Potent Therapy and Transcriptional Prole of Combined Erythropoietin Derived Peptide CHBP and Caspase-3 siRNA against Kidney Ischemia/Reperfusion Injury in mice

Cause-specic treatment and timely diagnosis are still not available for acute kidney injury (AKI) apart from supportive therapy and serum creatinine measurement. A novel erythropoietin-derived cyclic helix B surface peptide (CHBP) protects kidneys against AKI with different causes, but the underlying mechanism is not fully dened. Herein, we investigated the transcriptional prole of renoprotection induced by CHBP and its potential synergistic effects with siRNA targeting caspase-3, an executing enzyme of apoptosis and inammation, (CASP3siRNA) on ischemia/reperfusion (IR)-induced AKI. Utilizing a mouse model with 30-min renal bilateral ischemia and 48-h reperfusion, the renoprotection of CHBP or CASP3siRNA was demonstrated in renal function and structure, active caspase-3 and HMGB1 expression. Combined treatment of CHBP and CASP3siRNA further preserved kidney structure, and reduced active caspase-3 and HMGB1. Furthermore, differentially expressed genes (DEGs) were identied with fold change > 1.414 and P < 0.05. In IR kidneys, 281 DEGs induced by CHBP were mainly involved in promoting cell division and improving cellular function and metabolism (up-regulated STAT5B and SLC22A7). The additional administration of CASP3siRNA caused 504 and 418 DEGs in IR + CHBP kidneys with or without NCsiRNA, with 37 genes in common. These DEGs were associated with modulated apoptosis and inammation (up-regulated BCL6, SLPI and SERPINA3M), and immunity, injury and microvascular homeostasis (up-regulated CFH and GREM1, and down-regulated ANGPTL2). This proof-of-effect study indicated the potent renoprotection of CASP3siRNA upon CHBP at the early stage of IR-induced AKI. Underlying genes, BCL6, SLPI, SERPINA3M, GREM1 and ANGPTL2, might be potential new biomarkers for clinical applications.


Introduction
Acute kidney injury (AKI) is a public health problem and has attracted much attention in recent years (Mehta et al., 2016). In worldwide, AKI affects about 2% patients in hospital admissions with a rate of mortality about 12%, both of which were increased to around 20% in the intensive care unit (Bouchard et al., 2015;Yang et al., 2015). There is no speci c treatment for AKI apart from passive support or renal replacement therapy such as volume control or dialysis in clinic (Moore et al., 2018). It is urgent, therefore, to develop speci c and effective treatment for AKI to reduce mortality and prevent its progression to chronic kidney disease (Mehta et al., 2015;Noble et al., 2020).
Renal ischemia/reperfusion (IR) injury is a major cause of AKI, characterized by apoptosis, in ammation and immune responses associated damage (Bellomo et al., 2012;Dong et al., 2019). Recently, the innate repair mechanism in AKI has attracted great attention, which is highlighted by an innate repair receptor, a heterodimer of erythropoietin (EPO) receptor and β common receptor (EPOR/βcR) . EPO, a natural ligand of EPOR/βcR, is defective in tissue protection due to low a nity, but high a nity to a homodimer receptor (EPOR) 2 in erythropoiesis (Gobe et al., 2014;Wang et al., 2017;Shi et al., 2018). EPO-derived helix B surface peptide (HBSP) and cyclic HBSP (CHBP, more stable and potent than HBSP (Yang et al., 2014b)) only bind with EPOR/βcR, so remaining the tissue protective property without erythropoiesis, and have promising potential for clinical application (Brines et al., 2008;Patel et al., 2012;Wu et al., 2013;Yang et al., 2013). In the IR kidney, CHBP reduces endoplasmic reticulum stress  and increases autophagy (Yang et al., 2014b), leading to less apoptosis (Kaushal and Shah 2016). CHBP also ameliorated renal in ammation and reduced chronic deposition of extracellular matrix through inactivating forkhead box O 3a (FoxO3a) after IR (Yang et al., 2015c). Nevertheless, the exact underling mechanism in the renoprotection of CHBP is incompletely understood.
Caspase-3, up-regulated by IR in the kidney, is a major effector enzyme in the process of apoptosis, as well as in ammation (Yang et al., 2011a;Li et al., 2019). Evidence suggests that down-regulating the expression of active caspase-3 is presented by HBSP/CHBP treatment in IR kidneys Yang et al., 2015b). The contributing role of caspase-3 in IR kidneys was further veri ed by small interfering RNA (siRNA), showing that serum-stabilized siRNA targeting caspase-3 greatly reversed renal function and in ammation in a 2-week porcine kidney auto-transplantation model (Yang et al., 2014a). It is also intriguing to discover whether there are synergistic effects on IR-induced AKI by combined administration of HBSP/CHBP and caspase-3 siRNA (CASP3siRNA).
In the present study, the effect of CHBP was explored by a single peritoneal injection, as well as its cotreatment with CASP3siRNA injected via the tail vein in a 48-h mouse renal IR model. To delineate the possible mechanisms of single/simultaneous administration, the modern technology of transcriptomic microarray analysis was also used to disclose a transcriptional overview in an array of genes and their biological involvements.

