CIMB, Vol. 44, Pages 5655-5665: Deletion of Antigen-Presenting Cells in Lipopolysaccharide-Induced Acute Kidney Injury (AKI) Affects the Exacerbation and Repair in AKI

Conceptualization, Y.N.; Data curation, J.L.; Formal analysis, J.L.; Investigation, J.L.; Methodology, Y.N.; Project administration, Y.N.; Supervision, Y.N., K.K. (Kazuya Kishimoto) and I.M.; Validation, J.L., Y.N., K.K. (Kazuya Kishimoto) and K.K. (Koji Kinoshita); Visualization, J.L.; Writing—original draft, J.L.; Writing—review & editing, J.L., Y.N. and H.A. All authors have read and agreed to the published version of the manuscript.

Figure 1. Experimental protocol. At 48 h, mice were given either liposomal clodronate (LC) or saline by an intrapertoneal injection. At 0 h, a total of 40 C57BL/6 mice were injected intraperitoneally (i.p.) with 30 mg/kg of lipopolysaccharide (LPS). Mice were culled at 18 (n = 10 each) and 120 h (n = 10 each). Specimens were collected each hr. Asterisks indicate when specimens were collected.

Figure 1. Experimental protocol. At 48 h, mice were given either liposomal clodronate (LC) or saline by an intrapertoneal injection. At 0 h, a total of 40 C57BL/6 mice were injected intraperitoneally (i.p.) with 30 mg/kg of lipopolysaccharide (LPS). Mice were culled at 18 (n = 10 each) and 120 h (n = 10 each). Specimens were collected each hr. Asterisks indicate when specimens were collected.

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Figure 2. Liposomal clodronate depleted CD11b+ and CD11c+ cells gating with F4/80+cells. (A): At 18 and 120 h after the LPS injection, cell suspensions were obtained from the kidney of LPS + LC-treated and LPS-treated mice (n = 10 each). Representative data from the FACS analysis are shown. (B): The percentages of CD11b+ F4/80+ and CD11c+F4/80+ cells. The data are mean ± SEM. *** p < 0.005, **** p < 0.001, LPS + LC vs. LPS-treated group.

Figure 2. Liposomal clodronate depleted CD11b+ and CD11c+ cells gating with F4/80+cells. (A): At 18 and 120 h after the LPS injection, cell suspensions were obtained from the kidney of LPS + LC-treated and LPS-treated mice (n = 10 each). Representative data from the FACS analysis are shown. (B): The percentages of CD11b+ F4/80+ and CD11c+F4/80+ cells. The data are mean ± SEM. *** p < 0.005, **** p < 0.001, LPS + LC vs. LPS-treated group.

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Figure 3. Effect of LC on the survival and the BUN level in the development of AKI after LPS injection. (A): Survival of the LPS + LC-treated mice and LPS-treated mice subjected to AKI by LPS injection. Mice were evaluated until 120 h after the LPS injection. (B): Renal function of the LPS + LC-treated mice and LPS-treated mice at 18 and 120 h after LPS injection, assessed by BUN levels. LC-treated mice that received saline instead of LPS were designated as the sham group, and BUN levels were also measured. (C): The grade of renal injury was evaluated by periodic acid–Schiff staining. Kidneys were stained with periodic acid–Schiff reagent to evaluate tubular necrosis in a semiquantitative manner by determining the percentage of cortical tubules in which epithelial necrosis, loss of the brush border, cast formation, and tubular dilation were observed; ×400. (D): The grade of renal injury in the LPS + LC- and LPS-treated groups at 18 and 120 h after LPS injection was evaluated by the acute tubular necrosis (ATN) score. The data are mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.005, LPS + LC- vs. LPS-treated group. Scale bar, 100 µm. AKI: acute kidney injury, BUN: blood urea nitrogen.

