
(a) 50
Oxidative stress after I/R in WH and WF rats
*
To determine the level of oxidative stress, urinary H2O2 and malondialdehyde (MDA) excretion were measured. Values for both were similar under control conditions in both rat strains (Fig. 4a). Interestingly, 48 h after I/R, WF rats had significantly higher urinary H2O2 levels than WH rats (89 19 vs 32 5 nmol/mg creatinine, respectively; P < 0.05). Thereafter, H2O2 levels returned to baseline by Day 7 in both strains. As shown in Fig. 4b, urinary MDA excretion measured under control conditions was similar in both rat strains. Urinary MDA levels increased significantly 48 h after I/R in both WH and WF rats, with no significant difference between the two strains.
*†
40 30 20 10 0 0
(b) 12
168
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*
FENa (%)
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Renal NOS expression after I/R in WH and WF rats
8 6
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4 2 0
913
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168
Time after I/R (h) Fig. 2 Evaluation of renal injury. Temporal evolution of (a) proteinuria and (b) fractional excretion of sodium (FENa) in Wistar-Hanover (■, WH) and Wistar-Furth (□, WF) rats under control conditions (Time 0) and at different time points ischaemia–reperfusion (I/R). Results are the mean SEM (n = 6–9 per group). *P < 0.05 compared with Time 0; † P < 0.05 compared with WH rats (two-way ANOVA for repeated measurements (a) or two-way ANOVA (b)).
3.72 1.32%, respectively; P < 0.05), suggesting higher salt wasting in WH rats due to enhanced tubular dysfunction. Thereafter, on Day 7 after I/R, FENa levels had returned to baseline levels in both strains. Inflammatory responses after I/R in WH and WF rats To determine whether WF rats are also protected against renal inflammation occurring after an ischaemic insult, tissue levels of monocyte chemoattractant protein-1 (MCP-1), considered an early marker of inflammation, and macrophage and CD8 infiltration, considered chronic markers of inflammation, were investigated (Fig. 3). As shown in Fig. 3a, a significant increase in tissue MCP-1 levels was observed in WH rats 12 h after I/R (P < 0.05), whereas there was no significant change in MCP-1 levels in WF rats. Increased macrophage infiltration was observed in both WH and WF rats 48 h after I/R (P < 0.05), equating to approximately 300 ED1-positive cells/mm2 in both strains. Interestingly, on Day 7 after I/R, the number of ED1-positive cells had increased even further in WH rats but not in WF rats (1045 180 vs 762 126 ED1-positive cells/mm2, respectively; P < 0.05 for WF vs WH rats; Fig. 3b, d, e). Similarly, the increased CD8 infiltration 48 h and on Day 7 after I/R in WH rats was not seen in WF rats (P < 0.05; Fig. 3c,f,g).
To evaluate NOS expression in WF rats after renal ischaemic insult, nNOS, eNOS and iNOS mRNA levels were measured by real-time polymerase chain reaction in the renal cortex and outer medulla (OM). As indicated in Table 1, cortical mRNA levels for nNOS decreased significantly 48 h and 7 days after I/R in WH rats. However, in WF rats, nNOS mRNA levels were maintained throughout the observation period. In contrast, iNOS mRNA levels in WH rats were significantly increased 48 h after I/R in the cortex and the OM. In WF rats, iNOS expression was undetectable in the cortex and barely increased in the OM after I/R. There were no significant changes in eNOS expression. To confirm our data and better illustrate the localization of nNOS and iNOS in the kidney, immunostaining analysis was performed on paraffin-embedded kidney sections. Representative photomicrographs of nNOS staining in the cortex in WH and WF rats are shown in Fig. 5a–f. Under control conditions (Fig. 5a,b), nNOS was found in the macula densa. Forty-eight hours after I/R, positive nNOS staining was decreased significantly in WH rats compared with baseline (27.5 3.3% vs 76.7 3.6%, respectively; P < 0.05; Fig. 5c,g). In contrast, in WF rats, the immunolocalization of nNOS was preserved in the macula densa 48 h after I/R compared with baseline (62.8 6.2% vs 75.7 5%; NS; Fig. 5d,g). Seven days after I/R, this difference between strains was still evident (Fig. 5e–g). Representative photographs of iNOS-positive staining in WH and WF rats are shown in Fig. 6a–f. Under control conditions, iNOS was mostly localized in the collecting ducts (Fig. 6a,b). After I/R injury, iNOS expression increased significantly in WH rats after both 48 h (Fig. 6c) and 168 h (Fig. 6e). Furthermore, iNOS was no longer primarily localized in the collecting ducts, but was also strongly expressed in the proximal tubules. Although the same pattern for iNOS localization was observed in WF rats, the levels of expression were significantly less than in WH rats (Fig. 6d–f). Quantitative measurement of iNOS-positive staining revealed that the significant increase in staining in the cortex and OM reached 26 7% and 17 10%, respectively, in WH rats 48 h after I/R (P < 0.05 for both), compared with increases of 3 1% and 4 1%, respectively, in WF rats (P > 0.05 for both; Fig. 6h). These data demonstrate that nNOS expression is preserved in WF rats after I/R injury. This difference is associated with prevention of iNOS upregulation.
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(c)
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1200 1000 800 600
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*†
800
0 12
*
1200
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140
No. CD8+ cells (/mm2)
1600
No. ED1+ cells (/mm2)
MCP-1 (pg/mg tissue)
(a)
120
*
100 80
†
60 40 20
0
48
168
336
0 0
48
168
Time after I/R (h)
(d)
(e)
(f)
(g)
Fig. 3 Inflammatory response after ischaemia–reperfusion (I/R). (a) Changes in monocyte chemoattractant protein-1 (MCP-1) levels in renal tissue, (b) semiquantitative analysis of macrophage infiltration and (c) quantitative measurement of CD8 infiltration in Wistar-Hanover (■, WH) and Wistar-Furth (□, WF) rats after I/R. Data are the mean SEM (n = 6). *P < 0.05 compared with Time 0 (control conditions; one-way ANOVA followed by Dunnett’s test); †P < 0.05 compared with WH rats (two-way ANOVA). (d–g) Representative photomicrographs of (d, e) macrophage immunostaining and (f, g) lymphocyte CD8 on Day 7 in WH (d, f) and WF (e, g) rats.
Urinary excretion of PGE2 and TxB2 after I/R in WH and WF rats Because the mechanism underlying the protection of WF rats against renal I/R may be caused by factors other than changes in NO metabolism, we investigated changes in TxA2 levels by measuring the urinary excretion of TxB2, a stable metabolite of TxA2, and PGE2. Prostaglandin E2 is the main kidney prostaglandin produced by microsomal PGE synthase localized in the macula densa.30 As shown in Fig. 7a, under control conditions urinary PGE2 excretion was similar in both strains. Forty-eight hours after I/R, there was a significant increase in PGE2 excretion in both strains (P < 0.05 for both). In contrast, urinary TxB2 excretion (Fig. 7b) was lower in WF than WH rats under control conditions and after I/R injury (P < 0.05).
DISCUSSION Ischaemia–reperfusion injury is an unavoidable event after kidney transplantation. However, there is no current therapy to protect the kidney and guaranty a positive clinical outcome. An I/R episode causes a cascade of cellular events, leading to cellular damage and organ dysfunction. These adverse effects, which induce glomerular, tubular and endothelial alterations,1 occur together with repair and regeneration processes. Therefore, the renal outcome of I/R depends on the capability of the renal tissue to fully regenerate. In the present study, we demonstrated that the WF rat strain exhibits some degree of resistance against an I/R insult. Several studies have showed the resistance of this strain to the development of CKD.22,24,25,31 However, no data suggested potential protection against AKI. In the present study, we clearly showed
beneficial outcomes following renal I/R in WF rats, as evidenced by reduced creatininaemia and renal inflammation, maintenance of tubular function, preservation of nNOS expression and prevention of iNOS upregulation. In the CKD studies, the same data were reported with regard to nNOS expression, which places NO as a key player in this beneficial resistance of WF rats. Nitric oxide is a well-known vasoactive mediator that was shown to play a role in AKI and was identified as a key factor in the progression of renal I/R injury.19,32–34 Our renal I/R model has been demonstrated to induce a severe but reversible AKI.29 In AKI, creatininaemia is still considered as the classical clinical marker to determine kidney damage and renal outcome. In the present study, WF rats had lower creatininaemia levels than WH rats. This was also associated with lower levels of urinary NGAL, proteinuria and FENa. In the present study, NGAL was used as another marker of AKI. Indeed, although creatininaemia is the main diagnostic indicator of renal function, other biomarkers, such as NGAL, have demonstrated their usefulness in the early diagnosis of AKI.35,36 In the present study, urinary NGAL was also significantly lower in WF than WH rats after I/R. To better characterize tubular function, we measured FENa in both WF and WH rats. It is recognized that an I/R episode is accompanied by a decrease in tubular sodium reabsorption. Our data showed that the postischaemic increase in FENa in WH rats was significantly reduced in WF rats, reinforcing the hypothesis that WF rats are protected against impaired renal function in a model of I/R injury. In addition, we showed that WF rats were protected against renal inflammation. The renal inflammatory process is a common feature of kidney transplantation occurring after the I/R episode. The inflammatory process is a response of the renal tissue to hypoxia and injury. It is important to understand that inflamma-
© 2014 Wiley Publishing Asia Pty Ltd
Ischaemic kidney injury in Wistar-Furth rats
Urinary H2O2 (nmol/mg Cr)
(a) 120
915
Table 1 Gene expression of nitric oxide synthase isoforms in the cortex and outer medulla of Wistar-Hanover and Wistar-Furth rats under control conditions (Time 0) and at different time points after ischaemia–reperfusion, as determined by real-time polymerase chain reaction
*†
100 80 60
nNOS 2 DDCt
iNOS 2 DDCt
eNOS 2 DDCt
1.00 0.19 0.09 0.04* 0.09 0.03*
1.00 0.33 3.68 0.45* 11.20 6.09
1.00 0.37 0.39 0.16 0.83 0.29
1.00 0.60 1.14 0.39 1.12 0.62
Undetectable Undetectable Undetectable
1.00 0.29 1.90 0.71 1.20 0.26
40
Cortex Wistar-Hanover Control 48 h 168 h Wistar-Furth Control 48 h 168 h Outer medulla Wistar-Hanover Control 48 h 168 h Wistar-Furth Control 48 h 168 h
20 0
0
48
168
(b)
Urinary MDA (nmol/mg Cr)
* *
0
48
168
Time after I/R (h) Fig. 4 Oxidative stress after ischaemia–reperfusion (I/R). Urinary (a) H2O2 and (b) malondialdehyde (MDA) levels in Wistar-Hanover (■, WH) and Wistar-Furth (□, WF) rats under control conditions (Time 0) and at different time points after I/R. Data are the mean SEM. *P < 0.05 compared with Time 0 (control conditions; one-way ANOVA for repeated measurements); †P < 0.05 compared with WH rats (two-way ANOVA for repeated measurements). Cr, creatinine.
