Valproic Acid Prevents Renal Dysfunction and Inflammation in the Ischemia-Reperfusion Injury Model

Ischemia-reperfusion injury (IRI) is a major contributor to acute kidney injury (AKI). At present, there are no effective therapies to prevent AKI. The aim of this study was to analyse whether valproic acid (VPA), a histone deacetylase inhibitor with anti-inflammatory properties, prevents renal IRI. Male Wistar rats were divided into three groups: SHAM rats were subjected to a SHAM surgery, IRI rats underwent bilateral renal ischemia for 45 min, and IRI + VPA rats were treated with VPA at 300 mg/kg twice daily 2 days before bilateral IRI. Animals were euthanized at 48 hours after IRI. VPA attenuated renal dysfunction after ischemia, which was characterized by a decrease in BUN (mg/dL), serum creatinine (mg/dL), and FENa (%) in the IRI + VPA group (39±11, 0.5±0.05, and 0.5±0.06, resp.) compared with the IRI group (145±35, 2.7±0.05, and 4.9±1, resp.; p<0.001). Additionally, significantly lower acute tubular necrosis grade and number of apoptotic cells were found in the IRI + VPA group compared to the IRI group (p<0.001). Furthermore, VPA treatment reduced inflammatory cellular infiltration and expression of proinflammatory cytokines. These data suggest that VPA prevents the renal dysfunction and inflammation that is associated with renal IRI

Adipose-Derived Mesenchymal Stem Cells Modulate Fibrosis and Inflammation in the Peritoneal Fibrosis Model Developed in Uremic Rats

Peritoneal fibrosis (PF) represents a long-term complication of peritoneal dialysis (PD), affecting the peritoneal membrane (PM) function. Adipose tissue-derived mesenchymal stem cells (ASC) display immunomodulatory effects and may represent a strategy to block PF. The aim of this study was to analyze the effect of ASC in an experimental PF model developed in uremic rats. To mimic the clinical situation of patients on long-term PD, a combo model, characterized by the combination of PF and chronic kidney disease (CKD), was developed in Wistar rats. Rats were fed with a 0.75% adenine-containing diet, for 30 days, to induce CKD with uremia. PF was induced with intraperitoneal injections of chlorhexidine gluconate (CG) from day 15 to 30. 1×106 ASC were intravenously injected at days 15 and 21. Rats were divided into 5 groups: control, normal rats; CKD, rats receiving adenine diet; PF, rats receiving CG; CKD+PF, CKD rats with PF; CKD+PF+ASC, uremic rats with PF treated with ASC. PF was assessed by Masson trichrome staining. Inflammation- and fibrosis-associated factors were assessed by immunohistochemistry, multiplex analysis, and qPCR. When compared with the control and CKD groups, GC administration induced a striking increase in PM thickness and inflammation in the PF and CKD+PF groups. The development of PF was blocked by ASC treatment. Further, the upregulation of profibrotic factors (TGF-β, fibronectin, and collagen) and the increased myofibroblast expression observed in the CKD+PF group were significantly ameliorated by ASC. Beyond the antifibrotic effect, ASC showed an anti-inflammatory effect avoiding leucocyte infiltration and the overexpression of inflammatory cytokines (IL-1β, TNF-α, and IL-6) in the PM induced by GC. ASC were effective in preventing the development of PF in the experimental model of CKD+PF, probably due to their immunomodulatory properties. These results suggest that ASC may represent a potential strategy for treating long-term PD-associated fibrosis

Peritoneal membrane infiltration of macrophages and expression of macrophage-related chemokines.

<p><b>(A-C)</b> Representative photomicrographs of immunohistochemistry for macrophages (ED1⁺) (x200). <b>(A):</b> Only few ED1⁺ cells were present in the normal peritoneal membrane; <b>(B)</b> PF group showed a marked increase in the number of macrophages infiltrating peritoneal membrane; <b>(C)</b> Animals with PF treated with VPA exhibited a non-significant reduction of macrophage infiltration. <b>(D)</b> Quantitative analysis of the number of macrophages of all groups (n = 5/group). <b>(E-F)</b> Concentration of MCP-1 and MIP-2 in the peritoneal tissue, detected by Multiplex. VPA treatment reduced the protein levels of macrophage chemoattractant proteins (n = 4-5/group).</p

Expression of fibronectin, TGF-β, FSP-1, and BMP-7 genes in the peritoneal tissue by qPCR.

