New Therapeutic Strategy For Diabetic Nephropathy: CCR2 Antagonist
Mar 25, 2022
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PART Ⅱ：Effects of a CCR2 antagonist on macrophages and Toll-like receptor 9 expressions in a mouse model of diabetic nephropathy
Seigo Ito, Hiroyuki Nakashima,Takuya Ishikiriyama, Masahiro Nakashima, Akira Yamagata, & et al.
Abstract
The pathogenesis of diabetic nephropathy(DN) is related to macrophage (Mo) recruitment to the kidneys, tumor necrosis factor-αu (TNF-o) production, and oxidative stress. Toll-like receptor 9 (TLR9) activation is reportedly involved in systemic inflammation, and it exacerbates this condition in metabolic syndrome. Therefore, we hypothesized that TLR9 plays a role in the pathogenesis of DN(diabetic nephropathy). Two subsets of kidney Mos in DN diabetic nephropathy model (db/db) mice were analyzed using flow cytometry to evaluate their distribution and TLR9 expression and function. Mice were administered the CCR2 antagonist INCB3344 for 8 wk; changes in Mo distribution and function and its therapeutic effects on DN diabetic nephropathy pathology were examined. Bone marrow-derived CD11bhigh(BM-Mo) and tissue-resident CD11blow Mos(Res-Mo) were identified in the mouse kidneys. As DN diabetic nephropathy progressed， the BM-Mφ number， TLR9 expression， and TNF-α production increased significantly. In Res-Mos, reactive oxygen species (ROS) production and phagocytic activity were enhanced. INCB3344 decreased albuminuria， serum creatinine level， BM-Mφ abundance， TLR9 expression， and TNF-a production by BM-Mφs and ROS production by Res-Mos. Both increased activation of BM-Mo via TLR9 and TNF-α production and increased ROS production by Res-Mos were involved in DN(diabetic nephropathy) progression. Thus, inactivating Mos and their TLR9 expression by INCB3344 is a potential therapeutic strategy for DN(diabetic nephropathy).
NEW & NOTEWORTHY We classified kidney macrophages (Mos) into bone marrow-derived Mos(BM-Mos) expressing high CD1lb and tissue-specific resident Mo(Res-Mos) expressing low CD11b. In diabetic nephropathy(DN) model mice. Toll-like receptor 9(TLR9) expression and TNF-α production via TLR9 activation in BM-Mos and ROS production in Res-Mos were enhanced Furthermore, CCR2 antagonist suppressed the kidney infiltration of BM-Mos and their function and the ROS production by Res-Mos, with concomitant TLR9 suppression. Our study presents a new therapeutic strategy for DN(diabetic nephropathy).
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DISCUSSION
We presented three new findings in this study. First,in diabetic DB/DB mice, BM-Mos expressed TLR9 and released TNF-。 more abundantly than those in nondiabetic misty mice, whereas Res-Mos produced more ROS and showed phagocytic function. Second, INCB3344, a CCR2 antagonist, reduced TLR9 expression and TNF-α release from both types of MPS and decreased ROS production by Res-Mos. Last, these mechanisms could account for decreases in albuminuria and serum Cr levels in diabetic DB/DB mice as well as improvement in mesangial proliferation and podocyte expression.
Based on our findings, kidney Mos were divided into two populations, ie., BM-Mos highly expressing a bone marrow-derived marker and Res-Mos hardly expressing it. As they did not depend on the presence of diabetes, these were considered as unique properties of the two types of kidney Mos. In contrast, their various functions were enhanced in diabetic mice. Moreover, the production of TNF-α, a pro-inflammatory cytokine mainly produced by Mos, and IL-10, an anti-inflammatory cytokine, was also increased. This indicates that kidney Mos are simultaneously involved in not only the induction but also the convergence of inflammation in chronic inflammatory conditions such as DN(Diabetic nephropathy). Roles of Two Types of Kidney Macrophages
Since urinary albumin excretion, which reflects nephropathy severity, increased with the percentage of BM-Mos in DB/DB mice, we suggest that BM-Mos play major roles in DN(Diabetic nephropathy) development. Moreover, we found that TLR9 expression and TNF-α production were enhanced in BM-Mos, whereas ROS production and phagocytic activity were increased in Res-Mos with DN(diabetic nephropathy) progression. Since these observations were not made in the younger and older misty mice(data not shown), we think that they are associated with DN(diabetic nephropathy) progression and are not due to age-dependent changes. TLRs recognize pathogen-associated molecular patterns, such as lipopolysaccharides, bacterial DNA, and endogenous danger-associated molecular patterns, whose levels are increased in diabetes (33,34).In particular, TLR2 and TLR4 are reportedly involved in perpetuating inflammation in DN(diabetic nephropathy), presumably via Mos (35-38). TLR9 recognizes mitochondrial and cell-free DNA released into circulation by damaged/dead cells or adipocytes and common bacterial DNA motif CpG-ODN, thus activating innate immunity and suggesting its involvement in kidney injury (11,14).
ROS production and phagocytosis by Res-Mos were increased along with DN(Diabetic nephropathy) progression. Kidney Mos have reportedly been involved in DN(diabetic nephropathy) progression by releasing ROs through nicotinamide adenine dinucleotide phosphate (NADPH)oxidase activity (39,40), and our previous study showed that ROS derived from Res-Mos are involved in hepatic tissue injury (20). Therefore, in the kidney, Res-Mos may also be involved in ROS-induced tissue injury. Furthermore, TLR9 expression in Res-Mos was enhanced in the diabetic condition(Fig. 1E). As ROS produced by Res-Mos are affected by TLR9 activation and local TNF-a levels(20,41, 42), the increase in ROS production by Res-Mos with DN(Diabetic nephropathy) progression was due to the increase in their TLR9 expression or affected by the enhanced TNF-α pro-duction of BM-Mos. Therefore, we speculate that the increase in TNF-a production by BM-Mos and ROS production by Res-Mgps exacerbated DN(diabetic nephropathy).
