Treatment Of Diabetic Nephropathy: Carbon Monoxide

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Part Ⅱ： Carbon monoxide alleviates senescence in diabetic nephropathy by improving autophagy

Guibin Meil; Chunjie Jiang; Xueer Cheng; & at al.

ABSTRACT

Objectives: Senescence, characterized by permanent cycle arrest, plays an important role in diabetic nephropathy (DN). However, the mechanism of renal senescence is still unclear, and the treatment targeting it remains to be further explored.

Materials and Methods: The DN (diabetic nephropathy) mice were induced by HFD and STZ, and 3 types of renal cells were treated with high glucose (HG) to establish in vitro model. Senescence-related and autophagy-related markers were detected by qRT-PCR and Western blot. Further, autophagy inhibitors and co-immunoprecipitation were used to clarify the mechanism of Carbon monoxide (CO). Additionally, the specific relationship between autophagy and senescence was explored by immunofluorescence triple co-localization and ELISA.

Results: We unraveled that senescence occurred in vivo and in vitro, which could be reversed by CO (Carbon monoxide). Mechanistically, we demonstrated that CO (Carbon monoxide) inhibited the dysfunction of autophagy in DN (diabetic nephropathy) mice partly through dissociating the Beclin-1-Bcl-2 complex. Further results showed that autophagy inhibitors blocked the improvement of CO (Carbon monoxide) on senescence. In addition, the data revealed that autophagy regulated the degradation of senescence-related secretory phenotype(SASP)including-1β, Il-6, Tgf-βand Vegf. Conclusions: These results suggested that CO (Carbon monoxide) protects DN (diabetic nephropathy) mice from renal senescence and function loss via improving autophagy partly mediated by dissociating Beclin-1-Bcl-2 complex, which is possibly ascribed to the degradation of SASP. These findings bring new ideas for the prevention and treatment of DN (diabetic nephropathy) and the regulation of senescence.

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4 | DISCUSSION

Accumulating senescent cells are increasingly considered to be malignant zombie cells resistant to death, with the capacity of inducing tissue dysfunction and fueling age-related diseases. Researchers reported that transplantation of senescent precursor fat cells into 6-month-old mice caused a dose-dependent decline in body function.2 Conversely, removal of senescent cells improved a range of age-related diseases such as T2D,30osteoarthritis31, and Alzheimer's disease.32Our results displayed the occurrence of senescence in DN (diabetic nephropathy) mice and 3types of renal cells exposed to HG. These results and previous research consistently suggested the presence of senescence in DN (diabetic nephropathy). Interestingly, among the 9 kinds of tissue of 27-month-old SD rats, renal senescence was the earliest and most serious, indicating the kidney was susceptible to senescence.33 Based on these findings, we speculated that the therapies targeting senescent cells may protect against DN (diabetic nephropathy). Recent limited evidence showed that CO (Carbon monoxide) alleviated drug-induced senescence of endothelial cells. Similarly, we provided results that CO (Carbon monoxide) significantly down-regulated a great deal of senescent phenotype and accordingly recovered renal function in DN (diabetic nephropathy) mice. While CO (Carbon monoxide) has been proven to exert renoprotection against ischemia-reperfusion and kidney transplantation mice via anti-inflammatory, anti-apoptosis, and other classic pathways,4 our study proposed a novel mechanism that CO (Carbon monoxide) confers improvement from renal senescence of DN (diabetic nephropathy) mice.

