Sidt2 Is A Key Protein in The Autophagy-lysosomal Degradation Pathway And Is Essential For The Maintenance Of Kidney Structure And Filtration Function
Nov 07, 2023
The regulation and homeostasis of autophagy are essential for maintaining organ morphology and function. As a lysosomal membrane protein, the effect of Sidt2 on kidney structure and renal autophagy is still unknown. In this study, we found that the kidneys of Sidt2−/− mice showed changes in basement membrane thickening, foot process fusion, and mitochondrial swelling, suggesting that the structure of the kidney was damaged. Increased urine protein at 24 h indicated that the kidney function was also damaged. At the same time, the absence of Sidt2 caused a decrease in the number of acidic lysosomes, a decrease in acid hydrolase activity and expression in the lysosome, and an increase of pH in the lysosome, suggesting that lysosomal function was impaired after Sidt2 deletion. The accumulation of autophagolysosomes, increased LC3-II and P62 protein levels, and decreased P62 mRNA levels indicated that the absence of the Sidt2 gene caused abnormal autophagy pathway flow. Chloroquine experiment, immunofluorescence autophagosome, lysosome fusion assay, and Ad-mcherry-GFP-LC3B further indicated that, after Sidt2 deletion, the production of autophagosomes did not increase, but the fusion of autophagosomes and lysosomes and the degradation of autophagolysosomes were impaired. When incubating Sidt2−/− cells with the autophagy activator rapamycin, we found that it could activate autophagy, which manifested as an increase in autophagosomes, but it could not improve autophagolysosome degradation. Meanwhile, it further illustrated that the Sidt2 gene plays an important role in the smooth progress of autophagolysosome processes. In summary, the absence of the Sidt2 gene caused impaired lysosome function and a decreased number of acidic lysosomes, leading to the formation and degradation disorders of the autophagolysosomes, which eventually manifested as abnormal kidney structure and function. Sidt2 is essential in maintaining the normal function of the lysosomes and the physiological stability of the kidneys.
INTRODUCTION
The traditional view is that lysosomes are the cells’ garbage disposals to remove wastes produced by cells [1–3]. However, many studies have shown that the role of lysosomes is far more complex, and may involve cell signal transduction, tumorigenesis, development, and other aspects that affect the life activities of the body [4–10]. Lysosomes are usually enriched in tissues such as the liver and kidney [11]. Therefore, lysosomal dysfunction is also closely related to tissue diseases of the liver and kidney, such as Gaucher’s disease [12], mucopolysaccharidosis [13], Niemann-Pick disease [14], delayed glomerulosclerosis [15], idiopathic membranous nephropathy [16], etc. Lysosomal membrane proteins (LMPs) are the membrane components whose function is not only to maintain the integrity of the lysosome but which are also involved in various aspects such as intracellular signal transduction and regulation that are essential for maintaining the function of the lysosome and cell life activities [17–19].
To date, more than 100 LMPs have been discovered, but the functions of most of them are still unknown [20]. Transmembrane 7 superfamily member 1 (TM7SF1) is essential for the maintenance of renal podocyte function [21] and plays an important role in the process of kidney development [22]. Chloride Voltage-Gated Channel 5 (ClC-5) is a chloride ion (Cl(−)) channel expressed in renal tubules, which is essential for normal renal tubular function [23]; when it is mutated, it may cause Dent’s disease [24]. Overexpression of Chloride Voltage-Gated Channel 7 (ClC-7) prevents the apoptosis of renal tubular epithelial cells caused by impaired redox state [25]. These studies demonstrate that, as components of lysosome-enriched kidney tissue, LMPs are essential to maintain normal function, but the specific mechanism of their related pathogenicity is still unclear.
SID1 transmembrane family, member 2 (Sidt2) is a newly discovered LMP that is highly expressed in liver and kidney tissues [26]. Previous studies have shown that Sidt2 deletion can cause liver-related diseases, which manifest as liver steatosis and liver lipid metabolism disorders [11, 18]. In a recent study, we found that the kidneys of Sidt2−/−mice also experienced damage, but the specific mechanism is unclear. Lysosomes are important executive organelles for autophagy [10]. Is autophagy regulation involved? In this study, we examined the correlation between lysosomal function (autophagy) and disease and explored the underlying mechanism involving Sidt2 that causes kidney damage, which will be of great help for the study of the correlation between LMP and disease.
