It is well known that injury to podocytes, the outermost layer of the glomerular filtration barrier, is the most common pathologic phenomenon and a dominant player in the disease progression of CKD. As highly differentiated epithelial cells, podocytes are connected to FPs by the slit diaphragm (SD). Recently, published research has shown that the SD is a special cholesterol-rich raft structure . As an important component of the cell membrane, lipid rafts are involved in regulating cell membrane fluidity, membrane protein exchange, signal transduction and other functions . Intracellular cholesterol homeostasis (such as the normal secretion, inflow and outflow of cholesterol in cells) is an important part of ensuring the normal function of cells . To date, a great deal of progress has been made regarding the mechanism of podocyte lipid metabolism disorder.
ANGPTL3 is necessary to regulate the activity of LPL. More recent studies have demonstrated that this molecule is not only expressed in podocytes but is also related to podocyte injury, such as increased podocyte motility and FPs fusion . In the previous study, we reported that serum ANGPTL3 was significantly enhanced in patients with hyperlipidemia-related kidney injury . In this study, the data further confirm that glomerular ANGPTL3 expression is enhanced in a mouse model of renal injury associated with hyperlipidemia. Recently, we also reported that Angptl3 transgenic mice exhibited hyperlipidemia and proteinuria, and TEM showed that the podocyte FPs were effaced . In this hyperlipidemia-related renal injury mouse model, with prolonged high-fat feeding time, the expression of ANGPTL3 gradually increased, and podocyte FPs fusion was increased accordingly. Importantly, compared with the wild-type mice, in the mice without the angptl3 gene that were fed the same high-fat diet, the serum lipids did not increase significantly, proteinuria also obviously recovered, and the severity of podocyte FPs fusion was prominently relieved.
These results suggested that ANGPTL3 not only affects hyperlipidemia induced by a high-fat diet but also participates in renal injury in this model. ANGPTL3 may be involved in the glomerular injury induced by hyperlipidemia. Currently, inhibitors of ANGPTL3 have been extensively studied in cardiovascular disease, and suppression of ANGPTL3 is protective in nephrotic syndrome [16, 17]; in the future, this inhibitor would be a candidate for hyperlipidemia-related kidney injury.
Podocytes are terminally differentiated epithelial cells with abundant cytoskeletal proteins. The rearrangement of cytoskeletal actin includes the polarity loss of the arrangement of stress filaments and the abnormal expression of cytoskeletal proteins. At present, it is generally believed that cytoskeletal actin rearrangement is the molecular basis of podocyte motility. When rearrangement occurs, podocyte junctions are weakened, and podocyte adhesion is lost . The loss of podocyte adhesion is an important pathological basis of glomerulosclerosis.
Following the widespread clinical use of second-generation sequencing technology, the actn4 gene mutation was found in patients who suffered from congenital kidney disease. The abnormality of the actn4 gene led to the occurrence of steroid-resistant congenital kidney disease. The pathological type was FSGS . Recent published studies have shown that podocytes with homozygous mutation of the actn4 gene may have an irrecoverable decrease in intercellular connectivity and the occurrence of cytoskeleton rearrangement . In our published studies, we also confirmed that ANGPTL3 can affect the expression levels of podocyte lipid raft-associated molecules such as nephrin and ACTN4 in an adriamycin-induced nephropathy model and can then aggravate the cytoskeleton rearrangement and cell motility of podocytes [15, 21]. ANGPTL3 can play a role through ACTN4 in a hyperlipidemia-related kidney injury model has not been reported.
The IHC data primarily revealed that the ACTN4 staining intensity was weakened in hyperlipidemia-related glomeruli with prolonged high-fat diet feeding, suggesting that ACTN4 may be involved in podocyte injury caused by hyperlipidemia. After gene knockout of angptl3, the decrease in ACTN4 was significantly inhibited in the glomeruli of mice treat with high-fat diet, suggesting that ACTN4 is a potential molecular signal in ANGPTL3’s mechanism for hyperlipidemia-related renal injury. ACTN4 is a potential signaling molecule for ANGPTL3 in the mechanism of hyperlipidemia-related kidney injury. ANGPTL3 can bind to and activate the podocyte integrin β3 receptor, phosphorylate FAK, and ultimately change ACTN4 expression in ADR-treated podocytes in vitro . However, how ANGPTL3 impacts ACTN4 in podocyte injury induced by high fat has not been well clarified.
Currently, so many studies have reported that mutations in the NPHS2 gene can trigger congenital kidney disease, and the protein podocin encoded by the NPHS2 gene is an intrinsic molecule of the podocyte SD. Schermer B et al. found that podocin must bind with cholesterol to activate transient receptor potential channel 6 and then complete signal transduction and participate in podocyte function regulation . This study discovered that a high-fat diet induced a noteworthy fading of podocin staining intensity in wild-type glomeruli. However, without the angptl3 gene did not reverse the fate of the podocin decline after a high-fat diet. This means that podocin might not be a candidate for the ANGPTL3 mechanism in lipid-related podocyte injury.
As an adapter molecule, CD2AP could contact the cytoplasmic domain of nephrin and form the podocyte SD . CD2AP is related to cytoskeletal actin rearrangement via the interplay between nephrin and the actin cytoskeleton . Unexpectedly, we did not find any significant changes in glomerular CD2AP expression in WT + HF mice. We also did not find any differences in glomerular CD2AP expression in the mice with knockout of the angptl3 gene with or without a high-fat diet. There were no obvious changes in CD2AP expression in glomeruli between wild-type and angptl3 knockout mice. Therefore, we think that the reactions between various podocyte molecules are quite complex . The interactions between some podocyte molecules need further exploration.
Finally, mice after high-fat feeding, the degree of glomerular injury was slight, and only podocyte FPs fusion occurred. The biochemical and pathological changes in the model were only observed within 21 weeks. Prolonging the observation time is necessary to further understand the kidney injury in this model.
In the later stage of mice on a high-fat diet, renal function gradually deteriorated, accompanied by a gradual decrease in ACTN4 in the kidney. However, after knockdown of the angptl3 gene, ACTN4 was not decreased, indicating that there might be some interaction between them and that ANGPTL3 promoted the expression of ACTN4 or inhibited the degradation of ACTN4 in some manner.
Study strength and limitations
This study has several strengths. WT and ANGPTL3 knockout mice were divided into groups based on a normal diet or a high-fat diet. Renal tissue was taken for pathological evaluation by H&E and transmission electron microscopy. Ultrastructural results showed that knockout of the angptl3 gene could alleviate podocyte damage in mice caused by high-fat feeding. In addition, immunohistochemistry staining showed that the decrease in ACTN4 expression is related to podocyte injury in mice. There are also some limitations. ANGPTL3 and ACTN4 are involved in podocyte injury, but the pathways of ANGPTL3 and ACTN4 in glomerular podocyte injury and the specific relationship between these two proteins are not explained in detail.