Female and male Wistar rats (8–12 weeks old) from Envigo (the Netherlands) were maintained at 23 ± 2°C, with a 12 h light and dark cycle and food and water ad libitum. The animals were cared for in accordance with the Principles of Laboratory Animal Care (NIH Publication No. 86-23, revised 1985) and the Dutch Law on Experimental Animals Care. This study was approved by the Institutional Animal Care and Use Committee of the University of Groningen (IvD number: 171245-01-004).

The animals were allocated into groups: male and female naive, control animals that did not undergo any surgical procedure (n = 4); male and female BD, animals that underwent BD induction without IPK (n = 8); male and female IPK, animals that underwent BD, followed by IPK without treatment (n = 8); and male and female IPK + Treat, animals that underwent BD, followed by IPK with the combined treatment of 17β-estradiol and methylprednisolone (n = 8).

The female animals were selected by estral cycle phase, identifying the higher estradiol period (estrous and proestrous). Vaginal lavage was performed with a Pasteur pipette filled with 10 μl of saline solution (0.9% NaCl). The cellular profile was identified by optical microscopy observation.

All animals were placed in a closed chamber, anesthetized with 5% isoflurane, and maintained with 2% isoflurane during the surgical procedure. The temperature was maintained at 37°C via a heating mat. The jugular vein was cannulated for fluid administration and influx of vasoactive drugs for hemodynamic stabilization; the carotid artery was used for blood pressure measurements. The animals were connected to a small animal ventilator after tracheostomy (Harvard Apparatus, model 683, USA) and kept at a frequency of 70 breaths/min and a tidal volume of 10 ml/kg. For BD induction, a Fogarty 4F catheter was inserted into the intracranial space and slowly inflated for 30 min. BD was confirmed by bilateral mydriasis and apnea. After BD confirmation, anesthesia was stopped, and fluid administration was initiated (2 ml/h) until the end of the experiment. Blood samples were collected at the beginning and the end of the 4 h period. After 4 h, the animals were exsanguinated, and a whole-body flush (maximum pressure of 30 mmHg) was performed with 40 ml of cold saline. Blood, urine, and organs were collected. The left kidney (LK) was prepared for perfusion, while the right kidney was immediately snap-frozen. For the LK, a nephrectomy was performed. The ureter and the renal artery were cannulated, and the kidney was then flushed with 5 ml of cold saline solution and directly transferred to the isolated kidney perfusion (IPK) system.

For the IPK, pressure-controlled normothermic machine perfusion was performed via a roller pump (Ismatec ISM404, Switzerland). The pressure was constantly set at 100 mmHg, and a pressure sensor (Edwards Lifesciences, USA) was placed close to the renal artery. For the perfusion solution, 100 ml of William's medium E supplemented with 30 mmol/L HEPES, 7 mmol/L creatinine, and 50 g/L albumin (Sigma-Aldrich, USA) was used. Carbogen (95% O₂, 5% CO₂) was used to oxygenate the perfusate at a flow rate of 0.5 L/min. Perfusate gas analyses were performed at the initial and final points of IPK, and the values were maintained at approximately 525 mmHg. A water bath and heat exchanger were used to heat the solution. In the first 30 min of perfusion, temperature was slowly increased (1°C/2 min) during the rewarming phase. After that, the temperature was kept at 37°C. Flow measurements were recorded during perfusion.

Perfusate and ultrafiltrate (urine) samples were collected at T15, T30, T60, and T90 (Tmin). After 90 min, the LK was stored for further analysis.

For treatment, 40 mg of methylprednisolone and 5 μg/ml of 17β-estradiol were added to the IPK perfusion solutions.

lactate dehydrogenase (LDH) was measured in the IPK perfusate. Creatinine and Na⁺ levels were determined in the IPK perfusate and ultrafiltrate. Measurements were performed in accordance with the Clinical Laboratory, University Medical Center Groningen, following standard biochemical methods.

The creatinine and Na⁺ concentrations in the ultrafiltrate and perfusate obtained via biochemical analysis were used to estimate creatinine clearance and Na⁺ excretion via the following calculations:Creatinineclearance=(ultrafililtratecreatinineconcentration[μmol/L]×ultrafiltrateproductionflow[ml/min]/perfusionsolutioncreatinineconcentration[μmol/L])/kidneyweight[g]).FENa+(%)=(ultrafiltrateNa+[mmol/L]×creatinineperfusate[mmol/L]/perfusateNa+[mmol/L]×ultrafiltratecreatinine[mmol/L])×100

IL-6 was measured in kidney homogenate and IPK perfusate. Quantifications were performed via Duo Set commercial ELISA kits (R&D Systems, USA) in accordance with the manufacturer's specifications.