tory cells or cytokines responding to the stress signal contribute to tissue injury at one stage, but also provide signals for the resolution of the injury and so are also involved in tissue repair. The inflammatory response is a complex mechanism between the beneficial and harmful issues. Thus, persistent inflammation characterized by phagocyte infiltration, such as macrophages, is detrimental for survival of the kidney transplant.4,37 In experimental I/R models, several studies have highlighted the persistence of a few damaged tubules associated with persistent inflammatory cells in the renal tissue despite normalization of plasma creatinine. This phenomenon proves that the initial injury can induce chronic damage to the kidney.34,38,39 In the present study, we observed an early significant increase in MCP-1 in renal ischaemic tissues of WH but not WF rats. Monocyte chemoattractant protein-1 is recognized as an early mediator of inflammation.40–42 The inflammatory response was also associated with increased macrophage and lymphocyte CD8 cells in both WH and WF rats 48 h after I/R. Later, on Day 7 after I/R, these populations of inflammatory cells were still enhanced in WH but not WF rats, further indicating a better renal outcome for the WF rat strain. Although it is clear that WF rats exhibit an evident protection against I/R injury, the underlying mechanisms remain unclear. In experimental CKD models using WF rats, investigators
1.00 0.37 18.6 10.8* 3.65 1.02
1.00 0.14 0.75 0.14 1.93 0.67
1.00 0.29 1.52 0.55 2.47 0.83
1.00 0.15 1.25 0.36 1.37 0.46
Data are the mean SEM (n = 6). *P < 0.05 compared with control (one-way ANOVA followed by the Holm–Sidak test). Neuronal nitric oxide synthase (nNOS) expression was not quantified in the outer medulla because nNOS is located in macula densa cells. iNOS, inducible nitric oxide synthase; eNOS, endothelial nitric oxide synthase.
hypothesized that NO could somehow be associated with this protection. Indeed, NO is an important renal mediator under both normal and pathological conditions. Its functions may depend on the site and the level of production, as well as on the local cellular environment.11 Nitric oxide synthesis occurs through activation of NOS. Most of the physiological effects of NO are produced by activation of constitutive isoforms of NOS, namely nNOS (NOS1) and eNOS (NOS3), whereas the third isoform (iNOS, NOS2) is inducible mostly in an inflammatory environment.18 In the kidney, NO is known to be involved in the regulation of vascular resistance, GFR, renin secretion and water and sodium excretion, as well as in the maintenance of renal structural integrity.9,10 In addition, NO may have beneficial or deleterious effects depending on its concentration, duration of release and site of production.9,11,12 To determine whether NO could also be involved in the protection of WF rats against I/R injury, NOS expression was measured. Similar to previous studies in models of CKD,24,26,31 our data demonstrated that nNOS expression was maintained at the mRNA and protein levels in the renal tissue of WF rats after I/R injury, whereas nNOS expression was decreased in vulnerable WH rats. As proposed by Erdely et al.31 the preserved nNOS expression in WF rats may be explained by blockade of the renin–angiotensin system (RAS). Indeed, in a model of renal mass reduction using WF rats, it has been reported that aldosterone levels, expected to increase, remained unchanged.24 In addition, another study showed that inhibition of the RAS was associated with
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916 WH
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WF
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iNOS (%)
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5
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Time after I/R (h)
Fig. 5 Intrarenal neuronal nitric oxide synthase (nNOS) expression after ischaemia–reperfusion (I/R). Representative photomicrographs of nNOS immunostaining in the macula densa of (a, c, e) Wistar-Hanover (WH) and (b, d, f) Wistar-Furth (WF) rats under control conditions (a, b) and 48 h (c, d) and 7 days (e, f) after I/R. Sections were counterstained with haemalun and luxol fast blue. Arrowheads indicate macula densa localization. (g) Quantitative analysis of nNOS-positive staining in the macula densa in WH (■) and WF (□) rats under control conditions (Time 0) and at different time points after I/R. Data are the mean SEM (n = 6). *P < 0.05 compared with control (one-way ANOVA followed by Dunnett’s test); †P < 0.05 compared with WH rats (two-way ANOVA).
Fig. 6 Intrarenal inducible nitric oxide synthase (iNOS) expression after ischaemia–reperfusion (I/R). Representative photomicrographs of iNOS immunostaining in kidney tissues of (a, c, e) Wistar-Hanover (WH) and (b, d, f) Wistar-Furth (WF) rats under control conditions (a, b) and 48 h (c, d) and 7 days (e, f) after I/R. Expression of iNOS was localized to the collecting duct (CD) under control conditions (a, b) and was enhanced in the proximal tubule (PT) 48 h after I/R (c, d). On Day 7 after I/R (e, f), renal tissue from both strains exhibits many cystic tubules (CT), as well as attenuation of iNOS expression. (g, h) Quantitative analysis of iNOSpositive staining in WH (■) and WF (□) rats under control conditions (Time 0) and at different time points after I/R in the cortex (g) and outer medulla (h). Data are the mean SEM (n = 6). *P < 0.05 compared with control (one-way ANOVA followed by Dunnett’s test); †P < 0.05 compared with WH rats (two-way ANOVA).
maintenance of renal nNOS production.43 In the kidney, nNOS is specifically expressed in the macula densa, which plays a major role in the regulation of renal haemodynamics. Indeed, following activation of TGF, local production of NO induces a vasodilator response of afferent arterioles,13 preventing a decrease in GFR and thus a decline in renal function. Tubular injury can also be linked to filtration failure through mechanisms such as tubular obstruction, which is common after I/R injury.44 In the present study, the postischaemic increase in FENa in WH rats, which reflects functional tubular impairment with reduced reabsorptive capacity, was significantly reduced in WF rats. Furthermore, our data demonstrated that, as reported
previously in various models of CKD,24–26,31 nNOS expression was maintained in the macula densa of WF rats after I/R injury, whereas nNOS expression was decreased in WH rats. Together, the data suggest that, in WF rats, activation of TGF is reduced due to less tubular impairment and that the counteracting effect of nNOS-derived NO on the TGF response remains effective, thus contributing to improved postischaemic renal function. With regard to iNOS expression, the results of the present study indicate that iNOS expression is significantly induced after I/R in WH rats. This was shown at both the mRNA and protein level (by immunolocalization of iNOS in the ischaemic renal parenchyma). In contrast, this increase was not observed in WF
0
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© 2014 Wiley Publishing Asia Pty Ltd
Ischaemic kidney injury in Wistar-Furth rats
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Time after I/R (h) Fig. 7 Urinary excretion of (a) prostaglandin (PG) E2 and (b) thromboxane (Tx) B2 in Wistar-Hanover (■, WH) and Wistar-Furth (□, WF) rats under control conditions (Time 0) and at different time points after ischaemia–reperfusion (I/R). Data are the mean SEM. *P < 0.05 compared with control (one-way ANOVA followed by Dunnett’s test); † P < 0.01 compared with WH rats (t-test).
