Inhibition of PKC‐δ reduce rhabdomyolysis‐induced acute kidney injury

Abstract Despite extensive research, the mechanisms underlying rhabdomyolysis‐induced acute kidney injury (AKI) remain largely elusive. In this study, we established both cell and murine models of rhabdomyolysis‐induced AKI by using myoglobin and glycerin, respectively, and provided evidence that protein kinase Cδ (PKC‐δ) was activated in both models and subsequently promoted cell apoptosis. Moreover, we found that this detrimental effect of PKC‐δ activation can be reversed by its pharmaceutical inhibitor rottlerin. Furthermore, we detected and confirmed the existence of PKC‐δ‐mediated myoglobin‐induced cell apoptosis and the expression of TNF‐α and IL1‐β via regulation of the p38MAPK and ERK1/2 signalling pathways. In summary, our research revealed the role of PKC‐δ in renal cell apoptosis and suggests that PKC‐δ is a viable therapeutic target for rhabdomyolysis‐induced AKI.


| INTRODUC TI ON
Rhabdomyolysis is a syndrome caused by the breakdown of skeletal muscle and necrosis resulting from various conditions. The release of intracellular contents and breakdown products into the bloodstream can lead to acute kidney injury (AKI), a life-threatening complication associated with poor outcomes including end-stage renal disease (ESRD) and even death. [1][2][3] Trauma patients often endure direct muscle injury; damaged myocytes can release myoglobin, creatine phosphokinase, and lactate dehydrogenase into the circulation, thus creating a heavy burden on the kidneys. [4][5][6] At present, the mechanisms underlying rhabdomyolysis-related AKI are not well understood despite significant effort, leaving us with limited options for the prevention and treatment of this condition. Tubular damage, and more specifically apoptosis of the tubule cells, is the main pathological change occurring during AKI. Apoptosis is the main form of programmed cell death and renal cell apoptosis is a necessary aspect of normal renal function. 7,8 Under normal circumstances, apoptotic cells turn into small apoptotic bodies that are either ingested by phagocytes or taken up by the epithelia into the tubular lumen. 9,10 However, apoptosis causes the loss of parenchymal cells during the process of acute renal injury. This is because there are more apoptotic cells than neighbouring cells; these cells progress into a necrosis phase, spilling toxic and immunogenic contents that cause further damage in the kidney. 10 Exploring the mechanisms underlying renal cell apoptosis might provide us with an interventional method for AKI at an earlier stage.
Protein kinase C is a subgroup of serine/threonine kinases that when activated regulate multiple cellular functions, including cell differentiation, survival and death. [11][12][13] Phosphorylated protein kinase C has been reported to influence several renal diseases in manners that both expedite or impede apoptosis; furthermore, isoform-specific inhibitors and agonists have been proven to counter or promote these effects. 14,15 PKCδ, one of the key PKC isoforms, is known to play an anti-apoptosis role in tumour cells. 16 However, recent studies showed that PKCδ promotes renal cell apoptosis under treatments with cisplatin and vancomycin, and in cold storage transplantationinduced AKI. [17][18][19] At present, the specific role and mechanisms underlying PKCδ in rhabdomyolysis-induced AKI remain unclear.
In the current study, we found that PKCδ was induced by myoglobin and glycerin both in vitro and in vivo. We also found that the inhibition of PKCδ by both rottlerin and PKCδ-KD attenuated the progression of rhabdomyolysis-induced AKI and improved the survival rate. Mechanistically, we used mouse proximal tubular cells to demonstrate that the inhibition of PKCδ ameliorated myoglobininduced renal cell apoptosis via the inactivation of the p38MAPK and ERK1/2 pathways. In summary, our data suggested that the inhibition of PKCδ ameliorates rhabdomyolysis-induced apoptosis in renal epithelial cells and kidney injury.

| Animal model of RM-induced AKI
C57BL/6 mice were purchased from Hunan SJA Laboratory Animal Co., Ltd (Changsha, China). Male C57BL/6 mice aged 8-10 weeks were injected 50% glycerol at a dose of 8ml/kg as previous described 20 ; the control group was injected with an equal volume of saline. The mice were injected intraperitoneally with Rottlerin were 10 mg/kg/day to suppress the PKC. Animal experimental protocols were approved by the Care and Use of Laboratory Animals Institutional Committee from Second Xiangya Hospital, China. The mice were housed at stable room temperature in a 12-h light/dark cycle and provided adequate supplies of standard rodent chow and water. We randomly divide twenty mice aged 6-8 weeks into two groups, and mice were intraperitoneally injected with saline or glycerol. We observe the survival of mice within 48 h to obtain the survival curve of mice after injection of glycerol.

| Cell culture and treatment
We obtained the BUMPT cell line (mouse renal proximal tubular epi-

| Real-time PCR
We use TRIzol (Takara, T9108) to extract RNA from tissues and cells according to the instructions and use a reverse transcription kit (Takara, RR037A) to prepare cDNA as previously described, and then, use

| Statistics
All data were presented as means ± SD. Two-tailed Student t tests were used for two group comparisons. Two-way ANOVA was used for the multiple group comparisons. p < 0.05 was considered statistically significant.