Total RNA was extracted from kidney tissue via TRIzol reagent (Invitrogen, USA). The yield of extracted RNA was analyzed with a NanoDrop 1000 spectrophotometer (NanoDrop Technologies, USA), and the quality was assessed via RNA electrophoresis. The extracted RNA was reverse transcribed with random primers at 37°C for 50 min. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted with specific primers (Table 1). SYBR Green (Applied Biosystems, the Netherlands) and a QuantStudio 7 Flex qPCR machine (Applied Biosystems, the Netherlands), which was configured with 1 cycle of 10 min at 95°C and 40 consecutive cycles of 15 s at 95°C and 1 min at 60°C. CT values were corrected for β-actin levels.

For the immunohistochemical analyses, the right and left kidneys were placed in formalin and subsequently paraffin-embedded. Slices (3 mm) were prepared and placed on glass slides. Deparaffinization was performed via xylene and ethanol. Antigen retrieval was performed via the use of an ethylenediaminetetraacetic acid (EDTA) (pH 8) solution. The slides were incubated in this solution for 3 h at 60°C. Later, endogenous peroxidase was blocked with H2O2 (2%) for 15 min. Non-specific blockage was performed with TBST-BSA (2%) solution for 1 h at 37°C. Antibodies against myeloperoxidase (MPO), inducible nitric oxide synthase (iNOS), kidney injury molecule-1 (KIM-1), endothelial nitric oxide synthase (eNOS), and endothelin-1 (ET-1) (Table 2) were incubated with the samples overnight at 4°C. The sections were then incubated with secondary HRP-conjugated antibodies (1:200, BA1054; Boster Biological Technology, USA) for 2 h at 37°C. A peroxidase substrate was subsequently used. Hematoxylin was used for counterstaining. For the slide analyses and image acquisition, NIS-Elements BD (Nikon, Japan) software was used.

Kidney morphology was analyzed via periodic acid–Schiff (PAS) staining of paraffin-embedded kidney sections (5 μm). The scoring system used is presented in Table 3.

The data are expressed as the means ± standard errors of the means (SEMs). The data were analyzed with GraphPad Prism Version 10, and the groups were compared via two-way analysis followed by the post hoc test of the two-stage linear step-up procedure of Benjamin, Krieger, and Yekutieli or three-way ANOVA.

IL-6 (Figure 1) was quantified in kidney homogenates (A) and IPK perfusates (B). Gene expression in renal tissue was also evaluated (C). Compared with non-perfused kidneys, all groups presented reduced expression of IL-6 in kidney homogenates after perfusion. For males, the treated group presented even lower concentrations than those in the non-treated group. In females, no difference was observed between the IPK and IPK + Treat groups (A). According to the results of the perfusate analyses, female IPK + Treat mice presented lower release of IL-6 than IPK-treated mice did. No difference was observed in males (B). Finally, gene expression was increased in all groups after IPK, regardless of sex and treatment (C).

The number of infiltrating neutrophils and macrophages was analyzed via quantification of MPO-marked cells and iNOS protein expression (Figure 2). The pre-IPK kidneys of males presented higher MPO levels than did those of females. After IPK, all groups presented similar numbers of infiltrated cells (A). Similarly, in the iNOS analyses, protein expression was increased in males and was reduced after perfusion in all the groups.

There was a reduction in the protein expression of KIM-1 with perfusion. With respect to gene expression, no difference was observed among the groups (Figure 3).

Perfusion flow was constantly measured during IPK. Females presented with slightly increased flow at the beginning of perfusion. Compared with the control group, the IPK + Treat group presented significantly reduced flow. The male groups presented similar values throughout perfusion (Figure 4).

The protein expression of eNOS and endothelin-1 (ET-1) was analyzed in kidney tissue (Figure 5). In females, eNOS expression was reduced in pre-IPK kidneys, and eNOS expression was increased in the IPK + Treat group (A). No difference was observed in the male groups. With respect to ET-1 (B), females also presented reduced expression in pre-IPK kidneys compared with males, and no difference was observed with perfusion or treatment.

Creatinine clearance was lower in males than in females, and female IPK + Treat presents lower values than female IPK does. No difference was observed over time (A). Fractional excretion of sodium (FENa) in the perfusate revealed greater reabsorption of Na⁺ in the female IPK + Treat group than in the female IPK group. Both male groups presented similar values at all 90 min (B). Over time, all groups presented a reduction in FENa⁺ (Figure 6).

LDH was measured in the perfusate at several time points (A). Lactate was quantified at the beginning and end of the perfusion (B). There is an increase in LDH in both male and female IPK + Treat groups compared with IPK. All groups presented an increase in LDH throughout perfusion. Lastly, all groups presented increased lactate from T15 to T90 (Figure 7).

Necrosis of the proximal and distal tubules and glomerular space was quantified via a scoring system (Figure 8). Both proximal and distal necrosis were reduced after perfusion in all groups (A and B). Compared with non-perfused kidneys, all perfused kidneys presented increased glomerular space (C).