In total, 30 healthy Wistar rats (males, 180–220 g) were obtained from the Animal Experiment Center of Soochow University and housed in SPF-grade cages with free food and water access under controlled conditions (24–26 °C, 40–60% relative humidity, 12 h light-dark cycle). All animal studies were in accordance with the NIH Guide for the Care of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Soochow University (Suzhou, China).

After intraperitoneal anesthesia using pentobarbital sodium (60 mg/kg), the skin and muscle were cut 1 cm away from the spin and 1 cm away from the lower rib edge such that the bilateral kidneys were visible. After ligating the right kidney at the pedicle with a silk thread, the pedicle was cut and the right kidney was excised. After separating the left renal artery, a vascular clip was applied to arrest blood flow for 1 h. After this ischemic period, the clip was removed to permit reperfusion such that the kidney changed in color from dark to bright red. The wound site was sutured and rats were injected (i.p.) with 0.5 mL of warm normal saline and returned to the incubator until awake, at which time they were transferred into clean cages. At 24 h post-surgery, rates received a pentobarbital sodium (60 mg/kg, i.p.) for anesthesia and were sacrificed, after which samples of blood and renal tissue were harvested.

For this study, rats were randomly assigned to six groups (n = 5/group): (1) A sham group in which all procedures other than kidney excision and left renal artery clamping were performed; (2) A DEX + sham group in which sham rats received an i.p. injection of DEX (30 ug/kg; Yangzijiang Pharmaceutical Company); (3) An ischemia-reperfusion (I/R) model group that underwent the surgical procedure detailed above; (4) A DEX + I/R group in which DEX (30 ug/kg, i.p.) was injected 30 min prior to the clamping of the left renal artery; (5) An ATI + DEX + I/R group in which the $\alpha$2-AR inhibitor atipamezole (ATI; 250 ug/kg; HY-12380, MCE) was intraperitoneally injected 30 min prior to DEX injection as in the DEX + I/R group [18]; and (6) A 3-MA + DEX + I/R group wherein the autophagy inhibitor 3-methyladenine (3-MA; 15 mg/kg; HY-19312, MCE) was intraperitoneally injected 30 min prior to DEX injection as in the DEX + I/R group [19].

Blood samples were rapidly collected via opening the ventricle apex, and were stored for 30 min at room temperature followed by centrifugation (10 min, 3500 cm, 4 °C) to collect the serum samples. Serum creatinine (Cre) and blood urea nitrogen (BUN) levels were analyzed with a fully automatic biochemical analyzer (C8000, ARCHITECH, Kyoto, Japan).

Kidney samples were fixed using 4% paraformaldehyde, paraffin-embedded, prepared into 5 µm sections, and subjected to hematoxylin and eosin (H&E) staining followed by imaging under light microscopy (BX-FM; Olympus Corporation, Tokyo, Japan). The percentages of tubules exhibiting brush border shedding and dilation were evaluated in 10 renal cortical regions to score the magnitude of renal injury as follows: 0 (0%), 1 (1–19%), 2 (20–49%), 3 (50–69%), and 4 ($\ge$70%) [20]. Two observers independently scored all samples.

Commercial ELISA kits were used to assess serum IL-1$\beta$ (E-EL-R0012, Elabscience, Wuhan, China), IL-6 (E-EL-R0015, Elabscience), and TNF-$\alpha$ (Multisciences, Hangzhou, China) concentrations based on provided directions.

Serum malondialdehyde (MDA) levels were detected using a lipid oxidation detection kit (S0131S, Beyotime, Shanghai, China). This assay reflects the extent of lipid peroxidation based on the production of a red product from the reaction between MDA and thiobarbituric acid (TBA). Levels of the reduction marker glutathione (GSH) were assessed in serum with GSH and GSSG test kits (S0053, Beyotime).

Beclin1, LC3, and p62 expression in kidney tissue samples were detected via qPCR, Western immunoblotting, and immunohistochemical (IHC) staining.

