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, 9 (1), 18445

Impact of Aging on Transition of Acute Kidney Injury to Chronic Kidney Disease

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Impact of Aging on Transition of Acute Kidney Injury to Chronic Kidney Disease

Myung-Gyu Kim et al. Sci Rep.

Abstract

Acute kidney injury (AKI) increases the risk of end stage renal disease among the elderly, but the precise underlying mechanism is unknown. We investigated the effects of aging on AKI-to-chronic kidney disease (CKD) transition, focusing on renal inflammation. Aged and young C57BL/6 mice were subjected to bilateral ischemia-reperfusion injury (IRI). Baseline proinflammatory cytokine levels of kidneys were elevated in aged mice. After IRI, aged mice also showed persistent M1 dominant inflammation, with increased proinflammatory cytokines during the recovery phase. Persistent M1 inflammation was associated with blunted activation of CSF-1/IRF4 signal for M1/M2 polarization, but in vitro macrophage polarization with cytokine stimulation was not different between young and aged mononuclear cells. The tubular expressions of cell cycle arrest markers increased in aged mice during recovery phase, and in vitro transwell experiments showed that mononuclear cells or M1 macrophages co-cultured with arrested proximal tubular cells at G1 phase significantly impaired M2 polarization, suggesting that prolonged G1 arrest might be involved in persistent M1 inflammation in aged mice. Finally, M1 dominant inflammation in aged mice resulted in fibrosis progression. Our data show that impaired M2 polarization partially driven by senescent tubule cells with cell-cycle arrest may lead to an accelerated progression to CKD in the elderly.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Renal functional deterioration and histologic damage following ischemia-reperfusion injury (IRI) of young and aged mice. There is no difference in (A) serum creatinine, (B) NGAL, and (C) tubular injury scores on day 1 and 3 after IRI between young and aged mice. Magnification: ×100, n = 4–6 per group.
Figure 2
Figure 2
Renal inflammation during recovery phase of young and aged mice. (A) In immunohistochemical staining, infiltration of F4/80+ macrophages increased during recovery phase in both young and aged mice and remained for a significantly longer duration in aged mice than in young mice, (B) Prior to IRI, the renal expression of TNF-α, IFN-γ, and IL-12 was higher in aged mice than in young mice, suggesting the presence of low-grade inflammation. After IRI, they were increased more in aged mice than in young mice and the difference was greater on day 28, (C) The number of neutrophils was also significantly higher in aged kidneys. Magnification: ×100, *p < 0.05 compared to young mice, n = 4–6 per group.
Figure 3
Figure 3
Impaired M1-M2 polarization during recovery phase in aged mice. (A) Renal macrophages of aged mice were skewed from the “F4/80 + CD206 + M2” to “F4/80 + CD206- M1” compared to those in young mice during recovery phase, (B) The increase in mRNA expressions of colony stimulating factor-1 (CSF-1), interferon regulatory factor-4 (IRF4), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) were blunted in aged mice, but mRNA expressions of STAT6 and IL-1 receptor-associated kinase-M (IRAK-M) were not. *p < 0.05 compared to young mice, n = 4–6 per group.
Figure 4
Figure 4
Cytokine-induced M2 polarization of young and aged bone marrow derived mononuclear cells. The differentiation of aged bone marrow (BM)-derived mononuclear cells into (A) F4/80+ CD206+ M2a or (B) F4/80+ B7H4+ M2c macrophages after treatment with IL-4 or IL-10/TGF-b, was not impaired compared to that of young BM derived cell.
Figure 5
Figure 5
Tubular cell arrest at G1 or G2 phase during recovery phase of young and aged mice. In immunohistochemistry, tubular cells showed significantly elevated (A) tissue inhibitor of metalloproteinase-2 (TIMP-2) and (B) phospho-Histone H3 (pH3) levels during the recovery phase, along with (C) increased p53 and p21 levels in aged mice. Cropped gels are used in the figure, and the full-size gels are presented in Supplementary Fig. S2. Magnification: ×100, *p < 0.05 compared to young mice, n = 4–6 per group.
Figure 6
Figure 6
Arrested tubular cells at G1 phase and impaired M2 conversion of aged monocytes and M1 macrophages. Bone marrow derived monocytes or M1 macrophages co-cultured with mouse tubular cells for 72 h using a transwell assay, were converted to M2 macrophages, but (A) when monocytes were co-cultured with tubular cells pretreated with PD0332991 (PD), a selective cyclin dependent kinase 4/6 inhibitor, M2 conversion was much decreased dependent on the dose of PD0332991, and when monocytes were co-cultured with tubular cells pretreated with RO3306 (RO), a selective cyclin‐dependent kinase 1 inhibitor, M2 conversion was decreased but not in a dose-dependent manner. M2 conversion from M1 macrophage was also significantly decreased when co-cultured with tubular cells pretreated with PD0332991 (n = 3 per group). (B) In immunohistochemistry, tubular cells in aged mice showed low CSF-1 expression, (n = 3–5 per group). (C) Mouse tubular cells treated with PD0332991 (PD) showed low mRNA expression of CSF-1, and neutralization of CSF-1 during co-culture of M1 macrophages and mouse tubular cells impaired M2 polarization (n = 3 per group). Magnification: ×100, *p < 0.05 compared to young mice, #p < 0.05 compared to vehicle, Ɨp < 0.05 compared to PD 5 µM.
Figure 7
Figure 7
Persistent M1 predominant inflammation and progression to chronic kidney disease. (A) In addition to increased M1 related inflammation of aged mice, higher α-smooth muscle actin, severe renal fibrosis and lower glomerular filtration rate (GFR) were observed on day 28 post-IRI in aged mice compared to young mice, Cropped gels are used in the figure, and the full-size gels are presented in Supplementary Fig. S3, GFR loss was calculated as follows: 100 x (GFR difference between pre-IRI and day28 post-IRI) / (pre-IRI GFR), (B) Higher mRNA expression of TGF-β and α-smooth muscle actin was observed in tubular cells upon co-culture with M1 macrophages. Magnification: × 40, 100, #p < 0.05 compared to young, *p < 0.05 compared to tubular cells without M1, n = 3–6 per group.

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