RESULTS
Sidt2−/− model shows that kidney damage is associated with autophagolysosome accumulation
The method of constructing Sidt2−/− mice is shown in Fig. 1A. The obtained homozygous mouse tail DNA was sequenced and the sequencing result was unimodal (Fig. 1B), and a 199 bp fragment loss occurred in the second exon. Through PCR genotype identification, we found that Sidt2+/+(Wild Type, WT) mice had 685 bp DNA segments, whereas Sidt2−/− mice had 486 bp (Fig. 1C). As shown in Fig. 1D, the Sidt2 protein could hardly be detected in Sidt2−/− mice than that in WT, which suggested that the model was successfully constructed. By urine test, it was found that the 24 hurine protein was significantly increased in Sidt2−/− mice compared with WT (Fig. 1E), suggesting that the kidney filtration barrier was impaired after the Sidt2 was deleted. Transmission electron microscopy observations (Fig. 1F) showed that, compared with WT mice (a–f), Sidt2−/− mouse kidneys exhibited diffuse fusion of foot processes, thickening of the glomerular basement membrane (g, h), renal tubular epithelial cell edema, microvilli damage (i, j), mitochondrial edema, vacuole-like changes, and the disappearance of spines (k, l). Interestingly, Sidt2−/− mice have also exhibited a large number of autophagolysosome accumulations (shown by red arrows, m, n), and the number was significantly higher than that of the control (Fig. 1G). At the cellular level, we found that Sidt2 was also essential for the survival of kidney cells. The deletion of the Sidt2 gene causes decreased proliferation and increased apoptosis of MPC5 and SV40 MES 13 cells (Supplementary Fig. 1).
Sidt2 gene deletion leads to changes in the number and function of acidic lysosomes in mouse kidney cells
Crispr-Cas9 technology was used to knock out the Sidt2 gene in MPC5 and SV40 MES 13 cells to obtain the cell models. The mRNA and protein level verifications (Fig. 2A–C) were performed by qRTPCR and western blotting respectively, which show that the models were successfully constructed. The lysosome-mediated degradation system is a key step in autophagy degradation. As an indispensable LMP, will the knockout of the Sidt2 affect the expressions of lysosome-related proteins? We measured the major LMP lysosomal-associated membrane protein 1(LAMP1). The results showed that the expression of LAMP1 decreased after Sidt2 deletion in MPC5 and SV40 MES 13 cells (Fig. 2D, E). We further used LysoTracker to label the acidic lysosomes and found that the number of acidic lysosomes decreased after the Sidt2 deletion in both types of cells (Fig. 2F, G). Subsequently, we measured the lysosomal cathepsin B (CTSB) and found that in mouse kidney tissue (Fig. 2H, I), MPC5 cells (Fig. 2J, K) and SV40 MES 13 cells (Fig. 2L, M), the expressions of CTSB in the Sidt2−/− models were all reduced, indicating that the proteolytic enzyme activity in the lysosomes was decreased when the Sidt2 gene was deleted. Similarly, the expression of the precursor CTSB in the two types of cells of the Sidt2−/− group was significantly reduced, indicating that the CTSB was also affected after Sidt2 deletion. We further used LysoSensor to detect the acidic environment of the lysosomes. The lower the fluorescence intensity ratio, the higher the pH value in the lysosome. The results showed that after Sidt2 deletion, the pH value increased (Fig. 2N, O) and the acidification was abnormal. To further explore whether the above lysosomal abnormalities were due to abnormal numbers of lysosomes or abnormal lysosomal functions, we investigated the cells by electron microscopy (Fig. 2P). We found that there are no obvious changes in the number of primary lysosomes and the total number of lysosomes (including primary and secondary lysosomes) after Sidt2 deletion (Fig. 2Q).
