Phosphoprotein expression profiles in rat kidney injury: Source for potential mechanistic biomarkers

Acute kidney injury (AKI) is defined by the Acute Kidney Injury Network as “functional and structural disorder or signs of renal damage including any defect from blood or urine test, or tissue imaging that is less than 3 months.” AKI is the most common cause of renal dysfunction.1 Understanding of molecular determinants of AKI induction and development could help to diagnose and monitor kidney injury in the clinic as well as to select safe drug candidates in preclinical drug development. 
 
Protein phosphorylation plays a significant role in a wide range of cellular processes and can directly indicate an active/inactive state of cellular enzymes and the signalling pathways in injury initiation and progression. Alteration of phosphorylation of some proteins was already described for kidney injury: MAPK activation in cisplatin‐induced nephrotoxicity,2 increased phosphorylation of moesin and HSP90ɑ in tubular cells in response to TGF‐β3 and increased phosphorylation of lamin A and phospholamban in a salt‐load rat model of kidney damage.4 
 
Our study aimed at identifying changes in phosphoprotein expression and creating comprehensive phosphoprotein profiles of nephron‐specific injuries in a time‐dependent manner. To meet this objective, we utilized rat models of drug‐induced kidney injury (DIKI), which were induced with three nephrotoxicants over a 28‐day period: cisplatin (proximal tubules); puromycin (glomerulus) and N‐phenylanthranylic acid (NPAA, collecting ducts). The phosphoproteins were studied by antibody microarrays in kidney tissue on days 7, day 14 and day 28. We established changes in phosphoprotein expression in response to kidney injury and associated these changes with biological processes. Up‐regulation of MEK2 (pThyr394) on day 14 in cisplatin and puromycin‐induced injuries was confirmed by immunohistochemistry (IHC). Identified phosphoprotein signatures provide a link to molecular mechanisms of kidney injury/recovery as well as a source for potential mechanistic biomarkers.


| INTRODUCTION
Acute kidney injury (AKI) is defined by the Acute Kidney Injury Network as "functional and structural disorder or signs of renal damage including any defect from blood or urine test, or tissue imaging that is less than 3 months." AKI is the most common cause of renal dysfunction. 1 Understanding of molecular determinants of AKI induction and development could help to diagnose and monitor kidney injury in the clinic as well as to select safe drug candidates in preclinical drug development.
Protein phosphorylation plays a significant role in a wide range of cellular processes and can directly indicate an active/inactive state of cellular enzymes and the signalling pathways in injury initiation and progression. Alteration of phosphorylation of some proteins was already described for kidney injury: MAPK activation in cisplatin-induced nephrotoxicity, 2 increased phosphorylation of moesin and HSP90ɑ in tubular cells in response to TGF-β 3 and increased phosphorylation of lamin A and phospholamban in a salt-load rat model of kidney damage. 4 Our study aimed at identifying changes in phosphoprotein expression and creating comprehensive phosphoprotein profiles of nephron-specific injuries in a time-dependent manner. To meet this objective, we utilized rat models of drug-induced kidney injury (DIKI), which were induced with three nephrotoxicants over a 28-day period: cisplatin (proximal tubules); puromycin (glomerulus) and Nphenylanthranylic acid (NPAA, collecting ducts). The phosphoproteins were studied by antibody microarrays in kidney tissue on days 7, day 14 and day 28. We established changes in phosphoprotein expression in response to kidney injury and associated these changes with biological processes. Up-regulation of MEK2 (pThyr394) on day 14 in cisplatin and puromycin-induced injuries was confirmed by immunohistochemistry (IHC). Identified phosphoprotein signatures provide a link to molecular mechanisms of kidney injury/recovery as well as a source for potential mechanistic biomarkers.

| In vivo study design and kidney collection
The study design is described in detail by Obajdin et al. 5

| Data analysis
The signal intensities of phosphorylated proteins were normalized to non-phosphorylated proteins and the rations were compared between treatments and vehicle controls to calculate fold changes (FC) at matched time-points using t test. The fold change of 1.5 and P ≤ 0.05 was applied to select significantly dysregulated phosphoproteins. To analyse the implication of selected candidates in different biological processes, PhosphoSitePlus was utilized.
Immunostaining was performed by using an Omni Map DAB kit (Roche, 760-4310). Slides were scanned using a NanoZoomer XR scanner (Hamamatsu, Japan) at a 20× magnification for whole slide imaging and visualized using NDP.view 2 software (Hamamatsu, Japan).

| IHC analysis of MEK2 (pThr394) expression in kidney tissue
We selected MEK2 (pThr394) to further investigate its expression in kidney tissue by IHC. No changes were observed in MEK2 (pThr394) phosphorylation for any treatment compared to control on day 7 after drug administration (data not shown). On day 14, an increase in MEK2 (pThr394) phosphorylation was detected after cisplatin and puromycin administration. Increased nuclear staining was observed in dilated tubules after puromycin treatment and cytoplasmic staining in the affected tubules after cisplatin treatment (Figure 1).

| Analysis of differentially expressed phosphoproteins
Based on the phosphorylation sites of the selected phosphoproteins, we determined their implication in different biological processes by PhosphoSitePlus ( Figure S1). Main processes affected in all treatments on day 7 were apoptosis and cytoskeletal reorganization.

| 2253
Available data on phosphoproteins dysregulation in the context of kidney injury are scarce and focuses mainly on MAPK activation in AKI. 2 Here, we analysed three rat models of DIKI using phosphoprotein microarrays. Cisplatin, puromycin and NPAA were selected based on their ability to induce proximal tubule, glomerular and collecting duct injury respectively. [5][6][7] This allowed us to identify phosphoprotein signatures associated with region-specific kidney injury.
Cisplatin-induced injury on day 7 resulted in altered phosphorylation of p53, MAPK3K7, JNK1, NFkB and IKK. Previously, activation of p53 was associated with tubular apoptosis in cisplatin-induced injury in mice. 8 It was also shown that cisplatin-induced activation of ERK p38 and JNK/SAPK in vivo preceded the development of AKI. 9 Phosphorylation of p38-MAPK and NF-κB was found to be up-regulated in rat kidneys following gentamicin treatment. 10  Daily administration of NPAA also induced significant changes in phosphoprotein expression which were related to apoptosis and cytoskeletal reorganization. Interestingly, on day 14, several phosphoproteins involved in transcription were down-regulated. We also observed up-regulation of phosphorylation of metabotropic glutamate receptor 1 (mGluR1), which is part of glutamatergic system in the brain. Interestingly, renal receptors for glutamate, including mGluR1, were shown to be modulated in AKI. 11 In summary, we established DIKI-associated phosphoprotein signatures in the rat by using kidney tissue and antibody-based arrays.
These phosphoprotein profiles varied depending on a time-point and a nephron-specific injury analyzed. Altered phosphoprotein levels were confirmed for pMEK (Thyr394) by IHC. Linking phosphoprotein changes and biological processes in the kidney upon injury helps to understand different molecular mechanisms underlying kidney damage/recovery. Selected candidates can be used for further investigation and validation as mechanistic biomarkers in vitro models.

ACKNOWLEDG EMENTS
This work was supported by a grant from the Walloon Region (Belgium) -[DGO6 (Convention N°7245)].

CONFLI CT OF INTEREST
The authors confirm that there are no conflicts of interest.