In this study, we found that the incubation of L6 cells with high Pi-containing medium induced cell atrophy in a time- and dose-dependent way, indicating that Pi overload could affect skeletal muscle cells directly. Most previous studies have examined the effect of Pi on calcification or osteogenic differentiation. Prior studies in vascular smooth muscle cells (VSMC) have shown an important role for BMP-2, OPN, and other osteogenic markers in phosphate-induced VSMC calcification [
11]. We tested the effect of high-Pi medium on calcification and osteogenic differentiation of L6 cells, but found no significant effect.
Autophagy, the lysosomal-dependent cellular turnover of organelles and proteins, has been shown to play a crucial role in muscle atrophy. The autophagy-lysosome system is a proteolytic pathway that mainly degrades long-lived proteins and organelles, such as mitochondria and the sarcoplasmic reticulum. It operates alongside the ubiquitin-proteasome system, which has been shown to play an important role in muscle atrophy and its activation has been well documented in CKD-associated muscle atrophy [
12,
13]. There are roughly three classes of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy. In macroautophagy (hereafter referred to as autophagy), membrane vesicles form autophagosomes by sequestering a small portion of the cytoplasm. These autophagosomes then fuse with lysosomes to degrade the materials within them [
14]. In recent years, the activation of skeletal muscle autophagy has been demonstrated under various conditions and disease states ranging from fasting [
15,
16], oxidative stress [
17], denervation [
18,
19], and drug effects [
20,
21] to systemic diseases, such as sepsis [
22], merosin-deficient congenital muscular dystrophy (MDC1A) [
23], and cancer [
16]. In a previous study, we reported the upregulation of mRNA and protein expression of Bnip3 and LC3B in the rectus abdominis of CKD patients [
24]. Thus, in this study, we investigated whether Pi overload induces cell atrophy through autophagy upregulation. During autophagosome formation, the cytosolic LC3-I protein is converted to LC3-II through lipidation. Western blot analysis showed that high Pi induces a dose- and time-dependent increase in the LC3-II/LC3-I ratio, which has been widely used as an indicator of autophagic activity. The levels of p62, a protein known to be incorporated into autophagosomes and efficiently degraded, decreased with high Pi. While PCR analysis of
LC3 and
p62 mRNA also suggested a transcription mechanism of Pi effect. We further used a pharmacological agent and genetic method to study the effect of autophagy on cell atrophy. The inhibition of autophagy by wortmannin or Atg5 knockdown significantly inhibited high Pi-induced atrophy. These findings indicate a stimulatory effect of high Pi on autophagy in skeletal muscle and suggest that autophagy may participate in the development of muscle atrophy induced by high Pi in CKD.
A low protein diet has been recommended for patients with CKD to maintain or improve nutritional status [
25]. Other studies have found that a very-low-protein diet [
26] or a low protein diet supplemented with ketoacids [
27,
28] can also improve calcium and phosphorus metabolism. Together with our previous report [
29], we hypothesize that hyperphosphatemia is an independent risk factor of CKD skeletal muscle wasting and proper low Pi diet may be a promising approach to maintain or improve skeletal muscle wasting in CKD patients. However, further studies are still needed to confirm this hypothesis.
Yamada et al. [
30] used dietary Pi overload to induce hyperphosphatemia in rats with CKD. serum albumin level, body weight, and muscle mass in these rats were decreased by Pi-overload-related chronic inflammationassociated with. However, there are still conflicting results on the effect of hyperphosphatemia in CKD skeletal muscle wasting in vivo. This study is only a preliminary study on Pi toxicity in skeletal muscle cells, being limited to in vitro experiments. Further studies are still needed to investigate the in vivo effects of adjusting Pi levels.