Effects of Valproic Acid and Dexamethasone Administration on Early Bio-Markers and Gene Expression Profile in Acute Kidney Ischemia-Reperfusion Injury in the Rat

<div><p>Renal ischemia-reperfusion (IR) causes acute kidney injury (AKI) with high mortality and morbidity. The objective of this investigation was to ameliorate kidney IR injury and identify novel biomarkers for kidney injury and repair. Under general anesthesia, left renal ischemia was induced in Wister rats by occluding renal artery for 45 minutes, followed by reperfusion and right nephrectomy. Thirty minutes prior to ischemia, rats (n = 8/group) received Valproic Acid (150 mg/kg; VPA), Dexamethasone (3 mg/kg; Dex) or Vehicle (saline) intraperitoneally. Animals were sacrificed at 3, 24 or 120 h post-IR. Plasma creatinine (mg/dL) at 24 h was reduced (P<0.05) in VPA (2.7±1.8) and Dex (2.3±1.2) compared to Vehicle (3.8±0.5) group. At 3 h, urine albumin (mg/mL) was higher in Vehicle (1.47±0.10), VPA (0.84±0.62) and Dex (1.04±0.73) compared to naïve (uninjured/untreated control) (0.14±0.26) group. At 24 h post-IR urine lipocalin-2 (μg/mL) was higher (P<0.05) in VPA, Dex and Vehicle groups (9.61–11.36) compared to naïve group (0.67±0.29); also, kidney injury molecule-1 (KIM-1; ng/mL) was higher (P<0.05) in VPA, Dex and Vehicle groups (13.7–18.7) compared to naïve group (1.7±1.9). Histopathology demonstrated reduced (P<0.05) ischemic injury in the renal cortex in VPA (Grade 1.6±1.5) compared to Vehicle (Grade 2.9±1.1). Inflammatory cytokines IL1β and IL6 were downregulated and anti-apoptotic molecule BCL2 was upregulated in VPA group. Furthermore, kidney DNA microarray demonstrated reduced injury, stress, and apoptosis related gene expression in the VPA administered rats. VPA appears to ameliorate kidney IR injury via reduced inflammatory cytokine, apoptosis/stress related gene expression, and improved regeneration. KIM-1, lipocalin-2 and albumin appear to be promising early urine biomarkers for the diagnosis of AKI.</p></div

Histopathology of hematoxylin and eosin stained kidney sections.

<p>A, B, C = Renal cortex at 3 hours (h) post ischemia-reperfusion (IR); D, E, F = Renal outer medulla at 24 h post-IR; A, D = Vehicle (saline control); B, E = Valproic acid (VPA); C, F = Dexamethasone (Dex) treated animals. Three high power fields (400x) representing approximately 50 tubules from cortex and outer medulla of each kidney were evaluated for ischemic changes (injury), tubular necrosis and regenerative changes. Collectively kidney injury and regeneration were graded (0–4) based on the mean percentage of tubules affected: 0, None; 1, <25%; 2, ≥25 but <50%; 3, ≥50 but <75%; 4, >75–100%. Ischemic changes included nuclear condensation <b>(nc)</b>, cytoplasmic eosinophilia, individual cell necrosis and tubular dilation <b>(td)</b>; tubular necrosis <b>(tn)</b> included confluent cell necrosis or sloughing of the tubular epithelium; and regenerative changes included tubular dilation, cytoplasmic basophilia and contraction of the cytoplasm, as well as vesicular chromatin with nucleoli. Hemorrhage <b>(hg)</b> was predominant in the vehicle control group. G, H, I = represent Histopathology quantification: renal cortex (black bars ■) and renal outer medulla (white bars □). The histologic injury score was significantly (P<0.05) lower in the VPA treated group compared to the Vehicle control at 3 h post-IR (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126622#pone.0126622.t003" target="_blank">Table 3</a>).</p

Gene transcription analysis as determined by Affymetrix Gene Array Technology.

<p>The Venn diagrams presented show the number of transcriptomes expressed and overlaps in the kidneys of animals treated with valproic acid (VPA), dexamethasone (Dex) or untreated (None; Vehicle control) at 3, 24 and 120 hours (h) post ischemia-reperfusion (IR). All relative changes in gene expression are derived from comparison with naïve rats. Fewer genes were up-regulated in the VPA group compared to untreated Vehicle control group at 3 h post-IR. At 120 h post-IR only two genes were upregulated and none was downregulated in VPA treated animals. Specific gene expression information is presented in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126622#pone.0126622.s001" target="_blank">S1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126622#pone.0126622.s006" target="_blank">S6</a> Tables</b>. The DNA microarray data have been deposited in NCBI’s Gene Expression Omnibus (GEO) repository and are accessible through Accession No. GSE58438 (<a href="http://www.ncbi.nlm.nih.gov/geo/" target="_blank">http://www.ncbi.nlm.nih.gov/geo/</a>).</p

Experimental design.

