FTY-720 pre-treatment attenuates acute lung injury following bilateral renal ischemia/reperfusion but not bilateral nephrectomy

Abstract

Acute kidney injury (AKI) frequently coexists with acute lung injury (ALI) in critically ill patients, resulting in high mortality rates in intensive care units. This study aimed to investigate the role of lymphocytes in ALI following bilateral renal ischemia and bilateral nephrectomy, using FTY-720 (Fingolimod), which inhibits lymphocyte release into the circulation. The study was conducted on 75 male Sprague–Dawley rats, which were divided into five groups: three untreated groups subjected to bilateral renal ischemia/reperfusion (BIR), bilateral nephrectomy (BNX), or a sham operation, and two groups pre-treated with FTY-720 followed by either BIR (BIR + FTY) or BNX (BNX + FTY). One hour of bilateral renal ischemia followed by 24 h of reperfusion caused severe kidney tissue damage and impaired renal hemodynamic, excretory, and urine-concentrating functions. These alterations were accompanied by significant increases in plasma creatinine, urea, K⁺, H⁺, osmolality, as well as elevated plasma MDA and NO metabolites, ultimately leading to ALI. Notably, FTY-720 pretreatment markedly attenuated AKI, reduced the accumulation of plasma markers, and prevented the development of ALI in the ischemia model. Bilateral nephrectomy also induced profound elevations in plasma creatinine, urea, K⁺, H⁺, and osmolality after 24 h; however, there was no difference in ALI severity between untreated and FTY-720–treated animals in this model of AKI. FTY-720 induced lymphopenia in BNX + FTY and BIR + FTY groups, but only mitigated ischemic AKI and ALI together in the BIR + FTY group. Therefore, lymphocytes are unlikely to mediate directly the link between AKI and ALI.

Introduction

Acute kidney injury (AKI), formerly known as acute renal failure, accounts for 2–5% of all hospital admissions and 30–50% of intensive care unit admissions^1–4. Ischemia/reperfusion (I/R) injury is a leading cause of AKI, frequently observed in patients undergoing cardiac, thoracic, or aortic surgeries, as well as in kidney transplantation^5–8. During renal ischemia, the abrupt deprivation of oxygen and nutrients is accompanied by increased production of reactive oxygen species (ROS), reactive nitrogen species (RNS), vasoactive compounds, proteolytic enzymes, chemokines, and cytokines, which collectively contribute to endothelial, epithelial, and interstitial injury^9–13. The restoration of blood flow is essential for the survival of the ischemic kidney, as it delivers oxygen and substrates required for energy metabolism and facilitates the clearance of accumulated toxic metabolites and injurious substances. However, reperfusion itself can exacerbate injury by increasing the workload of tubular cells through the resumption of glomerular filtration, as well as by amplifying oxidative stress and promoting the recruitment of inflammatory mediators and immune cells¹⁴.

AKI is unfortunately associated with a high mortality rate exceeding 50%, mainly due to the development of multiple organ dysfunction^1,15,16. The failure of kidney function results in the accumulation of nitrogenous metabolic wastes, water and electrolyte imbalance, and the development of metabolic acidosis, a condition collectively referred to as uremia. The distant effects of renal I/R on other organs can be conducted by inducing systemic uremia, impairing clearance of chemokine/cytokines, and releasing injurious factors from damaged kidneys, such as inflammatory mediators, ROS, nitric oxide (NO), exosomes, and cell debris/fragments. To study the links between AKI and remote organ dysfunctions, different experimental models of AKI have been developed via bilateral renal I/R (BIR; renal ischemia with uremia), unilateral renal I/R (UIR; renal ischemia without uremia), bilateral nephrectomy (BNX; uremia without renal ischemia), and unilateral nephrectomy (UNX; without uremia and renal ischemia)^17,18.

Our previous studies have shown that neither UNX nor 2-h unilateral renal ischemia in rats induces acute lung injury (ALI) within 24 h^19,20. However, UIR that was associated with modest increases in plasma creatinine, urea, malondialdehyde (MDA), and NO metabolites had distant effects on contralateral kidney to dampen its compensatory overfunction²⁰ and brain to cause blood–brain barrier disruption, hippocampal neuronal loss, and memory impairment²¹. BNX and 1-h bilateral renal ischemia markedly increased plasma urea and creatinine levels after 24 h in rats, but only BIR led to elevated plasma MDA and NO metabolites¹⁹, as well as profound blood–brain barrier disruption, hippocampal apoptosis, electrophysiological impairments, and deficits in learning and memory²¹. Furthermore, BIR and BNX induced ALI with distinct characteristics because of differences between the accumulated systemic mediators after 24 h^17,19. In consistent, Hoke et al. demonstrated elevations in serum cytokines with differing profiles during 24 h of BIR and BNX^17,19, and Hasson et al. reported that global lung gene expression patterns at 6 and 36 h after bilateral renal ischemia and BNX were clearly distinguishable²². In addition, cardiomyocyte apoptosis was observed following BIR but not BNX²³, whereas both BIR and BNX induced liver and intestinal injuries^24,25. Therefore, it can be suggested that the acutely accumulated systemic mediators following UIR, BIR, and BNX differ, leading to distinct effects on remote organs. Moreover, each distant organ may respond differently to these mediators.

The importance of inflammatory cell infiltration, particularly T lymphocytes of the CD4⁺ phenotype, as key mediator of I/R-induced AKI has been demonstrated in several studies^26–28. FTY720 (Fingolimod) is a sphingosine-1-phosphate (S1P) receptor agonist that functions as an immunomodulator by promoting lymphocyte sequestration in lymph nodes, resulting in subsequent lymphopenia. S1P, a bioactive sphingolipid metabolite, is generated through phosphorylation of sphingosine by sphingosine kinases 1 and 2. It acts both as an intracellular second messenger and as an extracellular signaling ligand for a family of five specific G protein-coupled receptors (S1PR1–5)^29,30. S1P signaling pathways regulate diverse biological processes, including cell survival and proliferation, cytoskeletal organization, extracellular matrix assembly, maintenance of endothelial barrier integrity, and immune cell trafficking^31,32.

FTY-720 is an agent approved by the U.S. Food and Drug Administration for the treatment of multiple sclerosis³³. Several studies have demonstrated its beneficial effects in experimental models of AKI³⁴, ALI^35,36, stroke^37,38, hepatic encephalopathy³⁹, cardiac I/R⁴⁰ and Alzheimer disease⁴¹. FTY-720 administration has been shown to attenuate I/R-induced AKI not only through T-lymphocyte depletion in peripheral blood^42–45, but also via activation of S1P receptors on renal endothelial and tubular cells^46,47. Additionally, FTY-720 treatment has been reported to mitigate ALI induced by paraquat⁴⁸, lipopolysaccharide^35,36, hind limb I/R⁴⁹, and liver injury induced by renal I/R⁵⁰. Shi et al. reported that intraperitoneal administration of FTY-720, given 15 min after the onset of renal I/R in rats, attenuated kidney tissue injury and the rise in plasma creatinine. Moreover, FTY-720 conferred protection against renal I/R-induced ALI by reducing pulmonary pro-inflammatory cytokines, neutrophil infiltration, apoptosis, oxidative stress, and sphingosine kinase expression³⁵. However, it remains unclear whether FTY-720 protects against renal I/R-induced ALI primarily by blocking the mediatory role of lymphocytes and S1P receptors in the AKI-ALI axis or indirectly by attenuating AKI itself. The present study was designed to investigate (i) the protective effects of FTY-720 pre-treatment on renal tissue injury induced by BIR, as well as on associated alterations in hemodynamics, solute excretion, and urine-concentrating ability; and (ii) the mediatory role of lymphocytes in the interaction between AKI and ALI. To this end, blood differential leukocyte counts, plasma MDA and NO levels, blood gas and acid–base parameters, along with pulmonary histopathology, neutrophil and macrophage infiltration, capillary leakage, and edema were assessed following AKI induction by BIR or BNX, with or without FTY-720 pre-treatment.

