INTRODUCTION
Acute kidney injury (AKI) is one of a number of conditions that affect kidney structure and function which results in a sudden episode of kidney failure that can occur over a matter of hours or days. It is a common complication of critically ill patients that is independently associated with increased mortality. Glomerular disease is a related cause of AKI which results in acute inflammation of blood vessels and glomeruli and is often seen in severe cases of acute glomerulonephritis [
1]. It has been reported that podocyte injury is a pivotal factor of glomerular diseases and, in particular, it is also associated with the occurrence of AKI [
2,
3]. Podocytes are terminally differentiated cells in the Bowman’s capsule of the kidneys that wrap around the capillaries of the glomerulus. They are attached to the outside of the glomerular basement membrane (GBM), a structure which is very important for the filtration of proteins and other molecules from the blood.
Injuries to podocytes (necrosis [
4], apoptosis [
5], and altered autophagy [
6]) are considered to be the major contributors to the development of glomerular disease [
7,
8] as their loss causes proteinuria and is associated with progressive AKI [
2]. Foot processes (FP, derived from major processes) form a characteristic interdigitating pattern with FP of neighboring podocytes and are found to be in between the filtration slits that are bridged by the slit diaphragm (SD). The SD is a highly specialized intercellular junction between the podocyte FP and is crucial in the formation of the filtration barrier in the renal glomeruli. Nephrin, podocin, CD2-associated protein (CD2AP), and synaptopodin (SYNPO) are proteins considered to be the critical components of the epithelial SD and FP, and these can help maintain the integrity of the podocyte for avoiding proteinuria [
9‐
11]. Recently, some investigators have reported that the levels of the SD and FP proteins, nephrin, podocin, CD2AP, and SYNPO, decrease in glomerular diseases [
12‐
15]. Furthermore, these authors have highlighted the association between the expression of nephrin, podocin, CD2AP, and SYNPO proteins and the development of glomerular disease [
9,
16,
17].
Lipopolysaccharides (LPS), also known as lipoglycans and endotoxins, are derived from the outer membrane of gram-negative bacterial cell walls, are released from proliferating or dying bacteria, and these are recognized by a variety of host cells. After bacterial infections, LPS is one of the major factors that lead to cell and tissue injury and is an inducing virulence factor for the pathogenesis of inflammation-associated diseases [
18,
19]. In the kidney, it has been reported that LPS could be the underlying cause of albuminuria and aggravate glomerulonephritis in MRL/lpr mice [
20]. LPS can stimulate the expression of inflammatory cytokines and activation of the NF-κB pathway in the pathogenesis of renal disease caused by injury, infection, and autoimmune factors [
21‐
23]. Moreover, LPS is also one of the most important factors that lead to AKI [
24,
25].
Fractalkine (FKN/CX3CL1) is a unique member of the CX3C chemokine family and participates in inflammatory response in several biological systems [
26,
27]. It is also implicated in progression of a variety of renal diseases (glomerular inflammation and endothelial injury) [
28,
29]. Park et al. found that LPS can upregulate expression levels of FKN and may contribute to renal inflammation leading to chronic renal allograft rejection [
30]. In our previous studies, we showed that LPS can induce the expression of FKN increased in HK-2 cells and MRL/lpr mice which can cause cell damage, leading to proteinuria, renal dysfunction, and severity of renal pathology [
31]. Therefore, our previous studies indicated that FKN induced by LPS is one of the major factors that lead to renal cell and tissue damage.
In addition, recent research has also shown that upregulation of Wnt/β-catenin signaling expression plays an essential role in kidney disease and is associated with podocyte injury both
in vivo and
in vitro [
32‐
34]. The Wnt/β-catenin signaling pathway is considered to be an important developmental signaling pathway implicated in organogenesis and disease development in multicellular organisms, and kidney is one of these organs. In the kidney, it can regulate cell proliferation, survival, and cell behavior in both embryos and adults. However, in the mature kidney, the Wnt signaling pathway appears to be silenced, but it can be re-activated upon renal injury. Wang et al. confirmed that AKI in patients are caused by the activation of the Wnt/β-catenin signaling pathway [
35]. Furthermore, it has been reported that activation of the Wnt/β-catenin pathway can aggravate LPS-induced inflammation [
36,
37]. In addition, modulation of the Wnt/β-catenin pathway plays an important role in suppressing LPS-induced inflammatory response .
In this study, we detected expression of FKN and activation of Wnt/β-catenin signaling pathway in podocytes induced by LPS in vitro and in vivo and examined whether the expression of FKN occurring through regulation of Wnt/β-catenin pathway leads to podocyte injury by reducing expression of podocyte-specific proteins and contributes to risk factors that affect the development of AKI. Thus, our findings may constitute a key step in the pathogenesis of podocyte injury and could be a novel target for therapeutic intervention of AKI.
DISCUSSION
The present study revealed that LPS can cause inflammatory responses both in vitro and in vivo of glomerular podocytes by means of enhancing FKN, wnt-4, β-catenin, cyclin-D1, and c-myc mRNA and protein synthesis. Moreover, the signal-transducing mechanisms of LPS-induction regulate FKN expression, leading to activation of Wnt/β-catenin pathway. These findings indicate that LPS may induce FKN synthesis and activate the Wnt/β-catenin signaling pathway in podocytes, resulting in a decrease in the expression of podocyte-specific mRNA and proteins and is involved in the occurrence and development of AKI.
