A new mouse model of hemorrhagic shock-induced acute kidney injury

Abstract

Current animal models of hemorrhagic shock-induced acute kidney injury (HS-induced AKI) require extensive surgical procedures and constant monitoring of hemodynamic parameters. Application of these HS-induced AKI models in mice to produce consistent kidney injury is challenging. In the present study, we developed a simple and highly reproducible mouse model of HS-induced AKI by combining moderate bleeding and renal pedicle clamping, which was abbreviated as HS-AKI. HS was induced by retroorbital bleeding of 0.4 ml blood in C57BL/6 mice. Mice were left in HS stage for 30 min, followed by renal pedicle clamping for 18 min at 36.8–37.0°C. Mean arterial pressure (MAP) and heart rate were monitored with preimplanted radio transmitters throughout the experiment. The acute response in renal blood flow (RBF) triggered by HS was measured with transonic flow probe. Mice received sham operation; bleeding alone and renal pedicle clamping alone served as respective controls. MAP was reduced from 77 ± 4 to 35 ± 3 mmHg after bleeding. RBF was reduced by 65% in the HS period. Plasma creatinine and kidney injury molecule-1 levels were increased by more than 22-fold 24 h after reperfusion. GFR was declined by 78% of baseline 3 days after reperfusion. Histological examination revealed a moderate-to-severe acute tubular damage, mostly at the cortex-medulla junction area, followed by the medullar and cortex regions. HS alone did not induce significant kidney injury, but synergistically enhanced pedicle clamping-induced AKI. This is a well-controlled, simple, and reliable mouse model of HS-AKI.

Acute kidney injury (AKI) is characterized by a rapid loss of renal function, including a sudden and sustained fall in glomerular filtration rate (GFR) with retention of nitrogenous waste products (13, 16, 18, 30). AKI is responsible for ~2 million annual deaths worldwide and costs ~10 billion dollars each year in the United States (7–9, 12, 32). Recent epidemiology studies demonstrated that patients who survive an episode of AKI have a significant increase in risk for progression to chronic kidney disease and long-term mortality (5, 22, 29, 36, 37). Despite the fact that AKI is a major health problem, there is currently no specific guideline to prevent AKI or effective therapeutic remedy for AKI. Therefore, further understanding the pathophysiological mechanism of AKI is crucial to find a potential target of treatment and prevention for AKI.

Hemorrhagic shock (HS) and ischemia-reperfusion injury (IRI) are common causes of AKI. HS leads to reduced tissue perfusion and inadequate delivery of oxygen and nutrient, which subsequently leads to multiple systemic organ ischemia. On therapeutic treatment to reestablish tissue perfusion, further injuries are induced and result in IRI (5, 28, 45). Kidneys are highly susceptible to IRI (14, 29).

Various animal models of AKI have been generated by either applying toxic, hypoxic, or septic insult to the kidneys (11, 22, 33, 36, 37). Among them, IRI induced by obstruction of renal blood flow (RBF) is the most commonly used method. A graded injury response can be achieved using IRI (1–3, 37). While these widely used IRI-induced AKI models mimic the clinical situations of kidney transplantation, abdominal and cardiac surgeries, and cardiac arrest in clinical situations (22, 37), these AKI models do not simulate HS-induced AKI, because there is no significant blood volume reduction (6, 19).

Current animal models of HS-induced AKI are induced by either fixed pressure or fixed blood volume reduction (41). Although some recent reported HS-induced AKI models have been associated with moderate kidney injury in rats (20, 35, 46) and mice (24, 25), these models required extensive surgeries and continuous monitoring of hemodynamic parameters. In addition, the long duration of experimental process is accompanied with high mortality rate and variability in kidney injury.

In the present study, we described a simple and a highly reproducible HS-AKI mouse model induced by moderate bleeding plus bilateral clamping of renal pedicles. The AKI in these mice were associated with consistent kidney injury, reduction in GFR, and systemic responses to hemorrhage.

METHODS

Animals.

All procedures and experiment protocols were approved by the Institutional Animal Care and Use Committee at the University Of South Florida College Of Medicine. Male C57BL/6J mice, aged 10–12 wk (22–25 g), were obtained from Jackson Laboratory (Indianapolis, IN). After arrival, the mice were allowed to acclimatize in a temperature-controlled environment with 12:12-h light-dark cycle for 1 wk and ad libitum access to mouse chow and tap water.