<p>VPA treatment reduced the upregulation of the profibrotic genes observed in the PF group and increased BMP-7 expression depressed in the PF group (n = 6/group).</p

Anti-fibrotic effects of valproic acid in experimental peritoneal fibrosis - Fig 1

<p><b>Effect of VPA on the peritoneal thickness in experimental PF model (A-C):</b> Representative photomicrographs of peritoneal samples stained with Masson’s trichrome (x200)<b>. (A)</b> Control group (n = 8) showed a thin submesothelial layer of the peritoneum, without any morphological changes. <b>(B)</b> The peritoneal membrane in the PF group (n = 10) rats showed a marked thickening of the submesothelial compact zone. <b>(C):</b> VPA prevented the development of submesothelial thickening in the PF+VPA group (n = 10). <b>(D)</b> Quantification analysis of the effect of VPA on peritoneal thickness, at day 30.</p

Representative photomicrographs of parameters related to experimental peritoneal fibrosis and effect of treatment with VPA (x200).

<p><b>(A-C)</b> Effect of VPA on the peritoneal expression of α-SMA (myofibroblasts) by immunohistochemistry. <b>(A):</b> No α-SMA expression was detected in the peritoneal membrane of the Control group. <b>(B)</b> Induction of PF was associated with a marked increase in α-SMA expression. <b>(C)</b> A striking reduction of myofibroblasts was observed with VPA treatment. <b>(D-F)</b> Effect of VPA on the peritoneal expression of pSmad3, by immunohistochemistry. <b>(D)</b> Only a few positive cells were noted in the peritoneal membrane of the Control group; <b>(E)</b> PF group showed an increased number of pSmad3 positive cells (stained in brown) infiltrating peritoneal membrane; <b>(F)</b> pSmad3 expression was significantly reduced by VPA treatment. <b>(G-H)</b> Quantification analysis of the effect of VPA in experimental PF, regarding α-SMA staining area, and the number of pSmad3 positive cells in all groups (n = 5-6/group). <b>(I)</b> Quantification analysis of the effect of VPA in the peritoneal membrane expression of mRNA Smad3 in all groups (n = 6/group).</p

Capillary density and VEGF mRNA expression in the peritoneum.

<p><b>(A-C)</b> Representative immunofluorescence photomicrographs of Isolectin-B₄ (stained in red), an endothelium marker, and DAPI+ cells (stained in blue) in the peritoneal membrane. While virtually no vessels were identified in Control Group <b>(A),</b> an intense neoangiogenesis (white arrows) in the submesothelial zone was observed in the PF group <b>(B)</b>. <b>(C)</b> VPA treatment attenuated this increase. <b>(D)</b> Graph comparing the vascular density of all groups(n = 3-4/group). <b>(E)</b> Expression of mRNA VEGF in peritoneal tissue by PCR (n = 6/group).</p

Expression of proinflammatory cytokines in the peritoneal membrane of the different groups.

<p>VPA treated animals showed a significant lower gene (n = 6/group) and protein expression (n = 4-5/group) of TNF-α in peritoneal tissue than the PF group.</p

Evaluation of Intermittent Hemodialysis in Critically Ill Cancer Patients with Acute Kidney Injury Using Single-Pass Batch Equipment.

BACKGROUND:Data on renal replacement therapy (RRT) in cancer patients with acute kidney injury (AKI) in the intensive care unit (ICU) is scarce. The aim of this study was to assess the safety and the adequacy of intermittent hemodialysis (IHD) in critically ill cancer patients with AKI. METHODS AND FINDINGS:In this observational prospective cohort study, 149 ICU cancer patients with AKI were treated with 448 single-pass batch IHD procedures and evaluated from June 2010 to June 2012. Primary outcomes were IHD complications (hypotension and clotting) and adequacy. A multiple logistic regression was performed in order to identify factors associated with IHD complications (hypotension and clotting). Patients were 62.2 ± 14.3 years old, 86.6% had a solid cancer, sepsis was the main AKI cause (51%) and in-hospital mortality was 59.7%. RRT session time was 240 (180-300) min, blood/dialysate flow was 250 (200-300) mL/min and UF was 1000 (0-2000) ml. Hypotension occurred in 25% of the sessions. Independent risk factors (RF) for hypotension were dialysate conductivity (each ms/cm, OR 0.81, CI 0.69-0.95), initial mean arterial pressure (each 10 mmHg, OR 0.49, CI 0.40-0.61) and SOFA score (OR 1.16, CI 1.03-1.30). Clotting and malfunctioning catheters (MC) occurred in 23.8% and 29.2% of the procedures, respectively. Independent RF for clotting were heparin use (OR 0.57, CI 0.33-0.99), MC (OR 3.59, CI 2.24-5.77) and RRT system pressure increase over 25% (OR 2.15, CI 1.61-4.17). Post RRT blood tests were urea 71 (49-104) mg/dL, creatinine 2.71 (2.10-3.8) mg/dL, bicarbonate 24.1 (22.5-25.5) mEq/L and K 3.8 (3.5-4.1) mEq/L. CONCLUSION:IHD for critically ill patients with cancer and AKI offered acceptable hemodynamic stability and provided adequate metabolic control

Parameters of intermittent hemodialysis sessions (n = 448).

<p>Parameters of intermittent hemodialysis sessions (n = 448).</p