The renal autophagy pathway is abnormal after Sidt2 removal
As mentioned above, a large number of autophagolysosome accumulations were observed in the kidney cells of Sidt2−/− mice, suggesting that the autophagy pathway was abnormal. Using western blot analysis, it was shown that there was a significant increase in the protein level of LC3-phosphatidylethanolamine conjugate (LC3-II) and Sequestosome 1(P62) in the kidney of Sidt2−/− mice, and an increase in autophagy-related protein expressions of Autophagyrelated 5(Atg5), Autophagy-related 7(Atg7), and Autophagy-related 12(Atg12) (Fig. 3A, B). However, the P62 mRNA level was significantly decreased (Fig. 3C), suggesting that the autophagy pathway was abnormal after the elimination of Sidt2 in vivo. Then we found that the expressions of the autophagy proteins LC3-II, P62, Atg5, Atg7, and Atg12 were also increased (Fig. 3D–G) in the two types of kidney cells after Sidt2 deletion, which was consistent with the in vivo. The increase in LC3-II indicated an increase in autophagosomes in the kidney cells after Sidt2 deletion, which may be caused by the activation of autophagy or the blocked degradation of autophagosomes. P62 immunofluorescence (Fig. 3H) clearly showed an increase in P62 content in the Sidt2−/− cells (Fig. 3I). Contrary to the protein level, qRT-PCR showed that the P62 decreased significantly at the mRNA level (Fig. 3J), indicating that the increase in P62 was related to insufficient degradation. The increases in Atg5, Atg7, and Atg12 indicated that autophagy began and formed normally, but the accumulation of P62 also suggested autophagy process obstacles, and this contradiction required us to further explore the true situation of autophagy flux.
In vitro, chloroquine application indicates reduced autophagy flux after Sidt2 loss The chloroquine experiment is one of the classic experiments for observing autophagy flux. To understand the reasons for the increases in LC3-II and P62 after Sidt2 deletion, we used chloroquine (CQ), a downstream inhibitor of autophagy, to act on the cells to observe the autophagy flux. First, we used different concentration gradients to confirm that the saturation concentration inhibited by CQ was 50 μM (Fig. 4A–F) in the MPC5 and SV40 MES 13 cells. On top of this concentration, when the CQ concentration continued to increase, LC3-II and P62 did not increase further to reach saturation. Therefore, we used 50 μM CQ stimulation over 16 h to completely inhibit autophagy flux, and at this time, the LC3-II differences between the Sidt2+/+group and Sidt2−/− group disappeared in the MPC5 and SV40 MES 13 cells. Similarly, after 50 μM CQ processing, the P62 differences caused by the deletion of Sidt2 also disappeared (Fig. 4G, H, J, K). The statistics on autophagy flux showed that it decreased when Sidt2 was missing (Fig. 4I, L), which further confirmed that the increases in LC3-II and P62 expression after the Sidt2 deletion were due to the failure of autophagy clearance rather than the increase of the level in autophagy (the activation of autophagy).
Disruption of autophagosome-lysosome fusion in vitro after Sidt2 deletion Clearance and degradation are the middle and late stages in the autophagy process, involving the fusion of the autophagosome with decreased after the deletion of the Sidt2 (Fig. 5D), indicating that the fusion of autophagosomes and lysosomes was impaired.
Fig. 1 Kidney damage and autophagolysosome accumulation in Sidt2−/− mice. A Cre-LoxP system gene targeting schematic; B above is the exported sequence diagram of Sidt2 gene knockout mouse tail DNA; below is the sequencing map, the arrow indicating the location of the missing gene. Compared with WT mice, a 199 bp gene loss occurs in exon 2; C DNA level verification of Sidt2 (extracted from tail tissue). The primeramplified product contains the base knockout region, shown as Sidt2+ /+(WT), Sidt2+/−, or Sidt2−/−; D protein level verification to detect Sidt2 protein expression levels by western blot; E kidney 24 h urine protein in WT and Sidt2−/− mice; F ultra-micro-morphological structure of the kidneys of WT mice (a, f) and Sidt2−/− mice (g–n). Compared with the WT mouse, the Sidt2−/− mouse kidney displays foot process fusion, basement membrane thickening (g, h), renal tubular epithelial cell edema, microvilli damage (i, j), mitochondrial destruction (k, l), and autophagolysosome accumulation (m, n); G total number of renal autophagolysosomes in WT and Sidt2−/− mice. *P < 0.05, **P < 0.01.
Fig. 3 Sidt2 deletion disrupts the autophagy pathway. A Detection of WT and Sidt2−/− cell autophagy-related protein expression levels by western blot; B statistical charts of the western blot test results; C P62 mRNA expression level in kidney tissues of WT and Sidt2−/− mice; D expression levels of MPC5 cell autophagy pathway proteins before and after Sidt2 knockout; E statistical graph of (D) chart; F expression levels of autophagy pathway proteins in SV40 MES 13 cells before and after Sidt2 knockout; G statistical chart of the (F) chart; H P62 immunofluorescence in MPC5 and SV40 MES 13 cells before and after Sidt2 knockout; I statistical charts of the immunofluorescence results; J P62 mRNA expression levels in MPC5 and SV40 MES 13 cells before and after Sidt2 knockout. *P < 0.05, **P < 0.01, ***P < 0.001.