CHBP
The sequence of CHBP was the same with HBSP, QEQLERALNSS, and it was thioether-cyclized (molecule weight 1416.7). The detailed structure of CHBP was described previously, which was designed and synthesized by Shanghai Institute of Materia Medica, Chinese Academy of Sciences, China (Yang et al., 2014b).

Renal IR surgery
Male C57BL/6 mice, 8-12 weeks, were purchased from the Experimental Animal Center of Yangzhou University, China. All animal experiments were performed according to the guidelines of the Laboratory Animal Monitoring Committee of Jiangsu Province.
The renal IR surgical procedures were performed under general anesthesia by intraperitoneal (i.p.) injection of pentobarbital sodium at 75 mg/kg body weight (BW). Bilateral kidneys were exposed via dorsal incisions sequentially, and the renal pedicle was carefully isolated and occluded using a nontraumatic vascular clamp for 30 min. The e cacy of occlusion was con rmed by the color change of kidney surface and to dark red eventually. Followed by removing the clamps, patched blanching appeared to the kidney surface and then normal pink, indicating blood reperfusion. Sham operation was performed in the similar manner, except clamping of renal pedicles. Mice were randomly divided into 7 groups (n = 6): (1) Sham; (2) IR; (3) IR + CASP3siRNA; (4) IR + NCsiRNA; (5) IR + CHBP; (6) IR + CHBP + CASP3siRNA; (7) IR + CHBP + NCsiRNA. Six animals in each group was determined using power calculation according to the change of the key parameter in our previous IR time course model  and CHBP intervention study (Yang et al., 2014). The experimental design was shown in Fig. 1a. 0.03 mg/kg BW of siRNA (dissolved in saline) was injected into the tail vein 2 h pre-surgery. 24 nmol/kg BW of CHBP (dissolved in saline) was given through i.p. at 15 min after clamps were released.

Sample collection
At 48 h of renal IR injury, animals were anaesthetized with pentobarbital sodium, followed by cardiac puncture for drawing whole blood. The serum sample from each animal was then obtained by centrifuging at 10,000 rpm for 15 min and stored individually at -80 o C. Kidneys were removed and transversally cut at the midplane, following crosscutting from the middle. One quarter of each kidney was xed in 10% neutral formalin for 24 h, while two quarters were rapidly frozen in liquid nitrogen and the fourth part was preserved in RNAlater (Life Technologies).
Biochemistry analysis Serum creatinine (SCr) level of each animal was determined using a QuantiChrom TM Creatinine Assay Kit separately (BioAssay Systems, Hayward, USA). Brie y, thirty μl of standard or sample serum were transferred into a 96-well plate followed by adding in 200 μl working reagent per well, a mixture of reagent A and B. Absorbance at 510 nm was read immediately and 5 min later. Calculation was performed according to the manufacturer's instruction. The detection was performed three times independently.

