Transcriptional Profiles in Ischemia/Reperfusion-Injured Murine Kidneys Synergistically Protected by Erythropoietin Derived Peptide CHBP and Caspase-3 siRNA


 Background: Target-specific treatment is not available for acute kidney injury (AKI). A novel erythropoietin-derived cyclic helix B surface peptide (CHBP) protects kidneys against AKI subjected to different causes. Herein, we investigated the transcriptional profile of renoprotection induced by CHBP and its potential synergistic effects with caspase-3 siRNA (CASP3siRNA) on ischemia/reperfusion (IR) injury associated AKI. Methods: A mouse renal IR model was established by clamping bilateral pedicles for 30 min and reperfusion for 48 h. 0.03 mg/kg of CASP3siRNA/negative control (NCsiRNA) was injected via tail vein 2 h pre-surgery, with/without 24 nmol/kg of CHBP administered to peritoneal cavity at 15 min post reperfusion. The transcriptomic profile in kidneys was assessed by affymetrix gene chips, along with renal function, histology, active caspase-3 and HMGB1.Results: CHBP or CASP3siRNA significantly improved renal function and structure, with decreased caspase-3 and HMGB1 in IR kidneys. Combined treatment of CHBP and CASP3siRNA further preserved kidney structure, and reduced active caspase-3 and HMGB1. Furthermore, fold change > 1.414 and P < 0.05 were used to identify differentially expressed genes (DEGs). 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 inflammation (up-regulated BCL6，SLPI and SERPINA3M), and immunity, injury and microvascular homeostasis (up-regulated CFH and GREM1, and down-regulated ANGPTL2). Conclusions: This proof-of-effect study indicated that the synergistic renoprotection of CHBP and CASP3siRNA 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 [1]. In worldwide, AKI affects about 2% patients in hospital admissions with a rate of mortality about 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 Scientific, Waltham, Massachusetts, USA) and a Molecular Imager Chemi Doc XRS+system (Bio-Rad, Berkeley, USA).

Microarray analysis
The kidney stored in RNAlater was performed microarray analysis to reveal the profile of whole genomic transcripts by Shanghai Biotechnology Corporation, China. The detection was done in 4 groups (n = 3 in each group): IR, IR + CHBP, IR + CHBP + CASP3siRNA and IR + CHBP + NCsiRNA. The RNA integrity and quantity were monitored by the 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA) and NanoDrop One (Thermo Scientific), respectively. Two μg RNA with an Integrity Number of no less than 8 was required for the genomic profile analysis. The Agilent Whole Mouse Genome Oligo Microarray was applied to interrogate about 41,174 transcripts targeting 34,000 wellestablished 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 significant 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 amplified 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 significant DEGs identified between groups was performed using Gene Ontology (GO, http://geneontology.org/) [26]. The resulting GO terms with P value less than 0.05 were considered significantly enriched.

Statistical analysis
Data was expressed as mean ± standard error of the mean (SEM). Statistical analysis of the data was performed using GraphPad Prism v8.0 software. One-way ANOVA analysis was used to check the homogeneity of variance for more than two groups. Unpaired student TTEST was then carried out to compare between parameters from two groups. P value < 0.05 was considered as statistically significant.

Improved kidney function and structure
At 48 h, the SCr level raised by IR was significantly reduced by the treatment of CHBP, CASP3siRNA or CHBP + CASP3siRNA (Fig. 1b). However, no significant difference was observed among these treatments. IR mice treated with CASP3siRNA or CHBP + CASP3siRNA demonstrated a significant lower SCr level than those treated with NCsiRNA or CHBP + NCsiRNA, respectively. CHBP, CASP3siRNA and CHBP+CASP3siRNA treatment significantly decreased the extent of TID in IR kidneys ( Fig. 1c, d). Furthermore, IR mice with co-treatment of CHBP and CASP3siRNA exhibited a lower level of TID in contrasted to those treated with CHBP only or CASP3siRNA only (1.43 ± 0.15 versus 2.72 ± 0.09 or 2.49 ± 0.13, P < 0.01). In addition, CASP3siRNA-treated IR or IR+CHBP mice demonstrated a significant decrease of TID compared with corresponding NCsiRNA controls.

Alleviated apoptosis
As anti-apoptosis is a shared renoprotective effect used by CHBP and CASP3siRNA, immunolabelling was performed to examine their effectiveness on reducing kidney IR-induced apoptosis. IR significantly raised the number of ISEL+ cells compared with sham controls, but greatly lowered by CHBP, CASP3siRNA and CHBP + CASP3siRNA (Fig. 2a, b). Nevertheless, comparable levels of ISEL+ cells were found among these treatments. In contrast to NCsiRNA controls, CASP3siRNA reduced apoptosis in either IR kidneys or CHBP-modified IR kidneys.