| PKC-δ was activated by glycerine-induced AKI
Prior to detecting PKCδ, we wanted to establish and validate a murine model of rhabdomyolysis-induced AKI. To investigate survival rates, C57BL/6 mice were intramuscularly injected with glycerin at a dose of 14 µl/g for 48 h; resultant data suggested that C57BL/6 mice began to die at 24 h and all mice had died by the 40 h timepoint ( Figure 1A). To create a model of AKI, C57BL/6 mice were intramuscular injected with glycerin a dose of 14 µl/g for 12 and 24 h. Functionally, the levels of serum creatinine and blood urine were notably increased at 12 h and reached a peak at 24 h ( Figure 1B,C). H&E staining showed that glycerin induced tubular damage and tube type at 12 h and induced moderate levels of damage by 24 h ( Figure 1D); this was supported by the tubular damage score ( Figure 1F). TUNEL staining results indicated that glycerine induced renal cell apoptosis at 12h, and that the levels of apoptosis had increased further by 24 h; this effect was demonstrated by the counting of TUNEL-positive cells ( Figure 1E,G). Also, PI staining indicated elevated cell death in a time-dependent manner in mice kidney ( Figure S1A,B). Finally, immunoblotting results indicated that glycerine induced the activation of PKCδ and caspase3 at 12 h; with notable increased levels at 24 h ( Figure 1H,I). Further immunoblotting detection showed phosphorylated p38 and ERK1/2 expression were increased too in glycerin-treated mice ( Figure S1 C,D). We complementarily detected the expression of other PKCα and PKCη and found that they were also activated by glycerin ( Figure E,F). Taken together, these data suggested that PKCδ was activated by glycerine in C57BL/6 mice. We then injected mice with rottlerin after glycerin treatment and found that rottlerin significantly inhibited the activation of phosphorylated p38 and ERK1/2 ( Figure S2). In summary, these data suggested that the inhibition of PKCδ attenuates AKI caused by glycerine.

| Rottlerin and PKCδ-KD plasmid attenuated renal damage caused by Glycerin and MYO in mice
We then tried to pin the function of PKCδ in rhabdomyolysis induced AKI in vivo. We injected the mice with saline or rottlerin (10 mg/kg/day) for three days before they underwent intramuscular glycerin injection. 24 h later, mice kidneys were collected, H&E staining showed markedly decreased tubular damage ( Figure S3A,C), and TUNEL staining detected far less apoptosis in mice treated with rottlerin ( Figure S3B,D), indicating that inhibition of PKC kinase exerts a renal protective function. To specify the role of PKCδ, we injected mice with 25 μg PKCδ-KD plasmid through tail vein per day for three day before intramuscular glycerin injection and found that the activation of PKCδ p38 MAPK and ERK1/2 were all downregulated significantly in PKCδ-KD-treated mice by immunoblotting ( FigureS 3B,D). These results together confirmed that PKCδ inhibition can protect kidney from crush syndrome-related renal injury,

| PKC-δ was activated by myoglobin in BUMPT cells
To further investigate the role of PKCδ, we used myoglobin in a range of in vitro experiments. Immunoblotting results indicated that myoglobin induced the activation of PKCδ and caspase3 in a dose-dependent manner (5 and 10 mg/ml) ( Figure 3A,B). In addition, myoglobin (10 mg/ ml) induced the activation of PKCδ and caspase3 in a dose-dependent manner (6, 12, and 24 h) ( Figure 3C,D). These data showed that myoglobin induced the activation of PKCδ in BUMPT cells.

| Rottlerin attenuated myoglobin-induced apoptosis in BUMPT cells
Hoechst staining demonstrated that rottlerin attenuated myoglobininduced apoptosis in BUMPT cells. This was confirmed by determining the rate of apoptosis ( Figure 4A,B). Rottlerin notably attenuated glycerine-induced activation of PKCδ and caspase3 at 24 h ( Figure 4C,D). These findings were consistent with the in vivo results.