Results

Total and differential leukocytes count of blood

Table 1 presents the total leukocyte count, as well as the number and percentage of lymphocytes and neutrophils, in blood samples collected prior to the first injection of FTY-720 in the pre-treated groups or saline in the non-treated groups (Day − 1), before second injection of FTY-720 or saline (pre-values) followed by performing BIR, BNX or sham-operation, and 24 h after these challenges (post-values). Total and differential leukocytes count at day − 1 were not different between the 5 groups and 24 h after first injection of saline (pre-values) between the sham, BIR, and BNX groups and also from their values at day − 1 in each of these 3 groups. However, there were large reductions in the leukocytes and lymphocytes counts and percentage of lymphocytes at 24 h after the first injection of FTY-720 in the BIR + FTY and BNX + FTY groups from their levels at day − 1 and also the pre-values of the 3 non-treated groups (all P < 0.001). In the BIR-FTY and BNX-FTY groups, the pre-values of neutrophils counts were not changed but percentages of neutrophils were markedly increased with respect to their values at day − 1 and also the pre-values of the 3 non-treated groups (all P < 0.001).

Table 1.

Total and differential white blood cells count before and after bilateral renal ischemia/reperfusion or bilateral nephrectomy in FTY-treated and non-treated groups

Groups	Total leukocytes (mm−3)	Total lymphocytes (mm−3)	Total neutrophils (mm−3)	Percentage of lymphocytes (%)	Percentage of neutrophiles (%)
Sham
Day -1	17157 ± 629	13,948 ± 554	2813 ± 117	81.20 ± 0.53	16.46 ± 0.52
Pre-values	16802 ± 752	13,703 ± 646	2673 ± 136	81.46 ± 0.51	16.00 ± 0.62
Post-value	8117 ± 255 c	4779 ± 255 c	3123 ± 236	59.13 ± 2.67 c	38.33 ± 2.36 c
BIR
Day -1	17016 ± 535	13,950 ± 394	2701 ± 155	82.13 ± 0.68	15.86 ± 0.72
Pre-values	16903 ± 565	13,871 ± 406	2648 ± 177	82.26 ± 0.76	15.60 ± 0.80
Post-values	7500 ± 268 c	3871 ± 238 c*	3537 ± 301 c	52.00 ± 3.07 c*	46.80 ± 3.03 c*
BIR + FTY
Day -1	18100 ± 1131	14,994 ± 967	2943 ± 261	82.73 ± 1.02	16.33 ± 1.05
Pre-values	5790 ± 845 c *** †††	3000 ± 341 c *** †††	2757 ± 545	53.40 ± 159 c *** †††	45.86 ± 1.64 c *** †††
Post-values	3424 ± 221 c *** †††	539 ± 64 c *** †††	2853 ± 190	15.60 ± 1.51 c *** †††	83.60 ± 1.76 c *** †††
BNX
Day -1	17574 ± 344	14,225 ± 337	3000 ± 89	80.87 ± 0.58	17.13 ± 0.54
Pre-values	17498 ± 407	14,233 ± 354	2917 ± 113	81.33 ± 0.57	16.67 ± 0.53
Post-values	7483 ± 345 c	4280 ± 283 c	3074 ± 290	57.47 ± 2.97 c	40.87 ± 3.03 c
BNX + FTY
Day -1	17326 ± 1070	14,476 ± 793	2574 ± 306	84.20 ± 1.31	14.40 ± 1.19
Pre-values	5750 ± 574 c *** †††	3067 ± 358 c †††	2644 ± 475	54.13 ± 3.41 c *** †††	44.87 ± 3.37 c *** †††
Post-values	3113 ± 208 c *** †††	574 ± 48 c †††	2506 ± 201	19.13 ± 1.61 c *** †††	79.73 ± 1.78 c *** †††

The values are expressed as means ± SEM for total leukocytes and also total and percentage of lymphocytes and neutrophils in blood taken before first injection (Day -1), before second injection followed by performing surgery (Pre-values) and 24 h after (Post-values) sham-operation (sham group), a 1-h bilateral renal ischemia without pre-treatment (BIR group) or pre-treated with FTY-720 (BIR + FTY group), and bilateral nephrectomy without pre-treatment (BNX group) or pre-treated with FTY-720 (BNX + FTY group), n = 15 in each group. ^cP < 0.001, versus the previous period in each group. *P < 0.05, ***P < 0.001, versus the Sham group at each period. ^†††P < 0.001, BIR + FTY group versus BIR group or BNX + FTY group versus BNX group at each period.

At 24 h after the end of sham-operation, bilateral renal ischemia or bilateral nephrectomy, the post-values in comparison to the pre-values became lower for leukocytes and lymphocytes counts and percentages of lymphocytes but higher for percentages of neutrophils in all 5 groups and neutrophils number only in the BIR group (all P < 0.001). The post-values of total and differential leukocytes counts were not different between the sham and BNX groups and also between the BIR and BNX group, but the BIR group compared to the sham group had lower post-values of lymphocytes number and percentage and higher post-values of neutrophils percentage (all P < 0.05). Importantly, there were much lower post-values of leukocytes and lymphocytes counts and percentages of lymphocytes and also much higher post-values of neutrophils percentages in the BIR + FTY and BNX + FTY groups than the sham and the respective non-treated group (all P < 0.001).

Renal histopathology

In the sham group, tubular and vascular structures were normal at the cortex (Fig. 1A1), OM-os (Fig. 1B1), OM-is (Fig. 1C1), and IM (Fig. 1D1). One-hour renal ischemia followed by 24-h reperfusion led to tissue injuries in all renal regions of the BIR group (Fig. 1A2–D2) and the BIR + FTY group (Fig. 1A3–D3), but they were considerably attenuated by pre-treatment with the FTY-720. The average grades of cellular necrosis, cytoplasmic vacuolization, and exfoliated cells in the BIR + FTY group versus the BIR group were 0.38 ± 0.10 versus 1.36 ± 0.07 in proximal tubules (PT) at the cortex, 1.36 ± 0.19 versus 4.02 ± 0.12 in PT at the OM-os, 0.06 ± 0.06 versus 1.00 ± 0.07 in thick ascending limbs (TAL) at the cortex, 0.64 ± 0.10 versus 3.96 ± 0.13 in TAL at the OM-os, and 0.00 ± 0.00 versus 1.0 ± 0.00 in TAL at the OM-is (all P < 0.01). There were also scanty tubular damages in the cortical and outer medullary collecting ducts and distal tubules of the BIR group, which were not observed in the BIR + FTY group. Moreover, the BIR + FTY group in comparison to the BIR group had lower grades of Bowman’s space enlargement (2.06 ± 0.11 vs. 3.22 ± 0.11), as well as formation of intratubular proteinaceous casts in the cortex (0.00 ± 0.00 vs. 0.60 ± 0.24), OM-os, (0.22 ± 0.12 vs. 1.02 ± 0.02), OM-is (0.76 ± 0.17 vs. 1.50 ± 0.15), and IM (0.02 ± 0.02 vs. 0.80 ± 0.20) and vascular congestion in the cortex (0.00 ± 0.00 vs. 0.80 ± 0.20), OM-os, (0.24 ± 0.10 vs. 1.02 ± 0.02), OM-is (1.48 ± 0.43 vs. 2.00 ± 0.29), and IM (0.00 ± 0.00 vs. 1.03 ± 0.03) (all P < 0.01). Finally, the pre-treatment with FTY-720 caused the renal total histopathological score to become much lower in the BIR + FTY group than the BIR group (7.30 ± 0.34 vs. 23.85 ± 0.69, P < 0.001).

Fig. 1.

Representative light microphotographs of the renal (A1–3) cortex (B1–3) juxtamedullary cortex, (C1–3) outer medulla, and (D1–3) inner medulla of rats at 24 h after sham-operation (sham group), a 1-h bilateral renal ischemia without pre-treatment (BIR group) or pre-treated with FTY-720 (BIR + FTY group). n = 5 in each group. Hematoxylin and eosin (H&E) staining, magnification 400, with indications of white arrow to tubular cell damage, black arrow to vascular congestion, white triangle to tubular cell debris, black triangle to intra tubular cast, and asterisk to Bowman’s space. In Figs. C1 and C2, the images were obtained from vertical tissue sections, whereas in Fig. C3, the image represents a transverse section.