Histological assessment
Hematoxylin & eosin (H&E) staining of kidney tissues was performed to observe and evaluate the degree of tubulointerstitial damage (TID) in the cortex using a scoring system by assessing tubular damage (degeneration and detachment from basement membrane), interstitial expansion (edema or in ammatory cell in ltration), and dilation of tubular lumina. Histological changes were graded based on the percentage of damaged area involved: < 5% area was scored 0; 5% -25% area was scored 1; 25% -50% area was scored 2; 50% -75% area was scored 3; and area exceeding 75% was scored 4. Kidney sections were blindly reviewed by two researchers independently. The scores from three compartments (tubular and interstitial areas, tubular lumina) of each kidney were obtained from 12 elds at 200 magni cations.
The average scores per eld of three compartments were then summed up for each kidney. The nal score of animal was then calculated by averaging the scores from left and right kidneys.
In Situ End-Labeling (ISEL) of apoptotic cells Apoptotic cells were detected using a TUNEL Apoptosis Detection Kit (Millipore, MA, USA) by ISEL, as previously described (Wu et al., 2013). Para n-embedded kidney sections were de-waxed and digested by proteinase K at 20 μg/ml for 10 min at 37 o C. The sections were then applied with equilibration buffer, terminal deoxynucleotidyl transferase (TdT) and anti-digoxigenin-peroxidase sequentially. The labeling of apoptotic cells was then revealed with 3-amino-9-ethylcarbazole (AEC, dark red color).
Apoptotic cells were examined at 400 magni cations in up to 20 elds of tubulointerstitial areas in the cortex. The number of positively stained cells in each animal was calculated by averaging the average number per eld from left and right kidneys. This was blindly reviewed by two researchers independently.

Immunostaining of active caspase-3 in kidneys
Active 17 kDa subunit of caspase-3 was stained on kidney para n sections using the method described before (Yang et al., 2011). Brie y, sections were de-waxed and performed antigen retrieval before incubation with a rabbit-anti-mouse 17 kDa caspase-3 antibody (1:100 dilution, R&D System, Abingdon, United Kingdom). For negative control, normal rabbit immunoglobulin G was applied at the same concentration of primary antibody. 17 kDa caspase-3+ cells were counted at 400 magni cations in up to 20 cortical elds of each kidney blindly by two researchers independently. The number of apoptotic cells for each animal was obtained by averaging the numbers from all elds in both kidneys.

Western blot analysis
Twenty-ve μg of kidney homogenate was separated in reduced SDS-PAGE (sodium dodecyl sulfatepolyacrylamide gel electrophoresis) gels and electroblotted onto a PVDF membrane. The membrane was then blocked in 5% (weight/volume) non-fat milk, following by probing with an anti-full length caspase-3 antibody (CST, Danvers, USA) at 1: 400 dilution, an anti-high mobility group box 1 (HMGB1) antibody (CST) at 1:1000 or an anti-β-actin antibody (Abcam, Cambridge, UK) at 1:8000 dilution for overnight at 4°C . The corresponding secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, USA) was then applied to the membrane for 2 h at room temperature. Afterwards, antibody binding was revealed using ECL substrate (Thermo Scienti c, Waltham, Massachusetts, USA) and a Molecular Imager Chemi Doc XRS+system (Bio-Rad, Berkeley, USA). The experiment was performed three times independently.

Microarray analysis
The kidney stored in RNAlater was performed microarray analysis to reveal the pro le of whole genomic transcripts by Shanghai Biotechnology Corporation, China. The detection was done in 4 groups (n = 3): IR, IR + CHBP, IR + CHBP + CASP3siRNA and IR + CHBP + NCsiRNA. The chosen kidney tissue samples from 3 animals in each group were nearest to the average level of the group in renal function and structure. The total 12 kidney samples were analyzed individually. The RNA integrity and quantity were monitored by the 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA) and NanoDrop One (Thermo Scienti c), respectively. Two μg RNA with an Integrity Number of no less than 8 was required for the genomic pro le analysis. The Agilent Whole Mouse Genome Oligo Microarray was applied to interrogate about 41,174 transcripts targeting 34,000 well-established annotated genes. The criteria of fold change (FC) > 1.414 (up-regulated genes) or FC < -1.414 (down-regulated genes) and P < 0.05 was used for sorting signi cant differentially expressed genes (DEGs). The cutoff value of FC was based on the fact that 0.5 cycle was the minimum number of polymerase chain reaction (PCR) cycle to distinguish the expressional differences between two samples.

Validation of candidate DEGs by quantitative PCR (qPCR)
Total RNAs were extracted by Trizol reagent from the kidney tissues of the same animals selected for microarray analysis. One μg total RNA was used for reverse transcription in a 20 μl reaction system supplemented with 4 μl 5x HiScript II qRT SuperMix and RNase-free water using a kit of HiScript II Q RT SuperMix for qPCR (Vazyme, Nanjing, China). The temperature setting was 50°C 15 min, followed by 85°C 2 min. One μl of cDNA product was ampli ed within a SYBR reaction system (Bioline, London, UK) containing 200 nM forward and reverse primers (Table 1, Biomics, Nantong, China) at 95 o C for 10 min followed by 40 cycles of 95 o C for 15 s and 55 o C for 60 s. The level of β-actin mRNA was used as an endogenous control.