Ad-mCherry-GFP-
LC3B fluorescence double-labeling suggests that autophagolysosome formation and its degradation pathway were blocked after Sidt2 deletion The Ad-mCherry-GFP-LC3B transfection experiment can be used to detect changes in dynamic autophagy flux within cells. During the process of autophagy, mCherry-GFP-LC3B gathers on the autophagosome membrane and manifests itself in the form of yellow spots under a fluorescence microscope. When autophagosomes and lysosomes are fused to form autophagolysosomes, the acidic environment within the lysosome will quench the fluorescence of GFP so that it manifests itself in the form of red spots. Therefore, GFP bound to LC3 can only be used to detect autophagosomes, while mCherry can detect autophagosomes and autophagolysosomes at the same time. When the green and red fluorescence spots are combined and displayed as yellow fluorescence spots, this corresponds to autophagosomes. At this time, the red fluorescence can indicate autophagolysosomes, and it can also indicate the smoothness of autophagolysosome formation [27]. MPC5 cells and SV40 MES 13 cells in both the Sidt2+/+ and Sidt2−/− groups were transfected with Ad-mCherryGFP-LC3B adenovirus and photographed with a confocal laser microscope (Fig. 6A). It was found that the number of yellow fluorescent dots increased and the number of red fluorescent dots decreased (Fig. 6B, C) after Sidt2 deletion, shown that the number of autophagosomes increased and the autophagolysosomes decreased in the Sidt2−/− group. This is because there were obstacles to autophagolysosome formation, and the autophagolysosome degradation pathways were blocked, which led to autophagosome degradation obstacles.
Rapamycin did not improve the P62 accumulation caused by Sidt2 deletion but rather exacerbated it Rapamycin (RAPA) is an inhibitor of mTOR and activates autophagy. When RAPA acted on MPC5 and SV40 MES 13 cells of the Sidt2+/+and Sidt2−/− groups, it increased the amounts of LC3-II in both groups. This meant that RAPA could activate the upstream pathway of autophagy. Similarly, we found that the increase in LC3-II level in the Sidt2−/− group with RAPA was more obvious than that in the control (Sidt2+/+group), showing that when RAPA acted on Sidt2−/− group, the degradation of autophagosomes still was not improved. Meanwhile, we also observed the changes in P62 and found that when RAPA acted on the Sidt2+/+ group of MPC5 and SV40 MES 13 cells, P62 did not change significantly, indicating that the autophagy flux was normal. When RAPA acted on the Sidt2−/− group, the expression level of P62 was further increased compared to that before administration and was also more obvious than that in control after RAPA treatment (Fig. 7A, B, D, E). Subsequently, in order to explore the reasons for the inconsistent expression of P62 protein after the Sidt2+/+ and the Sidt2−/− group were treated with RAPA, we measured the mRNA levels of P62 and found that the level increased after RAPA treatment in both the Sidt2+/+ and the Sidt2−/− groups, and the Sidt2+/+ group had a higher level than that in the Sidt2−/− group (Fig. 7C, F), which could further prove the autophagy flow was smooth in the Sidt2+/+ group, while the autophagy flow disorder occurred at the end of autophagy in the Sidt2−/− group, that is, the autophagolysosomes degradation link. We also studied the effect of Sidt2 on the proliferation and apoptosis of kidney cells under the condition of activated autophagy and found that after serum-free medium-induced cell activation of autophagy, compared with the control group, the proliferation of the Sidt2−/− group was further inhibited, and the apoptosis was more obvious (Supplementary Fig. 2).
Fig. 5 Autophagosome and lysosome fusion is prevented after Sidt2 deletion. A Immunofluorescence co-localization of LC3B and LAMP1 in MPC5 and SV40 MES 13 cells before and after Sidt2 knockout, analysis with Pearson’s correlation coefficient; B comparison of LC3B fluorescence points before and after Sidt2 knockout in MPC5 and SV40 MES 13 cells; C comparison of Pearson’s correlation coefficient before and after Sidt2 knockout in MPC5 and SV40 MES 13 cells; D comparison of the fusion rate before and after Sidt2 knockout in MPC5 and SV40 MES 13 cells (the ratio of the number of co-localized fluorescent spots of LAMP1 and LC3B to the number of fluorescent spots of LAMP1); *P < 0.05, **P < 0.01.
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