The Role of M2 Macrophages in the Progression of Chronic Kidney Disease following Acute Kidney Injury

<div><p>Introduction</p><p>Acute kidney injury (AKI) is a major risk factor in the development of chronic kidney disease (CKD). However, the mechanisms linking AKI to CKD remain unclear. We examined the alteration of macrophage phenotypes during an extended recovery period following ischemia/reperfusion injury (IRI) and determine their roles in the development of fibrosis.</p><p>Methods</p><p>The left renal pedicle of mice was clamped for 40 min. To deplete monocyte/macrophage, liposome clodronate was injected or CD11b-DTR and CD11c-DTR transgenic mice were used.</p><p>Results</p><p>Throughout the phase of IRI recovery, M2-phenotype macrophages made up the predominant macrophage subset. On day 28, renal fibrosis was clearly shown with increased type IV collagen and TGF-β. The depletion of macrophages induced by the liposome clodronate injection improved renal fibrosis with a reduction of kidney IL-6, type IV collagen, and TGF-β levels. Additionally, the adoptive transfer of the M2c macrophages partially reversed the beneficial effect of macrophage depletion, whereas the adoptive transfer of the M1 macrophages did not. M2 macrophages isolated from the kidneys during the recovery phase expressed 2.5 fold higher levels of TGF-β than the M1 macrophages. The injection of the diphtheria toxin into CD11b or CD11c-DTR transgenic mice resulted in lesser depletion or no change in M2 macrophages and had little impact on renal fibrosis.</p><p>Conclusion</p><p>Although M2 macrophages are known to be indispensible for short-term recovery, they are thought to be main culprit in the development of renal fibrosis following IRI.</p></div

Macrophage depletion by liposome clodronate (LC) administration.

<p>(A) In a flow cytometric analysis, both M1 and M2 macrophages were depleted by LC in the spleen and kidneys on day 7 (B) The mRNA expression of the M1/M2 markers showed that the relative reduction of the M2 marker, Arginase-1, was significantly greater than that of the M1 marker, iNOS, in kidneys. (n = 4–6 per group), *p < 0.05 compared to IRI+PBS, ^†p < 0.05 compared to the previous time point.</p

The impact of macrophage depletion on kidneys during the recovery phase.

<p>(A) Treatment with LC during the recovery phase improved renal fibrosis (magnification, ×100) and it was associated with a significant reduction in (B) the expression of type IV collagen in kidneys and (C) the level of the pro-inflammatory cytokine IL-6, (n = 3–5 per group), (D) As seen by immunohistochemical staining, the TGF-β expression decreased significantly in the kidneys of clodronate-treated mice on day 28. The TGF-β mRNA expression in kidneys also significantly decreased following liposome clodronate injection during the recovery phase. Magnification: ×100, (n = 4–6 per group), *p<0.05 compared to sham, ^#p < 0.05 compared to IRI+PBS, ^†p < 0.05 compared to the previous time point.</p

Renal infiltration of macrophages during the recovery phase.

<p>(A) In the immunohistochemical staining of F4/80, the amount of F4/80-positive macrophage infiltration increased significantly throughout the recovery phase. Magnification: ×100. (n = 4–5 per group), *p < 0.05 compared to day 1, ^†p < 0.05 compared to the previous time point, (B) In a flow cytometric analysis to differentiate M1 and M2 macrophages using a CD206 Ab, the macrophage phenotype shifted from being predominantly M1 on day 3 to being predominantly M2 on day 7, 14 and 28 following IRI. (n = 4 per group), *p < 0.05 compared to M1 macrophages, ^†p < 0.05 compared to the previous time point. As seen by immunohistochemistry, more F4/80-positive cells were shown to be CD206-positive on day 7 (c and d) than on day3 (a and b). Magnification: ×100. (C) An analysis of the mRNA expression level of Arginase-1, an M2 marker, showed a persistently increased expression of more than 100-fold throughout the recovery phase, compared to that of iNOS, an M1 marker. (n = 3–5 per group), *p < 0.05 compared to the mRNA expression of iNOS, ^†p < 0.05 compared to the previous time point.</p

Diphtheria toxin injection in CD11c DTR transgenic mice.