Renal hemodynamic, excretory and urine concentrating functions

The percentage of kidney weight per body weight (KW/BW, Fig. 2A) in the BIR and BIR + FTY groups were higher (P < 0.001 and P < 0.05, respectively) in comparison to that of the sham group (0.67 ± 0.02%). However, KW/BW was much lower in the BIR + FTY group than the BIR group (1.09 ± 0.05 vs. 0.77 ± 0.03, P < 0.001). One hour of renal ischemia led to marked reductions in renal blood flow (RBF; Fig. 2B) and creatinine clearance (C_Cr; Fig. 2C) during the last 60 min of 24 h reperfusion period in the BIR and BIR + FTY groups with respect to their levels at the equivalent period in the sham group (9.32 ± 0.66 ml min⁻¹ gkw⁻¹ and 1226.7 ± 93.0 μl min⁻¹ gkw⁻¹, respectively, all P < 0.001). The pre-treatment with FTY-720 caused the BIR + FTY group compared to the BIR group to have higher values of RBF (6.40 ± 0.25 vs. 4.32 ± 0.34 ml min⁻¹ gkw⁻¹, P < 0.01) and C_Cr (284.6 ± 72.9 vs. 37.5 ± 4.3 μl min⁻¹ gkw⁻¹, P < 0.05).

Fig. 2.

The percentage of kidney weight per body weight (KW/BW, A), blood flow (RBF; B), creatinine clearance (C_Cr; C), fractional Na⁺-excretion (FE_Na; D), absolute Na⁺-excretion (U_NaV°; E) and absolute K⁺-excretion (U_KV°; F) at 24 h after sham-operation (sham group), a 1-h bilateral renal ischemia without pre-treatment (BIR group) and pre-treated with FTY-720 (BIR + FTY group), n = 10 in each group. The values are expressed as means ± SEM. *P < 0.05 and ***P < 0.001, versus the Sham group. ^†P < 0.05, ^††P < 0.01, ^†††P < 0.001, BIR + FTY group versus BIR group.

The increase of fractional Na⁺-excretion (FE_Na; Fig. 2D) during the clearance period in the BIR + FTY group (2.38 ± 0.24%) from the sham group (0.44 ± 0.08%) was not statistically significant, whereas FE_Na of both groups were much lower than that of the BIR group (14.1 ± 2.94%, both P < 0.001). The levels of absolute Na⁺-excretion (U_NaV°; Fig. 2E) during the clearance period in the sham, BIR and BIR + FTY groups (0.84 ± 0.12, 0.67 ± 0.12 and 0.93 ± 0.09 μmol min⁻¹ gkw⁻¹, respectively) were not statistically different from each other. However, absolute K⁺-excretion (U_KV°; Fig. 2F) was lower in the BIR and BIR + FTY groups (both P < 0.001) than the sham group (1.33 ± 0.14 μmol min⁻¹ gkw⁻¹) and also in the BIR group than the BIR + FTY group (0.34 ± 0.04 vs. 0.65 ± 0.04 μmol min⁻¹ gkw⁻¹, P < 0.05).

During the 60-min clearance period, the sham group had higher amounts of absolute urea-excretion (U_UreaV°, 3.97 ± 0.36 μmol min⁻¹ gkw⁻¹; Fig. 3A), osmolar clearance (C_osm, 37.24 ± 2.53 μl min⁻¹ gkw⁻¹; Fig. 3B), urine osmolality (U_osm 1461.2 ± 71.2 mosm kgH₂O⁻¹; Fig. 3D), free-water reabsorption (T^C_H2O, 30.09 ± 2.20 53 μl min⁻¹ gkw⁻¹; Fig. 3E), and effective free-water reabsorption (^effT^C_H2O, 16.59 ± 1.80 μl min⁻¹ gkw⁻¹; Fig. 3F) than those of the two ischemic groups (all P < 0.001). Moreover, the BIR + FTY group compared to the BIR group had larger levels of U_UreaV° (2.35 ± 0.28 vs. 0.94 ± 0.15 μmol min⁻¹ gkw⁻¹, P < 0.01), C_osm (18.24 ± 1.81 vs. 9.63 ± 1.02 μl min⁻¹ gkw⁻¹, P < 0.01), urine osmolality (649.4 ± 91.1 vs. 343.1 ± 11.8 mosm kgH₂O⁻¹, P < 0.01), T^C_H2O (9.37 ± 1.77 vs. 0.69 ± 0.19 μl min⁻¹ gkw⁻¹, P < 0.01), and ^effT^C_H2O (2.92 ± 1.29 vs. − 1.26 ± 0.40 μl min⁻¹ gkw⁻¹, P < 0.05). While, the amounts of urine flow rate (V°; Fig. 3C) during clearance period did not statistically differ between the sham, BIR and BIR + FTY groups (6.88 ± 0.41, 8.94 ± 1.00 and 8.56 ± 0.79 μl min⁻¹ gkw⁻¹, respectively).

Fig. 3.

The absolute urea-excretion (U_UreaV°, A), osmolar clearance (C_osm, B), urine flow rate (V°, C), urine osmolality (U_osm, D), free water reabsorption (T^C H₂O, E) and effective free water reabsorption (^effT^C H₂O, F) at 24 h after sham-operation (sham group), a 1-h bilateral renal ischemia without pre-treatment (BIR group) and pre-treated with FTY-720 (BIR + FTY group), n = 10 in each group. The values are expressed as means ± SEM. ***P < 0.001, versus the Sham group. ^†P < 0.05 and ^††P < 0.01, BIR + FTY group versus BIR group.

Plasma variables

Table 2 shows that the levels of plasma creatinine, urea, glucose, Na⁺, K⁺, and osmolality before performing renal ischemia, nephrectomy or sham-operation (pre-values) were not different among the 5 groups. The post-values of all plasma variables did not significantly change from their pre-values after 24-h from the end of sham-operation in the sham group. However, the post-values of plasma creatinine, urea, K⁺, and osmolality were raised after elapsing 24 h from the bilateral renal ischemia or nephrectomy in the non- and pre-treated groups with respect to their pre-values and also their post-values in the sham group (P < 0.05–0.001). Moreover, the post-values of plasma creatinine and K⁺ were higher in the BNX group than the BIR group (P < 0.05 and P < 0.01, respectively). Importantly, the post-values of plasma creatinine, urea, K⁺, and osmolality were not different between BNX and BNX + FTY groups, but they were much lower in BIR + FTY group than the BIR and BNX + FTY groups (all P < 0.001). There were significant decreases of post-values from the pre-values for plasma glucose in the BIR, BNX and BNX + FTY groups (P < 0.05, P < 0.001 and P < 0.01, respectively) and plasma Na⁺ in the BNX and BNX + FTY groups (both P < 0.001), while post-values of plasma glucose and Na⁺ were lower in the BNX group than the sham and BIR groups and also the BNX + FTY group than the sham and BIR + FTY group (P < 0.05–0.001).

Table 2.

The levels of plasma variables before and after bilateral renal ischemia/reperfusion or bilateral nephrectomy in FTY-treated and non-treated groups.

Groups	Plasma creatinine (mg dl-1)	Plasma urea (mg dl−1)	Plasma glucose (mg dl−1)	Plasma potassium (μmol ml−1)	Plasma sodium (μmol ml−1)	Plasma osmolality (mosm kgH2O−1)	Plasma nitrite/nitrate (μmol l−1)	Plasma MDA (μmol l−1)
Sham
Pre-values	0.35 ± 0.03	18.3 ± 0.8	183.4 ± 5.0	3.87 ± 0.06	144.6 ± 0.5	294.7 ± 1.6	55.3 ± 6.0	1.25 ± 0.22
Post-values	0.39 ± 0.03	18.8 ± 1.0	169.9 ± 17.0	3.81 ± 0.11	145.9 ± 0.5	296.3 ± 1.8	55.1 ± 6.4	1.27 ± 0.20
BIR
Pre-values	0.37 ± 0.02	19.7 ± 1.0	191.9 ± 9.3	3.83 ± 0.09	145.6 ± 0.4	295.1 ± 2.6	59.8 ± 7.0	1.51 ± 0.16
Post-values	3.75 ± 0.13 c ***	142.9 ± 4.1 c ***	150.8 ± 9.6 a	5.28 ± 0.16 c ***	145.2 ± 0.9	328.6 ± 3.6 c ***	110.4 ± 9.3 c ***	3.87 ± 0.41 c ***
BIR + FTY
Pre-values	0.35 ± 0.02	18.5 ± 0.9	171.5 ± 9.3	3.76 ± 0.09	144.4 ± 0.5	293.4 ± 2.3	56.9 ± 4.9	2.15 ± 0.24**†
Post-values	1.73 ± 0.19 C*** †††	79.0 ± 6.0 c *** †††	162.8 ± 17.8	4.30 ± 0.09 c * †††	144.3 ± 0.6	303.5 ± 2.3 b * †††	58.9 ± 6.6 †††	2.29 ± 0.28 * ††
BNX
Pre-values	0.33 ± 0.02	19.1 ± 0.8	177.0 ± 11.6	3.77 ± .0.10	145.4 ± 0.5	296.1 ± 1.6	53.8 ± 6.0	1.24 ± 0.26
Post-values	4.21 ± 0.15 c *** #	140.5 ± 4.8 c ***	83.0 ± 6.5 c *** ##	5.85 ± 0.22 c *** ##	140.6 ± 0.9 c *** ###	332.3 ± 2.5 c ***	61.3 ± 6.3 ###	1.69 ± 0.29 c ###
BNX + FTY
Pre-values	0.38 ± 0.01	19.6 ± 1.0	167.7 ± 15.3	3.71 ± 0.05	145.3 ± 0.4	293.9 ± 2.6	60.1 ± 4.7	2.34 ± 0.20**††
Post-values	4.09 ± 0.12 c *** ###	142.6 ± 5.2 c *** ###	116.5 ± 13.4 b ** #	5.60 ± 0.11 c *** ###	140.9 ± 0.6 c *** ##	329.5 ± 2.9 c *** ###	58.2 ± 4.6	2.39 ± 0.23 *