Trizol (Invitrogen, Waltham, MA, USA) was used to isolate total RNA from renal tissue samples based on provided directions, after which the RrimeScriptRT kit (RR036A, Takara, Kyoto, Japan) was used to prepare cDNA. Then, a TB Green Premix Ex TaqII kit (RR820A, Takara) and a 480 II Detection System (Roche) were used to assess relative gene expression via qPCR using primers compiled in Table 1. GAPDH served as a normalization control, and relative expression was quantified via the 2${}^{-\mathrm{\Delta }\mathrm{\Delta }\text{Ct}}$ method.

For Western immunoblotting, minced kidney tissue samples were suspended in RIPA buffer (P0013C, Beyotime, China) containing PMSF (P0100, Solarbio, Beijing, China), protease inhibitors (05892791001, Roche, Basel, Switzerland), and phosphatase inhibitors (04906845001, Roche) and homogenized in a tissue grinder (F6/10 F013200021, JingXin, Xincang, China). After centrifugation (10 min, 3000 rpm, 4 °C) supernatant protein levels were detected via BCA assay (P0012, Beyotime, China). Equal protein amounts were separated via SDS-PAGE and transferred onto PVDF blots that were blocked for 2 h at room temperature with 5% skim milk followed by overnight incubation with primary antibodies (diluted 1:1000 unless otherwise noted) specific for the following proteins: Beclin 1 (1:2000; ab207612, abcam, Cambridge, UK); LC3B (2775S, Cell Signaling Technology, Danvers, MA, USA); p62 (5114S, Cell Signaling Technology); GAPDH (60004-1-Ig, Proteintech Group, Wuhan, China); $\beta$-Actin (3700, Cell Signaling Technology). Blots were then incubated for 2 h at room temperature with goat anti-rabbit IgG (GAR0072, Multisciences, China) or goat anti-mouse IgG antibody (GAM0072, Multisciences, China), and signal was subsequently detected with a chemiluminescence system (FluorChem M, USA) with high-sensitivity ECL substrate (180501, Tanon, Shanghai, China). Image J (V1.51, Bethesda, Maryland, USA) was then used to analyze protein band intensity.

For IHC staining, xylene was used to deparaffinize 3 µm thick kidney sections that were dehydrated with an ethanol gradient and treated with 3% H${}_2$O${}_2$ for endogenous peroxidase inactivation. After microwaving samples for 15 min in citric acid, these sections were blocked at room temperature for 15 min with goat serum and probed overnight at 4 °C with appropriate primary antibodies specific for Beclin1 (1:100; Servicebio, Wuhan, China), LC3 (1:100; Servicebio), p62 (1:100; Servicebio). After rinsing thrice with PBS, sections were stained using a DAB horseradish peroxidase chromogen kit (Beyotime) and counterstained with hematoxylin, followed by visualization with a PD37 microscope (Olympus). Five random fields per section were analyzed at 200$\times$ magnification.

AKI-related ultrastructural changes and autophagosome numbers were evaluated via transmission electron microscopy (TEM). Briefly, kidney tissue samples were washed using chilled PBS (pH 7.4), cut into 2 mm${}^3$ cubes, and fixed using 2.5% glutaraldehyde (Servicebio). Ultrathin 70 nm sections were then cut, stained using uranyl acetate and lead citrate, and visualized via TEM (HT7700; Japan).

Western immunoblotting was used to detect AMPK and mTOR levels in renal tissue samples as above using the following primary antibodies (diluted 1:1000): anti-AMPK (5831S, Cell Signaling Technology), anti-mTOR (CY5306, Abways, Shanghai, China). Proteins were detected with the same secondary antibodies used above.

Data are reported as means $\pm$ SEM and were analyzed using Student’s t-tests and ANOVAs in GraphPad Prism 9.20 (GraphPad Inc, San Diego, CA, USA). p 0.05 served as the cut-off for statistical significance.