Radiotelemetry transmitter implantation.

Radiotelemetry transmitters were implanted subcutaneously to monitor mean arterial pressure (MAP) and heart rate (HR) of mice, as previously described by our laboratory (23, 38, 47). In brief, the mice were anesthetized via inhalation of isoflurane (2% in air; flow 200 ml/min). A small incision was made in the middle of the neck, and a telemetry transmitter (PA-C10) with catheter attached was inserted into the left carotid artery and advanced down to the aortic arch. The body of the transmitter was placed subcutaneously in the right ventral flank of the animal. The incision was closed with suture, and the mice were allowed to recover for 10 days. MAP and HR were measured continually for the first 48 h after HS-AKI and then every 2 min for 4 h from 1 PM to 5 PM each day for the rest of the experiment.

HS-AKI induction.

Mice were divided into survival and nonsurvival groups. There were four subgroups in the survival group: sham operation, HS alone, IRI alone, and HS-AKI. The nonsurvival group includes two subgroups: sham operation and HS-AKI. The mice in the survival group were implanted with radio transmitters to monitor MAP and HR throughout the experiment. Measurement of GFR, kidney injury markers, inflammation markers, and kidney histological examination were conducted in mice from the survival group. Mice in the nonsurvival group were used for RBF measurement under anesthesia.

Basal MAP and HR were monitored for 5 days with telemetry before AKI induction. The mice were then anesthetized with pentobarbital (50 mg/kg ip). HS was induced by withdrawing 0.4 ml blood through the right retroorbital sinus with a standard heparinized micro-hematocrit capillary tube within 3 min. The body temperature was monitored through a rectal probe and controlled in the range of 36.8–37.0°C. A midventral abdominal incision was performed to expose the kidneys. Thirty minutes after the bleeding procedure, bilateral renal pedicles were isolated and clamped for 18 min. The clamps were then released to allow kidney reperfusion. A vicryl suture was used to close the incision. The animal was kept on a heating pad until it gained full consciousness. MAP was monitored throughout the experiment. To compare HS-AKI with HS-alone-induced AKI and IRI-alone-induced AKI, the experimental procedure of bleeding alone or pedicle clamping alone were conducted into two separated groups of mice to serve as respective controls. The sham-operated group was subject to the same procedure and duration of experiment, except that bleeding and bilateral renal pedicle clamping were not performed.

For the nonsurvival group, mice were anesthetized with pentobarbital (50 mg/kg ip). The right common carotid artery was cannulated with PE-10 tubing for MAP measurement. MAP was monitored with an AD Instrument Bridge Amp pressure transducer (AD Instrument) and recorded on a Laboratory Chart (AD Instrument). Sequence of surgical procedure and time course were the same as for HS-AKI and sham-operated mice, except that the RBF was measured as described below.

RBF measurement.

A midventral abdominal incision was performed, and the left kidney was exposed. The left renal artery was carefully dissected from the connective tissue. A perivascular flow probe (MA-0.5PSB) was positioned on the left renal artery. The probe was connected to a transonic flowmeter (TS420), and the blood flow signal was collected and stored digitally (PowerLab, LabChart Pro version 7 software; ADInstruments, Colorado Springs, CO). The renal flow probe was calibrated to 0 and 1.5 ml/min before each experiment. After the flow probe was positioned, the mice were allowed to equilibrate for 30 min. RBF and MAP were measured before and during HS and 1 h after reperfusion. RBF was expressed as milliliters per minute (ml/min).

Plasma creatinine and kidney injury molecule-1 measurement.

Plasma creatinine and kidney injury molecule-1 (KIM-1) were measured at 24 h and 3 days after operation. Blood (30 µl) was collected with a heparinized microcapillary tube from the tail vein and was immediately centrifuged. Plasma (15 µl) was collected and stored in a −80°C freezer until analyzed. Plasma creatinine levels were measured with HPLC-mass spectrometry at O'Brien Center at the University of Alabama at Birmingham and were expressed as milligrams per deciliter (mg/dl).

KIM-1 was measured using an ELISA kit (R&D Systems, Minneapolis, MN; RKM100), according to the manufacturer's instructions, and was expressed as picograms per liter (pg/l).