The values are expressed as means ± SEM before (Pre-values) and 24 h after (Post-values) sham-operation (sham group), a 1-h bilateral renal ischemia without pre-treatment (BIR group) or pre-treated with FTY-720 (BIR + FTY group), and bilateral nephrectomy without pre-treatment (BNX group) or pre-treated with FTY-720 (BNX + FTY group), n = 15 in each group. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, post-value versus pre-value in each group. *P < 0.05, **P < 0.01, ***P < 0.001, versus the Sham group at each period. ^†P < 0.05, ^††P < 0.01, ^†††P < 0.001, BIR + FTY group versus BIR group or BNX + FTY group versus BNX group at each period. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001,BNX group versus BIR group or BNX + FTY group versus BIR + FTY group at each period.

There were no differences between the pre-values of plasma nitrite/nitrate (NO-metabolites) in all groups and the pre-values of plasma MDA in the sham, BIR and BNX groups. However, the pre-values of plasma MDA elevated at 1 day after the first injection of FTY-720 in the BIR + FTY and BNX + FTY groups in comparison to sham group and their respective non-treated group (P < 0.05–0.01). The post-value of plasma nitrite/nitrate was higher only in BIR groups from its pre-value and also the post-values of the sham, BIR-FTY and BNX groups (all P < 0.001). The post-values in comparison to pre-values of plasma MDA increased in the BIR and BNX group (both P < 0.001) but not in the BIR + FTY and BNX + FTY groups, while BIR, BIR + FTY and BNX + FTY groups had higher post-values of plasma MDA than that of the sham group (P < 0.001, P < 0.05 and P < 0.05, respectively). In addition, the post-value of plasma MDA was higher in the BIR group than the BIR + FTY and BNX groups (P < 0.001 and P < 0.01, respectively).

Mean arterial pressure, heart rate, and arterial acid–base and gases

Table 3 shows that the levels of mean arterial pressure (MAP) and heart rate (HR) during clearance period in the BIR, BNX and BNX + FTY groups, but not the BIR + FTY group, became lower than those of the sham group (P < 0.05–0.001). The BIR + FTY group had higher values of MAP and HR than the BNX + FTY group (both P < 0.001), and HR than the BIR group (P < 0.05). In addition, MAP and HR were lower in the BNX group than the BIR group (both P < 0.001). At 24 h after performing the related challenge in the BIR, BIR + FTY, BNX, and BNX + FTY groups with respect to the sham group, there were large decrements of arterial blood pH (pH_a), bicarbonate concentration ([HCO₃⁻]_a), oxygen tension (P_aO₂), and carbon dioxide tension (P_aCO₂, except in the BIR + FTY group), as well as ratio of arterial oxygen tension to inspired fraction of oxygen (P_aO₂/F_iO₂) (all P < 0.001). Importantly, all of these blood variables had higher values in the BIR + FTY group than the BIR group (all P < 0.001), whereas they did not show any difference between the BNX and BNX + FTY groups. Moreover, the BIR group compared to the BNX group showed higher pH_a (P < 0.05), ([HCO₃⁻]_a (P < 0.05) and P_aCO₂ (P < 0.01), and also lower P_aO₂/F_iO₂ (P < 0.05).

Table 3.

The levels of mean arterial pressure, heart rate and arterial blood variables, and lung tissue damages after bilateral renal ischemia/reperfusion or bilateral nephrectomy in FTY-treated and non-treated groups.

Groups	MAP (mmHg)	HR (beats min−1)	pHa	[HCO3−]a (μmol ml−1)	PaCO2 (mmHg)	PaO2 (mmHg)	PaO2/FiO2
Sham	106.2 ± 1.9	411.8 ± 6.1	7.36 ± .01	23.03 ± 0.43	38.3 ± 0.6	98.9 ± 3.9	416.6 ± 4.3
BIR	99.0 ± 3.5 *	370.8 ± 9.3 ***	7.23 ± .01 ***	13.58 ± 0.28 ***	31.2 ± 0.7 ***	65.9 ± 1.9 ***	279.7 ± 4.9 ***
BIR + FTY	104.5 ± 1.9	394.3 ± 6.8 †	7.28 ± .02 ** †	18.77 ± 1.24 *** †††	38.1 ± 1.2 †††	88.2 ± 2.4 *** †††	379.6 ± 9.9 *** †††
BNX	81.7 ± 1.8 *** ###	330.0 ± 5.4 *** ###	7.19 ± .01 *** #	10.94 ± 0.45 *** #	27.7 ± 0.9 *** ##	72.6 ± 2.1 ***	303.1 ± 2.9 *** #
BNX + FTY	80.8 ± 2.4 *** ###	332.6 ± 9.9 *** ###	7.18 ± .01 *** ###	11.06 ± 0.66 *** ###	28.8 ± 0.7 *** ###	72.7 ± 3.3 *** ###	296.4 ± 8.0 *** ###

Values are means ± SEM for mean arterial pressure (MAP), heart rate (HR), arterial pH (pH_a), bicarbonate concentration ([HCO₃⁻]_a), carbon dioxide tension (P_aCO₂), oxygen tension (P_aO₂), and ratio of arterial oxygen tension to inspired fraction of oxygen (P_aO₂/F_iO₂) after 24 h of sham-operation (sham group), a 1-h bilateral renal ischemia without pre-treatment (BIR group) or pre-treated with FTY-720 (BIR + FTY group), and bilateral nephrectomy without pre-treatment (BNX group) or pre-treated with FTY-720 (BNX + FTY group), n = 15 in each group. *P < 0.05, **P < 0.01, ***P < 0.001, versus the Sham group. ^†P < 0.05 and ^†††P < 0.001, BIR + FTY group versus BIR group or BNX + FTY group versus BNX group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, BNX group versus BIR group or BNX + FTY group versus BIR + FTY group.

Pulmonary histopathology

Figure 4A1 (magnification × 400) and Fig. 4B1 (magnification × 1000) show the normal appearance of the lung tissue in the sham group, with usual interstitial thickness and number of resident macrophages and without any vascular congestion and alveolar hemorrhage. Bilateral renal ischemia or nephrectomy led to obvious pulmonary tissue injuries after 24 h in the non-treated groups, which total histopathological score was slightly higher in the BNX group than the BIR group (9.55 ± 0.69 vs. 7.66 ± 0.70, P < 0.05). It was observed that the BIR group (Fig. 4A2 and B2) in comparison to the BNX group (Fig. 4A4 and B4) had somewhat lower grades of some histopathological manifestations, as vascular congestion (1.67 ± 0.19 vs. 2.59 ± 0.19, P < 0.01), alveolar hemorrhage (1.24 ± 0.12 vs. 1.61 ± 0.17, P < 0.05), and increased interstitial thickness (1.14 ± 0.14 vs. 1.92 ± 0.20, P < 0.01), but interstitial neutrophils increased more in the BIR group than the BNX group and interstitial macrophages equally increased in both groups.