Except for the widely recognized p53/p21 and p16 pathways, senescence has been described to be regulated by autophagy positively. These results and previous research consistently suggested the presence of senescence in DN (diabetic nephropathy) Consistent with this, the reduction of autophagy was found in kidneys with aging.5 Moreover, there was a negative correlation between Atg5 and renal aging of DN (diabetic nephropathy) mice, suggesting the potentiality of anti-senescence of autophagy in DN (diabetic nephropathy). Our results showed that the dysfunction of autophagy and autophagy flow in vivo and vitro were positively associated with accumulated senescent cells. Interestingly, CO (Carbon monoxide) has been considered to be an effective autophagy activator. In our study, CO (Carbon monoxide) activated autophagy but decreased autophagosomes. The further data displayed that CO (Carbon monoxide) ameliorated lysosomal function and autophagy flow by the transfection of RFP-GFP-LC3. Furthermore, autophagy inhibitors and siAtg7 restrained the improvement of senescence by CO (Carbon monoxide) in vitro, supporting that CO (Carbon monoxide) slowed senescence via autophagy.

Not only that, the dissociation of Beclin-1-Bcl-2 complex by CO (Carbon monoxide) was observed in DN (diabetic nephropathy) mice. To our knowledge, Beclin-1 functions as an autophagy initiator when dissociated from Bcl-2. The disruption of the complex may be a potential target for autophagy activated by CO (Carbon monoxide).In addition, knocking out the Beclin-1 and Bcl-2 binding sites not only activated autophagy but improved aging and the lifespan of mice,21 suggesting the complex coordinately regulated autophagy and senescence. Our experiments showed that this complex dissociation agent ABT737 further enhanced the activation of autophagy and improvement of senescence by CO (Carbon monoxide) in vitro. Consistent with our results, disrupting the interaction of the Beclin-1-Bcl-2 complex positively influences multiple diseases, including protecting against cardiac injuries of diabetic cardiomyopathy mice3° and exerting renoprotection on mice challenged by ischemia-reperfusion.37 These observations indicated that CO (Carbon monoxide) may activate autophagy by dissociating the Beclin-1-Bcl-2 complex to relieve senescence and renal injuries.

FIGURE 5 Activated autophagy may degrade SASP in DN (diabetic nephropathy) mice.

A, Immunohistochemical images and quantification of p65 expression and nuclear translocation (n = 3). Scale bar: 50 μm. B, Western blot analysis of GATA4 (n = 3).

C, Representative immunofluorescence images of mTOR, LC3 puncta and LAMP2 co-localization in the kidney. Scale bar: 25 μm.

D-G, Representative immunofluorescence images showed the triple co-localization of IL-1β, IL-6, TGF-β, and VEGF with LC3 and LAMP2 (the red arrows) and the white arrows indicated the location with LC3. Scale bar: 25 μm.

H, The total VEGF protein level (cell lysate and supernatant) in HPC treated with different formulations (HG, ABT737: 1 mmol/L, rapamycin: 10 nmol/L, and CORM-2).

I, VEGF protein expression in the supernatant of HPC treated differently. J, VEGF protein release ratio of HPC (supernatant/ total lysate) (n = 6). *P < .05, **P < .01, ***P < .001

What's more, the specific relationship between autophagy and senescence requires further elucidation. Autophagy is a cleansing process to deliver enclosed constituents to the lysosome for degradation. Researchers evidenced that autophagy reduced the secretion of Il-1β, a major SASP component, through the degradative pathway. Surprisingly, the co-localization of IL-1β, LC3 puncta, and LAMP2 was observed in kidneys, suggesting the degradation of IL-1β by CO (Carbon monoxide) treatment in DN (diabetic nephropathy) mice. Besides, intracellular I-6, TGF-β, and VEGF, respectively,co-localized with LC3 puncta and LAMP2, indicating the intersection between autophagy and the degradation of SASP. Consistently, the drug-activated autophagy was proven to reduce the secretion of IL-6 in chondrocytes,3" while the suppression of autophagy by deleting Atg7 caused increased export of IL-16.IL-6 and IL-8 in adipocytes and breast cancer cells.0 However, IL-1α was reported not to be a direct substrate for autophagic elimination in macrophages,1suggesting that SASP was partly degraded by autophagy.