Gene function analysis
Functional enrichment analysis of signi cant DEGs identi ed between groups was performed using Gene Ontology (GO, http://geneontology.org/, (Harris et al., 2004). The resulting GO terms with P value less than 0.05 were considered signi cantly enriched.

Statistical analysis
Data was expressed as mean ± standard deviation (SD). The statistical analysis of the data was performed using IBM SPSS Statistics v26.0 software. One-way ANOVA analysis was used to check the homogeneity of variance and then post hoc LSD test for multiple comparisons. The data of QPCR were analyzed using the two-tailed unpaired Student's t-test between two groups. Statistical signi cance was de ned as P < 0.05.

Identi cation of differentially expressed genes and re-validation
To disclose the mechanism of renoprotection induced by CHBP and/or CASP3siRNA, transcriptomic microarray analysis was conducted to identify DEGs affected in the IR kidneys. The 3 chosen samples from each group could best represent biochemistry and pathological changes in the group. 281 DEGs (153 up-regulated, 128 down-regulated) were identi ed in the CHBP treated IR kidneys versus IR kidneys ( Fig. 5A). 418  were shown by the additional administration of CASP3siRNA to CHBP-treated IR kidneys versus IR+CHBP kidneys with 46 genes in common to the comparison of IR+CHBP versus IR groups. In contrast to the NCsiRNA treatment to IR+CHBP kidneys, CASP3siRNA produced 504 DEGs (218 up-regulated, 286 down-regulated) in IR+CHBP kidneys, of which 9 genes were commonly altered with the IR+CHBP kidneys versus IR kidneys, and 37 genes in common with the comparison of IR+CHBP+CASP3siRNA versus IR+CHBP. Among above three comparisons, there were only 3 genes affected universally. The top 5 genes of up-regulated and down-regulated in three comparisons were listed (Table 2-4). Among DEGs, up-regulated BCL6 was associated with the negative regulation of apoptosis (Table 2), up-regulated SLPI and SERPINA3M were related to in ammation (Table  3), and up-regulated GREM1 and down-regulated ANGPTL2 linked to injury, in ammation and microvascular homeostasis (Table 4).
To validate the outcome of microarray analysis, 4 DEGs were selected for qPCR detection: up-regulated SLC22A7 by CHBP compared with the IR group (FC = 2.996), associated with the epithelial function of organic anion transport; up-regulated CFH by CASP3siRNA compared with NCsiRNA (FC = 1.949), a negative regulator in the alternative pathway of complement activation; and ANGPTL2 and GREM1 as described above. QPCR results showed that the level of SLC22A7 was greatly up-regulated by CHBP (Fig.  5B), so were CFH and GREM1 increased (Fig. 5, C and D), but Angptl2 was decreased by CASP3siRNA compared with NCsiRNA (Fig. 5E). Thus, all results from qPCR were consistent with the output of microarray data.

GO analysis of the DEGs
The identi ed DEGs were subjected to GO functional enrichment analysis to elucidate biological processes altered by CHBP and/or CASP3siRNA in the IR kidneys at 48 h. Top 30 items of biological process (P<0.05) with enrich factors are presented (Fig. 6, A-C). DEGs induced by CHBP were mainly involved in positive regulation of mitotic cell cycle, regulation of protein tyrosine kinase activity, acyl-CoA/glucose/cholesterol metabolic processes, positive regulation of cellular component biogenesis and organic anion transport (Fig. 6A). For instance, up-regulated signal transducer and activator of transcription 5B (STAT5B, FC = 1.478), a positive regulator of mitotic cell cycle; up-regulated SLC22A7, mediating organic anion transport, as well as positive regulator of cellular component biogenesis and glucose metabolic process. Further altered genes by CASP3siRNA treatment in IR + CHBP kidneys versus IR+CHBP involved in the negative regulation of immune response (Fig. 6B). Compared with the NCsiRNA control, CASP3siRNA further affected biological processes including regulation of interleukin-1 beta (IL-1β) production, positive regulation of cAMP metabolic process, positive regulation of phosphatidylinositol 3-kinase signaling and release of cytochrome c from mitochondria (Fig. 6C).