rats. Several studies have reported induction of iNOS expression after renal ischaemic insult along with increased kidney damage.18,45,46 Moreover, in many studies, the use of specific iNOS inhibitors reduced postischaemic injury.17,18,32,34,47 Similar data have been reported in iNOS-knockout mice,33 demonstrating the adverse effect of iNOS-derived NO. In experimental I/R models, iNOS is usually related to inflammation and oxidative stress. After I/R insult, an increase in reactive oxygen species (ROS), such as the free radical superoxide, is observed.48,49 The superoxide is known to interact with NO-derived from iNOS to produce peroxynitrite, a highly toxic reactive nitrosative species (RNS), thereby contributing to the pathogenesis of renal postischaemic injury.19 In the present study, the lower inflammatory response in WF rats may be related to the lower induction of iNOS. Unfortunately, we were not able to determine whether the oxidative stress response after the I/R injury was also improved in WF rats. Superoxide is tricky to measure because of its short half-life. Currently, there is a lack of available and reliable techniques to measure oxidative stress. Malondialdehyde, another marker used in the present study to evaluate lipid peroxidation, did not reveal any differences between the two strains. Therefore, we evaluated levels of H2O2, also known as a marker of oxidative stress. Unexpectedly, we showed that H2O2 levels were increased in WF compared with WH rats. The reasons for this result are unclear, but it could be due to faster formation of peroxynitrite from the reaction of superoxide and iNOS-derived NO against
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lower superoxide degradation by superoxide dismutase (SOD).50 However, this requires further investigation. Finally, assuming that not just preserved nNOS and reduced iNOS induction can protect the kidney against I/R injury, we investigated the involvement of another vasoactive factor, namely TxA2. In the kidney, TxA2 acts as a vasoconstrictor agent and is known to play a role in renal haemodynamics by modulating vascular resistance. In the kidney, TxA2 is generated by specific enzymes, such as TxA2 synthase, acting on products of COX-1 and COX-2.51 However, TxA2 is very unstable and quickly degrades to a biologically inactive product, TXB2, which can be then easily measured. Our data clearly indicated that urinary TxB2 was lower in both the normal and postischaemic kidney of WF rats, whereas excretion of the vasodilator PGE2, the main prostanoid in the kidney, was similarly increased in both WH and WF rats. The ratio between vasodilator (PGE2) and vasoconstrictor (TxA2) autacoids, which is routinely used as an indicator of the direct role of vasoconstrictor factors in the development of renal failure,52–54 appeared to be higher in WF than WH rats after I/R. Interestingly, COX-2 activity has been reported to depend on nNOS activity.55 During acute inhibition of COX-2, renal haemodynamics were maintained due to a reduction in TxA2 production along with increased nNOS activity.20 It has also been reported in an in vitro study that NO inhibits TxA2 synthase activity in a concentration-dependent manner.56 Therefore, we may hypothesize that, in WF rats, the preserved production of nNOS-derived NO, along with decreased TxA2 synthesis, maintains a balance between vasoactive mediators, promoting decreased renal vascular resistance and hence reducing renal injury. In conclusion, the data of the present study demonstrate that the WF rat strain also exhibits marked protection against ischaemic AKI, as evidenced by lowered creatininaemia and renal inflammation, maintenance of tubular function, preservation of nNOS expression and prevention of iNOS upregulation This protection may be associated with a complex interplay between paracrine mediators such as NO and TxA2. Perspectives Future investigations are needed to better understand the protection against renal failure in WF rats. To this end, it will be of interest to evaluate the potential effects of specific and selective inhibitors of the different NOS isoforms. In this regard, several in vitro and in vivo studies have demonstrated that specific inhibition of the expression or activity of iNOS, as well as the absence of iNOS expression in knockout mice, can reduce postischaemic injury,17,18,32,33,47,48 suggesting a deleterious effect of iNOS-derived NO. Conversely, the use of specific inhibitors, such as S-methyl-L-thiocitrulline (SMTC), a specific inhibitor of nNOS, or sulotroban, a specific antagonist of the thromboxane (TP) receptor,57 should be considered as pharmacological tools to delineate the relative involvement of nNOS and TxA2 in the protection of postischaemic AKI in WF rats. Finally, the interplay between NO and endothelin in the postischaemic kidney of WF rats should be addressed to further investigate the imbalance between vasoactive factors, because endothelin has been demonstrated previously to be increased after renal I/R and to participate in renal damage.58
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METHODS Animals The present study conformed to the American Society of Physiology’s Guiding Principles in the Care and Use of Animals (http:// www.the-aps.org/mm/sciencepolicy/about/policy-statements/guiding-principles-.html, accessed 5 September 2014) and the protocols were approved by the local Ethics Commission on Animal Welfare. Experiments were performed on 49 male WH and 47 male WF rats (Harlan, Horst, the Netherlands), weighing 220– 260 g. In the first experiment, rats from each strain were randomly distributed in three groups, as detailed below. 1. Group I: I/R 7 days (WH, n = 9; WF, n = 11). Rats were anaesthetized with 60 mg/kg, i.p., sodium pentobarbital (Nembutal; CEVA, Libourne, France) and 0.15 mg/kg buprenorphine hydrochloride (Ecuphar, Oostkamp, Belgium), placed on a heated table and subjected to a right nephrectomy followed by occlusion of the left renal artery for 60 min, as described previously.34 Rats were allowed to recover and were placed in metabolic cages for daily weighing and urine sampling over a period of 7 days. Finally, on Day 7, blood samples were collected and the remaining kidney was harvested. 2. Group II: control (WH, n = 8; WF, n = 8). Control rats were anaesthetized and subjected to sham surgery, including laparotomy and manipulation of the left renal vascular pedicle without the induction of left renal ischaemia or contralateral nephrectomy. 3. Group III: I/R group 12 h and 48 h (WH, n = 20; WF, n = 20). Rats were subjected to the same surgical procedures as rats in Group I. Blood samples and the left kidney were collected 12 h and 48 h after I/R. The right kidney was considered as the control kidney. The kidneys were then processed for further experiments. Portions of kidneys were dissected into the cortex and OM before being snap-frozen in liquid nitrogen for RNA and protein isolation. An additional portion of kidney was fixed in Duboscq-Brazil solution for immunostaining. Urine and blood data Plasma creatinine concentration was measured by the Jaffe reaction. Urine creatinine concentrations were measured using a commercially available kit (Assay Designs, Antwerpen, Belgium). Urinary NGAL was determined using an ELISA kit (Bioporto Diagnostics, Hellerup, Denmark). Total urine protein levels were evaluated by using the Bradford dye binding assay with bovine serum albumin as the standard (Sigma-Aldrich, Diegem, Belgium). As an index of oxidative stress, urine samples were also analysed for hydrogen peroxide by the Amplex red assay (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s instructions. Urinary and plasma sodium concentrations were measured by flame emission spectrophotometry (IL 943; Instrumentation Laboratory, Zaventem, Belgium) to calculate FENa according to the standard formula. Finally, urinary MDA levels were determined by measuring the thiobarbituric acid-reactive substances. All urinary markers were normalized against creatinine to obviate any losses in collection.
Immunostaining for ED1 (mouse anti-rat monoclonal antibodies (mAbs); 1 : 50; Serotec, Puchheim, Germany), CD8 (mouse antirat mAbs; 1 : 50; Serotec), nNOS (mouse anti-rat mAbs; 1 : 75; Transduction Laboratories, Leuven, Belgium) and iNOS (1 : 200; NeoMarkers, Fremont, CA, USA) was performed on paraffinembedded rat kidney sections as described previously.34 Briefly, after dewaxing and rehydration, microwave pretreatment in citrate buffer (pH 6.2) was performed to unmask antigens present in the renal tissue. Tissue sections were then incubated for 1 h with different primary antibodies. After rinsing in phosphate-buffered saline (PBS), slides were exposed for 30 min to the appropriate secondary antibody. Kidney sections were finally incubated with ABC complex (Vector Laboratories, Burlingame, CA, USA) for 30 min and bound peroxidase activity was detected with a diaminobenzidine (DAB) kit. The number of ED1- or CD8-positive cells was counted under a light microscope at 9400 magnification in five squares fields (0.084 mm2/field) randomly taken in each renal zone. ImageJ (National Institutes of Health, Bethesda, MD, USA) was used to quantify the percentage of the NOS-positive area. Twenty random fields in the renal tissue were analysed at 9200 magnification. Renal tissue MCP-1 Samples of renal tissue were homogenized in PBS (1 mg/20 nL). The concentration of MCP-1 was measured in renal homogenates using the OptEAI Set for MCP-1 (BD Biosciences Pharmingen, Antwerpen, Belgium) according to the manufacturer’s instructions, and is expressed as pg MCP-1/mg renal tissue. Reverse transcription–polymerase chain reaction analysis Total RNA from the cortex and OM was extracted using the guanidinium thiocyanate method. All samples were quantified by spectrophotometric analysis at 260 nm. Total RNA (2 lg) was reverse transcribed using random hexamers and Superscript II MMLV-reverse transcriptase (InVitrogen, Merelbeke, Belgium). Real-time PCR reactions were performed on ABI PRISM 7000 Sequence Detection System using Taqman Universal PCR Master Mix and Assays-on-demand Gene expression Probes (Applied Biosystems, Applera, France) for the different isoforms of NOS genes (NOS1 assay ID: Rn 00583793_m1; NOS2 assay ID: Rn00561646_m1; NOS3 assay ID: Rn02132634_S1; and 18S assay ID: HS99999901_S1). Analysis of relative target gene expression was performed using the comparative Ct method. Results were normalized against data for the 18S rRNA internal control. The amount of target gene mRNA, normalized against an internal control and relative to a calibrator, is given by 2 DDCt . The change in gene expression after I/R was calculated as the ratio between the average 2 DDCt value corresponding to I/ R rats and the average 2 DDCt value corresponding to control rats. Calculations and statistical analysis Results are presented as the mean SEM and further statistical analysis was performed using SIGMASTAT (Systat Software,
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Ischaemic kidney injury in Wistar-Furth rats Erkrath, Germany). The level for statistical significance was set at P < 0.05, which refers to two-sided probability. Student’s ttest was used to determine significant differences between two groups, one-way ANOVA was used for multiple inter-group comparisons followed by Dunnett’s test and two-way ANOVA was used for repeated measurements when appropriate, followed by a Holm–Sidak test.56
ACKNOWLEDGEMENTS The authors thank Dr G Toubeau (University of Mons, Mons, Belgium) and Dr Y. Poumay (University of Namur, Namur, Belgium) for their technical support. This work was supported, in part, by grants from Fonds de la Recherche Scientifique, Belgium.
DISCLOSURE No conflicts of interest, financial or otherwise, are declared by the authors.