Figure 3. Effect of LC on the survival and the BUN level in the development of AKI after LPS injection. (A): Survival of the LPS + LC-treated mice and LPS-treated mice subjected to AKI by LPS injection. Mice were evaluated until 120 h after the LPS injection. (B): Renal function of the LPS + LC-treated mice and LPS-treated mice at 18 and 120 h after LPS injection, assessed by BUN levels. LC-treated mice that received saline instead of LPS were designated as the sham group, and BUN levels were also measured. (C): The grade of renal injury was evaluated by periodic acid–Schiff staining. Kidneys were stained with periodic acid–Schiff reagent to evaluate tubular necrosis in a semiquantitative manner by determining the percentage of cortical tubules in which epithelial necrosis, loss of the brush border, cast formation, and tubular dilation were observed; ×400. (D): The grade of renal injury in the LPS + LC- and LPS-treated groups at 18 and 120 h after LPS injection was evaluated by the acute tubular necrosis (ATN) score. The data are mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.005, LPS + LC- vs. LPS-treated group. Scale bar, 100 µm. AKI: acute kidney injury, BUN: blood urea nitrogen.

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Figure 4. Effect of LC on the accumulation of inflammation F4/80+cells in the interstitium after LPS injection. (A): Representative photograph of F4/80+ cells in the kidney from an LPS + LC-treated mouse and an LPS-treated mouse after LPS injection. The infiltrations of F4/80+ cells in the interstitium in the LPS + LC and LPS-treated groups after LPS injection. In the periglomerular area, F4/80+ cells infiltrated and are presented as brown dots. (B): The numbers of F4/80+ cell infiltrates in the renal interstitium are shown at 18 and 120 h. The data are mean ± SEM. **** p < 0.001, LPS + LC vs. LPS-treated group. c/hpf: cells per high-power field.

Figure 4. Effect of LC on the accumulation of inflammation F4/80+cells in the interstitium after LPS injection. (A): Representative photograph of F4/80+ cells in the kidney from an LPS + LC-treated mouse and an LPS-treated mouse after LPS injection. The infiltrations of F4/80+ cells in the interstitium in the LPS + LC and LPS-treated groups after LPS injection. In the periglomerular area, F4/80+ cells infiltrated and are presented as brown dots. (B): The numbers of F4/80+ cell infiltrates in the renal interstitium are shown at 18 and 120 h. The data are mean ± SEM. **** p < 0.001, LPS + LC vs. LPS-treated group. c/hpf: cells per high-power field.

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Figure 5. Effect of LC on T-cell and DC accumulation and KIM-1 expression in the glomerulus and interstitium after LPS injection. (A,B): At 18 and 120 h post-LPS injection, the numbers of CD4+ and CD8+ T cells and the numbers of CD68+ and CD11c+cells as macrophages and DCs (c/hpf) in the glomeruli and interstitia in the LPS + LC- and LPS-treated groups. (C): KIM-1 expression was detected in many tubules in the injured interstitium after LPS injection, localized to the apical side of the epithelium with some diffuse cytoplasmic staining (brown) (×400 magnification). (D): KIM-1+ cells were also counted and the numbers of positive cells in the LPS + LC- and LPS-treated groups were compared. Dotted lines: Mean values from the saline-injected group without LPS. The data are mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.005, LPS + LC vs. LPS-treated group. KIM-1: kidney injury molecule-1.

Figure 5. Effect of LC on T-cell and DC accumulation and KIM-1 expression in the glomerulus and interstitium after LPS injection. (A,B): At 18 and 120 h post-LPS injection, the numbers of CD4+ and CD8+ T cells and the numbers of CD68+ and CD11c+cells as macrophages and DCs (c/hpf) in the glomeruli and interstitia in the LPS + LC- and LPS-treated groups. (C): KIM-1 expression was detected in many tubules in the injured interstitium after LPS injection, localized to the apical side of the epithelium with some diffuse cytoplasmic staining (brown) (×400 magnification). (D): KIM-1+ cells were also counted and the numbers of positive cells in the LPS + LC- and LPS-treated groups were compared. Dotted lines: Mean values from the saline-injected group without LPS. The data are mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.005, LPS + LC vs. LPS-treated group. KIM-1: kidney injury molecule-1.