GFR measurement.

GFR was measured in conscious mice from the survival group using a single-bolus intravenous injection of FITC labeled inulin, as our laboratory described recently with a modification (42). FITC-inulin (5% in 0.9% NaCl) was dialyzed for 24 h against 0.9% NaCl, which resulted in an ~2% solution and was used to establish the standard curve. In previous studies, the penile vein was used for FITC-inulin injection (42). In the present study, we utilized the retroorbital sinus as the injection site. The dialyzed FITC-inulin solution (3.74 μl/g body wt) was filtered and injected via retroorbital sinus of the mice under a light anesthesia with isoflurane. Blood was collected (≈5 μl/mouse each) from a small tail nick the end of the tail into a heparinized microcapillary tube at 3, 7, 10, 15, 35, 55, 75, and 90 min after injection. The blood samples were centrifuged, and plasma fractions (2 μl/each) were collected. FITC-inulin fluorescent intensities of the plasma samples were measured using a plate reader (Cytation5, BioTek) with the excitation at 485 nm and the emission at 538 nm. GFR was calculated using a two-compartment model of two-phase exponential decay (39) (GraphPad Prism, San Diego, CA) and was presented as microliters per minute (µl/min).

Plasma renin concentration measurement.

Plasma renin concentration (PRC) was measured as the amount of ANG I generated after incubation with excess porcine angiotensinogen using the ANG I enzyme immunoassay kit (Bachem, San Carlos, CA), as described by Ramkumar et al. (34). A blood sample (≈20 μl) was collected from the tail tip at 1, 3, 12, and 24 h after HS and immediately spun down using a cooled centrifuge, whereby plasma was isolated and frozen in −80°C freezer until use. Ten microliters of plasma were incubated with excess porcine angiotensinogen (0.4 μmol/l; Sigma, St. Louis, MO) for 20 min in a 50-μl reaction containing sodium acetate (50 mmol/l, pH 6.5), 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (2 mmol/l), 8-hydoxoyquinoline (1 mmol/l), and EDTA (5 mmol/l). ANG I generated was measured using a commercially available enzyme immunoassay (Bachem). PRC was expressed as the amount of ANG I generated per hour per microliter of plasma (ng·ml⁻¹·h⁻¹).

Histology.

Some animals in the survival group were killed 24 h after AKI for histological examination. Kidneys were removed and dissected in the longitudinal axis. Kidney samples were fixed in 10% formaldehyde for 24 h and then embedded in paraffin, cut into 4-µm sections, and stained with periodic acid Schiff. Kidney injury was evaluated based on percentage of necrotic tubules, as reported (26). Ten randomly chosen fields were captured under ×200 magnifications from the cortex, cortico-medullary region, and medulla. The percentage of necrotic tubules in each image was quantified. All morphometric analyses were performed in a blinded manner.

Inflammatory factors in renal homogenates.

Proinflammation cytokines tumor necrosis factor α (TNF-α), interleukin (IL)-6, and keratinocyte-derived chemokine (KC) mRNA expression levels in the renal homogenates were determined by RT-PCR at the end of experiment. Total RNAs were extracted from whole kidney, according to the manufacturer's instructions. After digestion with RNase-free DNase (Promega) to eliminate the genomic contamination, the cDNAs were synthesized with reverse transcription system using corresponding primer sets (IL-6: 5′-CTCTGGGAAATCGTGGAAAT-3′ and 5′-CCAGTTTGGTAGCATCCATC-3′; TNF: 5′-ATGAGAAGTTCCCAAATGGC-3′ and 5′-CTCCACTTGGTGGTTTGCTA-3′; KC: 5′-GCTGGGATTCACCTCAAGAA-3′ and 5′-TGGGGACACCTTTTAGCATC-3′), and used as templates. To evaluate if there is any genomic contamination, a β-actin primer set (5′-GTCCCTCACCCTCCCAAAAG-3′ and 5′-GCTGCCTCAACACCTCAACCC-3′) amplifying a region, including an intron, was used. PCR primer sets specific for the β-actin and related genes were designed using the primer3Plus based on the sequences deposited in the GenBank. The housekeeping β-actin gene was used as the reference for internal standardization. After qualification of the cDNA template, quantitative PCR analysis was performed using a supermix (iTaq SYRB, Bio-Rad) and Real-Time Detection System (Chromo4, Bio-Rad), according to the manufacturer's protocol. Reaction conditions were set as follows: 95°C for 1 min, followed by 40 cycles of 95°C for 15 s, and 60°C for 30 s. Reaction of each sample was performed in triplicate. Dissociation analysis was performed at the end of each PCR reaction to confirm the amplification specificity. After the PCR program, data were analyzed and quantified with the comparative Ct method (2–ΔΔCt) based on Ct values for complement genes and β-actin to calculate the relative mRNA expression level.