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16. Ichihara A, Hayashi M, Navar LG, Saruta T. Inducible nitric oxide synthase attenuates endothelium-dependent renal microvascular vasodilation. J. Am. Soc. Nephrol. 2000; 11: 1807–12. 17. Chatterjee PK, di VBR, Sivarajah A, McDonald MC, Cuzzocrea S, Thiemermann C. Pyrrolidine dithiocarbamate reduces renal dysfunction and injury caused by ischemia/reperfusion of the rat kidney. Eur. J. Pharmacol. 2003; 482: 271–80. 18. Korkmaz A, Kolankaya D. Inhibiting inducible nitric oxide synthase with rutin reduces renal ischemia/reperfusion injury. Can. J. Surg. 2013; 56: 6–14. 19. Walker LM, Walker PD, Imam SZ, Ali SF, Mayeux PR. Evidence for peroxynitrite formation in renal ischemia-reperfusion injury: Studies with the inducible nitric oxide synthase inhibitor L-N6-(1iminoethyl) lysine. J. Pharmacol. Exp. Ther. 2000; 295: 417–22. 20. Araujo M, Welch WJ. Cyclooxygenase 2 inhibition suppresses tubuloglomerular feedback: Roles of thromboxane receptors and nitric oxide. Am. J. Physiol. Renal Physiol. 2009; 296: F790–4. 21. Araujo M, Welch WJ. Tubuloglomerular feedback is decreased in COX-1 knockout mice after chronic angiotensin II infusion. Am. J. Physiol. Renal Physiol. 2010; 298: F1059–63. 22. Sciotti V, Gallant S. Resitence to mineralocorticoid-induced hypertensive vascular disease. Hypertension 1987; 10: 176–80. 23. Bruner CA. Vascular responsiveness in rats resistant to aldosteronesalt hypertension. Hypertension 1992; 20: 59–66. 24. Fitzgibbon WR, Greene EL, Grewal JS et al. Resistance to remnant nephropathy in the Wistar-Furth rat. J. Am. Soc. Nephrol. 1999; 10: 814–21. 25. Erdely A, Freshour G, Smith C, Engels K, Olson JL, Baylis C. Protection against puromycin aminonucleoside-induced chronic renal disease in the Wistar-Furth rat. Am. J. Physiol. Renal Physiol. 2004; 287: F81–9. 26. Erdely A, Freshour G, Tain YL, Engels K, Baylis C. DOCA/NaClinduced chronic kidney disease: A comparison of renal nitric oxide production in resistant and susceptible rat strains. Am. J. Physiol. Renal Physiol. 2007; 292: F192–6. 27. Boersema M, Katta K, Rienstra H et al. Local medial microenvironment directs phenotypic modulation of smooth muscle cells after experimental renal transplantation. Am. J. Transplant. 2012; 12: 1429–40. 28. Cassis P, Gallon L, Benigni A et al. Erythropoietin, but not the correction of anemia alone, protects from chronic kidney allograft injury. Kidney Int. 2012; 81: 903–18. 29. Decleves AE, Caron N, Nonclercq D et al. Dynamics of hyaluronan, CD44, and inflammatory cells in the rat kidney after ischemia/reperfusion injury. Int. J. Mol. Med. 2006; 18: 83–94. 30. Bell PD, Komlosi P, Zhang ZR. ATP as a madiator of macula densa cell sigalling. Purinergic Signal. 2009; 5: 461–71. 31. Erdely A, Wagner L, Muller V, Szabo A, Baylis C. Protection of Wistar Furth rats from chronic renal disease is associated with maintained renal nitric oxide synthase. J. Am. Soc. Nephrol. 2003; 14: 2526–33. 32. Chatterjee PK, Patel NS, Kvale EO et al. Inhibition of inducible nitric oxide synthase reduces renal ischemia/reperfusion injury. Kidney Int. 2002; 61: 862–71. 33. Ling H, Gengaro PE, Edelstein CL et al. Effect of hypoxia on proximal tubules isolated from nitric oxide synthase knockout mice. Kidney Int. 1998; 53: 1642–6. 34. Noiri E, Peresleni T, Miller F, Goligorsky MS. In vivo targeting of inducible NO synthase with oligodeoxynucleotides protects rat kidney against ischemia. J. Clin. Invest. 1996; 97: 2377–83. 35. Omerika L, Rasic S, Serdarevic N. Importance of determination of urine neutrophile gelatinase associated lipocalin in early detection of acute kidney injury. Coll. Antropol. 2014; 38: 161–6. 36. Hosgood SA, Hunter JP, Nicholson ML. Early urinary biomarkers of warm and cold ischemic injury in an experimental kidney model. J. Surg. Res. 2011; 174: e85–90.
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37. Burne-Taney MJ, Liu M, Baldwin WM, Racusen L, Rabb H. Decreased capacity of immune cells to cause tissue injury mediates kidney ischemic preconditioning. J. Immunol. 2006; 176: 7015–20. 38. Basile DP, Donohoe DL, Roethe K, Mattson DL. Chronic renal hypoxia after acute ischemic injury: Effects of L-arginine on hypoxia and secondary damage. Am. J. Physiol. Renal Physiol. 2003; 284: F338–48. 39. Forbes JM, Hewitson TD, Becker GJ, Jones CL. Ischemic acute renal failure: Long term histology of cell and matrix changes in rat. Kidney Int. 2000; 57: 2375–85. 40. Jo SK, Sung SA, Cho WY, Go KJ, Kim HK. Macrophages contribute to the initiation of ischaemic acute renal failure in rats. Nephrol. Dial. Transplant. 2006; 21: 1231–9. 41. Vinuesa E, Hotter G, Jung M, Herrero-Fresneda Torras J, Sola1 A. Macrophage involvement in the kidney repair phase after ischaemia/ reperfusion injury. J. Pathol. 2008; 214: 104–13. 42. Decleves AE, Mathew AV, Cunard R, Sharma K. AMPK mediates the initiation of kidney disease induced by a high-fat diet. J. Am. Soc. Nephrol. 2011; 22: 1846–55. 43. Roczniak A, Fryer JN, Levine DZ, Burns KD. Downregulation of neuronal nitric oxide synthase in the rat remnant kidney. J. Am. Soc. Nephrol. 1999; 10: 704–13. 44. Singh P, Okusa MD. The role of tubuloglomerular feedback in the pathogenesis of acute kidney injury. Contrib. Nephrol. 2011; 174: 12–21. 45. Betz B, Schneider R, Kress T, Schick MA, Wanner C, Sauvant C. Rosiglitazone affects nitric oxide synthases and improves renal outcome in a rat model of severe ischemia/reperfusion injury. PPAR Res. 2012; 2012: 219319. 46. Vi~ nas JL, Sola A, Genesca M, Alfaro V, Pı F, Hotter G. NO and NOS isoforms in the development of apoptosis in renal ischemia/ reperfusion. Free Radic. Biol. Med. 2006; 40: 992–1003. 47. Mark LA, Robinson AV, Schulak JA. Inhibition of nitric oxide synthase reduces renal ischemia/reperfusion injury. J. Surg. Res. 2005; 129: 236–41.
48. Noiri E, Nakao A, Uchida K et al. Oxidative and nitrosative stress in acute renal ischemia. Am. J. Physiol. Renal Physiol. 2001; 281: F948–57. 49. Tan X, Zhang L, Jiang Y et al. Postconditioning ameliorates mitochondrial DNA damage and deletion after renal ischemic injury. Nephrol. Dial. Transplant. 2013; 28: 2754–65. 50. Huie RE, Padmaja S. The reaction of NO with superoxide. Free Radic. Res. Commun. 1993; 18: 195–9. 51. Qi Z, Cai H, Morrow JD, Breyer MD. Differentiation of cyclooxygenase 1- and 2-derived prostanoids in mouse kidney and aorta. Hypertension 2006; 48: 323–8. 52. Fukuzaki A, Morrissey J, Klahr S. Role of glomerular eicosanoid production in the obstructed kidney. Int. Urol. Nephrol. 1993; 25: 525–31. 53. Papanikolaou N, Darlametsos I, Tsipas G et al. Effects of OKY046 and nifedipine in cyclosporine-induced renal dysfunction in rats. Prostaglandins Leukot. Essent. Fatty Acids 1996; 55: 249– 56. 54. Darlametsos I, Morphake P, Bariety J et al. Effect of ketanserine in cyclosporine-induced renal dysfunction in rats. Nephron 1995; 70: 249–54. 55. Ichihara A, Hayashi M, Hirota N, Saruta T. Superoxide inhibits neuronal nitric oxide synthase influences on afferent arterioles in spontaneously hypertensive rats. Hypertension 2001; 37: 630–4. 56. Wade ML, Fitzpatrick FA. Nitric oxide modulates the activity of the hemoproteins prostaglandin I2 synthase and thromboxane A2 synthase. Arch. Biochem. Biophys. 1997; 347: 174–80. 57. Kramer HJ, Mohaupt MG, Pinoli F, Backer A, Meyer-Lehnert H, Schlebusch H. Effects of thromboxane A2 receptor blockade on oliguric ischemic acute renal failure in conscious rats. J. Am. Soc. Nephrol. 1993; 4: 50–7. 58. Abu-Saleh N, Ovcharenko E, Awad H et al. Involvement of the endothelin and nitric oxide systems in the pathogenesis of renal ischemic damage in an experimental diabetic model. Life Sci. 2012; 91: 669–75.