Fig. 4.

Representative light microphotographs of lung tissue at 24 h after sham-operation (sham group), a 1-h bilateral renal ischemia without pre-treatment (BIR group) or pre-treated with FTY-720 (BIR + FTY group), and bilateral nephrectomy without pre-treatment (BNX group) or pre-treated with FTY-720 (BNX + FTY group). Hematoxylin and eosin (H&E) staining, magnifications × 400 (A₁–A₅) and × 1000 (B₁–B₅) with indications of white arrows to interstitial thickness, black arrow to alveolar haemorrhage, white arrowhead to neutrophil infiltration, black arrowhead to macrophage, black triangle to alveolar edema.

The pre-treatment with FTY-720 caused all the pulmonary tissue injuries to become considerably lower in the BIR-FTY group than the BIR group (P < 0.05–0.001), but did not have any improving effect on them in the BNX-FTY group with respect to the BNX group. Therefore, the BIR-FTY group (Fig. 4A3 and B3) compared to the BNX-FTY group (Fig. 4A5 and B5) had lower grades of vascular congestion (0.28 ± 0.09 vs. 2.20 ± 0.18, P < 0.01), alveolar hemorrhage (0.00 ± 0.00 vs. 1.34 ± 0.12, P < 0.05), increased interstitial thickness (0.12 ± 0.07 vs. 1.70 ± 0.19, P < 0.01), accumulated interstitial macrophages (0.26 ± 0.06 vs. 2.14 ± 0.07, P < 0.01), and total histopathological score (2.54 ± 0.14 vs. 9.18 ± 0.33, P < 0.001).

Pulmonary leukocytes infiltration

Figure 5A shows that the numbers of resident macrophages in the lung interstitium of the sham and BIR + FTY groups were statistically equal (238.5 ± 19.0 and 254.6 ± 6.6 counts mm⁻², respectively). However, the numbers of pulmonary interstitial macrophages equally increased in the BIR, BNX and BNX + FTY groups (401.0 ± 19.1, 388.1 ± 14.6 and 353.5 ± 11.8 counts mm⁻², respectively) and became higher than those of the sham and BIR + FTY groups (all P < 0.001). The numbers of neutrophils in pulmonary interstitium (Fig. 5B) of the BIR + FTY, BNX and BNX + FTY groups (13.57 ± 1.84, 14.21 ± 2.96 and 13.92 ± 2.84 counts mm⁻², respectively) were similar and higher (all P < 0.05) than that of the sham group (2.43 ± 0.61 counts mm⁻²). The BIR group had considerable increase of lung interstitial neutrophils (64.50 ± 5.59 counts mm⁻²) in comparison to the sham, BIR + FTY and BNX groups (all P < 0.001). The total leukocytes numbers in BALF (Fig. 5C) of the BIR, BNX and BNX + FTY groups (374.2 ± 16.4, 317.8 ± 8.2 and 293.2 ± 13.5 counts mm⁻³, respectively), but not the BIR + FTY group (137.6 ± 17.8 counts mm⁻³), were much higher (all P < 0.001) than that of the sham group (130.0 ± 11.4 counts mm⁻³). In addition, the total leukocytes number in BALF was lower in the BIR + FTY group than the BIR and BNX + FTY (both P < 0.001) and in the BNX group than the BIR group (P < 0.01).

Fig. 5.

The numbers of resident macrophages (A) and neutrophils (B) in the lung, and the total leukocytes number in BALF (C) at 24 h after sham-operation (sham group), a 1-h bilateral renal ischemia without pre-treatment (BIR group) or pre-treated with FTY-720 (BIR + FTY group), and bilateral nephrectomy without pre-treatment (BNX group) or pre-treated with FTY-720 (BNX + FTY group), n = 5 in each group. The values are expressed as means ± SEM. *P < 0.05 and ***P < 0.001, versus the Sham group. ^†††P < 0.001, BIR + FTY group versus BIR group. ^##P < 0.01, ^###P < 0.001, BNX group versus BIR group or BNX + FTY group versus BIR + FTY group.

Pulmonary capillary permeability and edema

Figure 6A shows that the ratios of BALF to plasma protein concentrations ([Pr]_BALF/[Pr]_P × 1000) in the BIR, BNX and BNX + FTY groups (3.38 ± 0.14, 3.34 ± 0.18 and 3.14 ± 0.12%, respectively) did not statistically differ from each other and all of them were higher than that of the sham group (2.68 ± 0.13%, P < 0.01, P < 0.01, and P < 0.05, respectively). The ratio of BALF to plasma protein concentrations in the BIR + FTY group (2.93 ± 0.06%) was equal to the sham group and lower than the BIR group (P < 0.05). The measured capillary filtration coefficient (K_fc) in the isolated-perfused lung model (Fig. 6B), as a direct indicator of pulmonary capillary permeability, in the BIR, BNX and BNX + FTY groups (0.63 ± 0.14, 0.70 ± 0.08 and 0.82 ± 0.04 cm³ [sec mmHg g 10⁴]⁻¹, respectively) were equally elevated in comparison to that of the sham group (0.27 ± 0.05 cm³ [sec mmHg g 10⁴]⁻¹, P < 0.01, P < 0.01, and P < 0.001, respectively). However, the value of K_fc in the BIR + FTY group (0.32 ± 0.03 cm³ [sec mmHg g 10⁴]⁻¹) was not different from that of the sham group, and it was lower than those of the BIR and BNX + FTY group (P < 0.05, and P < 0.001, respectively). The lung wet/dry weight (Fig. 6C) increased in the BIR, BNX and BNX + FTY groups (5.74 ± 0.14, 6.34 ± 0.21 and 6.29 ± 0.14 ratio, respectively), but not the BIR + FTY group (5.25 ± 0.15 ratio), with respect to the sham group (5.20 ± 11.4 ratio, P < 0.05, P < 0.001, and P < 0.001, respectively). Moreover, the lung wet/dry weight was lower in the BIR + FTY group than the BIR and BNX + FTY (P < 0.05 and P < 0.001, respectively) and in the BIR group than the BNX group (P < 0.05).

Fig. 6.

The plasma protein concentrations ([Pr]BALF/[Pr]P × 1000), capillary filtration coefficient (K_fc) and the lung wet/dry weight ratios at 24 h after sham-operation (sham group), a 1-h bilateral renal ischemia without pre-treatment (BIR group) or pre-treated with FTY-720 (BIR + FTY group), and bilateral nephrectomy without pre-treatment (BNX group) or pre-treated with FTY-720 (BNX + FTY group), n = 5 in each group. The values are expressed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, versus the Sham group. ^†P < 0.05, BIR + FTY group versus BIR group. ^#P < 0.05 and ^###P < 0.001, BNX group versus BIR group or BNX + FTY group versus BIR + FTY group.

Discussion

This study investigated the role of lymphocytes in the AKI–ALI axis using FTY-720 pre-treatment in animal models of renal I/R injury and nephrectomy. Bilateral renal ischemia followed by 24 h of reperfusion caused severe kidney tissue damage, along with impaired renal hemodynamics, excretory function, and urine-concentrating ability. These alterations were associated with significant increases in plasma creatinine, urea, K⁺, H⁺, and osmolality, as well as elevated levels of plasma MDA and NO metabolites, ultimately leading to ALI. Notably, FTY-720 pre-treatment in the bilateral renal ischemia model substantially attenuated AKI, reduced the accumulation of plasma markers, and prevented the development of ALI. Kidney removal also led to pronounced elevations in plasma creatinine, urea, K⁺, H⁺, and osmolality after 24 h and resulted in ALI. However, FTY-720 pre-treatment did not confer any protective effect against nephrectomy-induced ALI.

The marked reductions in circulating lymphocytes and, consequently, total WBC counts observed 24 h after anesthesia and surgery in the sham group suggest that lymphocytes had migrated from the circulation into injured tissues at the surgical sites. This finding is consistent with the study by Hoke et al., which demonstrated certain chemokines and cytokines are elevated 24 h after sham operation¹⁷. At 24 h after the respective interventions, the number and percentage of circulating lymphocytes were comparable between the BNX and sham groups but were significantly lower in the BIR group than in the sham group, suggesting enhanced migration of lymphocytes from the circulation into the injured post-ischemic kidneys⁵¹. Notably, FTY-720 pre-treatment in the BIR + FTY and BNX + FTY groups led to marked reductions in circulating lymphocyte counts, decreasing them to approximately 20% and 4% of their baseline values after 24 h from the first and second injections, respectively. These findings clearly indicate the induction of lymphopenia, attributable to FTY-720-mediated inhibition of lymphocyte egress from lymphoid organs in these groups.