<p>(A) An RT-PCR analysis showed no significant difference in the level of mRNA expression of arginase and iNOS in the kidneys. (n = 4–5 per group), (B) Diphtheria toxin injection resulted in no significant change in renal fibrosis on day 28, as seen by Masson’s trichrome staining and (C) in TGF-β mRNA expression throughout the recovery phase. Magnification: ×100. (n = 4–5 per group), *p < 0.05 compared to WT+DT, ^†p < 0.05 compared to the previous time point.</p

Renal histology following a unilateral ischemia reperfusion injury.

<p>(A) As seen by periodic acid-Schiff (PAS) staining, the tubular injury was partially restored from day 3 to day 28 (D3 to D28). (B) As seen by Masson’s trichrome (MT) staining, we observed the development and progression of interstitial fibrosis in the kidneys during the 28 days. (n = 5–6 per group), (C) In immunohistochemical staining, we detected the upregulation of TGF-β. Magnification: ×100, (n = 4–6 per group), *p < 0.05 compared to day 0, ^†p < 0.05 compared to the previous time point.</p

Adoptive transfer of M1 or M2c macrophages following liposome clodronate (LC) treatment.

<p>(A) As seen by Masson’s trichrome staining, transferring M2c cells following the LC treatment led to the complete reversion of the improvement of interstitial fibrosis, a. IRI+PBS (IRI), b. liposome clodronate (IRI+LC), c. M2 (IRI+LC+M2c) Magnification: ×100, (n = 4–5 per group), (B) As seen by Masson’s trichrome staining, transferring M1 macrophages into LC-treated mice did not affect the degree of fibrosis, a. IRI+PBS (IRI), b. liposome clodronate (IRI+LC), c. M1 (IRI+LC+M1) Magnification: ×100, (n = 4–5 per group), *p < 0.05 compared to sham, **p < 0.05 compared to IRI, ^#p < 0.05 compared to LC.</p

Diphtheria toxin injection into CD11b DTR transgenic mice.

<p>(A) An RT-PCR analysis showed the relative reduction in the mRNA expression level of arginase-1. However, on day 7, it was less significant than the reduction observed in kidney of liposome clodronate-treated mice. (n = 4–5 per group), (B) Diphtheria toxin injection resulted in no significant change in renal fibrosis on day 28, as seen by Masson’s trichrome staining, and (C) in TGF-β mRNA expression throughout the recovery phase. Magnification: ×100. (n = 4–5 per group), *p < 0.05 compared to WT+DT, ^†p < 0.05 compared to the previous time point.</p

TGF-β mRNA expression of M1 and M2 macrophages.

<p>We used a FACs aria analysis to sort M1 and M2 macrophages and compared their TGF-β mRNA expression. The expression of TGF-β was about 2.5 fold higher in M2 macrophages than in M1 macrophages. (n = 4 per group), *p < 0.05 compared to F4/80⁺CD206^- M1 macrophages.</p

CD11c⁺ Cells Partially Mediate the Renoprotective Effect Induced by Bone Marrow-Derived Mesenchymal Stem Cells

<div><p>Previous studies have shown that induction of immune tolerance by mesenchymal stem cells (MSCs) is partially mediated via monocytes or dendritic cells (DCs). The purpose of this study was to determine the role of CD11c⁺ cells in MSC-induced effects on ischemia/reperfusion injury (IRI). IRI was induced in wildtype (WT) mice and CD11c⁺-depleted mice following pretreatment with or without MSCs. In the in-vitro experiments, the MSC-treated CD11c⁺ cells acquired regulatory phenotype with increased intracellular IL-10 production. Although splenocytes cocultured with MSCs showed reduced T cell proliferation and expansion of CD4⁺FoxP3⁺ regulatory T cells (Tregs), depletion of CD11c⁺ cells was associated with partial loss of MSCs effect on T cells. In in-vivo experiment, MSCs’ renoprotective effect was also associated with induction of more immature CD11c⁺ cells and increased FoxP3 expression in I/R kidneys. However all these effects induced by the MSCs were partially abrogated when CD11c⁺ cells were depleted in the CD11c⁺-DTR transgenic mice. In addition, the observation that adoptive transfer of WT CD11c⁺ cells partially restored the beneficial effect of the MSCs, while transferring IL-10 deficient CD11c⁺ cells did not, strongly suggest the important contribution of IL-10 producing CD11c⁺ cells in attenuating kidney injury by MSCs. Our results suggest that the CD11c⁺ cell-Tregs play critical role in mediating renoprotective effect of MSCs.</p> </div