Although Res-Mos might play a deleterious role through enhanced ROS production, their strong phagocytosis ability could exert a protective effect on DN(Diabetic nephropathy) mice. Res-Mos may phagocytose damaged kidney cells during disease progression and participate in kidney tissue repair. Furthermore, the increase in ROS production by Res-Mos might occur owing to the digestion of the phagocytosed damaged tissue. As we evaluated ROS produced by kidney Mos in an unstimulated environment, ROS were produced at the stage where Mos phagocytosed and digested the damaged kidney tissue. Therefore, increased ROS production in Res-Mos may not be a negative effect in DN(diabetic nephropathy).
Effects of the CCR2 Antagonist INCB3344 on Diabetic Nephropathy
CCL2 is overexpressed in the kidneys of diabetic model mice(43-46) and is implicated in the exacerbation of albuminuria owing to its action on glomerular podocytes(47). Furthermore, monocyte infiltration at the inflammatory sites is mainly regulated by CCL2, and CCL2 attracts Ms to the kidney tissue (through CCR2-mediated signaling) and plays an important role in DN(Diabetic nephropathy) pathogenesis(46,47). CCL2is produced by glomerular podocytes, mesangial cells, and tubular epithelial cells and induces interstitial inflammation, tubular atrophy, and fibrosis in the kidney(44, 48,49). Administration of several CCR2 antagonists in diabetic model mice was recently shown to suppress Mo infiltration into the kidney and prevent DN(diabetic nephropathy) progression (50-53). However, CCR2 antagonists have not been clinically used in humans, probably owing to issues such as a short pharmacological half-life and low affinity for human CCR2. In contrast, INCB3344 can bind to human monocytes with high selectivity for CCR2 and has a long pharmacokinetic half-life in the human or mouse body (8,9).
In the present study, INCB3344 could suppress the migration of BM-Mos to the kidney in DN(Diabetic nephropathy); however, it did not have a pronounced effect on Res-Migos, suggesting that the BM- and Res-Mφ populations had different properties. Moreover， INCB3344 polarized Mgs into the M2 phenotype(M2 polarization), suggesting an effect of converting proinflammatory to anti-inflammatory phenotype.
Notably, INCB3344 significantly reduced TNF-α production in both types of Mos, which may have contributed the most to the improvement of DN(Diabetic nephropathy). TNF-α is involved in kidney hypertrophy, albumin excretion, and decreased kidney function in DN(Diabetic nephropathy) (3, 54) and is a pleiotropic cytokine that stimulates the production of other cytokines in an autocrine and paracrine manner(55-58). Therefore, inhibition of TNF-o may serve to block a variety of other inflammatory mediators that could account for its beneficial effects on DN(diabetic nephropathy). As CCR2 stimulation leads to TNF-a production via the activity of intracellular glycogen synthase kinase 3(GSK3)(59), we speculated that INCB3344 inhibited TNF-α production by blocking this pathway in the kidney Mos. However, we believe that the GSK3 levels in this model should be evaluated, and further studies need to be conducted to confirm this speculation. Furthermore, our results showed that most glomerular Mos were BM-Mos, and TNF-production in glomerular BM-Mos was higher than that in other BM-Mos in the kidney. These results suggest that INCB3344, which sup-pressed the BM-Mφ counts （more specifically）， mainly contributed to the amelioration of glomerular lesions by decreasing mesangial proliferation and protecting podocytes (increasing WT-1).
CCR2 inhibition has been reported to decrease TNF-α pro-duction through TLR9 suppression (60,61), and TLR9 expression was not observed in the kidney parenchyma, including tubules, interstitium, and glomeruli, except for cells morphologically presumed to be Mos in the present study. Therefore, INCB3344 administration also reduced TLR9 expression in both types of Mos. Taken together, the results suggested that INCB3344 suppressed not only the inflammatory infiltration of BM-Mg into the kidney but also their harmful inflammation-related functions and may contribute to the alleviation of DN(diabetic nephropathy).
In conclusion, BM-Mos expressed TLR9 and released TNF-a more abundantly in diabetic mice than in nondiabetic mice, whereas Res-Mos produced more ROS and showed increased phagocytic activity in diabetic mice. INCB3344, a CCR2 antagonist, reduced TLR9 expression and TNF-α release from both types of Mos and decreased ROS production by Res-Mos. These changes explain the decreases in albuminuria and serum Cr levels as well as the pathological improvement in diabetic mice. We suggest that INCB3344 has a robust potential to treat DN(diabetic nephropathy) in the clinic.
Perspectives and Significance
We demonstrated that the CCR2 antagonist INCB3344 can decrease albuminuria and serum Cr level and improve mesangial proliferation and podocyte expression in mice with DN(diabetic nephropathy). These beneficial effects were exerted through a reduction in TNF-α production from BM-Mos and ROS release from Res-Mφs， both of which were based on a decrease in TLR9 expression in both types of Mos and M2 polarization. In the future, this receptor antagonist can be used as a robust alternative or a partner to angiotensin II receptor blocker and sodium-glucose cotransporter-2 to treat patients to prevent the progression of DN(diabetic nephropathy).
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