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doi: 10.1111/1440-1681.12298
ORIGINAL ARTICLE
Protection of Wistar-Furth rats against postischaemic acute renal injury: Role for nitric oxide and thromboxane? Viginie Voisin,* Anne-Emilie Decleves,* Virginie Hubert,* Vanessa Colombaro,* Laetitia Giordano,* Isabelle Habsch,* Nadine Bouby,† Denis Nonclercq‡ and Nathalie Caron* *Molecular Physiology Research Unit–Namur Research Institute for Life Sciences, Univerity of Namur, Namur, Belgium, † UMRS872 Team no. 2, Cordelier Research Center, Paris, France and ‡Department of Histology, University of Mons, Mons, Belgium
SUMMARY The Wistar-Furth (WF) rat strain is usually used in models of full major histocompatibility complex-mismatched kidney transplantation. Because these rats have been demonstrated to be resistant to several models of chronic kidney disease, the aim of the present study was to investigate their potential resistance to renal ischaemia–reperfusion (I/R) injury compared with another strain, namely Wistar-Hanover (WH) rats. Anaesthetized male WH and WF rats were submitted to I/R by occlusion of the left renal artery and contralateral nephrectomy. Urine, blood and tissue samples were collected at different time points after I/R to evaluate renal function, inflammation and tubular injury, along with determination of nitric oxide synthase (NOS) expression and thromboxane A2 (TxA2) production. Post-ischaemic renal function was better preserved in WF than WH rats, as evidenced by reduced levels of creatininaemia, urinary neutrophil gelatinase-associated lipocalin excretion and proteinuria. In addition, WF rats had less intrarenal inflammation than WH rats after I/R injury. These observations were associated with maintenance of neuronal NOS expression, along with lower induction of inducible NOS expression in WF versus WH rats. Moreover, WF rats excreted a significantly lower amount of TxB2. The results indicate that WF rats are more resistant to an I/R injury than WH rats in terms of renal function and inflammation. These observations are associated with differential regulation of intrarenal NOS expression, as well as a reduction in thromboxane production, which could contribute to a better outcome for the postischaemic kidney in WF rats. Key words: acute renal injury, nitric oxide synthase, renal ischaemia–reperfusion, thromboxane, Wistar-Furth rats.
Correspondence: Nathalie Caron, 61 rue de Bruxelles, B-5000, Namur, Belgium. Email: [email protected] Received 13 November 2013; revision 6 July 2014; accepted 7 August 2014. © 2014 Wiley Publishing Asia Pty Ltd
INTRODUCTION Acute kidney injury (AKI) is a very common clinical complication contributing to significant morbidity and mortality in the intensive care unit.1–3 Renal ischaemia–reperfusion (I/R) injury is a well-known experimental model used to investigate the pathogenesis of AKI, particularly because I/R insult is a prominent negative factor in the survival of renal allografts.4 Specifically, I/ R injury causes cellular hypoxia, increased oxidative stress, leucocyte infiltration and tubular and endothelial injuries. In particular, endothelial dysfunction is characterized by an imbalance in vasoactive factors in favour of the synthesis of endothelin, angiotensin II and thromboxane (Tx) A2, along with reduced nitric oxide (NO) production, thereby enhancing the degree of vasoconstriction.5,6 Furthermore, even a short episode of AKI can lead to long-term complications, including chronic kidney disease (CKD) or end-stage renal disease.7 These complications are usually associated with incomplete tubular repair, persistent tubulointerstitial inflammation, proliferation of fibroblasts and excessive accumulation of extracellular matrix.1 This highlights the urgent need to better characterize the pathogenesis of AKI to develop potent therapeutic approaches. One of the cellular responses to an ischaemic insult is decreased NO production, limiting the pool of vasodilator factors.5,8 Especially in the kidney, NO is involved in the regulation of vascular resistance and glomerular filtration rate (GFR). It also maintains renal structural integrity.9,10 However, NO has multiple actions: both beneficial and deleterious.9,11,12 Constitutive NO production by endothelial (e) NO synthase (NOS) induces vasodilation and reduces endothelial dysfunction,11 whereas neuronal (n) NOS-derived NO, produced in the macula densa in the kidney, plays a role in the regulation of renal haemodynamics by modulating tubuloglomerular feedback (TGF).10,13–15 In contrast, NO produced by inducible (i) NOS is primarily involved in inflammation or oxidative stress.16 Several studies have shown that inhibition of iNOS improves renal injury and reduces oxidative stress.17–19 Another potent mediator of renal function is TxA2. It has been shown that TxA2 controls afferent arteriole tone and is therefore involved in TGF.20,21 Hence, there is a balance between NO produced by nNOS and TxA2 produced by cyclo-oxygenase-(COX) 2. Indeed, COX2 can generate TxA2 and the free radical
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RESULTS
(a) 700
Creatininaemia (µmol/L)
superoxide, leading to a simultaneous reduction in NO bioavailability.20 This may impair the TGF response. The aim of the present study was to investigate the potential resistance of Wistar-Furth (WF) rats to I/R injury and to determine whether this could be linked to NO production. Indeed, several studies have reported that WF rats are resistant to the development of CKD in experimental rodent models, such as mineralocorticoid hypertension-induced renal injury,22,23 the 5/6 nephrectomy model,24 puromycin-aminonucleoside (PAN)induced CKD25 and DOCA salt-induced CKD.26 In these studies, it was suggested that NO production, and particularly nNOS expression, were maintained after renal injury in WF rats, which could explain their resistance to CKD. Interestingly, another study reported that WF rats were protected against renal microcirculation injury after chronic NOS inhibition.26 The findings of that study suggest that in addition to NO other mechanisms may be involved in the protection of WF rats against progression to CKD.26 Thereby, demonstrating reduced susceptibility to AKI in WF rats could be of particular interest to investigate therapeutic approaches. Moreover, because this rat strain is often used in rat models of full major histocompatibility complex-mismatched kidney transplantation,27,28 this potential resistance to AKI should be taken into consideration when interpreting the results of such studies. In the present study, we sought to determine whether WF rats were resistant to AKI in a model of renal I/R. The renal response to I/R injury in WF rats was then compared with that in another rat strain, namely Wistar-Hanover (WH) rats. To further characterize the potential resistance, we also evaluated renal NOS expression and TxA2 production after I/R.
*
600 500 400
†
300 200 100
0 0 (b)
NGAL (×105 U/24 h)
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*
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Time after I/R (h) Fig. 1 Evaluation of renal function. Temporal evolution of (a) creatininaemia and (b) 24 h urinary neutrophil gelatinase-associated lipocalin (NGAL) excretion in Wistar-Hanover (■, WH) and Wistar-Furth (□, WF) rats under control conditions (Time 0) and at different time points after ischaemia–reperfusion (I/R). Results are the mean SEM (n = 6–9 per group). *P < 0.05 compared with Time 0; †P < 0.05 compared with WH rats (two-way ANOVA (a) or two-way ANOVA for repeated measurements (b)).
General observations The mortality rate in WF and WH rats after I/R was 1.4% and 3.9%, respectively. The weight of the right kidney was similar in all groups, with a mean ( SEM) of 1.01 0.03 g. In control WH and WF rats, left kidney weight did not differ significantly from that of the right kidney. However, after I/R, the weight of the left kidney in WH rats increased progressively from 0.93 0.05 g on Day 0 to 2.16 0.07 g on Day 7 after I/R (P < 0.05). The progressive increase in postischaemic left kidney weight in WF rats was less pronounced, with an increase from 0.93 0.05 g on Day 0 to 1.76 0.06 g on Day 7 after I/R (P < 0.05); the difference between the WH and WF groups was significant on Day 7 (P < 0.05). Renal function and renal injury after I/R To determine whether WF rats may be protected against renal ischaemic insult, we measured levels of two markers of AKI,5,29 namely plasma creatinine and urinary neutrophil gelatinase-associated lipocalin (NGAL). As shown in Fig. 1, under control conditions, creatininaemia and NGAL excretion were similar in WH and WF rats. However, 48 h after I/R, creatininaemia (Fig. 1a) had increased nearly fivefold in WH rats (P < 0.001), but thereafter returned to baseline levels by 72 h. In WF rats, despite a slight (non-significant) increase in creatin-
inaemia at 48 h after I/R, there were no significant changes in creatininaemia throughout the observation period. There was a 130-fold increase in NGAL excretion in WH rats (Fig. 1b) 48 h after I/R (P < 0.05), but NGAL excretion had returned to baseline levels 72 h after I/R. In WF rats, there was also a significant increase in NGAL excretion 48 h after I/R, but this increase corresponded only to a 30-fold change (P < 0.05) compared with baseline, and NGAL excretion had returned to baseline levels by 72 h after I/R. Proteinuria was also determined in WH and WF rats (Fig. 2a). Under control conditions, levels were similar in both strains. A significant increase in proteinuria was observed 48 h after I/R in both strains (P < 0.05), but the magnitude of the increase was less in WF than WH rats (P < 0.05). To further characterize renal function, and particularly tubular function, we evaluated renal sodium handling by measuring fractional excretion of sodium (FENa), which enables determination of the ability of renal tubules to reabsorb sodium properly (Fig. 2b). As indicated in Fig. 2b, FENa under control conditions was similar in WH and WF rats (1.17 0.07% vs 1.49 0.06%, respectively; NS). Forty-eight hours after I/R, FENa was increased significantly in both strains, but the increase was 2.5-fold higher in WH than WF rats (9.20 2.09% vs
© 2014 Wiley Publishing Asia Pty Ltd
Ischaemic kidney injury in Wistar-Furth rats
Proteinuria (mg/24 h)
(a) 50
Oxidative stress after I/R in WH and WF rats
*
To determine the level of oxidative stress, urinary H2O2 and malondialdehyde (MDA) excretion were measured. Values for both were similar under control conditions in both rat strains (Fig. 4a). Interestingly, 48 h after I/R, WF rats had significantly higher urinary H2O2 levels than WH rats (89 19 vs 32 5 nmol/mg creatinine, respectively; P < 0.05). Thereafter, H2O2 levels returned to baseline by Day 7 in both strains. As shown in Fig. 4b, urinary MDA excretion measured under control conditions was similar in both rat strains. Urinary MDA levels increased significantly 48 h after I/R in both WH and WF rats, with no significant difference between the two strains.