Renal I/R causes damage to both the tubular and vascular systems. Vascular injury results in disintegration of the capillary endothelium and activation of endothelial cells, leading to the expression of adhesion molecules that promote the attachment of leukocytes, red blood cells, and platelets, thereby contributing to vascular congestion. In the BIR group, vascular congestion has been observed throughout the kidney, with greater prominence in the medulla—particularly in the outer medulla, which contains dense capillary plexuses. The renal I/R-induced endothelial disruption and increased capillary permeability²⁰ also led to renal edema in the BIR group, as indicated by the elevated kidney weight-to-body weight ratio. Vascular congestion combined with increased renal arteriolar resistance, due to an imbalance between vasoconstrictor and vasodilator production⁵², maintained low RBF during the reperfusion period in the BIR group. During the reperfusion period, the reduced RBF and impaired oxygen delivery to the post-ischemic kidney enhanced the production of ROS and activated inducible nitric oxide synthase (iNOS)²⁰. This led to elevated plasma levels of MDA and NO metabolites in the BIR group, changes that were not observed in the absence of kidneys in the BNX group. Tubular injury in the post-ischemic kidney may be either lethal—manifesting as apoptosis and necrosis—or sublethal, characterized by cytoskeletal and intercellular junctional protein disintegration, detachment of viable cells, shedding of brush borders, and formation of intratubular casts^14,52. Sloughed cells, remnants of the shed brush border, cellular debris, and proteins aggregate to form intratubular proteinaceous casts, which obstruct the tubular lumen, leading to increased pressure within Bowman’s space and its subsequent enlargement⁵³. The most severe epithelial cell damage in BIR animals occurred in the PT and TAL, due to their high active transport activity—particularly in the OM, which is primarily supplied by venous blood from the ascending vasa recta and remains hypoxic even under normal conditions^54–57.

There is broad consensus that infiltration of inflammatory cells into the post-ischemic kidney is a key factor in the progression of AKI. While earlier studies primarily focused on the roles of neutrophils and macrophages, subsequent research has highlighted the critical involvement of lymphocytes—particularly the CD4⁺ subset—as principal mediator of I/R-induced AKI^26,45,58. It has also been shown that renal I/R stimulates the migration of lymphocytes from their aggregation in the hilar region into the cortical periarterial spaces within the first 4 h of reperfusion. Subsequently, these lymphocytes gradually infiltrate the renal interstitium over the following 24 h, contributing to the progression of structural and functional kidney damage⁵¹. Therefore, pre-treatment with FTY-720, which blocks lymphocyte egress from lymphoid organs and induces lymphopenia, prevented lymphocyte migration into the post-ischemic kidneys during 24 h of reperfusion. This resulted in significantly improved tissue integrity across all renal regions in the BIR + FTY group. Similarly, other studies have demonstrated that administration of FTY-720^43,44 or SEW2871, an S1PR1-selective agonist^45,59, attenuates I/R-induced AKI through T-lymphocyte depletion in peripheral blood. Additionally, activation of S1P receptors on renal endothelial and tubular cells has been found to confer protection in ischemic AKI^46,47.

Pre-treatment with FTY-720 resulted in the BIR + FTY group exhibiting higher RBF and C_Cr values compared to the BIR group. Consequently, the rise in plasma creatinine at the end of the 24-h reperfusion period was significantly attenuated in the BIR + FTY group relative to the BIR group. Renal I/R-induced severe tubular cell death, exfoliation of viable cells, shedding of brush borders, and loss of tight junction integrity and cell polarity resulted in profoundly impaired Na⁺ reabsorption in the BIR group^60,61. This was evidenced by a 32.7-fold increase in FE_Na compared to the sham group. The protective effects of FTY-720 pre-treatment against I/R-induced vascular and tubular injury significantly improved renal hemodynamics and tubular Na⁺ reabsorption in the BIR + FTY group, which exhibited a FE_Na 5.4-fold higher than that of the sham group. Despite impaired tubular Na⁺ reabsorption, the U_NaV° levels in both the BIR and BIR + FTY groups remained comparable to the sham group, likely due to proportional reductions in GFR and, consequently, the filtered load of Na⁺. As a result, the levels of V° and plasma Na⁺ remained unchanged in both the BIR and BIR + FTY groups compared to the sham group. However, the smaller reduction in GFR observed in the BIR + FTY group relative to the BIR group mitigated the decreases in U_KV° and U_UreaV°, thereby attenuating the elevations in plasma K⁺ and urea.

The U_osm near to isosthenuria observed in the BIR group reflects a disruption of medullary interstitial hyperosmolality, attributable to renal I/R-induced damage in the TAL, reduced GFR, and decreased Na⁺ delivery to the loop of Henle. The disruption of medullary interstitial hyperosmolality in the BIR group severely diminished the driving force for water reabsorption in the medullary CD. This, along with reduced C_osm and decreased fluid delivery to the distal nephron—limiting water availability for reabsorption in the medullary CD led to an approximately 98% reduction in T^C_H2O and a negative value of ^effT^C_H2O. Notably, FTY-720 pre-treatment alleviated TAL injury and attenuated the GFR decline, thereby improving urine-concentrating ability in the BIR + FTY group and increasing U_osm, T^C_H2O, and ^effT^C_H2O.

AKI can impact distant organs, particularly the lungs—which receive the entire cardiac output—through the acute accumulation of systemic mediators such as soluble uremic toxins, ROS, iNOS-derived NO, and pro-inflammatory cytokines including interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-α). Additionally, immune cells such as neutrophils, macrophages, and T-lymphocytes contribute to this process. These systemic mediators primarily target the pulmonary capillaries, leading to disintegration of the endothelial layer and increased vascular permeability. They also activate capillary endothelial cells to express adhesion molecules, promoting the attachment of RBCs, WBCs, and platelets, which contributes to vascular congestion. Subsequently, adherent neutrophils and macrophages transmigrate into the pulmonary interstitium, where—together with activated resident macrophages—they induce widespread injury to the capillary endothelium and alveolar epithelium. Damage to the capillary endothelium and alveolar epithelium promotes interstitial and alveolar leukocyte infiltration, alveolar hemorrhage, interstitial edema, and protein-rich alveolar edema. These changes collectively impair gas exchange between air and blood, leading to severe hypoxemia and a reduction in the P_aO₂/F_iO₂ ratio to below 300 in ALI^1–4. In the present study, the comparable elevations of K_fc in the isolated-perfused lung experiments and protein levels in BALF indicate that both the BIR and BNX groups exhibited similarly increased pulmonary capillary permeability. However, the severity of vascular congestion, alveolar hemorrhage, interstitial thickening observed in histopathological analysis, and pulmonary edema—evidenced by an increased lung wet/dry weight ratio—were moderately greater in the BNX group than in the BIR group. In contrast, bilateral renal ischemia induced more pronounced neutrophil infiltration into the lung interstitium and leukocyte accumulation in BALF, which was associated with a greater reduction in P_aO₂/F_iO₂ after 24 h.

The BIR-induced pulmonary structural and functional impairments, along with plasma accumulation of creatinine, urea, K⁺, H⁺, MDA, and NO metabolites, were significantly attenuated in the BIR + FTY group compared to the BIR group. However, these parameters remained similar between the BNX + FTY and BNX groups. Therefore, the protective effects of FTY-720 pre-treatment on I/R-induced AKI, which mitigated the accumulation of systemic mediators, likely prevented the development of ALI in the BIR + FTY group. This treated-group showed no increases in pulmonary capillary permeability, edema formation, or interstitial macrophage infiltration, alongside significantly reduced lung tissue damage, neutrophil infiltration, and preservation of the P_aO₂/F_iO₂ ratio. Since FTY-720 pre-treatment induced lymphopenia in both the BNX + FTY and BIR + FTY groups, it can be inferred that lymphocytes are unlikely to mediate the link between AKI and ALI. Therefore, the protective effect of FTY-720 against ALI observed in the renal I/R model by Shi et al. is likely attributable to its amelioration of post-ischemic kidney tissue injury and dysfunction³⁵. Conversely, Lie et al. reported that 1 h of bilateral renal ischemia followed by 24 h of reperfusion induced pulmonary apoptosis and microvascular barrier dysfunction in wild-type mice, but not in T cell-deficient (T_nu/nu) mice⁶². Since they also observed trafficking of CD8⁺ T cells into the lungs of wild-type mice 24 h after renal ischemia, it was hypothesized that T lymphocytes activated during renal I/R migrate to the lungs and mediate pulmonary apoptosis and capillary endothelial disruption. However, they noted that the activated CD8⁺ lymphocyte population differs from the CD4⁺ lymphocytes infiltrating the kidney during I/R, highlighting an intriguing area for future research.