Noteworthily, we observed that SASP co-located with autophagosomes in the kidney of DN (diabetic nephropathy) mice (Figure 5D-G, the white arrow), indicating SASP may be engulfed by autophagosomes but escape lysosomes. This is potentially attributed to secretory autophagy in that autophagosomes are not delivered to lysosomes but instead fuse directly with the plasma membrane, leading to the release of cytosolic components(SASP) to the extracellular space. However, the co-localization of SASP and autolysosomes increased greatly in normal and CO (Carbon monoxide)-intervened mice(Figure 5D-G, the red arrow). The possible explanation for this phenomenon is that both autophagy-mediated secretion and degradation participate in SASP transportation.3 Although the regulation of the divergence between excretion versus degradation of the intracellular contents remains to be fully understood, we found both CO (Carbon monoxide) and autophagy agonists blocked the export of VEGF in HPC treated with HG, suggesting activated autophagy contributed to SASP degradation. Additionally, the impaired lysosomal function was reported to cause the release of autolysosome materials in the microglial cell.4 Thus, CO (Carbon monoxide)-induced activation of autophagy and restoration of lysosomal function may lead to more degradation of SASP than secretion.

Much evidence demonstrated that secreted SASP played a negative role in some diseases. For example, the SASP was reported to facilitate retinal vascular damage by transmitting senescence in retinopathy mice,4 induce senescence and injure of non-senescent biliary cells.46 However, it is unknown whether SASPis responsible for DN (diabetic nephropathy) progression. Omics results showed that inflammatory cytokines were up-regulated in early and especially in late DN (diabetic nephropathy).7Consistently, we found lots of SASP was developed in DN (diabetic nephropathy) mice, among which IL-1βwas increased most significantly compared to normal mice and VEGF was co-localized with autolysosomes most obviously in normal and CO (Carbon monoxide)-intervened mice. And the treatment of anti-IL-β lgG attenuated kidney injuries including fibrosis in db/db mice.48 The deletion of VEGF-B protected against DN (diabetic nephropathy) by reducing renal lipid accumulation, while the overexpression of VEGF-A in adult mice exacerbated renal dysfunction and structure change.50The above data on the regulation of SASP further suggested the key role of SASPin the DN (diabetic nephropathy). Our study also showed that the administration of CO (Carbon monoxide) inhibited the expression and secrete of SASP, subsequently improving renal fibrosis and structural changes. Hence, we inferred that the block-age of SASP-mediated senescence through the degradation pathway of autophagy may be one of the mechanisms by which CO (Carbon monoxide) protects HFD+ STZ-induced DN (diabetic nephropathy).

Taken together, the present findings indicated that CO (Carbon monoxide) alleviated renal senescence of DN (diabetic nephropathy) through the improvement of autophagy-mediated by dissociating Beclin-1-Bcl-2 complex, which was partly attributed to the degradation of SASP. Our study sheds new light on the prevention and treatment of DN (diabetic nephropathy) and the regulation of renal senescence. Expectantly, the specific effects of senescence on DN (diabetic nephropathy) are worthy of further investigation. On the other hand, anti-senescence drugs may become a breakthrough in the treatment of DN (diabetic nephropathy) and other age-related diseases.

FIGURE 6 CO activated autophagy partly by dissociating the Beclin-1-Bcl-2 complex.

A, Co (Carbon monoxide)-immunoprecipitation of Beclin-1 and Bcl-2 and quantification of the ratio of Beclin-1 to Bcl-2 in the kidney (n = 3).

B, Western blot analysis of a series of autophagy proteins (LAPM2, p62 and LC3) in HBZY-1 treated differently (HG, CORM-2 and Beclin-1-Bcl2 complex dissociation agent ABT737) (n = 5).

C, Quantification of the expression of p53, p21 and p16 (n = 5 or 6).

D, The SA-β-gal staining images of 3 kinds of renal cells treated differently and statistical results (n = 3). Scale bar: 50 μm. *P < .05, **P < .01, ***P < .001

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