Statistical analysis.

All data were presented as means ± SE. The significance of differences in mean values between and within groups was determined by analysis of variance (ANOVA) for repeated measures and a post hoc Fisher least significant difference test or a paired t test where appropriate. A P value of <0.05 was considered to be statistically significant.

RESULTS

MAP and HR.

In the survival group, basal MAP was 102 ± 2 mmHg in conscious state and was decreased by 12 mmHg under anesthesia. MAP was decreased to 35 ± 3 mmHg after withdrawal of 0.4 ml blood during the bleeding phase (*P < 0.01 vs. sham, n = 6; Fig. 1A). MAP returned to 60% of baseline (60 ± 3 mmHg) in 3 h and to 90% of baseline (89 ± 4 mmHg) in 8 h after HS-AKI (n = 6; Fig. 1B). MAP returned to baseline (100 ± 3 mmHg) in 2 days after HS-AKI (n = 6; Fig. 1C). For the HS-alone group, MAP changes were similar as in the HS-AKI group. For the IRI-alone group, MAP did not change significantly during the experiment (data not shown). In the HS-AKI nonsurvival group, MAP changes followed a similar pattern as the survival group during the 1st h of HS-AKI (*P < 0.01 vs. baseline and sham, n = 5; see Fig. 3A). For the sham-operated mice from both survival and nonsurvival groups, MAP remained constant during the experiments, except that the MAP decreased ~12 mmHg when under anesthesia (n = 6, Fig. 1; n = 5, see Fig. 3A).

Fig. 1.

MAP measurement of the survival group with telemetry system. MAP was monitored throughout the experiment with radio transmitters for the sham-operated mice and HS-AKI mice. Figure only showed the data for 3 days after AKI, as MAP remained stable thereafter until the end of the experiment. A: MAP recording during the procedure (zoom of the block area in B). B: MAP changes during the first 8 h after HS-AKI (zoom of the block area in C). C: MAP changes of the sham-operated mice and HS-AKI mice from the beginning of the experiment until 3 days after the operation. Values are means ± SE; n = 6. *P < 0.01 vs. sham.

Fig. 3.

MAP and RBF measurement of the nonsurvival group. A: MAP of the nonsurvival group from the beginning of the experiment to 1 h after reperfusion for the sham-operated mice and mice in the HS-AKI group. B: RBF decreased dramatically after HS. The recording of RBF was terminated during the ischemia. RBF were continuously measured for 1 h after release of the clips. In the sham-operated mice, RBF remained stable during the experiment. C: mean RBF during different phases of the experiment: preshock (baseline), HS, and 1 h of reperfusion. Values are means ± SE; n = 5. *P < 0.01 vs. baseline and sham.

The HR were also monitored with radiotelemetry. HS-AKI was accompanied by tachycardia, which demonstrated the activation of the sympathetic response to blood loss. The HR slowly returned to the baseline in 10 h after bleeding. While there was no obvious change of the HR in the sham-operated group throughout the experiment (*P < 0.01 vs. sham, n = 6; Fig. 2). Similar tachycardia was found in the HS-alone group, and no obvious changes in HR were in the IRI-alone group (data not shown).

Fig. 2.

HR measurement. The HRs were also monitored with radiotelemetry. HS was accompanied by tachycardia. The HRs were almost double at the end of bleeding, which were from 443 to 734 beats/min (bpm) for the HS-AKI group. HR slowly recovered, which took ~10 h to return to basal rate. There was no significant change of HR for the sham-operated mice and IRI-alone mice (data did shown). Values are means ± SE; n = 6. *P < 0.01 vs. sham.

RBF.