To explore the impact of DEX on renal function in IRI-AKI model rats, serum creatinine (Cre) and urea nitrogen (BUN) levels were analyzed as functional biomarkers. Both Cre and BUN levels in the I/R group were significantly elevated relative to sham control rats, while they were significantly reduced in the DEX + I/R group as compared to the I/R model group. This suggests that DEX is capable of reversing IRI-AKI-associated reductions in kidney function. Strikingly, both Cre and BUN levels were significantly elevated in the ATI + DEX + I/R and 3-MA + DEX + I/R groups as compared to the DEX + I/R group, suggesting that $\alpha$2-AR signaling is responsible for the protective benefits of DEX in this experimental system, and that autophagic activity plays a role in IRI-AKI pathogenesis. No differences were observed between the sham and DEX + sham groups, indicating that DEX alone had no significant effects in animals exhibiting healthy renal function (Fig. 1A,B).

Fig. 1.

DEX alleviates renal functional impairment in IRI-AKI model rats. (A,B) Serum Cre (A) and BUN (B) levels. (C) H&E staining of renal cortical tissues (400$\times$, Scale bar: 20 µm). (D) Renal injury scores based on histopathological findings. Data are means $\pm$ SEM (n = 5). *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001. DEX, dexmedetomidine; IRI, ischemia-reperfusion injury; AKI, acute kidney injury; ATI, atipamezole; 3-MA, autophagy inhibitor.

To test the effects of DEX in IRI-AKI model rats, H&E staining and associated scores were used to evaluate the extent of renal pathology. Significantly higher scores were evident in I/R model rats relative to sham controls, while these scores were significantly reduced in the DEX + I/R group, supporting the ability of DEX to reverse I/R-associated kidney damage. Renal scores for ATI + DEX + I/R and 3-MA + DEX + I/R rats were also significantly elevated as compared to the DEX + I/R group, supporting the ability of ATI or 3-MA to eliminate the protective benefits of DEX against IRI-AKI (Fig. 1C,D).

To better understand the effects of DEX on IRI-AKI pathogenesis, inflammatory cytokine production was next evaluated at serum. Significant increases in serum IL-6, IL-1$\beta$, and TNF-$\alpha$ levels were evident in the I/R group relative to sham control rats, with all three being found at significantly lower levels in DEX + I/R rats relative to those in the I/R model group, consistent with the ability of DEX to suppress inflammatory activity. Both 3-MA and ATI reversed these DEX-mediated reductions in inflammatory cytokine levels (Fig. 2A–C).

Fig. 2.

DEX suppresses renal and systemic inflammation in IRI-AKI rats. Serum levels of IL-6 (A, n = 4), IL-1$\beta$ (B, n = 4), TNF-$\alpha$ (C, n = 3). Data are means $\pm$ SEM. *p 0.05, **p 0.01, ****p 0.0001. DEX, dexmedetomidine; IRI, ischemia-reperfusion injury; AKI, acute kidney injury; ATI, atipamezole; 3-MA, autophagy inhibitor.

The impact of DEX on IRI-AKI-associated oxidative stress was next evaluated. Serum MDA levels in I/R model rats were significantly elevated over sham controls, while these levels were significantly reduced in DEX + I/R rats, demonstrating the ability of DEX to abrogate oxidative stress induced in response to I/R. However, significantly increased MDA levels were evident in both the ATI + DEX + I/R and 3-MA + DEX + I/R groups relative to the DEX + I/R group. Changes in serum GSH levels were the inverse of those observed for serum MDA in these rats. Overall, these data are consistent with the ability of DEX to regulate oxidative stress via $\alpha$2-AR and autophagic signaling (Fig. 3).

Fig. 3.

DEX alleviates oxidative stress in IRI-AKI model rats. (A,B) Serum MDA (A) and GSH (B) levels (n = 4). Data are means $\pm$ SEM. *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001. DEX, dexmedetomidine; IRI, ischemia-reperfusion injury; AKI, acute kidney injury; ATI, atipamezole; 3-MA, autophagy inhibitor.