A reduction in podocyte number after an injury or through apoptosis induced by external factors or drugs is a hallmark in the development of glomerulopathies [
44,
45]. Furthermore, the inciting injury to the podocyte may vary among these glomerular diseases and the inevitable consequence of podocyte injury is loss of SD proteins, actin cytoskeleton derangement, and loss of structural integrity, leading to eventual FP effacement and podocyte apoptosis. These SD- and FP-specific proteins, especially nephrin, podocin, CD2AP, and SYNPO are crucial in signal transduction regulating a number of cell processes such as cell polarity, cytoskeleton organization, and survival of podocytes. Recent studies have demonstrated that a disruption of the FP and SD can lead to loss of their main protein components: nephrin, podocin, CD2AP, and SYNPO [
46,
47].
Nephrin is synthesized by glomerular podocytes and is localized at the SD area between the podocyte FP, and it helps to maintain the interaction between the basement membrane and the podocytes of the epithelial cells. While podocin interacts with the cytosolic tail of nephrin. CD2AP serves as an adaptor protein in the structural organization of the SD and participates in a common signaling pathway necessary to maintain crucial podocyte functions [
48]. SYNPO is a podocyte FP-specific actin-binding protein, which plays a crucial role in actin-based cell shape and motility and is also critical for stabilizing SD integrity because it is bound to nephrin through its direct interaction with CD2AP [
49]. Thus, these proteins play major roles in maintaining the structural and functional integrity of the GBM and reduced expression of these proteins may contribute to the development of glomerular disease and lead to the pathogenesis of AKI.
Recently, studies demonstrated that LPS has the ability to cause a robust change in transcriptional activity of NF-κB and to increase the expression of IL-6, IL-8, IL-1β, and TNF-α markedly, leading to cell apoptosis. It has also has been reported that alterations in NF-κB activity might contribute to podocyte disorders in idiopathic nephrotic syndrome. According to a recent report, LPS is able to inhibit podocyte autophagy, which contribute to LPS-induced injury of podocytes. Tarak et al. reported that LPS can deform morphology and disrupt the actin cytoskeleton of podocytes and induce it proceed to injury [
50]. Furthermore, LPS is recognized as the cause of kidney injury and it is regarded to be linked to the mechanisms of pathogenesis of AKI in mice [
38,
51]. Therefore, LPS is one of the causes of podocyte injury and is a critical determinant in the pathogenesis and progression of AKI. The results from this study indicate that LPS inhibited podocyte proliferation and induced their apoptosis by regulating the expression of nephrin, podocin, CD2AP, and SYNPO levels and led to the injury of podocytes, which plays an important role in the development and progression of AKI. Most importantly, research has also found that LPS-induced typical changes in podocytes by upregulating the expression of FKN and activating the Wnt/β-catenin pathway.
FKN is a chemokine that plays an important role in modulating inflammation in the kidneys. Studies have shown that in humans and rodents, FKN expression is prominent in renal diseases, particularly in glomerular inflammation and endothelial injury [
52,
53]. Zhuang et al. [
54] found that FKN was produced and increasingly expressed in various cells of kidney (such as podocytes, mesangial cells, endothelium, and tubular epithelium) when stimulated by inflammatory factors. It has been revealed that LPS stimulation increases FKN expression through the activation of the NF-κB signaling pathway
in vitro [
55]. In our previous studies, we showed that both
in vitro and
in vivo, LPS can induce the expression of FKN and this can lead to inflammation of cells and tissues, which in turn causes proteinuria, renal dysfunction, and structural damage. Our data demonstrate that LPS induced FKN expression and led to a decrease of nephrin, podocin, CD2AP, and SYNPO levels, resulting in podocyte damage or loss, and this was a causal factor in the pathogenesis of AKI. Moreover, FKN is critically involved with the Wnt/β-catenin pathway in inducing podocyte injury and promoting the progression of kidney damage in AKI.
Previous studies proved overexpression of CX3CR1, the receptor for FKN, is closely related to the pathogenesis of osteoarthrosis by regulation of chondrocyte proliferation and apoptosis
via the Wnt/β-catenin pathway [
56]. In the spine, inhibition of the Wnt pathway could reduce FKN expression in remifentanil-induced post-operative hyperalgesia [
57]. In HUVECs, regulation of the Wnt pathway plays an important role in inhibiting the expression of FKN in anti-inflammatory conditions induced by high glucose [
58]. In the kidney, upregulation of the Wnt/β-catenin pathway is associated with podocyte injury and it can be involved in the pathogenesis of AKI. Furthermore, inhibition of the Wnt pathway plays an important role in suppressing the LPS-induced inflammatory response [
59]. Consequently, the Wnt/β-catenin pathway is closely related to the expression of FKN induced by LPS. These results suggest that the expression of FKN and Wnt/β-catenin pathways was significantly increased
in vitro and
in vivo after LPS, which can lead to podocyte injury and resulting in the occurrence of AKI.
In summary, the present study suggests that nephrin, podocin, CD2AP, and SYNPO form a signaling complex that is important for supporting the functional and structural integrity of glomerular podocytes. In addition, LPS is one of the major factors that reduces expression of these podocyte-specific markers thereby mediating podocyte apoptosis and plays an important role in the occurrence and development of AKI by means of enhancing FKN and Wnt/β-catenin signaling pathway expression. This report may help shed light on one of causes of podocyte injury that contributes to the pathogenesis of AKI by upregulating the expression of FKN and Wnt/β-catenin pathway. However, we currently lack a full comprehensive understanding of the factors implicated in upregulating the expression of FKN through regulation of Wnt/β-catenin pathway. Further studies are required to determine the mechanisms and relationship between of FKN and the Wnt/β-catenin pathway, and how they are regulated in podocytes during AKI. This may potentially present new opportunities for the treatment and management of podocyte injury in AKI.
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