Discussion
The present study demonstrated that a single dose of CHBP or CASP3siRNA markedly ameliorated IRinduced kidney injury in terms of preserving renal function and structure, reducing active caspase-3 and HMGB1 expression. The combination of both further decreased TID, active caspase-3 and HMGB1. In addition, genomic microarray analysis identi ed DEGs induced by CHBP were mainly involved in preserving cell division, cellular function and metabolism. DEGs modi ed by CASP3siRNA were associated with inhibiting in ammation and maintaining vascular function. Certain genes such as BCL6, SLPI, SERPINA3M, GREM1 and ANGPTL2 might be potential biomarkers in IR-induced AKI.
The present study demonstrated that a single dose of CHBP, plasma half-life 300 min, (Yang et al., 2014b) administrated 15 min after reperfusion greatly ameliorated renal IR injury at the early stage of 48 h. This result was consistent with the evidence that a single dose of CHBP protected the kidney from IR injury at 12-week (Yang et al., 2015c). Linear HBSP, plasma half-life about 2 min, administered at 1 h, 6 h and 12 h protected the kidney against IR injury at 24 h (Brines et al., 2008). Our previous study also showed that daily injection of HBSP protected the kidney from immunosuppressant cyclosporine Ainduced damage upon IR injury, but did not affect IR injury alone in a 2-w rat model (Wu et al., 2013). It has been also reported that CHBP protected against aristolochic acid induced AKI (Zeng et al., 2017). These data imply a variety of potential clinical applications of CHBP or HBSP.
It is the rst time verifying that a single dose of CASP3siRNA was comparable to CHBP in renal protection. siRNA is a potent and speci c tool that can silence detrimental genes under disease conditions so siRNA therapy provides prospective in the development of precision medicine (Hawgood et al., 2015). Although there are over 30 siRNA-related clinical trials that have been completed, no siRNA treatment against AKI is available in clinical practice. The result from this study implies that caspase-3 gene may be one of major affected genes by CHBP in renoprotection, therefore, CASP3siRNA might be an alternative treatment additional to CHBP for IR-induced renal injury.
The transcriptomic pro le, moreover, demonstrated that CHBP altered genes in biological processes were mainly linked to cell division cellular function and metabolism. For example, STAT5B up-regulated by CHBP was involved in cell proliferation in rodent kidneys (Chen et al., 2007;Fragiadaki et al., 2017), while SLC22A7, enriched in organic anion transport, was associated with the extrusion of creatinine from TECs and maintaining SCr level (Shen et al., 2015). BCL6, among the top 5 DEGs up-regulated by CHBP (Table 2), has a broad role on anti-apoptosis and cell survival (Baron et al., 2010), promoting the expression of organic anion transporter 1 in TECs and maintaining the secreting function of TECs (Wegner et al., 2014). In addition, metabolic processes were greatly enriched by CHBP including glucose metabolism, which is bene cial for energy production (Wei et al., 2014). It has also been reported that the proteome pro le in IR kidneys at 48 h changed by CHBP treatment was mainly related to the oxidative stress (Yang et al., 2015a). There may be differentiations between transcriptional and translational changes, as well as the mouse strain (BALB/c) and dose of CHBP (8 nmol/kg).
Intriguingly, in contrast to single CHBP or CASP3siRNA treatment, co-treatment with CHBP and CASP3siRNA contributed to further preservation in renal structure, with lower active caspase-3 and HMGB1 in IR kidneys. The negative regulation of immune responses was also revealed by microarray analysis, verifying the effectiveness of further CASP3siRNA against renal IR. SLPI, secretory leukocyte peptidase inhibitor among the top 5 DEGs up-regulated by CASP3 siRNA (Table 3), was renoprotective in experimental ischemia AKI (Ochi et al., 2017). SLPI inhibits nuclear factor kappa beta (NF-κB) signaling pathway (Tang et al., 2020) and the maturation of IL-1β (Zakrzewicz et al., 2019), and showed as a biomarker candidate in AKI (Averdunk et al., 2019;Averdunk et al., 2020). In human, SERPINA3, a member of the serpin superfamily of protease inhibitors, could limit in ammation by targeting cathepsin family (proin ammatory enzymes) (Horvath et al., 2005;Lannan et al., 2012). SERPINA3 expression was also found in rat kidneys, which can detect renal in ammation and brosis after IR injury and also serve as a urinary marker for early detection of AKI to CKD transition (Sanchez-Navarro et al., 2019). Because murine SERPINA3M (FC = 8.289, Table 3) is a likely orthologue of human SERPINA3, the two proteins may have similar structural and kinetical characterization. The role of SERPINA3M in renal IR injury is worthy of further exploring.
