Acute Kidney Injury Results in Long-Term Diastolic Dysfunction That Is Prevented by Histone Deacetylase Inhibition

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  Figure 1. Study Schematic Baseline tGFR and echocardiograms were obtained. Mice were randomized to AKI or sham cohorts on day 0. Male mice underwent 25 min of bilateral renal ischemia or sham procedure. Blood was collected retro-orbitally on day 1 to assess post-operative BUN and SCr, and mice in the AKI cohorts that did not meet inclusion criteria were sacrificed. In the serial study, mice were sacrificed 1, 3, 7, 14 days, 1, 3, 6, 9 and 12 months after the sham or AKI procedure. In the histone deacetylase inhibitor (HDACi) study arm, mice were started on chow containing ITF2357 on post-operative day 3 and maintained on treatment throughout the study; mice were sacrificed at 12 months and tissue was collected for analysis. AKI = acute kidney injury; BP = blood pressure; BUN = blood urea nitrogen; SCr = serum creatinine; tGFR = transcutaneous glomerular filtration rate.

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  Figure 2. Long-Term Kidney Sequelae in Mice After AKI Male mice underwent 25 min of bilateral renal ischemia or sham procedure and were sacrificed 1, 3, 7, 14 days, or 1, 3, 6, 9, or 12 months later. (A) Blood urea nitrogen remained elevated after acute kidney injury (AKI) at all time points. (B) Serum creatinine improved and normalized after AKI. (C) Measured transcutaneous glomerular filtration rate, normalized for weight, remained decreased after AKI throughout the study. (D) Urine neutrophil gelatinase-associated lipocalin (NGAL) was elevated after AKI until1 month after the procedure. (E) AKI cohorts demonstrated an increase in protein expression for KIM-1 7 days and 1 month after AKI, and an increase in α-SMA at 14 days and 1 month (F). (G) Quantification of kidney fibrosis via Picrosirius Red showed an increase in fibrosis in the AKI cohorts from day 14 throughout the 1 year study. (H) Quantification of immunohistochemistry in the kidneys for Collagen 3 showed an increase in the AKI cohorts at the day 7, and months 1, 3, 6, 9, and 12 time points. (I) Hydroxyproline content of the kidneys was assessed in the 12 month samples and showed an increase in the AKI cohort compared to both…

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  Figure 3. Cardiac Outcomes After AKI At 6, 9, and 12 months, AKI causes diastolic dysfunction (low E’/A’) (A) and hypertension (B) but not in mice treated with ITF (AKIITF); At 1 month, AKI causes diastolic dysfunction (C) but not hypertension (D), indicating that the diastolic dysfunction precedes the hypertension. One year after AKI, ejection fraction is preserved despite diastolic dysfunction and hypertension (E). (F) AKI did not result in left ventricular hypertrophy nor increased collagen deposition (G) at 12 months. (H) Representative echocardiogram images 6 months after AKI or sham and treatment with ITF shows abnormal E’ to A’ ratio in the AKI cohort, and normalization in the AKI cohort treated with ITF2357. (I) Plasma renin levels 1 year after AKI or sham show no significant difference between groups (n = 5 to 11). ∗#p < 0.05 with AKI compared to sham or control, respectively. Abbreviation as in Figures 1 and 2.

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  Figure 4. Long-Term Kidney Sequelae in Male Mice Treated With ITF2357 Long-term renal sequelae in male mice treated with ITF2357. Male mice underwent 25 min of bilateral renal ischemia or sham procedure, were started on chow containing ITF2357 3 days later, and were sacrificed 12 months later (A). Measured tGFR, normalized for weight, demonstrated no effect on measured tGFR throughout the year-long study between the AKI vs. AKIITF or Sham vs. ShamITF cohorts. (B) 12 months after AKIITF, blood urea nitrogen was elevated compared to shamITF while there was no significant difference between the AKI and AKIITF cohorts. There was no significant difference in measured serum creatinine (C) or α-SMA (D) amongst the 12 month groups. Quantification of fibrosis via Picrosirius Red (E), Collagen 3 immunohistochemistry (F) and Hydroxyproline content (G) showed an increase in fibrosis markers at 12 months in the AKIITF cohorts compared to both shamITF and AKI cohorts. (n = 5 to 11) In A, ∗ indicates a difference between both Sham vs. AKI and ShamITF vs. AKIITF∗ whereas # indicates there was only a difference between the Sham and AKI cohorts. Otherwise, ∗ and # signify p < 0.05 with AKI compared to sham…

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  Figure 5. Representative Picrosirius Red Staining 12 Months After AKI or Sham Procedure Without and With ITF2357 Treatment AKI results in increased fibrosis. ITF treatment did not improve kidney fibrosis. (Top panels) Light microscopy. (Bottom panels) Polarized images. Magnification, 100×. Scale bar = 10 μM. Abbreviation as in Figures 1 and 2.

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  Figure 6. AKI Affects Long-Term Body Composition AKI affects long-term body composition. (A) Serial body weight measurements in the male cohorts demonstrate a widening reduction in body weight starting at 3 months post-procedure. AKIITF cohorts weighed the least by the end of the study. (B) Gastrocnemius weight after sacrifice 12 months after AKI or sham show reduced muscle weight in cohorts treated with ITF (n = 5 to 14). ∗p < 0.05 compared to sham control (AKI vs. sham, or AKIITF vs. shamITF). Abbreviations as in Figures 1 and 2.

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  Figure 7. Lung Inflammation After AKI (A) Serial lung myeloperoxidase activity in the male acute kidney injury (AKI) versus sham cohorts demonstrates an increase in lung inflammation up to 14 days after kidney injury. (B) 1 year after AKI or sham procedure, there is no difference amongst cohorts with or without ITF2357 treatment (n = 5 to 14). ∗p < 0.05 compared to sham control.

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  Figure 8. Liver Function Tests After AKI (A) The AKI cohort demonstrates an increase in alanine aminotransferase 1 day post-procedure but at no other time point. (B) There is no statistically significant difference between aspartate aminotransferase levels in AKI versus sham cohorts at any time point. Nota bene, measurements were unable to be obtained for the 12 month time point secondary to hemolysis (n = 5 to 13). ∗p < 0.05. Abbreviation as in Figure 1.

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  Figure 9. Metabolomic Effects of ITF in AKI and/or Sham Heart Tissue at 1 Year Pathway diagram illustrating the relative concentrations of one-carbon metabolites (i.e., cysteine and methionine pathway related metabolites) shared (∗) or differentially between AKI and Sham cohorts, relative to ITF treatment in heart tissue. The pathway, enriched in metaboanalyst analysis, is adapted with permission from the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway 00270 (cysteine and methionine metabolism), 00480 (glutathione metabolism), and 00250 (alanine, aspartate, and glutamate metabolism). Red indicates lower concentrations in the ITF treated group, while green indicates higher concentrations. Abbreviations as in Figures 1, 2, and 3.

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  Figure 10. Treatment With HDACi Preserves Cardiac ATP Levels 1 Year After AKI Treatment with Histone deacetylase inhibition (HDACi) preserves cardiac adenosine triphosphate (ATP) levels 1 year after AKI. At 12 months, AKI resulted in reduced levels of cardiac ATP and mice treated with ITF (AKIITF) had normal ATP levels (n = 5 to 11). ∗#p < 0.05 with AKI compared to sham or control, respectively. Abbreviations as in Figures 1, 2, and 3.