RBF was measured in the nonsurvival group. Withdrawing 0.4 ml of blood decreased RBF from 0.68 ± 0.09 to 0.24 ± 0.09 ml/min, which remained at the same low level until renal pedicles were clamped. No RBF was detected during the ischemic phase while the renal pedicles were clamped. After releasing the clamps, RBF was measured again for the reperfusion phase, which was resumed to the preclamping level in seconds after releasing the clamps (*P < 0.01 vs. baseline and sham, n = 5; Fig. 3, B and C). In the sham-operated group, RBF remained stable at 0.66 ± 0.03 ml/min during the experiment (n = 5; Fig. 3, B and C).

GFR.

Three days after AKI, GFR declined to 35% of baseline (from 246 ± 22 µl/min to 85 ± 15 µl/min) in the IRI-alone group and to 22% of baseline (from 248 ± 18 µl/min to 55 ± 20 µl/min) in the HS-AKI group. The GFR in the HS-alone group was similar to the sham-operated group which were ~250 µl/min throughout the experiment (*P < 0.01 vs. sham and HS alone, n = 6; Fig. 4).

Fig. 4.

GFR. GFR was measured in conscious mice. GFR of the HS-alone and sham-operated groups did not change during the experiment. However GFR of other groups declined dramatically compared with sham-operated mice and increased gradually 3 days after HS-AKI. Values are means ± SE; n = 6. *P < 0.01 vs. sham and HS-alone.

Plasma creatinine concentration and KIM-1.

Plasma creatinine concentration (PCr) concentration was <0.1 mg/dl in the baseline of all groups and was significantly elevated to 1.59 ± 0.13 mg/dl in the IRI-alone group and 2.03 ± 0.12 mg/dl in the HS-AKI group after 24 h of reperfusion. The PCr concentration then declined gradually but was still ~10- and 13-fold higher than baseline in the IRI-alone group and HS-AKI groups, respectively, at 3 days postoperation. No significant change for the PCr concentration was found in the sham-operated group and HS-alone group during the experiment (*P < 0.01 vs. sham and HS alone, #P < 0.05 vs. IRI alone, n = 6; Fig. 5).

Fig. 5.

Plasma creatinine (PCr) concentration. HS-AKI significantly elevated the PCr by around 19-fold for IRI-alone mice and 22-fold for HS-AKI mice 24 h after reperfusion. The PCr concentration gradually decreased 3 days after AKI. The PCr level of the sham-operated and HS-alone mice did not change much during the experiments. Values are means ± SE; n = 6. *P < 0.01 vs. sham and HS-alone. #P < 0.05 vs. IRI-alone.

Changes in plasma KIM-1 levels showed a similar pattern as that of PCr. KIM-1 increased from 35 ± 7 to 980 ± 102 pg/l in the IRI-alone group and from 33 ± 6 to 1,278 ± 160 pg/l in the HS-AKI group after 24 h of reperfusion. Plasma KIM-1 in the IRI-alone and the HS-AKI groups decreased gradually and was still ~19- and 24-fold higher than baseline in the IRI-alone group and HS-AKI group, respectively, at 3 days postoperation. There was no significant change of KIM-1 levels in the HS-alone group and sham-operated group during the experiment (*P < 0.01 vs. sham and HS alone, #P < 0.05 vs. IRI-alone, n = 6; Fig. 6).

Fig. 6.

Plasma KIM-1. The plasma KIM-1 level of the sham-operated and HS-alone mice did not change much during the experimental period. Significant elevation of the KIM-1 has been observed for both IRI-alone and HS-AKI mice 24 h after reperfusion. Plasma KIM-1 was gradually reduced 3 days after AKI. Values are means ± SE; n = 6. *P < 0.01 vs. sham and HS-alone. #P < 0.05 vs. IRI-alone.

PRC measurement.

PRC was measured at 1, 3, 12, and 24 h after HS. In both the HS-AKI and HS-alone groups, PRC were increased approximately threefold within 1 h and remained at the high level until 3 h after bleeding. PRC returned to basal value in 12 h for both HS-alone and HS-AKI groups. The changes in PRC followed the same trend as MAP. No significant change in PRC was observed in the IRI-alone and sham-operated group (*P < 0.01, HS alone and HS-AKI vs. IRI alone and sham, n = 6; Fig. 7).

Fig. 7.

