Tubular epithelial C1orf54 mediates protection and recovery from acute kidney injury

Abstract Acute kidney injury (AKI) incidence among hospitalized patients is increasing steadily. Despite progress in prevention strategies and support measures, AKI remains correlated with high mortality, particularly among ICU patients, and no effective AKI therapy exists. Here, we investigated the function in kidney ischaemia‐reperfusion injury (IRI) of C1orf54, a newly identified protein encoded by an open reading frame on chromosome 1. C1orf54 expression was high in kidney and low in heart, liver, spleen, lung and skeletal muscle in healthy mice, and in the kidney, C1orf54 was expressed in tubular epithelial cells (TECs), but not in glomeruli. C1orf54 expression was markedly decreased on Day 1 after kidney IRI and then gradually recovered to baseline levels by Day 7. Notably, relative to wild‐type mice, C1orf54‐knockout mice exhibited impaired TEC proliferation and delayed recovery after kidney IRI, which led to deteriorated renal function and increased mortality. Conversely, adenovirus‐mediated C1orf54 overexpression promoted TEC proliferation and ameliorated kidney pathology, which resulted in accelerated renal repair and improved renal function. Mechanistically, C1orf54 was found to promote TEC proliferation through PI3K/AKT signalling. Thus, C1orf54 holds considerable potential as a therapeutic target in kidney IRI.

Local tissue oxygen supply and demand and accumulation of metabolism waste products are mismatched, and this results in tubular epithelial cell (TEC) injury, which, if severe, causes cell death by apoptosis and necrosis (acute tubular necrosis), coupled with organlevel functional impairment of water/electrolyte homeostasis and reduced excretion of metabolism waste products. 4,5 However, because the mechanism underlying kidney IRI is largely unknown, a treatment strategy is lacking.
The kidney can repair itself, 5 and a crucial pathologic feature of post-AKI repair is renal TEC proliferation and regeneration. 6 Cell proliferation repairs the damaged kidney by replacing TECs lost due to cell death. 4 Accumulating evidence indicates that paracrine signalling from endogenous surviving epithelial cells (eg distal TECs) could underlie anti-IRI effects, and multiple factors secreted from distal nephrons could produce paracrine effects to promote the proliferation and repair of surviving tubular cells through cell-to-cell crosstalk. 4,6 Chromosome 1 is the largest human chromosome and containş 8% of all human genetic information and thus might be more representative of the human genome than other chromosomes. 7 Chromosome 1 harbours C1orf54, which encodes a predicted secreted protein of mostly unknown function. As a secreted protein, we proposed that C1orf54 may have some function in the physiological or diseased states. Thus, we generated C1orf54 deficiency mice to examine its role in diseased state. In this study, we demonstrated that C1orf54 was expressed exclusively in renal TECs, and by gain and loss of function studies, we revealed that C1orf54 promoted renal repair and TEC proliferation through PI3K/AKT signalling, which alleviated kidney damage after IRI.  with loxp sites and a pGK-neomycin-polyA cassette. The vector was electroporated into B6/129 embryonic stem (ES) cells, which were then selected with 2 drugs, G418 and ganciclovir, to screen for homologous recombination clones. Long PCR and sequencing were used to identify and confirm the ES clones exhibiting correct homologous recombination, which was genotyped with these primers: 5′-A   CCCTTGGTGTCTATGCTGGTC-3′ and 5′-CTGGAAGATGTCCGTGG   TGTTA-3′, for correct 5′-homology-arm recombination; and 5′-CA   AAGAGGGTGAGAAGGTAAGC-3′ and 5′-CAGACATCAATAGGAGCA GGAAT-3′, for correct 3′-homology-arm recombination. Positive ES cell clones were expanded and microinjected into C57BL/6J blastocysts to generate chimeric mice, which were crossed with C57BL/6J mice to obtain C1orf54-floxed heterozygous (C1orf54 flox/+ ) mice. The C1orf54 flox/+ mice were mated with EIIA-Cre mice (Tg (EIIa-Cre); The Jackson Laboratory) expressing Cre recombinase in the early embryo to obtain heterozygous C1orf54-knockout (C1orf54 +/− ) mice, which were intercrossed to generate the homozygous C1orf54-knockout (C1orf54 −/− ) mice used here.

| AKI induction in mice
Kidney IRI was induced as described. 8 Mice were anesthetized by intraperitoneally injecting sodium pentobarbital (50 mg/kg bodyweight; Sigma-Aldrich, St. Louis, USA) before surgery, during which both kidneys were exposed through a flank incision and the kidney pedicles were clamped for 30 minutes at 37°C by a heat device.
Sham-operated animals underwent the same procedure except for the renal pedicle clamping. For the histological analysis of kidney from sham-operated mice, we killed the mice on Day 7 after operation.

| Histology and Immunohistochemistry
Kidneys were fixed in 4% paraformaldehyde (24 hours) and embedded in paraffin, and then, 5-μm sections were subject to PAS and Masson's trichrome staining as per standard protocols. At least 10 random fields from each sample were analysed. Immunohistochemical staining was performed with anti-C1orf54 (Sigma-Aldrich, St. Louis, USA) as described, 9 and C1orf54-positive cells were counted under a microscope (Olympus, 400× magnification).

| Renal-damage evaluation
The areas of PAS-stained debris (at corticomedullary junction) and brush borders (at corticomedullary junction and cortex region) in kidney specimens were quantified with NIH ImageJ software, and the percentages of these areas relative to the entire section in a slide are presented; 5 kidney sections from at least 3 mice from each group were examined. Specimens were evaluated in a manner blinded to the mouse strain.