Similar effects of renoprotection from CASP3siRNA were also revealed by comparing with NCsiRNA control in CHBP-treated IR kidneys. Microarray data revealed that further CASP3siRNA treatment altered 418 DEGs or 504 DEGs in IR + CHBP kidneys without or with NCsiRNA controls, which number was much higher than the 281 DEGs altered by CHBP in IR kidneys, with 46 or 9 genes in common respectively. It was indicating that CASP3siRNA may have additive effects on renoprotection upon CHBP treatment. GO analysis identi ed the further altered DEGs by CASP3siRNA mainly linked to regulation of renal in ammation and programmed cell death upon CHBP compared with that of NCsiRNA. CFH, a negative regulator of complement alternative pathway that plays crucial roles in IR injury (Goetz et al., 2018), was increased by further CASP3siRNA treatment. Notably, ANGPTL2 was the top one of down-regulated DEGs by CASP3siRNA (Table 4). Less Angptl2 could contribute to the reduction of renal in ammation as ANGPTL2 can activate resident macrophages and induce the secretion of proin ammatory cytokines (Umikawa et al., 2015;Amadatsu et al., 2016). Decreased ANGPTL2 may also ameliorate renal brosis in AKI-induced chronic kidney disease by depressing transforming growth factor-β (TGF-β) signaling . In addition, GREM1 was the top one of up-regulated DEGs by CASP3siRNA (Table  4), which activates vascular endothelial growth factor receptor 2 (VEGFR2) in endothelial cells to induce angiogenesis (Ravelli et al., 2013;Ji et al., 2016). The effective repair of endothelial cells in IR-injured kidney plays essential roles in maintaining the homeostasis of microvasculature and e cient renal blood ow (Kwon et al., 2010), and subsequently ameliorating tubular damage (Cantaluppi et al., 2012). The GREM1-VEGFR2 axis may be a novel therapeutic target for kidney in ammation and brosis (Mezzano et al., 2018). In addition, Yang and colleagues proposed that caspase-3 de ciency in mice reduced IR injury in kidneys through preserving microvascular density (Yang et al., 2018). However, whether the preservation of renal microvasculature in this study links to regulated GREM1 is worthy of further investigating. The above evidence indicates a promising strategy of silencing caspase-3 and administrating CHBP at the same time for optimized outcome in improving IR injury in kidneys.
Special attention should be paid to the toxicity of NCsiRNA in the present study, which was evidenced by further elevated SCr, TID and apoptotic levels in CHBP-modi ed IR kidneys. These data suggested that the synthetic siRNA duplexes may still modulate immunity and in ammation in IR kidneys, such as releasing cytokines and interferons, and activating toll-like receptors on immune and nonimmune cells (Judge et al., 2005;Forsbach et al., 2008;Robbins et al., 2009). It is indicating that NCsiRNA might down-regulate the in uence of CHBP treatment on IR kidneys, providing an ideal and necessary control for the speci c effects of CASP3siRNA in the context of CHBP-treated IR kidneys.
There are also limitations in this study. The additive renoprotective effect of CASP3siRNA upon CHBP should be further studied in the long-term prognosis of IR injury. In addition, DEGs identi ed by the present microarray analysis should be further validated in more comparisons with the groups including more samples. Moreover, to select and validate potential biomarkers from identi ed DEGs, different downstream biological events at translational and post-translational level should be further investigated in terms of its dynamic expression, regulation and intervention.
The co-treatment of CHBP and CASP3siRNA exhibited synergistic effects on preserving renal structure and reducing injury markers against 48-h renal IR in a mouse model. The DEGs induced by CHBP are associated with the preservation of cell division, function and metabolism, while the DEGs caused by CASP3siRNA are linked to improving in ammation and potential microvasculature.   Scale bar: 100 μm. (d) Semi-quantitative analysis of tubulointerstitial damage (TID) score (n = 6). The sections were blindly scored by two researchers independently. Data were shown as mean ± SD. n = 6: 6 animals per group; * P < 0.05; ** P < 0.01. Statistically comparisons were calculated by ANOVA followed by post hoc LSD test. protein corrected by β-actin was determined in each group (n = 6). The immunoblotting was performed three times independently. Data were shown as mean ± SD. n = 6: 6 animals per group; * P < 0.05; ** P < 0.01. Statistically comparisons were calculated by ANOVA followed by post hoc LSD test.