The role that autophagy plays in the protective benefits of DEX in this experimental system was next assessed by evaluating key autophagic biomarkers (Beclin1, LC3, and p62) and quantifying autophagosome numbers in renal tissue samples. Significantly lower Beclin1 levels and LC3-II/LC3-I ratio values were observed in I/R model rats relative to sham controls, with significantly higher levels of expression of these biomarkers in DEX + I/R rats as compared to the I/R model group. These changes were reversed in rats treated with ATI or 3-MA, which ablated the beneficial effects of DEX. Changes in relative p62 expression were the inverse of those for Beclin1 and the LC3-II/LC3-I ratio, suggesting that DEX can reverse the I/R-associated suppression of autophagy in a manner that can be overcome by ATI or 3-MA treatment (Fig. 4).

Fig. 4.

DEX enhances autophagy in a manner reversed by ATI or 3-MA treatment. (A–C) Beclin1 (n = 4), LC3 (n = 4), and p62 (n = 5) mRNA levels. (D–F) Beclin1, LC3, and p62 protein levels, with (G–I) corresponding quantification, using GAPDH as a normalization control (n = 4). Data are means $\pm$ SEM. *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001. DEX, dexmedetomidine; ATI, atipamezole; 3-MA, autophagy inhibitor.

IHC staining revealed Beclin1, LC3, and p62 expression levels consistent with the qPCR and Western immunoblotting data shown above (Fig. 5).

Fig. 5.

DEX induces enhanced autophagic activity. Beclin1, LC3, and p62 levels were detected via IHC staining with corresponding quantification (n = 4). Scale bar: 50 µm. Data are means $\pm$ SEM. *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001. DEX, dexmedetomidine; ATI, atipamezole; 3-MA, autophagy inhibitor.

Next, electron microscopy analyses were conducted that revealed a significant drop in autophagosome numbers in the I/R group as compared to sham control rats, whereas DEX + I/R rats exhibited significantly elevated autophagosome counts as compared to I/R model animals consistent with the ability of DEX to remediate I/R-associated autophagic inhibition. As compared to the DEX + I/R group, both ATI and 3-MA were able to reduce autophagosome numbers, thereby abrogating the protective benefits of DEX administration (Fig. 6).

Fig. 6.

DEX promotes enhanced autophagic activity. (A) Transmission electron microscopy images of renal tissue samples. Autolysosomes and autophagosomes are respectively indicated with blue and red arrows. Scale Bar: 2.0 µm. (B) Quantitative analyses of autophagosome numbers from 10 regions per group (n = 4). Data are means $\pm$ SEM. *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001. DEX, dexmedetomidine; ATI, atipamezole; 3-MA, autophagy inhibitor.

To determine the extent to which DEX influences AMPK/mTOR pathway activity, protein levels of both AMPK and mTOR were assessed via Western immunoblotting. Significant reductions in AMPK levels were detected in I/R model rats relative to sham controls, while AMPK levels in the DEX + I/R group were significantly elevated as compared to the I/R group. Compared with the DEX + I/R group, however, ATI or 3-MA administration resulted in significant decreases in AMPK expression, suggesting that IRI-AKI is associated with the suppression of AMPK/mTOR pathway activity, whereas DEX can facilitate the activation of this pathway in an $\alpha$2-AR-dependent manner to control autophagic flux (Fig. 7).

Fig. 7.

DEX promotes enhanced autophagic activity via the AMPK/mTOR pathway. (A,B) AMPK (A) and mTOR (B) protein levels. (C,D) The expression of AMPK (n = 4) (C) and mTOR (n = 3) (D) was normalized to $\beta$-Actin. Data are means $\pm$ SEM. *p 0.05, **p 0.01. DEX, dexmedetomidine; ATI, atipamezole; 3-MA, autophagy inhibitor.