Conclusion

FTY-720 pre-treatment in the BIR + FTY group significantly attenuated both AKI and BIR-induced ALI. However, ALI severity was similar between the BNX and BNX + FTY groups. Additionally, FTY-720 induced lymphopenia in both BNX + FTY and BIR + FTY groups. It can be concluded that lymphocytes are unlikely to mediate directly the link between AKI and ALI. Therefore, the protective role of FTY-720 in ALI induced by BIR can be linked to its ameliorating effect on post-ischemic kidney tissue injury and dysfunction.

Limitation of the study

In the present study, multiple in vivo, ex vivo, and biochemical parameters were evaluated; however, several limitations should be acknowledged. First, since it was not feasible to assess all pulmonary parameters in a single rat, each group was subdivided into three subgroups. As a result, given the large number of animals required for the experimental design, only the preventive, rather than the therapeutic, effects of FTY-720 were investigated. Second, due to methodological limitations, cytokine and chemokine concentrations in the blood were not determined, and immunohistochemical analysis of inflammatory cell infiltration in the kidney and lung tissues were not performed. Finally, because this study was conducted in rat models of acute kidney injury, the findings should be interpreted with caution when extrapolating to human or clinical settings.

Material and methods

Experimental animals and groups

This study was approved by the Ethics Committee of Shiraz University of Medical Sciences, Shiraz, Iran (approval codes: IR.SUMS.REC.1391.S6135 and IR.SUMS.REC.1391.S6010). All experimental procedures were performed in compliance with institutional guidelines and regulations for the care and use of laboratory animals.

This study was conducted on 75 male Sprague–Dawley rats weighing 280–320 g, obtained from the Centre of Experimental Animals of Shiraz University of Medical Sciences. The rats were housed in cages with free access to food and water, maintained in a temperature-controlled room (25 ± 1 °C) under a 12-h light/dark cycle. Animals were randomly assigned to five groups (n = 15 per group): three untreated groups subjected to bilateral renal ischemia/reperfusion (BIR), bilateral nephrectomy (BNX), or sham operation, and two groups pre-treated with FTY-720 followed by either BIR (BIR + FTY) or BNX (BNX + FTY).

Induction of bilateral renal ischemia/reperfusion and bilateral nephrectomy

One day prior to surgery, a small blood sample was taken from the tail of each rat to determine total and differential white blood cell (WBC) counts. Subsequently, 1 ml of either saline (NaCl 0.9%) containing FTY-720 (0.5 mg/kg; Sigma-Aldrich) for the pre-treated groups or saline alone for the non-treated groups was administered via intraperitoneal injection³⁵. The rats were then returned to their cages. For leukocyte counting, 25 µl of blood was mixed with 475 µl of Turk’s solution and gently shaken. Total WBC counts were performed using a hemocytometer. Additionally, blood smears were stained with Wright and Giemsa solutions, and differential WBC counts were conducted on 200 cells based on standard morphological criteria.

On the following day, each rat was anesthetized by intraperitoneal injection of pentobarbital sodium (60 mg/kg; Sigma, Poole, Dorset, UK) and placed supine on a heated surgical table maintained at 37 ± 1 °C. Body temperature was continuously monitored using a rectal probe connected to a thermistor (Physitemp BAT-12; USA). A 1 mL blood sample was collected from the tail, with a portion used for total and differential WBC counts. The remaining sample was centrifuged and plasma aliquots were stored for the measurement of baseline levels of relevant variables prior to the experimental challenge in each group, as their pre-values. One portion of the plasma was used immediately to measure Na⁺, K⁺, and osmolality; a second portion was refrigerated for later analysis of creatinine and urea; and a third portion was snap-frozen in liquid nitrogen and stored at –70 °C for subsequent determination of MDA and NO metabolites. The second intraperitoneal injection of FTY-720 in the pre-treated rats, or saline in the non-treated rats, was administered 24 h after the first injection⁶³. This was followed by intraperitoneal administration of heparin (100 units) to prevent intravascular coagulation. Following a midline electrosurgical laparotomy (Surgistat, Valleylab, USA), both renal pedicles were exposed under a surgical microscope (SZ6745TR, Optika, Via Rigla, Italy). BIR was induced by clamping both renal pedicles for 1 h, with successful reperfusion confirmed by the restoration of normal kidney color. In BNX rats, both kidneys were surgically removed. In the sham group, blood sampling and all surgical procedures were performed; however, the renal pedicles were only gently manipulated without clamping or removal. After completing the procedure, the abdominal incision was sutured, and the rats were allowed to recover from anesthesia. They were then returned to individual cages with ad libitum access to food and water, and routine monitoring revealed no signs of severe pain or distress.

Experimental protocols

Each group was further subdivided into three subgroups (n = 5 per subgroup). At 21 h’ post-surgery in subgroups A and B, and at 22 h in subgroup C, each rat was re-anesthetized with an intraperitoneal injection of pentobarbital sodium and placed supine on a heated surgical table. A tracheotomy was performed, followed by cannulation of the right femoral vein for continuous infusion of saline (3 ml/h) using a syringe infusion pump (SEP-10S; UAB Viltechmeda, Lithuania). Additional doses of pentobarbital sodium were administered as needed. The depth of surgical anesthesia was assessed by monitoring the pedal withdrawal reflex in response to a skin pinch. The right femoral artery was also cannulated and connected to a pressure transducer (MLT844; ADInstruments, Bella Vista, NSW, Australia) for arterial pressure recording using a PowerLab/8SP data acquisition system (ADInstruments).

In subgroups A and B, the midline abdominal suture was reopened, and the urinary bladder was cannulated. The left renal artery was carefully dissected under a surgical microscope, and a perivascular flow probe (MA1PRB) connected to a transonic flowmeter (T402-PB; Transonic Systems Inc., Ithaca, NY, USA) was positioned around it to measure renal blood flow (RBF) via the PowerLab system. After a 30-min equilibration period, a 1-h clearance period was conducted between 23 and 24 h’ post-surgery. During this period, urine was collected in a pre-weighed container, and arterial pressure, heart rate, and RBF were continuously recorded using the PowerLab. In subgroup C, only arterial pressure and heart rate were recorded over the same 23–24-h period.

In all subgroups, 24 h after the respective intervention, an arterial blood sample (1 mL) was collected into a chilled heparinized syringe from each rat to determine post-intervention values. The 1h ischemia/ 24 h reperfusion protocol was selected based on previous studies demonstrating that the peak inflammatory response and kidney injury occur at 24 h and gradually declines at 48 and 72 h²⁶. A 0.2 mL portion was immediately analyzed for acid–base status and blood gases, and a small aliquot was used for total and differential WBC counts. The remaining blood was centrifuged; part of the plasma was used immediately to measure electrolytes and osmolality, while the remainder was aliquoted and stored as previously described for later assessment of creatinine, urea, MDA, and NO metabolites. The red blood cells were resuspended in an equivalent volume of saline and reinfused into the animal. The tracheal tube was then aspirated and connected to a ventilator (SAR-830/AP; CWE Inc., Ardmore, USA) to deliver room air at a rate of 60 breaths per minute with a tidal volume of 1 mL per 100 g body weight. After 10 min of ventilation, arterial oxygen tension (P_aO₂) was measured and divided by the inspired oxygen fraction in room air (F_iO₂ = 21%) to calculate the P_aO₂/F_iO₂ ratio. Subsequently, animals were sacrificed under deep anesthesia with pentobarbital sodium and the following procedures were performed in each subgroup^19,20.