<p>Lewis rats were pre-medicated with Valproic acid (VPA; 150 mg/kg body weight) or Dexamethasone (Dex; 3 mg/kg/body weight) or saline (Vehicle control) intraperitoneally 30 minutes (min) prior to cross clamping left renal artery and inducing left renal ischemia. The cross clamp was removed after 45 min allowing kidney reperfusion, and at the same time a right nephrectomy was performed. Animals were sacrificed at 3, 24 and 120 hours post ischemia-reperfusion. Left kidney, blood and urine were collected for cellular and molecular analyses.</p

Comparisons of blood plasma and urine biomarkers (mean ± standard deviation) between time points (post kidney ischemia-reperfusion) in different treatment groups.^*

<p>*Creatinine and BUN (Blood Urea Nitrogen) were measured in blood plasma</p><p>Albumin, Lipocalin-2, Osteopontin, and KIM-1 (Kidney Injury Molecule-1) were measured in urine. <b>Normal/Naive,</b> plasma or urine drawn from naïve uninjured/untreated Wister rats served as base line. <b>VPA</b> (Valproic Acid), <b>Dex</b> (Dexamethasone) and <b>Vehicle</b> (saline control) treated groups at 3, 24 and 120 hour (h) reperfusion. Means with at least one common superscript (a, b or c) between 3, 24 or 120 h within each group (Vehicle, VPA or Dex) did not vary significantly (P>0.05).</p><p>Comparisons of blood plasma and urine biomarkers (mean ± standard deviation) between time points (post kidney ischemia-reperfusion) in different treatment groups.^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126622#t001fn001" target="_blank">*</a>}</p

Histopathologic scores (Mean± SD) of renal cortex and outer medulla at 3, 24 and 120 hours (h) post ischemia-reperfusion (IR) in Valproic Acid (VPA) and Dexamethasone (Dex) treated animals.

<p>Mean values within columns among treatment groups with at least one common <b><i>superscript letter (a or b)</i></b> did not vary significantly (P>0.05). Also, mean values in rows between cortex and medulla (within 3h, 24h or 120h post-IR) groups with a common <b><i>superscript number</i></b> did not vary significantly (P>0.05). Vehicle (saline) served as a control. N = 8/sub-group (3, 24 or 120 h).</p><p>Histopathologic scores (Mean± SD) of renal cortex and outer medulla at 3, 24 and 120 hours (h) post ischemia-reperfusion (IR) in Valproic Acid (VPA) and Dexamethasone (Dex) treated animals.</p

Kidney functional biomarker analysis.

<p>X-axis represents treatment groups (n = 8/group): Vehicle control (white bars □), Valproic Acid (VPA; black bars ■), and Dexamethasone (grey bars ■). N, Normal (naïve) uninjured and untreated rats (n = 5). Y-axis represents concentration of each biomarker in the blood (creatinine, blood urea nitrogen [BUN]) and urine (albumin, lipocalin-2, osteopontin, kidney injury molecule-1[KIM-1]). In VPA treated animals albumin level were markedly lower at three hours (h) while the creatinine and BUN values were lower at 24 h post ischemia-reperfusion (IR) compared to vehicle controls. However, lipocalin-2 and KIM-1 were higher at 24 h in all the groups (VPA, Dex, Vehicle) studied. See Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126622#pone.0126622.t001" target="_blank">1</a> & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126622#pone.0126622.t002" target="_blank">2</a> for statistical significance between treatment groups and with time post ischemia-reperfusion.</p

Histopathology of hematoxylin and eosin stained kidney sections.

<p>A, B, C = Renal cortex at 3 hours (h) post ischemia-reperfusion (IR); D, E, F = Renal outer medulla at 24 h post-IR; A, D = Vehicle (saline control); B, E = Valproic acid (VPA); C, F = Dexamethasone (Dex) treated animals. Three high power fields (400x) representing approximately 50 tubules from cortex and outer medulla of each kidney were evaluated for ischemic changes (injury), tubular necrosis and regenerative changes. Collectively kidney injury and regeneration were graded (0–4) based on the mean percentage of tubules affected: 0, None; 1, <25%; 2, ≥25 but <50%; 3, ≥50 but <75%; 4, >75–100%. Ischemic changes included nuclear condensation <b>(nc)</b>, cytoplasmic eosinophilia, individual cell necrosis and tubular dilation <b>(td)</b>; tubular necrosis <b>(tn)</b> included confluent cell necrosis or sloughing of the tubular epithelium; and regenerative changes included tubular dilation, cytoplasmic basophilia and contraction of the cytoplasm, as well as vesicular chromatin with nucleoli. Hemorrhage <b>(hg)</b> was predominant in the vehicle control group. G, H, I = represent Histopathology quantification: renal cortex (black bars ■) and renal outer medulla (white bars □). The histologic injury score was significantly (P<0.05) lower in the VPA treated group compared to the Vehicle control at 3 h post-IR (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126622#pone.0126622.t003" target="_blank">Table 3</a>).</p

Regulated transcription factors and their predicted genes that regulated up or down.

<p>TF, Transcription factor; P, Probability; FDR, False Discovery Rate; I, Interaction; TG, Target Gene; RC, Random Control; Tx, Treatment; H, Hours Post-ischemia reperfusion; None, No treatment; Dex, Dexamethazone; VPA, Valproic Acid. All treatment groups (None, Dex, and VPA) were queried with TFactS at each time point (3, 24, and 120 hours) by using the up and down regulated gene lists presented in Supplementary information <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126622#pone.0126622.s005" target="_blank">S5 Table</a></b>. To reduce the complexity of the data, only TF that has a random control <2%, p<0.01, FDR<0.01 and more than two gene targets were presented (see the full queries in supplementary information <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126622#pone.0126622.s006" target="_blank">S6 Table</a></b>). The total number of target genes for a given transcription factor (i.e. JUNB has 20 target genes) is a property of the TFactS database. The intersection (I) of the target genes (TGs) in the microarray analysis with the transcription factors (TFs) in the database is presented.</p><p>Regulated transcription factors and their predicted genes that regulated up or down.</p