| Serum BUN and creatinine measurement
At the end of experiments, mice were anesthetized, and blood was collected from the retro-orbital plexus. Serum creatinine (Cre) and BUN concentrations were measured with creatinine serum kit (2 whole plate kit, Arbor Assays, KB02-H1) and urea nitrogen (BUN) detection kit (2 plates, Arbor Assays, K024-H1).

| TUNEL assay
To examine apoptosis in damaged kidneys from WT and C1 KO mice post-IRI, we used an in situ cell death detection kit (Roche, 11684795910) for TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labelling) staining of apoptotic cells in renal sections.
TUNEL + cells were counted by researchers blinded to the mouse groups.

| Statistical analyses
Power was not calculated to predetermine sample sizes, and randomization was not used to determine samples or mice to be allocated to experiments. Areas were calculated in a blinded manner. In vitro experiments were repeated at least thrice. Data were analysed with GraphPad Prism (GraphPad Software Inc., San Diego, CA) and are presented as means ± SD unless specified otherwise. Paired results were assessed with parametric tests (eg Student's t test). Multiple groups were compared with 1-way or 2-way ANOVA followed by Bonferroni's post hoc test. For Kaplan-Meier curves, P values were determined with the log-rank test.

| C1orf54 was highly expressed in renal TECs and down-regulated after kidney IRI
No C1orf54 function in the body has been reported previously.
Here, we began by examining C1orf54 expression in different organs and tissues in healthy mice: Immunohistochemical staining (Figure 1A,B) revealed for the first time that C1orf54 expression was high in the kidney but low in heart, liver, spleen, lung and skeletal muscle and that renal C1orf54 was expressed in TECs but not glomeruli ( Figure 1A). Unexpectedly, C1orf54 was shown to be highly expressed in small intestine ( Figure S1A). Furthermore, double stainings with C1orf54 and villin confirmed that C1orf54 was exclusively in TECs, and specifically, it localized in both nucleus and cytoplasm ( Figure 1C).

| C1orf54 deficiency exacerbated renal dysfunction after kidney IRI
To assess C1orf54's pathophysiological role in kidney IRI, we gener-

| C1orf54 was required for hypoxia/ re-oxygenation-induced TEC proliferation
We examined the mechanism underlying C1orf54-mediated thus, we examined whether these signalling pathways participate in C1orf54-mediated TEC proliferation. We found that kidney IRI led to P38, JNK, STAT3 and β-catenin activation, while deficiency of C1orf54 did not alter phosphorylation of P38, JNK and STAT3, and expression level of β-catenin ( Figure 8A,B), indicating that these signalling pathways were not essential for TEC proliferation. Next, we revealed that IRIinduced AKT phosphorylation was considerably lower in C1 KO mice than in WT mice ( Figure 6). G, H, WT mice were transfected with C1orf54-overexpressing adenovirus, and wortmannin (PI3K inhibitor) was intraperitoneally injected into the mice once every other day, and serum BUN and Cre levels were examined at different time-points after kidney IRI (n = 6). Data are expressed as means ± SD. **P < .01, *P < .05 For kidney repair, "cell-to-cell crosstalk" and growth factors are reported to be essential, and secreted proteins could function as growth factors that facilitate tissue repair and regeneration. [15][16][17] Approximately 2000 proteins in the proteome have been reported to possess the structural features necessary for secretion into the extracellular milieu, but the biological functions of these proteins are largely unknown. Recently, the bone marrow cell secretome was screened, and after bioinformatic analysis was used to eliminate genes characterized or predicted to encode potential nonsecreted proteins, 150 genes were analysed 18 ; 2 proteins, C19orf10 ("myeloid-derived growth factor") and C1orf54, were The kidney IRI model is commonly used to study the AKI mechanism. By this model together with C1orf54 deletion and adenovirusmediated overexpression, we demonstrated that C1orf54 was essential for TEC proliferation and kidney repair, but did not affect TEC apoptosis. C1orf54 was reported to potentially function in the pathophysiology of diffuse congenital hyperinsulinism through the IGF-1/mTOR/AKT pathway, 19 and the PI3K/AKT pathway is known to play a critical role in cell proliferation. 20,21 Here, we demonstrated that AKT phosphorylation was inhibited in C1orf54-deficient mice after kidney IRI and that this could be reproduced in HK-2 cells subject to hypoxia/reoxygenation.
In the present study, on Day 5 post-IRI, about 50% of the C1orf54 KO mice were dead. However, kidney function as indicated by BUN and creatinine already started to drop on Day 3, which points to an additional risk factor beside AKI in the KO mice. This paradox may be due to the following: First, despite the gradual recovery of renal function, but it has not returned to normal, there is still a serious renal insufficiency; Second, acute renal insufficiency is often complicated with other organ injuries, namely multiple organ dysfunction, which may result in its death later after kidney ischaemia injury.
We suspect that C1orf54 acts as a protein that is secreted from TECs damaged after kidney IRI. Immunofluorescence imaging revealed certain C1orf54-positive particles around surviving and necrotic tubules, and Western blotting results showed that serum C1orf54 levels were low in sham-operated mice and markedly increased in mice subject to kidney IRI. However, further investigation is required to ascertain how C1orf54 is released from TECs and how C1orf54 regulates PI3K/AKT signalling.
In conclusion, this study demonstrated for the first time that C1orf54 was expressed exclusively in renal TECs and promoted TEC proliferation and kidney repair post-IRI by acting through the PI3K/ AKT signalling pathway. Therefore, our findings identify a potential target for AKI treatment.

ACKNOWLEDG MENTS
This study was supported by the National Natural Science Founda- There are no relationships with industry.

CONFLI CT OF INTEREST
The authors confirm that there are no conflict of interests.