Dr. Danielle E. Soranno is a pediatric nephrologist affiliated with Children's Hospital Colorado in Aurora, Colorado. She graduated from Case Western Reserve University School of Medicine with a medical degree. In this video Dr. Soranno discusses Acute Kidney Injury Results in Long-Term Diastolic Dysfunction That Is Prevented by Histone Deacetylase Inhibition.
Link to Abstract-
• This is the first long-term (1-year) study in mice to look at both the renal and systemic consequences of acute kidney damage. Transcutaneous glomerular filtration rate was used to track kidney function over time.
• Diastolic dysfunction was caused by AKI, which was followed by hypertension. The ejection fraction was kept. When compared to sham controls, myocardial ATP levels were lower one year following AKI.
• Mice treated with ITF2357, a histone deacetylase inhibitor, had normal diastolic function, blood pressure, and cardiac ATP after AKI.
• Metabolomics research suggests that ITF2357 therapy preserves energy metabolism pathways.
Acute kidney damage (AKI) is linked to long-term cardiovascular morbidity and mortality, according to growing epidemiological evidence. The authors provide a one-year investigation of cardiorenal outcomes in male mice after bilateral ischemia-reperfusion damage. These findings imply that AKI induces long-term cardiac metabolome dysfunction, which is linked to diastolic dysfunction and hypertension. Mice given ITF2357, a histone deacetylase inhibitor, had their heart function preserved and remained normotensive throughout the trial. ITF2357 was found to be ineffective in preventing renal fibrosis following AKI.
Acute kidney damage (AKI) affects both children and adults who are hospitalized (1,2). Patients with AKI have worse outcomes than those who do not, including higher morbidity and death, longer hospital stays, and higher hospitalization expenses (3–7). AKI induces nonrenal organ dysfunction, and systemic illnesses, not AKI, are the major cause of death in AKI patients (8–10). The short-term systemic effects of AKI have been documented in mouse models, including an increase in serum interleukin (IL)-6, lung inflammation, heart dysfunction, liver injury, and metabolic disturbances (11–14). These studies have focused on nonrenal consequences in the near term, usually analyzing results after 24 to 72 hours (15,16). AKI's long-term systemic consequences have yet to be studied in mouse models.
Patients who survive their episodes of AKI have an increased chance of acquiring a variety of long-term consequences, including cardiovascular disease, hence research into the long-term systemic sequelae of AKI in murine models is necessary (17,18). Patients with AKI have an increased risk of heart failure, according to a large body of evidence (19–23). Cho et al. recently published a study that found a link between AKI and diastolic dysfunction in elderly patients with femoral neck fractures (24). There is a scarcity of research on the pathophysiology that links AKI to a long-term risk of heart failure.
Histone deacetylase inhibitors (HDACi) have an impact on epigenetic gene regulation and nongenomic physiological processes (25–27). We recently showed that ITF2357/givinostat, an HDACi now in phase 3 clinical trials in Duchenne muscular dystrophy patients, improves diastolic function in a rat model of hypertension and protects animals from aging-induced diastolic failure (28).
We used our mouse model of bilateral ischemia reperfusion AKI to conduct a 1-year study to examine the systemic effects of one episode of AKI on the lung, liver, and heart, as well as circulating cytokines, metabolites, and kidney recovery and function. We also looked at the effect(s) of HDACi medication on cardiorenal outcomes such blood pressure, cardiac function, and metabolism. We predicted that AKI would have long-term systemic consequences and that inhibiting HDAC would improve cardiorenal outcomes.
The National Institutes of Health Guide for the Care and Use of Laboratory Animals was followed during the experiments. The Animal Care and Use Committee of the University of Colorado in Denver, Colorado, authorized the protocol. Male C57BL/6 mice aged six to seven weeks (Jackson Laboratories, Bar Harbor, Maine) were fed a regular diet and given free access to water. On post-operative day 3, mice in the ITF2357 therapy group were given chow. ITF2357 chow (Dyets Inc., Bethlehem, Pennsylvania) was custom made with 555 mg/kg of ITF2357 in Teklad 2920X Rodent Diet (Envigo, Indianapolis, Indiana), resulting in a daily dose of 50 mg/kg. Jackson Laboratories aged 12-month study controls, which came two weeks before sacrifice.
Rate of glomerular filtration
The transcutaneous filtration rate (tGFR) was measured using a transdermal continuous renal function monitor (MediBeacon GMBH, Mannheim, Germany) and a fluorescein isothiocyanate (FITC) tail-vein injection, as directed by the manufacturer. Mice were given at least three days to recover from baseline readings before surgery.
On 8-week-old mice, surgical operations were conducted. Mice were given either a sham or an AKI treatment. Ketamine (VetOne, MWI, Boise, Idaho) and Xylazine were used to anesthetize mice for all procedures (VetOne, Bimed-MTC Animal Health Inc., Cambridge, Ontario, Canada). Throughout the treatment, mice were placed on a heating pad that was kept at 36° C to 40°C. Both renal pedicles were clamped for 25 minutes using a ventral technique (12), and the abdomen was closed in two layers. Mice were given 750 l of warmed saline/buprenorphine hydrochloride (0.3 mg/ml, PAR Pharmaceuticals, Chestnut Ridge, New York) subcutaneously after surgery, followed by 750 l of normal saline for 5 days.