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Figure 6. Effect of LC on serum proinflammatory cytokines after LPS injection. Serum IL-18, IFN-γ, and TNF were measured as biomarkers in AKI. Dotted lines: Mean values from saline-injected group without LPS. The data are mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001, LPS- vs. LPS + LC-treated group.

Figure 6. Effect of LC on serum proinflammatory cytokines after LPS injection. Serum IL-18, IFN-γ, and TNF were measured as biomarkers in AKI. Dotted lines: Mean values from saline-injected group without LPS. The data are mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001, LPS- vs. LPS + LC-treated group.

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Table 1. Primer sequences (FastStart DNA Master Sybr Green) for analysis of mRNA expression.

Table 1. Primer sequences (FastStart DNA Master Sybr Green) for analysis of mRNA expression.

Gene NameForward Primer (5′-3′)Reverse Primer (5′-3′)18SrRNAGTAACCCGTTGAACCCCATTCGCCTCACTAAACCATCCAATCGIFN-γTGCTGATGGGAGGAGATGTCTTTTCTTTCAGGGACAGCCTGTTTNF-αCGATCACCCCGAAGTTCAGTAGGTGCCTATGTCTCAGCCTCTTIL-10GGTTGCCAAGCCTTATCGGAACCTGCTCCACTGCCTTGCTCCL2/MCP-1AAAAACCTGGATCGGAACCAACGGGTCAACTTCACATTCAAAGICAM-1CATCCCAGAGAAGCCTTCCTGTCAGCCACTGAGTCTCCAAGCT-betCCTGGACCCAACTGTCAACTAACTGTGTTCCCGAGGTGTCGATA3AGGGACATCCTGCGCGAACTGTCATCTTCCGGTTTCGGGTCTGG

Table 2. Primer sequences (TaqMan Gene Expression Assay) for analysis of mRNA expression.

Table 2. Primer sequences (TaqMan Gene Expression Assay) for analysis of mRNA expression.

Gene NameTaqMan Gene Expression Assay ID18SrRNA4310893EIL-6Mm00446190_m1IL-12p40Mm00434174_m1IL-18Mm00434226_m1KIM-1Mm00506686_m1

Table 3. Renal mRNA expression of cytokines, chemokines, KIM-1, Th cell subset transcription factors, and leukocyte adhesion molecules by real-time PCR.

Table 3. Renal mRNA expression of cytokines, chemokines, KIM-1, Th cell subset transcription factors, and leukocyte adhesion molecules by real-time PCR.

18 h120 h LPS + LC vs. LPS Treated GroupCytokines IFN-γ14.7 ± 1.6 vs. 23.6 ± 3.0 * 4.0 ± 0.6 vs. 3.0 ± 0.5TNF24.9 ± 2.6 vs. 31.5 ± 1.2 *12.2 ± 3.3 vs. 8.0 ± 1.7 IL-61314.0 ± 190.7 vs. 3900.0 ± 580.8 ****42.6 ± 9.8 vs. 17.4 ± 3.3 *IL-1056.2 ± 15.3 vs. 36.3 ± 11.15.8 ± 1.9 vs. 3.5 ± 0.5IL-12p400.6 ± 0.1 vs. 0.6 ± 0.10.9 ± 0.2 vs. 1.0 ± 0.1IL-185.9 ± 0.8 vs. 10.8 ± 0.9 **1.8 ± 0.4 vs. 1.3 ± 0.1Chemokines CCL2/MCP-175.0 ± 10.8 vs. 153.4 ± 18.5 **13.4 ± 2.2 vs. 6.5 ± 1.5 *KIM-189.8 ± 16.0 vs. 258.7 ± 45.1 **26.6 ± 16.8 vs. 2.7 ± 1.5 **Th cell subset transcription factors T-bet1.7 ± 0.3 vs. 3.5 ± 0.6 *3.7 ± 1.0 vs. 5.2 ± 0.6GATA30.9 ± 0.1 vs. 0.9 ± 0.10.9 ± 0.1 vs. 1.1 ± 0.1Leukocyte adhesion molecule ICAM-161.3 ± 6.7 vs. 101.5 ± 16.7 *1.9 ± 0.3 vs. 1.9 ± 0.3

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