| DISCUSS ION
Proximal tubular cell damage is closely associated with AKI.
Rhabdomyolysis refers to the breakdown of striated muscle resulting from traumatic events, genetic issues, exertional and non-exertional conditions and can cause renal damage due to the release of cellular contents from damaged muscle cells into the bloodstream; these secretions will eventually poison the kidney. The typical clinical presentation of this condition is pain and weakness in the muscle and the discoloration of urine. Another common factor that is toxic to renal cells is cisplatin. Cisplatin is a common treatment for multiple types of cancers and exerts a in rhabdomyolysis-induced AKI. 18 In the current study, we initially demonstrated that PKCδ was induced by glycerin and myoglobin in renal tubular cells and C57BL/6 mice, respectively. Interestingly, the inactivation of PKCδ by rottlerin markedly reduced glycerineinduced death in C57BL/6 mice and also attenuated the progression of glycerine-induced AKI. Mechanistically, we found that the PKCδ/p38MAPK and ERK1/2 signalling pathways mediated myoglobin-induced renal cell apoptosis and the expression of TNFα and IL1β. These data suggested that the inhibition of PKCδ has a beneficial effect on AKI induced by rhabdomyolysis.
The function of PKCδ in apoptosis depends on cell types and stimulating factors. 26,[31][32][33][34] In tumour cells, PKCδ usually antagonizes apoptosis. 16 In a previous study, we revealed that PKCδ induced renal cell apoptosis and then led to the progression of nephrotoxicity induced AKI. 17,18 A more recent study reported that PKCδ promoted the apoptosis in mitochondria and caused AKI induced by cold storage-associated kidney transplantation. 19 Consistently, we also found that PKCδ induced apoptosis in renal tubular cells to promote the progression of rhabdomyolysis-induced AKI. This effect was supported by two lines of evidence. First, rottlerin, a PKCδ pharmacological inhibitor, attenuated the rhabdomyolysis-induced AKI accompanied by a reduction of renal cells ( Figure 2). Secondly, rottlerin also ameliorated myoglobin-induced renal cell apoptosis ( Figure 4). Collectively, these data confirmed that PKCδ plays an apoptotic role during rhabdomyolysis-induced AKI.
Recent studies suggested that rottlerin non-selectively inhibits PKCδ and other enzymes, including PKCα and PKCη. 35,36 Therefore, this inhibition experiment alone is insufficient to illustrate the specific function of PKCδ. To further avoid the nonspecific effects of rottlerin, we used PKCδ-KD and PKCδ-CF to suppress or overexpress PKCδ, respectively. Previous studies reported that the P38MAPK and ERK1/2 pathways are related to apoptosis and inflammation. [37][38][39] The p38 MAPK (mitogen-activated protein kinase) signalling pathway conducts diverse cell functions by aiding cells to process various signals and is known to participate in cell apoptosis induced by different stimuli. 40 The ERK1/2 pathway is associated with the inflammatory responses of irritated cells. 41 In the present study, we demonstrated that PKCδ positively regulated the P38MAPK and ERK1/2 pathway to mediate apoptosis and inflammation. This was supported by specific evidence. First, PKCδ-KD attenuated myoglobin-induced apoptosis in renal cells and the expression of TNFα and IL1β by suppressing the p38MAPK F I G U R E 5 PKCδ-KD attenuates cell injury induced by myoglobin. BUMPT cells were treated with saline or myoglobin (10 mg/ml), with or without transfection of 1 μg kinase dead PKCδ (PKCδ-KD) for 24 h. (A) Representative immunoblots of PKCδ phosphorylation, Caspase3 activation, p38 MAPK phosphorylation and ERK1/2 phosphorylation in cell lysate. (B) Grayscale image analysis between them. (C,D) the mRNA level of TNFα and IL-1β. Data are expressed as mean ± SD (n = 6). * p < 0.05 versus Saline group. #p < 0.05 versus MYO-treated group and ERK1/2 signalling pathways ( Figure 5). Secondly, PKCδ-CF enhanced them by activating the p38MAPK and ERK1/2 signalling pathways ( Figure 6). These results suggested that the PKCδ/ p38MAPK and ERK1/2 axis mediated rhabdomyolysis-induced AKI.
Still, there are certain deficits in this research. Firstly, we used only inhibitors and plasmids to alter the expression of activated PKCδ, while more concrete evidence would be presented should specific genomic knockout of PKCδ in renal tubules was applied. Secondly, we did not investigate the functions of PKCα and PKCη even though they were also activated in glycerin-induced AKI. What's more, how glycerin/MYO caused PKCδ activation is yet to be discovered.
Further exploration is required to extend the findings in this study.
In conclusion, our research revealed that PKCδ mediates rhabdomyolysis-induced AKI via the activation of the P38MAPK and F I G U R E 6 PKCδ-CF aggravates cell injury induced by myoglobin. BUMPT cells were treated with saline or myoglobin (10 mg/ml), with or without transfection of 1 μg PKCδ active fragment (PKCδ-CF) for 24 h. (A) Representative immunoblots of PKCδ phosphorylation, Caspase3 activation, p38 MAPK phosphorylation and ERK1/2 phosphorylation in cell lysate. (B) Grayscale image analysis between them. (C,D) the mRNA level of TNFα and IL-1β. Data are expressed as mean ± SD (n = 6). *p < 0.05 versus Saline group. #p < 0.05 versus MYO-treated group F I G U R E 7 Mechanism of inhibiting PKCδ to reduce rhabdomyolysis-induced AKI. Myoglobin can activate PKCδ and promote cell apoptosis through p38 and ERK signalling pathways ERK1/2 pathways (Figure 7), thus providing evidence that PKCδ is a potent therapeutic target for rhabdomyolysis-induced AKI.

ACK N OWLED G EM ENTS
The study was supported in part by a grant from the National Natural

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used and/or analyzed are available from the corresponding author by reasonable request.