Rodent model of sepsis-induced AKI and serum creatinine analysis
All experimental procedures were approved by the Ethics in Research Committee of the Federal University of Sao Paulo (CEUA-UNIFESP #3083130317). Male Wistar rats weighing 150-200 g were used to establish the sepsis-induced AKI model. Animals were purchased from the animal facility of the Federal University of Sao Paulo, Brazil and were housed in collective cages (5 animals/cage) at room temperature with a 12 h light/dark cycle and free access to standard food and tap water. The basal parameters were obtained from blood sampled by venipuncture of the retro-orbital sinus under sedation. After 15-21 days, the animals received 7.5 mg/kg LPS from Escherichia coli (strain 011:B4) intraperitoneally (i.p.). Pilot experiments were performed to determine the ideal dose of LPS that was enough to induce AKI but not sufficient to kill the animals, and 7.5 mg/kg was determined to the most suitable dose.
Then, the animals were administered 7.5 mg/kg LPS, and blood samples were collected at different times (hours) after LPS administration (2 hr (n=5), 4 hr (n=6), 6 hr (n=2) and 8 hr (n=2) to determine the kinetics of the increases in serum creatinine. In the other group, rats (n=10) were anesthetized with xylazine/ketamine (5 mg/kg//75-95 mg/kg), and blood samples were collected (control). After 15-21 days, the same animals received 7.5 mg/kg LPS, and 2 hr later, the animals were anesthetized for aortic blood sampling. During this period the general aspect and behavior of the animals were considered as exclusion criteria. Except for 1 animal, that died after LPS administration, all were eligible (n=9). Animals were euthanized by anesthetic overdose. All blood samples were immediately centrifuged, and the serum was stored at -80 °C until use.
Serum creatinine concentrations were determined by Jaffe’s method. Serum concentrations of NGAL were measured using the rat NGAL ELISA kit (BioPorto, Denmark) according to the manufacturer’s instructions.
Exosome-derived RNA extraction, cDNA synthesis and preamplification of miRNAs
Total RNA, including miRNAs, was isolated from serum exosomes using the commercial exoRNeasy serum plasma midi kit (Qiagen, Germany) according to the manufacturer’s instructions. Exogenous cel-miR-39-3p (Qiagen) was added to the samples to measure the RNA isolation efficiency, as determined by the manufacturer.
The complementary DNA (cDNA) was then synthesized from 100 ng of total RNA by using the miScript® II RT kit (Qiagen), and miRNA preamplification was performed using the miScript® PreAmp PCR kit (Qiagen) according to the manufacturer’s instructions.
RT-qPCR array to analyze miRNA expression
The expression levels of each miRNA were determined by RT-qPCR in 5 serum samples from control animals and 5 serum samples from animals 2 h after LPS administration. PCR arrays were performed using the QuantiStudio 7 system (Life Technologies, EUA) according to the manufacturer’s instructions. The expression levels of the miRNAs were normalized to the cel-miRNA-39-3p, SNORD61, SNORD68, SNORD95, SNORD96A and RNU6-2 controls using Data Analysis Centersoftware (Qiagen).
Target genes and pathway analysis of the dysregulated miRNAs
The miRNA target gene databases miRDB (http://mirdb.org/) and TargetScan (http://www.targetscan.org/) were used to predict the target genes of the differentially expressed miRNAs. miRDB is an online bioinformatics tool for predicting molecular targets of microRNAs . TargetScan is another online bioinformatics tool for predicting biological targets of miRNAs that searches for the presence of conserved sites that can bind with the seed region of each miRNA .
The identified target gene set and the Enrichr database (http://amp.pharm.mssm.edu/Enrichr/) were used to analyze the biological process, molecular function, cellular component and pathways that were significantly enriched by the target genes. This online software integrates several databases and, from the target genes listed, is able to identify enriched transcription factors associated with these genes .
Analysis of exosome-enriched microvesicles
To obtain microvesicle fractions enriched with exosomes, serum was sequentially centrifuged at 300 g for 10 min, 2,000 g for 20 min, and 10,000 g for 30 min to remove dead cells, cellular debris, and large vesicles, respectively. The samples were then subjected to ultracentrifugation at 100,000 g for 120 min twice at 4 °C to obtain a pellet that was enriched with exosomes. The microvesicles were resuspended in PBS (Life Technologies) and stored at -80 °C until use.
The microvesicle size and concentration were determined using a Malvern Nanosight Tracking Analysis (NTA) system (NS300) (Worcestershire, UK). All samples were diluted according to the limit capacity of the equipment (106-109particles/mL), and the particle concentration (particles/mL) was calculated.
The paired t-test was used to compare mean creatinine levels between the LPS-treated animals and the respective basal values. The Shapiro-Wilk test was used to assess the distribution of NGAL, which did not show a normal distribution. Therefore, the Wilcoxon test was used to compare the NGAL levels in the serum of the animals 2 h after LPS injection compared to basal levels. The data were considered statistically significant when p <0.05.