*†
40 30 20 10 0 0
(b) 12
168
48
*
FENa (%)
10
Renal NOS expression after I/R in WH and WF rats
8 6
*†
4 2 0
913
0
48
168
Time after I/R (h) Fig. 2 Evaluation of renal injury. Temporal evolution of (a) proteinuria and (b) fractional excretion of sodium (FENa) in Wistar-Hanover (■, WH) and Wistar-Furth (□, WF) rats under control conditions (Time 0) and at different time points ischaemia–reperfusion (I/R). Results are the mean SEM (n = 6–9 per group). *P < 0.05 compared with Time 0; † P < 0.05 compared with WH rats (two-way ANOVA for repeated measurements (a) or two-way ANOVA (b)).
3.72 1.32%, respectively; P < 0.05), suggesting higher salt wasting in WH rats due to enhanced tubular dysfunction. Thereafter, on Day 7 after I/R, FENa levels had returned to baseline levels in both strains. Inflammatory responses after I/R in WH and WF rats To determine whether WF rats are also protected against renal inflammation occurring after an ischaemic insult, tissue levels of monocyte chemoattractant protein-1 (MCP-1), considered an early marker of inflammation, and macrophage and CD8 infiltration, considered chronic markers of inflammation, were investigated (Fig. 3). As shown in Fig. 3a, a significant increase in tissue MCP-1 levels was observed in WH rats 12 h after I/R (P < 0.05), whereas there was no significant change in MCP-1 levels in WF rats. Increased macrophage infiltration was observed in both WH and WF rats 48 h after I/R (P < 0.05), equating to approximately 300 ED1-positive cells/mm2 in both strains. Interestingly, on Day 7 after I/R, the number of ED1-positive cells had increased even further in WH rats but not in WF rats (1045 180 vs 762 126 ED1-positive cells/mm2, respectively; P < 0.05 for WF vs WH rats; Fig. 3b, d, e). Similarly, the increased CD8 infiltration 48 h and on Day 7 after I/R in WH rats was not seen in WF rats (P < 0.05; Fig. 3c,f,g).
To evaluate NOS expression in WF rats after renal ischaemic insult, nNOS, eNOS and iNOS mRNA levels were measured by real-time polymerase chain reaction in the renal cortex and outer medulla (OM). As indicated in Table 1, cortical mRNA levels for nNOS decreased significantly 48 h and 7 days after I/R in WH rats. However, in WF rats, nNOS mRNA levels were maintained throughout the observation period. In contrast, iNOS mRNA levels in WH rats were significantly increased 48 h after I/R in the cortex and the OM. In WF rats, iNOS expression was undetectable in the cortex and barely increased in the OM after I/R. There were no significant changes in eNOS expression. To confirm our data and better illustrate the localization of nNOS and iNOS in the kidney, immunostaining analysis was performed on paraffin-embedded kidney sections. Representative photomicrographs of nNOS staining in the cortex in WH and WF rats are shown in Fig. 5a–f. Under control conditions (Fig. 5a,b), nNOS was found in the macula densa. Forty-eight hours after I/R, positive nNOS staining was decreased significantly in WH rats compared with baseline (27.5 3.3% vs 76.7 3.6%, respectively; P < 0.05; Fig. 5c,g). In contrast, in WF rats, the immunolocalization of nNOS was preserved in the macula densa 48 h after I/R compared with baseline (62.8 6.2% vs 75.7 5%; NS; Fig. 5d,g). Seven days after I/R, this difference between strains was still evident (Fig. 5e–g). Representative photographs of iNOS-positive staining in WH and WF rats are shown in Fig. 6a–f. Under control conditions, iNOS was mostly localized in the collecting ducts (Fig. 6a,b). After I/R injury, iNOS expression increased significantly in WH rats after both 48 h (Fig. 6c) and 168 h (Fig. 6e). Furthermore, iNOS was no longer primarily localized in the collecting ducts, but was also strongly expressed in the proximal tubules. Although the same pattern for iNOS localization was observed in WF rats, the levels of expression were significantly less than in WH rats (Fig. 6d–f). Quantitative measurement of iNOS-positive staining revealed that the significant increase in staining in the cortex and OM reached 26 7% and 17 10%, respectively, in WH rats 48 h after I/R (P < 0.05 for both), compared with increases of 3 1% and 4 1%, respectively, in WF rats (P > 0.05 for both; Fig. 6h). These data demonstrate that nNOS expression is preserved in WF rats after I/R injury. This difference is associated with prevention of iNOS upregulation.
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V Voisin et al.
914 (b)
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*†
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0 12
*
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140
No. CD8+ cells (/mm2)
1600
No. ED1+ cells (/mm2)
MCP-1 (pg/mg tissue)
(a)
120
*
100 80
†
60 40 20
0
48
168
336
0 0
48
168
Time after I/R (h)
(d)
(e)
(f)
(g)
Fig. 3 Inflammatory response after ischaemia–reperfusion (I/R). (a) Changes in monocyte chemoattractant protein-1 (MCP-1) levels in renal tissue, (b) semiquantitative analysis of macrophage infiltration and (c) quantitative measurement of CD8 infiltration in Wistar-Hanover (■, WH) and Wistar-Furth (□, WF) rats after I/R. Data are the mean SEM (n = 6). *P < 0.05 compared with Time 0 (control conditions; one-way ANOVA followed by Dunnett’s test); †P < 0.05 compared with WH rats (two-way ANOVA). (d–g) Representative photomicrographs of (d, e) macrophage immunostaining and (f, g) lymphocyte CD8 on Day 7 in WH (d, f) and WF (e, g) rats.
Urinary excretion of PGE2 and TxB2 after I/R in WH and WF rats Because the mechanism underlying the protection of WF rats against renal I/R may be caused by factors other than changes in NO metabolism, we investigated changes in TxA2 levels by measuring the urinary excretion of TxB2, a stable metabolite of TxA2, and PGE2. Prostaglandin E2 is the main kidney prostaglandin produced by microsomal PGE synthase localized in the macula densa.30 As shown in Fig. 7a, under control conditions urinary PGE2 excretion was similar in both strains. Forty-eight hours after I/R, there was a significant increase in PGE2 excretion in both strains (P < 0.05 for both). In contrast, urinary TxB2 excretion (Fig. 7b) was lower in WF than WH rats under control conditions and after I/R injury (P < 0.05).
DISCUSSION Ischaemia–reperfusion injury is an unavoidable event after kidney transplantation. However, there is no current therapy to protect the kidney and guaranty a positive clinical outcome. An I/R episode causes a cascade of cellular events, leading to cellular damage and organ dysfunction. These adverse effects, which induce glomerular, tubular and endothelial alterations,1 occur together with repair and regeneration processes. Therefore, the renal outcome of I/R depends on the capability of the renal tissue to fully regenerate. In the present study, we demonstrated that the WF rat strain exhibits some degree of resistance against an I/R insult. Several studies have showed the resistance of this strain to the development of CKD.22,24,25,31 However, no data suggested potential protection against AKI. In the present study, we clearly showed
beneficial outcomes following renal I/R in WF rats, as evidenced by reduced creatininaemia and renal inflammation, maintenance of tubular function, preservation of nNOS expression and prevention of iNOS upregulation. In the CKD studies, the same data were reported with regard to nNOS expression, which places NO as a key player in this beneficial resistance of WF rats. Nitric oxide is a well-known vasoactive mediator that was shown to play a role in AKI and was identified as a key factor in the progression of renal I/R injury.19,32–34 Our renal I/R model has been demonstrated to induce a severe but reversible AKI.29 In AKI, creatininaemia is still considered as the classical clinical marker to determine kidney damage and renal outcome. In the present study, WF rats had lower creatininaemia levels than WH rats. This was also associated with lower levels of urinary NGAL, proteinuria and FENa. In the present study, NGAL was used as another marker of AKI. Indeed, although creatininaemia is the main diagnostic indicator of renal function, other biomarkers, such as NGAL, have demonstrated their usefulness in the early diagnosis of AKI.35,36 In the present study, urinary NGAL was also significantly lower in WF than WH rats after I/R. To better characterize tubular function, we measured FENa in both WF and WH rats. It is recognized that an I/R episode is accompanied by a decrease in tubular sodium reabsorption. Our data showed that the postischaemic increase in FENa in WH rats was significantly reduced in WF rats, reinforcing the hypothesis that WF rats are protected against impaired renal function in a model of I/R injury. In addition, we showed that WF rats were protected against renal inflammation. The renal inflammatory process is a common feature of kidney transplantation occurring after the I/R episode. The inflammatory process is a response of the renal tissue to hypoxia and injury. It is important to understand that inflamma-
© 2014 Wiley Publishing Asia Pty Ltd
Ischaemic kidney injury in Wistar-Furth rats
Urinary H2O2 (nmol/mg Cr)
(a) 120
915
Table 1 Gene expression of nitric oxide synthase isoforms in the cortex and outer medulla of Wistar-Hanover and Wistar-Furth rats under control conditions (Time 0) and at different time points after ischaemia–reperfusion, as determined by real-time polymerase chain reaction
*†
100 80 60
nNOS 2 DDCt
iNOS 2 DDCt
eNOS 2 DDCt
1.00 0.19 0.09 0.04* 0.09 0.03*
1.00 0.33 3.68 0.45* 11.20 6.09
1.00 0.37 0.39 0.16 0.83 0.29
1.00 0.60 1.14 0.39 1.12 0.62
Undetectable Undetectable Undetectable
1.00 0.29 1.90 0.71 1.20 0.26
40
Cortex Wistar-Hanover Control 48 h 168 h Wistar-Furth Control 48 h 168 h Outer medulla Wistar-Hanover Control 48 h 168 h Wistar-Furth Control 48 h 168 h
20 0
0
48
168
(b)
Urinary MDA (nmol/mg Cr)
* *
0
48
168
Time after I/R (h) Fig. 4 Oxidative stress after ischaemia–reperfusion (I/R). Urinary (a) H2O2 and (b) malondialdehyde (MDA) levels in Wistar-Hanover (■, WH) and Wistar-Furth (□, WF) rats under control conditions (Time 0) and at different time points after I/R. Data are the mean SEM. *P < 0.05 compared with Time 0 (control conditions; one-way ANOVA for repeated measurements); †P < 0.05 compared with WH rats (two-way ANOVA for repeated measurements). Cr, creatinine.