Decreased active caspase-3 staining positive cells
Cells labeled with active 17 kDa caspase-3 often having the morphological features of apoptosis as condensed nuclei were mainly located in tubular epithelia, tubular lumina and interstitial areas. The number of active caspase-3+ cells was significantly increased by IR, but decreased by CHBP, CASP3siRNA and CHBP + CASP3siRNA (Fig. 3a, b). No significant differences between these treatments were observed. Comparing with NCsiRNA, CASP3siRNA significantly reduced the number of 17 kDa caspase-3+ cells in IR kidneys, as well as in IR + CHBP kidneys.

Decreased expression of active caspase-3 and HMGB1 protein
Western blotting was used to determine whether there is a co-effect of CHBP and CASP3siRNA on the expression of 17 kDa active caspase-3. The level of 17 kDa caspase-3 was significantly increased by IR, but reduced by CHBP, CASP3siRNA and CHBP + CASP3siRNA (Fig. 4a, b). IR mice with the cotreatment of CHBP and CASP3siRNA showed an even lower expression of 17 kDa caspase-3 compared with IR mice treated with CASP3siRNA (0.29 ± 0.03 versus 0.52 ± 0.07, P < 0.05). Controlled by NCsiRNA, 17 kDa caspase-3 was reduced by CASP3siRNA in IR kidneys or IR + CHBP kidneys.
HMGB1 is a proinflammatory factor known to arouse profound innate responses by binding to toll-like receptor 4 on surface of tubular epithelial cells (TECs) and macrophages [27,28]. Western blotting using kidney homogenates demonstrated IR significantly increased HMGB1 expression compared with sham controls (Fig. 4c, d). However, treatments with CHBP, CASP3siRNA and CHBP + CASP3siRNA decreased the high level of HMGB1. Moreover, IR mice with co-treatment of CHBP and CASP3siRNA exhibited an even lower level of renal HMGB1 compared with CASP3siRNA-treated animals (0.81 ± 0.16 versus 1.57 ± 0.24, P < 0.05). In contrast to NCsiRNA, CASP3siRNA reduced HMGB-1 expression in IR kidneys as well as in IR + CHBP kidneys.

Identification 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. 281 DEGs (153 upregulated, 128 down-regulated) were identified in the CHBP treated IR kidneys versus IR kidneys (Fig. 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 inflammatory and cell death signaling in IR+CHBP kidneys, such as regulation of interleukin-1 beta (IL-1β) production, positive regulation of phosphatidylinositol 3-kinase signaling, 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 identified DEGs induced by CHBP were mainly involved in preserving cell division, cellular function and metabolism. DEGs modified by CASP3siRNA were associated with inhibiting inflammation 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 [13]) 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 [20]. 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 [14]. Our previous study also showed that daily injection of HBSP protected the kidney from immunosuppressant cyclosporine A-induced damage upon IR injury, but did not affect IR injury alone in a 2-w rat model [16]. It has been also reported that CHBP protected against aristolochic acid induced AKI [29]. These data imply a variety of potential clinical applications of CHBP or HBSP.
It is the first time verifying that a single dose of CASP3siRNA was comparable to CHBP in renal protection. siRNA is a potent and specific tool that can silence detrimental genes under disease conditions so siRNA therapy provides prospective in the development of precision medicine [30].
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 profile, moreover, demonstrated that CHBP altered genes in biological processes were mainly linked to cell division cellular function and metabolism. For example, STAT5B up- 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) subsequently ameliorating tubular damage [53]. The GREM1-VEGFR2 axis may be a novel therapeutic target for kidney inflammation and fibrosis [54]. In addition, Yang and colleagues proposed that caspase-3 deficiency in mice reduced IR injury in kidneys through preserving microvascular density [55]. 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-modified IR kidneys. These data suggested that the synthetic siRNA duplexes may still modulate immunity and inflammation in IR kidneys, such as releasing cytokines and interferons, and activating toll-like receptors on immune and nonimmune cells [56][57][58]. It is indicating that NCsiRNA might down-regulate the influence of CHBP treatment on IR kidneys, providing an ideal and necessary control for the specific effects of CASP3siRNA in the context Monitoring Committee of Jiangsu Province with established guidelines for the care and use of laboratory animals.

Consent for publication
Not applicable.

Availability of data materials
All data generated or analyzed during this study are included in this published article.

Competing interests:
There is no financial conflict in the information contained in this manuscript.  The gene in bold was discussed its function in IR-induced AKI. The gene in bold was discussed its function in IR-induced AKI. Genes in bold indicate analysis by quantitative polymerase chain reaction (qPCR) and discussed as biomarker candidates of IR-induced AKI. Semi-quantitative analysis of tubulointerstitial damage (TID) score (n = 6). * P < 0.05; ** P < 0.01.