In subgroup A, the abdomen and thorax were opened via a midline incision. The renal pedicles were ligated, and the kidneys were excised, decapsulated, longitudinally sectioned, and fixed in 10% buffered formaldehyde^51,60. Through the tracheal tube, 0.5% low-melting agarose in 10% buffered formaldehyde was instilled at a constant pressure of 25 cmH₂O to achieve uniform expansion of the lung parenchyma. The inflated lungs were then fixed in 10% buffered formaldehyde^19,22.

In subgroup B, bronchoalveolar lavage fluid (BALF) was collected by gently instilling 3 mL of pre-warmed (37 °C) PBS (pH = 7.4) into the lungs via the tracheal tube. After 30 s, the fluid was withdrawn by gentle suction, and the procedure was repeated twice. An aliquot of BALF (50 µL) was mixed with 50 µL of Turk’s solution, and total cell counts were performed using a hemocytometer. The remaining BALF was centrifuged at 450 g for 10 min at 4 °C, and the supernatant was separated. Both the BALF supernatant and a plasma sample collected from the rat prior to BALF retrieval were stored at − 20 °C for later analysis^25,64. Protein concentrations in the samples were determined using the Bradford method and expressed as the ratio of BALF to plasma protein concentrations multiplied by 1000 ([Pr]_BALF/[Pr]_P × 1000). Following BALF collection, the thorax was opened via median sternotomy, and all lung lobes were excised, rinsed with PBS to remove blood residues, and weighed to obtain the wet weight. The lungs were then dried in an oven at 90 °C for 72 h, and their dry weight was recorded. The lung wet-to-dry weight ratio (wet/dry) was calculated as an index of pulmonary edema^19,20.

In subgroup C, heparin (150 units per 100 g body weight) was administered intraperitoneally to anesthetized rats, which were then ventilated with a normoxic–normocapnic gas mixture (15.5% O₂ and 5.5% CO₂ balanced with N₂). As previously described^19,20 the chest was opened, and cannulas were inserted into the pulmonary artery and left atrium. The lungs were perfused using a cooled (4 °C), air bubble-free Krebs–Henseleit solution with the following composition: 120.0 mM NaCl, 1.1 mM K₂HPO₄, 1.3 mM MgCl₂, 4.3 mM KCl, 2.4 mM CaCl₂, 13.3 mM glucose, and 1 g dextran (MW 70,000; Sigma-Aldrich Chemie GmbH, Steinheim, Germany) per 100 mL. Perfusion was performed through the pulmonary artery cannula using a volumetric infusion pump (Model SM 2100; Jong Sang Techno Co. Ltd., Korea) at a pulsatile flow rate of 2 mL/min. The isolated, perfused lung was placed in a temperature-controlled housing chamber and suspended freely from a force transducer. After rinsing the lung with perfusate to remove residual blood, the perfusion circuit was closed with a total volume of 40 mL. The flow rate was gradually increased from 2 to 10 mL/min while the entire system—including double glass reservoirs, tubing, and housing chamber—was warmed from 4 to 40 °C. Simultaneously, the left atrial pressure was maintained at 2–3 cmH₂O by adjusting the height of the venous reservoir to ensure Zone 3 conditions within the lung. A positive end-expiratory pressure (PEEP) of 2 cmH₂O was applied to prevent regional alveolar collapse. Pressures in the left atrium, pulmonary artery, and airway were continuously measured using pressure transducers, while lung weight was monitored via a force transducer. All signals were recorded through the PowerLab system. To assess pulmonary capillary permeability, the capillary filtration coefficient (K_fc) was determined gravimetrically by a hydrostatic challenge. Briefly, the height of pulmonary venous outflow in the isolated system was elevated such that the left atrial pressure increased from 2 to 7.5 mmHg for 8 min and K_fc was calculated by a special software based on the slope of the lung weight-gain curve during the maneuver⁶⁵.

Measurement of blood and urine variables and renal functional parameters

Arterial pH (pH_a), carbon dioxide tension (P_aCO₂), P_aO₂, and bicarbonate concentration ([HCO₃⁻]_a) were measured using an Easy blood-gas analyzer (Medica Corporation, Bedford, MA, USA). Plasma and urine samples were analyzed for creatinine and urea nitrogen using an autoanalyzer (RA 1000; Technicon Instruments, USA), Na⁺ and K⁺ concentrations with an EasyLyte analyzer (Medica Corp., Bedford, MA, USA), and osmolality with a cryoscopic osmometer (Osmomat-010; Gonotec GmbH, Berlin, Germany). Urine volume collected during the clearance period was measured gravimetrically, and urine flow rate (V°) was calculated per gram of kidney weight. Creatinine clearance (as an estimate of glomerular filtration rate), absolute and fractional excretion of Na⁺, absolute excretion of K⁺ and urea, osmolar clearance, free-water reabsorption, and effective free-water reabsorption were calculated using standard formulas^20,66.

Measurement of MDA and NO metabolites

Plasma MDA levels were measured using the TBARS assay, as previously described in detail^20,67. The absorbance of plasma samples and serial concentrations of 1,1,3,3-tetraethoxy propane (Sigma-Aldrich), as the standard, were read using a microplate reader (Synergy HT, BioTeck Instruments; Winooski, VT, USA). Plasma MDA concentrations were calculated (µmol/L) based on the standard curve.

Plasma NO levels were determined by measuring the concentrations of nitrite and nitrate—the primary oxidation products of NO—using the Griess method, as previously described in detail^20,68. Total nitrite/nitrate concentrations were quantified (µmol/L) by generating a standard curve from serial dilutions of NaNO₂, with absorbance measured using a microplate reader.

Renal and pulmonary histopathological examinations

The preserved kidneys and lungs were embedded in paraffin, and 5-μm sections were prepared using a microtome. Sections were stained with hematoxylin and eosin (H&E) following standard protocols. Each kidney section was evaluated in the cortex, outer medulla–outer stripe (OM-os), outer medulla–inner stripe (OM-is), and inner medulla (IM) across 10 randomly selected non-overlapping fields under a light microscope. Renal histopathology was assessed based on Bowman’s space enlargement, tubular cell necrosis, cytoplasmic vacuolization, exfoliated cells within the lumen, vascular congestion, and intratubular cast formation. Lung morphological changes were assessed across 10 randomly selected fields based on the degree of vascular congestion, alveolar hemorrhage, interstitial thickening, and leukocyte infiltration⁶⁹. Each parameter was graded according to the extent of involvement: 0 (no changes), 1 (< 20%), 2 (20–40%), 3 (40–60%), 4 (60–80%), and 5 (> 80%). The total histopathological score for each group was calculated as the sum of all numerical grades. Additionally, the numbers of macrophages and neutrophils infiltrating the lung interstitium were counted separately in 20 randomly selected fields at × 1000 magnification under a light microscope. Data were expressed as cell counts per mm²¹⁹.

Statistical analysis

Data are presented as means ± SEM. Within-group comparisons were analyzed using the paired Student’s t-test, while between-group comparisons were performed using one-way analysis of variance (ANOVA) followed by Duncan’s post hoc test. The least significant difference (LSD) test was applied to determine exact P values. Histopathological scores were evaluated using the non-parametric Kruskal–Wallis test, followed by the Mann–Whitney U test for pairwise comparisons. All statistical analyses were conducted using SPSS software (version 11.5; SPSS Inc., Chicago, IL, USA), and a P value < 0.05 was considered statistically significant.

Author contributions

S.M.S., F.K. and S.O. conceived and designed research; N.A., Z.K. and S.O. performed experiments and analyzed data; S.M.S., F.K. and S.O. interpreted results of experiments; S.M.S., N.A. and Z.K. prepared figures; S.M.S. and F.K. drafted, edited and revised manuscript; S.M.S., F.K., S.O., N.A., and Z.K. approved final version of manuscript.

Funding

This study was supported by Shiraz University of Medical Sciences (Grant No. 91–6135 to N. Alebrahimdehkordi, MSc in Physiology; Grant No. 91–6010 to Z. Karimi, PhD in Physiology).

Data availability

The data generated and/or analyzed during the current study are not publicly available due to institutional policy but are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

All procedures conformed to the guidelines for the care and handling of animals prepared by the Ethics Committee of Shiraz University of Medical Sciences, Shiraz, Iran, (Codes: IR.SUMS.REC.1391.S6135 and IR.SUMS.REC.1391.S6010) and in accordance with the international conventions on animal experimentation. We confirm that all methods are reported in accordance with ARRIVE guidelines.