tory cells or cytokines responding to the stress signal contribute to tissue injury at one stage, but also provide signals for the resolution of the injury and so are also involved in tissue repair. The inflammatory response is a complex mechanism between the beneficial and harmful issues. Thus, persistent inflammation characterized by phagocyte infiltration, such as macrophages, is detrimental for survival of the kidney transplant.4,37 In experimental I/R models, several studies have highlighted the persistence of a few damaged tubules associated with persistent inflammatory cells in the renal tissue despite normalization of plasma creatinine. This phenomenon proves that the initial injury can induce chronic damage to the kidney.34,38,39 In the present study, we observed an early significant increase in MCP-1 in renal ischaemic tissues of WH but not WF rats. Monocyte chemoattractant protein-1 is recognized as an early mediator of inflammation.40–42 The inflammatory response was also associated with increased macrophage and lymphocyte CD8 cells in both WH and WF rats 48 h after I/R. Later, on Day 7 after I/R, these populations of inflammatory cells were still enhanced in WH but not WF rats, further indicating a better renal outcome for the WF rat strain. Although it is clear that WF rats exhibit an evident protection against I/R injury, the underlying mechanisms remain unclear. In experimental CKD models using WF rats, investigators
1.00 0.37 18.6 10.8* 3.65 1.02
1.00 0.14 0.75 0.14 1.93 0.67
1.00 0.29 1.52 0.55 2.47 0.83
1.00 0.15 1.25 0.36 1.37 0.46
Data are the mean SEM (n = 6). *P < 0.05 compared with control (one-way ANOVA followed by the Holm–Sidak test). Neuronal nitric oxide synthase (nNOS) expression was not quantified in the outer medulla because nNOS is located in macula densa cells. iNOS, inducible nitric oxide synthase; eNOS, endothelial nitric oxide synthase.
hypothesized that NO could somehow be associated with this protection. Indeed, NO is an important renal mediator under both normal and pathological conditions. Its functions may depend on the site and the level of production, as well as on the local cellular environment.11 Nitric oxide synthesis occurs through activation of NOS. Most of the physiological effects of NO are produced by activation of constitutive isoforms of NOS, namely nNOS (NOS1) and eNOS (NOS3), whereas the third isoform (iNOS, NOS2) is inducible mostly in an inflammatory environment.18 In the kidney, NO is known to be involved in the regulation of vascular resistance, GFR, renin secretion and water and sodium excretion, as well as in the maintenance of renal structural integrity.9,10 In addition, NO may have beneficial or deleterious effects depending on its concentration, duration of release and site of production.9,11,12 To determine whether NO could also be involved in the protection of WF rats against I/R injury, NOS expression was measured. Similar to previous studies in models of CKD,24,26,31 our data demonstrated that nNOS expression was maintained at the mRNA and protein levels in the renal tissue of WF rats after I/R injury, whereas nNOS expression was decreased in vulnerable WH rats. As proposed by Erdely et al.31 the preserved nNOS expression in WF rats may be explained by blockade of the renin–angiotensin system (RAS). Indeed, in a model of renal mass reduction using WF rats, it has been reported that aldosterone levels, expected to increase, remained unchanged.24 In addition, another study showed that inhibition of the RAS was associated with
© 2014 Wiley Publishing Asia Pty Ltd
V Voisin et al.
916 WH
WF
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WF
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iNOS (%)
nNOS staining (%)
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* *
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†
0
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5
†
0 0
20 0
25
5
40
*
25
*
30
100
48
168
0
48
168
Time after I/R (h)
Fig. 5 Intrarenal neuronal nitric oxide synthase (nNOS) expression after ischaemia–reperfusion (I/R). Representative photomicrographs of nNOS immunostaining in the macula densa of (a, c, e) Wistar-Hanover (WH) and (b, d, f) Wistar-Furth (WF) rats under control conditions (a, b) and 48 h (c, d) and 7 days (e, f) after I/R. Sections were counterstained with haemalun and luxol fast blue. Arrowheads indicate macula densa localization. (g) Quantitative analysis of nNOS-positive staining in the macula densa in WH (■) and WF (□) rats under control conditions (Time 0) and at different time points after I/R. Data are the mean SEM (n = 6). *P < 0.05 compared with control (one-way ANOVA followed by Dunnett’s test); †P < 0.05 compared with WH rats (two-way ANOVA).
Fig. 6 Intrarenal inducible nitric oxide synthase (iNOS) expression after ischaemia–reperfusion (I/R). Representative photomicrographs of iNOS immunostaining in kidney tissues of (a, c, e) Wistar-Hanover (WH) and (b, d, f) Wistar-Furth (WF) rats under control conditions (a, b) and 48 h (c, d) and 7 days (e, f) after I/R. Expression of iNOS was localized to the collecting duct (CD) under control conditions (a, b) and was enhanced in the proximal tubule (PT) 48 h after I/R (c, d). On Day 7 after I/R (e, f), renal tissue from both strains exhibits many cystic tubules (CT), as well as attenuation of iNOS expression. (g, h) Quantitative analysis of iNOSpositive staining in WH (■) and WF (□) rats under control conditions (Time 0) and at different time points after I/R in the cortex (g) and outer medulla (h). Data are the mean SEM (n = 6). *P < 0.05 compared with control (one-way ANOVA followed by Dunnett’s test); †P < 0.05 compared with WH rats (two-way ANOVA).
maintenance of renal nNOS production.43 In the kidney, nNOS is specifically expressed in the macula densa, which plays a major role in the regulation of renal haemodynamics. Indeed, following activation of TGF, local production of NO induces a vasodilator response of afferent arterioles,13 preventing a decrease in GFR and thus a decline in renal function. Tubular injury can also be linked to filtration failure through mechanisms such as tubular obstruction, which is common after I/R injury.44 In the present study, the postischaemic increase in FENa in WH rats, which reflects functional tubular impairment with reduced reabsorptive capacity, was significantly reduced in WF rats. Furthermore, our data demonstrated that, as reported
previously in various models of CKD,24–26,31 nNOS expression was maintained in the macula densa of WF rats after I/R injury, whereas nNOS expression was decreased in WH rats. Together, the data suggest that, in WF rats, activation of TGF is reduced due to less tubular impairment and that the counteracting effect of nNOS-derived NO on the TGF response remains effective, thus contributing to improved postischaemic renal function. With regard to iNOS expression, the results of the present study indicate that iNOS expression is significantly induced after I/R in WH rats. This was shown at both the mRNA and protein level (by immunolocalization of iNOS in the ischaemic renal parenchyma). In contrast, this increase was not observed in WF
0
48
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Time after I/R (h)
© 2014 Wiley Publishing Asia Pty Ltd
Ischaemic kidney injury in Wistar-Furth rats
Urinary PGE2 (pg/mg Cr)
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Time after I/R (h) Fig. 7 Urinary excretion of (a) prostaglandin (PG) E2 and (b) thromboxane (Tx) B2 in Wistar-Hanover (■, WH) and Wistar-Furth (□, WF) rats under control conditions (Time 0) and at different time points after ischaemia–reperfusion (I/R). Data are the mean SEM. *P < 0.05 compared with control (one-way ANOVA followed by Dunnett’s test); † P < 0.01 compared with WH rats (t-test).