Plasma renin concentration (PRC) change at the early phase post-HS. PRC was measured at 1, 3, 12, and 24 h after HS. PRC increased progressively as shock deepened and decreased as blood pressure returned to normal. In the HS-AKI and HS-alone groups, PRC increased ~3-fold at 1 and 3 h (from 68.5 ± 5.2 to 175.9 ± 21.9 ng·ml⁻¹·h⁻¹ for the HS-alone group and 65.5 ± 4.3 to 188.5 ± 18.9 ng·ml⁻¹·h⁻¹ for the HS-AKI group after HS), and returned to basal value in 12 h after HS. No obvious change in PRC in the IRI-alone group has been found during the experiment. Values are means ± SE; n = 6. *P < 0.01, HS-alone and HS-AKI vs. sham and IRI-alone.

Histology.

Periodic acid Schiff staining of kidney sections demonstrated that tubular damage and necrosis were found in mice from the IRI-alone and HS-AKI groups. The most severe tubular damage was observed at the cortex-medulla junction area, followed by the medullar and cortex regions. The mice from the HS-alone group and sham-operated group showed no obvious tubular damage in kidney sections (*P < 0.01 vs. sham, #P < 0.05 vs. IRI-alone, n = 6; Fig. 8B).

Fig. 8.

Histology. A: histology by PAS staining showed that all IRI-alone and HS-AKI mice had a significant increase in renal tubular damage and necrosis compared with sham-operated mice. B: most severe tubular damage was observed at the cortex-medulla junction area and then medulla and cortex. CMR, cortex-medulla region. Values are means ± SE; n = 6. *P < 0.01 vs. sham. #P < 0.05 vs. IRI-alone.

Inflammatory factors levels.

Except for the sham-operated group, all other groups of animals exhibited a significant elevation in the mRNA levels of IL-6, KC, and TNF-α. The HS-AKI group showed the highest levels, while the HS-alone group had the lowest levels of all the inflammation factors (*P < 0.01 vs. sham, #P < 0.05 vs. HS-alone, n = 6; Fig. 9).

Fig. 9.

Inflammation factors. All groups were associated with a significant elevation in the mRNA expression level of IL-6, KC, and TNF-α, except for the sham-operated group. HS-AKI showed the highest levels of all the inflammation factors measured in this study, followed by the IRI-alone group and HS-alone group. Values are means ± SE; n = 6. *P < 0.01 vs. sham. #P < 0.05 vs. HS-alone.

Body weight changes and survival rates.

The body weight dropped ~18% in the HS-AKI group and 16% in the HS-alone groups 3 days after the operation, as shown in Table 1. The survival rate was >90% for all of the mice.

Table 1.

Body weight change

	Baseline	1 Day	3 Days
Sham	24.3 ± 0.6	24.1 ± 0.4	24.3 ± 0.7
HS alone	23.8 ± 0.4	20.1 ± 0.2	20.0 ± 0.3
IRI alone	24.5 ± 0.5	24.0 ± 0.4	24.2 ± 0.5
HS-AKI	25.0 ± 0.3	21.8 ± 0.4	20.2 ± 0.6

Values are means ± SE in g; n = 6 mice. Mice in the sham-operated and IRI-alone groups gained body weight normally during the experiments. The body weight dropped ~18% in the HS-AKI group and 16% in the HS-alone group 3 days after the operation.

DISCUSSION

Hypovolemic shock is the most common reason for ischemic AKI in humans. When MAP decreases to <40 mmHg in humans, the supply of blood to the kidney is dramatically reduced or even terminated (21). It is a different scenario for rodents, especially for mice, where the renal blood supply occasionally continues, even when the MAP decreases to as low as 20–30 mmHg (27). To closely mimic clinical HS-induced AKI, we developed an HS-AKI mouse model by combining retroorbital bleeding and bilateral renal pedicle clamping. The mice exhibited consistent hemodynamic changes and robust kidney injury with a marked decrease in GFR and increase in plasma creatinine and KIM-1, as well as systemic responses to HS, including decreased MAP and RBF, increased HR and PRC, and enhanced inflammatory responses.