To determine inclusion status, 100 l of blood was obtained through retro-orbital bleed after 24 hours. Blood urea nitrogen (BUN) >70 mg/dl or serum creatinine (SCr) >0.7 mg/dl were the criteria for study inclusion in the AKI groups. The investigators were then blinded to the group assignment. Blood was taken through cardiac puncture, allowed to clot at room temperature, then centrifuged for 10 minutes at 4,000 g. The serum was taken and centrifuged for 1 minute at 4,000 g. Urine was collected and flash frozen at several study time points. At the time of sacrifice, the kidneys, liver, lung, heart, and muscle were collected; one-half was flash frozen, while the other half was formalin fixed and paraffin embedded. The heart was weighed, and the right and left ventricles were separated (LV). The upper portion of the right ventricle and the right ventricle were flash frozen. The caudal half of the LV was flash frozen after being placed in optimal cutting temperature (OCT) compound. At each study time point, the participants' weight was recorded. The tibia was dissected and measured, and the gastrocnemius muscle was cut, flash frozen, and weighed during sacrifice.
BioAssay Systems QuantiChrom Urea Assay Kit Cat: (DIUR-500) (BioAssay Systems, Hayward, California) and Pointe Scientific Creatinine (Enzymatic) Reagent Kit (Cat: C7548-480) were used to quantify BUN, serum, and urine creatinine (Pointe Scientific Inc, Canton, Michigan). The 12-month SCr was calculated utilizing the Comparative Pathology Core at UC and the Element DC Veterinary Chemistry Analyzer (Fujifilm, Serial # 73110474, Stamford, Connecticut [sponsored by Heska, Des Moines, Iowa]). The manufacturer's instructions were followed to assess neutrophil gelatinase-associated lipocalin (NGAL) using an enzyme-linked immunosorbent assay (ELISA) (R and D Systems, Minneapolis, Minnesota) (detection level: 8.8 pg/ml). Following the manufacturer's instructions, cytokines were quantified using a customized U-Plex Biomarker-1 (Cat # K15069L-S, Meso Scale Discovery, Rockville, Maryland). The Renin 1 Mouse ELISA Kit (Cat # EMREN1, Invitrogen, Carlsbad, California) was used to measure plasma renin, which was done according to the manufacturer's instructions.
Histology of the kidney
On the kidney, picrosirius red (PSR) was used, as well as normal histological staining methods with Sirius Red F3B. (Sigma-Aldrich, St. Louis, Missouri). An Olympus BX41 microscope (Olympus, Waltham, Massachusetts) with a linear polarizer was used to visualize ten cortical pictures at 100 magnification. The % area of polarization was calculated using imaging software (Image J). Immunohistochemistry for collagen type 3 was performed using a goat anti-type III collagen antibody (Cat # 1330-01, Southern Biotech, Birmingham, Alabama [1:100]), followed by rabbit antigoat horseradish peroxidase (Ref # P0449, Dako, Carpinteria, California [1:200]) and analyzed as previously described (29). PSR and collagen 3 levels were measured in both kidneys, and the average was used for analysis.
Western blot analysis
As previously described, kidneys were homogenized and analyzed with either -smooth muscle actin (SMA) primary antibody (ab32575, Abcam, Cambridge, United Kingdom, 1:5000) or KIM-1 primary antibody (Cat#: AF1817, R and D Systems, Minneapolis, Minnesota, 1:1000) using 1 RIPA buffer (Cell Signaling Technology, Danvers, Massachusetts) (29). Blots were normalized using the Revert Total Protein Stain (Cat # 926-11010, Licor, Lincoln, Nebraska) as a loading control and conducted as directed by the manufacturer.
Assay for hydroxyproline
Each sample's hydroxyproline content was determined in the left kidney, as previously described (29).
Blood pressure and echocardiography
Mean arterial blood pressure measurements were taken using a noninvasive computerized tail-cuff system (CODA High Throughput System, Kent Scientific Corp, Torrington, Connecticut), and transthoracic echocardiography and Doppler analyses were performed using Vevo2100 instruments (VisualSonics, Toronto, Ontario, Canada) (28).
Collagen portion of the LV
As previously mentioned, the percentage of collagen tissue in the LV was determined using 1-year cardiac tissue samples (28).
ATP (adenosine triphosphate) is a type of ATP
Pre-weighed LV tissue was treated according to manufacturer's instructions using commercially available reagents (Abcam, ab833355) and normalized to LV weight.
As previously reported, frozen heart and plasma samples were processed (14). The mean values for each group were determined, and pairwise group comparisons were done using the Student's t-test. p values were adjusted for the false discovery rate (FDR) in single-molecule analysis using Benjamini-ad Hochberg's hoc approach (30). The threshold for statistical significance was fixed at p 0.05. Metabolites with nominal p values 0.05 were entered into the Pathway Analysis feature of MetaboAnalyst 3.0 for pathway enrichment analysis (31). Compound Name was used to identify metabolites and was changed to conform to MetaboAnalyst's nomenclature limits.
Lung myeloperoxidase detection
MPO (myeloperoxidase) in the lungs was tested as described previously (12).
Analysis of variance (ANOVA) was used to compare groups, with Tukey post hoc adjustment for multiple comparisons and a significance level of 0.05. Unpaired Student's t-tests were used to compare two groups, assuming a Gaussian distribution with Welch's correction and a statistical significance of p0.05. The mean and standard error of the mean are used to present the data (SEM).
There were n = 5 to 11 people in each cohort. On post-operative days 1, 3, 7, 14 and months 1, 3, 6, 9, and 12, mice were sacrificed after AKI or sham (Figure 1). SCr (Figure 2B) recovered early but did not normalize until the 9-month time point, whereas BUN (Figure 2A) remained elevated throughout the 1-year research. tGFR was used to monitor serial kidney function; 1 year after AKI, there was a declining but still significant decline in tGFR (Figure 2C). Supplementary Figure 1 shows individual tGFR values for each time point. Figure 2D shows an increase in urine NGAL in the AKI cohort up to 1 month, after which there was no significant change.