rats. Several studies have reported induction of iNOS expression after renal ischaemic insult along with increased kidney damage.18,45,46 Moreover, in many studies, the use of specific iNOS inhibitors reduced postischaemic injury.17,18,32,34,47 Similar data have been reported in iNOS-knockout mice,33 demonstrating the adverse effect of iNOS-derived NO. In experimental I/R models, iNOS is usually related to inflammation and oxidative stress. After I/R insult, an increase in reactive oxygen species (ROS), such as the free radical superoxide, is observed.48,49 The superoxide is known to interact with NO-derived from iNOS to produce peroxynitrite, a highly toxic reactive nitrosative species (RNS), thereby contributing to the pathogenesis of renal postischaemic injury.19 In the present study, the lower inflammatory response in WF rats may be related to the lower induction of iNOS. Unfortunately, we were not able to determine whether the oxidative stress response after the I/R injury was also improved in WF rats. Superoxide is tricky to measure because of its short half-life. Currently, there is a lack of available and reliable techniques to measure oxidative stress. Malondialdehyde, another marker used in the present study to evaluate lipid peroxidation, did not reveal any differences between the two strains. Therefore, we evaluated levels of H2O2, also known as a marker of oxidative stress. Unexpectedly, we showed that H2O2 levels were increased in WF compared with WH rats. The reasons for this result are unclear, but it could be due to faster formation of peroxynitrite from the reaction of superoxide and iNOS-derived NO against
917
lower superoxide degradation by superoxide dismutase (SOD).50 However, this requires further investigation. Finally, assuming that not just preserved nNOS and reduced iNOS induction can protect the kidney against I/R injury, we investigated the involvement of another vasoactive factor, namely TxA2. In the kidney, TxA2 acts as a vasoconstrictor agent and is known to play a role in renal haemodynamics by modulating vascular resistance. In the kidney, TxA2 is generated by specific enzymes, such as TxA2 synthase, acting on products of COX-1 and COX-2.51 However, TxA2 is very unstable and quickly degrades to a biologically inactive product, TXB2, which can be then easily measured. Our data clearly indicated that urinary TxB2 was lower in both the normal and postischaemic kidney of WF rats, whereas excretion of the vasodilator PGE2, the main prostanoid in the kidney, was similarly increased in both WH and WF rats. The ratio between vasodilator (PGE2) and vasoconstrictor (TxA2) autacoids, which is routinely used as an indicator of the direct role of vasoconstrictor factors in the development of renal failure,52–54 appeared to be higher in WF than WH rats after I/R. Interestingly, COX-2 activity has been reported to depend on nNOS activity.55 During acute inhibition of COX-2, renal haemodynamics were maintained due to a reduction in TxA2 production along with increased nNOS activity.20 It has also been reported in an in vitro study that NO inhibits TxA2 synthase activity in a concentration-dependent manner.56 Therefore, we may hypothesize that, in WF rats, the preserved production of nNOS-derived NO, along with decreased TxA2 synthesis, maintains a balance between vasoactive mediators, promoting decreased renal vascular resistance and hence reducing renal injury. In conclusion, the data of the present study demonstrate that the WF rat strain also exhibits marked protection against ischaemic AKI, as evidenced by lowered creatininaemia and renal inflammation, maintenance of tubular function, preservation of nNOS expression and prevention of iNOS upregulation This protection may be associated with a complex interplay between paracrine mediators such as NO and TxA2. Perspectives Future investigations are needed to better understand the protection against renal failure in WF rats. To this end, it will be of interest to evaluate the potential effects of specific and selective inhibitors of the different NOS isoforms. In this regard, several in vitro and in vivo studies have demonstrated that specific inhibition of the expression or activity of iNOS, as well as the absence of iNOS expression in knockout mice, can reduce postischaemic injury,17,18,32,33,47,48 suggesting a deleterious effect of iNOS-derived NO. Conversely, the use of specific inhibitors, such as S-methyl-L-thiocitrulline (SMTC), a specific inhibitor of nNOS, or sulotroban, a specific antagonist of the thromboxane (TP) receptor,57 should be considered as pharmacological tools to delineate the relative involvement of nNOS and TxA2 in the protection of postischaemic AKI in WF rats. Finally, the interplay between NO and endothelin in the postischaemic kidney of WF rats should be addressed to further investigate the imbalance between vasoactive factors, because endothelin has been demonstrated previously to be increased after renal I/R and to participate in renal damage.58
© 2014 Wiley Publishing Asia Pty Ltd
V Voisin et al.
918
Immunohistochemistry
METHODS Animals The present study conformed to the American Society of Physiology’s Guiding Principles in the Care and Use of Animals (http:// www.the-aps.org/mm/sciencepolicy/about/policy-statements/guiding-principles-.html, accessed 5 September 2014) and the protocols were approved by the local Ethics Commission on Animal Welfare. Experiments were performed on 49 male WH and 47 male WF rats (Harlan, Horst, the Netherlands), weighing 220– 260 g. In the first experiment, rats from each strain were randomly distributed in three groups, as detailed below. 1. Group I: I/R 7 days (WH, n = 9; WF, n = 11). Rats were anaesthetized with 60 mg/kg, i.p., sodium pentobarbital (Nembutal; CEVA, Libourne, France) and 0.15 mg/kg buprenorphine hydrochloride (Ecuphar, Oostkamp, Belgium), placed on a heated table and subjected to a right nephrectomy followed by occlusion of the left renal artery for 60 min, as described previously.34 Rats were allowed to recover and were placed in metabolic cages for daily weighing and urine sampling over a period of 7 days. Finally, on Day 7, blood samples were collected and the remaining kidney was harvested. 2. Group II: control (WH, n = 8; WF, n = 8). Control rats were anaesthetized and subjected to sham surgery, including laparotomy and manipulation of the left renal vascular pedicle without the induction of left renal ischaemia or contralateral nephrectomy. 3. Group III: I/R group 12 h and 48 h (WH, n = 20; WF, n = 20). Rats were subjected to the same surgical procedures as rats in Group I. Blood samples and the left kidney were collected 12 h and 48 h after I/R. The right kidney was considered as the control kidney. The kidneys were then processed for further experiments. Portions of kidneys were dissected into the cortex and OM before being snap-frozen in liquid nitrogen for RNA and protein isolation. An additional portion of kidney was fixed in Duboscq-Brazil solution for immunostaining. Urine and blood data Plasma creatinine concentration was measured by the Jaffe reaction. Urine creatinine concentrations were measured using a commercially available kit (Assay Designs, Antwerpen, Belgium). Urinary NGAL was determined using an ELISA kit (Bioporto Diagnostics, Hellerup, Denmark). Total urine protein levels were evaluated by using the Bradford dye binding assay with bovine serum albumin as the standard (Sigma-Aldrich, Diegem, Belgium). As an index of oxidative stress, urine samples were also analysed for hydrogen peroxide by the Amplex red assay (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s instructions. Urinary and plasma sodium concentrations were measured by flame emission spectrophotometry (IL 943; Instrumentation Laboratory, Zaventem, Belgium) to calculate FENa according to the standard formula. Finally, urinary MDA levels were determined by measuring the thiobarbituric acid-reactive substances. All urinary markers were normalized against creatinine to obviate any losses in collection.
Immunostaining for ED1 (mouse anti-rat monoclonal antibodies (mAbs); 1 : 50; Serotec, Puchheim, Germany), CD8 (mouse antirat mAbs; 1 : 50; Serotec), nNOS (mouse anti-rat mAbs; 1 : 75; Transduction Laboratories, Leuven, Belgium) and iNOS (1 : 200; NeoMarkers, Fremont, CA, USA) was performed on paraffinembedded rat kidney sections as described previously.34 Briefly, after dewaxing and rehydration, microwave pretreatment in citrate buffer (pH 6.2) was performed to unmask antigens present in the renal tissue. Tissue sections were then incubated for 1 h with different primary antibodies. After rinsing in phosphate-buffered saline (PBS), slides were exposed for 30 min to the appropriate secondary antibody. Kidney sections were finally incubated with ABC complex (Vector Laboratories, Burlingame, CA, USA) for 30 min and bound peroxidase activity was detected with a diaminobenzidine (DAB) kit. The number of ED1- or CD8-positive cells was counted under a light microscope at 9400 magnification in five squares fields (0.084 mm2/field) randomly taken in each renal zone. ImageJ (National Institutes of Health, Bethesda, MD, USA) was used to quantify the percentage of the NOS-positive area. Twenty random fields in the renal tissue were analysed at 9200 magnification. Renal tissue MCP-1 Samples of renal tissue were homogenized in PBS (1 mg/20 nL). The concentration of MCP-1 was measured in renal homogenates using the OptEAI Set for MCP-1 (BD Biosciences Pharmingen, Antwerpen, Belgium) according to the manufacturer’s instructions, and is expressed as pg MCP-1/mg renal tissue. Reverse transcription–polymerase chain reaction analysis Total RNA from the cortex and OM was extracted using the guanidinium thiocyanate method. All samples were quantified by spectrophotometric analysis at 260 nm. Total RNA (2 lg) was reverse transcribed using random hexamers and Superscript II MMLV-reverse transcriptase (InVitrogen, Merelbeke, Belgium). Real-time PCR reactions were performed on ABI PRISM 7000 Sequence Detection System using Taqman Universal PCR Master Mix and Assays-on-demand Gene expression Probes (Applied Biosystems, Applera, France) for the different isoforms of NOS genes (NOS1 assay ID: Rn 00583793_m1; NOS2 assay ID: Rn00561646_m1; NOS3 assay ID: Rn02132634_S1; and 18S assay ID: HS99999901_S1). Analysis of relative target gene expression was performed using the comparative Ct method. Results were normalized against data for the 18S rRNA internal control. The amount of target gene mRNA, normalized against an internal control and relative to a calibrator, is given by 2 DDCt . The change in gene expression after I/R was calculated as the ratio between the average 2 DDCt value corresponding to I/ R rats and the average 2 DDCt value corresponding to control rats. Calculations and statistical analysis Results are presented as the mean SEM and further statistical analysis was performed using SIGMASTAT (Systat Software,
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Ischaemic kidney injury in Wistar-Furth rats Erkrath, Germany). The level for statistical significance was set at P < 0.05, which refers to two-sided probability. Student’s ttest was used to determine significant differences between two groups, one-way ANOVA was used for multiple inter-group comparisons followed by Dunnett’s test and two-way ANOVA was used for repeated measurements when appropriate, followed by a Holm–Sidak test.56
ACKNOWLEDGEMENTS The authors thank Dr G Toubeau (University of Mons, Mons, Belgium) and Dr Y. Poumay (University of Namur, Namur, Belgium) for their technical support. This work was supported, in part, by grants from Fonds de la Recherche Scientifique, Belgium.
DISCLOSURE No conflicts of interest, financial or otherwise, are declared by the authors.
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