Established HS-induced AKI animal models are generated either by controlling MAP (pressure controlled) or by bleeding a fixed volume (volume controlled). It has been suggested that severe and prolonged hypotension in rats does not typically induce severe renal injury and is, therefore, not suitable for use as a “single-insult” animal model (44). Although a few HS-induced AKI animal models in recent experiments have been generated in rats and mice with mild to moderate kidney injuries, all of these models require extensive surgeries, continuous hemodynamic monitoring, and long experimental duration. For pressure-controlled HS, constant MAP is maintained by frequent fluid infusion or blood withdrawal for 90 min to 120 min to induce kidney injury (10, 24, 28). For the volume-controlled HS, the variations of kidney injuries ranges from mild kidney injury to mortality due to low MAP (13, 14).

Previous animal models of IRI-induced AKI have been developed by clamping renal pedicles or arteries to interrupt RBF. In these IRI-induced AKI mouse models, the intervals of RBF obstruction used to induce AKI range from 18 to 90 min. The resulting plasma creatinine levels range from 1.4 to 2.8 mg/dl (10, 17, 31). We speculate that the huge variation in ischemia time required and plasma creatinine values achieved are mainly due to the variation of temperature when ischemia was induced. The environmental temperature during the experiment in these studies was controlled by monitoring either the heating pad or the body temperature. Some studies were conducted in ambient temperature. Renal injury is very sensitive to temperature, which is an important factor for determining the extent of kidney injury (43).

In the present study, we successfully developed a simple and reproducible HS-AKI mouse model with high survival rate by combining moderate bleeding plus renal pedicle clamping. We first tested different volumes of bleeding of 0.3, 0.4, and 0.5 ml from retroorbital sinus. Retroorbital sinus is an easy and very reliable route for bleeding and intravenous injection for both male and female mice. We found that the blood pressure recovered quickly after withdrawing 0.3 ml of blood from mice with a body weight of 22–25 g. Only 50% of mice survived when blood withdrawal volume was increased to 0.5 ml. Withdrawing 0.4 ml of blood reduced the MAP to <40 mmHg, and 90% of mice survived. However, bleeding 0.4 ml of blood alone did not induce significant kidney injury. There were no significant decreases in GFR, nor increases in plasma creatinine and KIM-1 concentration.

In our IRI-alone model, we maintained body temperature at 36.8–37.0°C using a heating pad and a heating lamp and monitored the body temperature through a rectal probe. We observed that bilateral clamping of pedicles for 18 min induced robust and consistent kidney injury. However, this IRI-induced AKI model did not trigger systemic responses, such as activation of the sympathetic nervous system and the renin-angiotensin-aldosterone system. By combining 0.4-ml bleeding and 18-min renal pedicle clamping, we found that HS significantly exacerbated the IRI-induced AKI, as demonstrated by significantly increased plasma creatinine and KIM-1 in the HS-AKI group compared with the IRI-alone group. These results indicated that the systemic responses to HS aggravated the IRI-induced kidney injury.

Inflammation has been demonstrated to play an essential role in the development of AKI, which is characterized by the generation of proinflammatory cytokines and chemokines. The mRNA levels of TNF-α, IL-6, and KC have been reported elevated in AKI in both humans and animal models. Inhibition of inflammatory response protects against the development of AKI and improves renal function (4, 15, 40). In the present study, we found that all groups of animals, except for the sham-operated group, demonstrated activation of inflammatory response. Interestingly, even though HS itself only induced a mild inflammatory response, it synergistically enhanced the IRI-induced inflammatory response of TNF-α and KC. These enhanced inflammatory responses may contribute to the more severe kidney injury observed in the HS-AKI group than the IRI-alone group.

In summary, we successfully developed and validated a new mouse model of HS-AKI by combining moderate bleeding with renal pedicle clamping for 18 min at 36.8–37°C. This new HS-AKI model closely represents the clinical scenario of HS-induced AKI and will provide a new tool to study the signaling pathway, such as inflammation, renin-angiotensin system, and sympathetic nervous system, as well as the significance of systemic responses in the development of HS-induced AKI.

GRANTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-099276 (R. Liu) and DK-098582 (R. Liu).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

L.W., J.W., S.W., G.Z. and J.Z. performed experiments; L.W., J.W., S.W., J.Z., and G.Z. analyzed data; L.W., D.K.Y., J.B., Y.L, J.S and R.L. interpreted results of experiments; L.W. and J.B. prepared figures; L.W. drafted manuscript; J.S., D.K.Y., J.B., and R.L. edited and revised manuscript; R.L. approved final version of manuscript.