SHP2 inhibitor PHPS1 ameliorates acute kidney injury by Erk1/2-STAT3 signaling in a hemorrhage followed CLP mice model CURRENT STATUS: REVIEW

Background Hypovolemic shock and septic challenge are two major causes of acute kidney injury (AKI) in the clinic course. Src homology 2 domain-containing phosphatase 2 (SHP2) is one of the major phosphatase protein tyrosine phosphatase (PTPs), which play a significant role in maintaining immunological homeostasis by regulating many facets of immune cell signaling. In this study, we explored whether SHP2 signaling contributed to AKI induced by hemorrhage (Hem) followed by cecal ligation and puncture (CLP) (Hem/CLP) and, further, if inactivation of SHP2 with the selective inhibitor, Phenylhydrazonopyrazolone sulfonate 1 (PHPS1), attenuated this injury. improved I/R-AKI via inhibition of the TLR4/NF-kB Pathway. revealed that the SHP2 inhibitor PHPS1 exerted a protective effect against atherosclerosis via suppression of the SHP2:ERK pathway activation. Our current study indicates that SHP2 suppression significantly reduced the levels of phosphorylated Erk1/2 and STAT3 in kidney. This is important as Erk1/2 and STAT3 signaling pathways are thought to be widely involved in the course of inflammation.


Introduction
Trauma patients often suffer with hypovolemic shock and septic challenge simultaneously, which results in severe organ dysfunction (Karasu et al. 2019, Spahn et al. 2019). The kidneys, are one of the most commonly affected organs, receiving 20-25% of the resting cardiac output, while their weight is less than 1% of the total body mass. Hypothetically, making them more vulnerable and susceptible to the effect of blood volume loss and fluid shifts associate with sepsis, resulting in acute kidney injury (AKI). And while significant aspects of the pathogenesis of AKI have lead to better diagnostics/identification of clinical risk factors, so that supportive therapies are provided more judiciously (Honore et al. 2015); the mortality rates of AKI remain unacceptably high (Barbar et al. 2018, Uchino et al. 2005, White et al. 2013). Inasmuch; it is important to focus on the pathological mechanism that allow us to identify novel therapeutic targets so that we might leverage them to better treat the pathological development of AKI in the critically ill/severly injured patient.
The pathophysiology of AKI is thought to be induced by ischemic and/or septic events that lead to metabolic derrangments (Dellepiane et al. 2016). These derrangments are thought to be, in part, a result of the dys-balance between the system inflammatory response syndrome (SIRS) and/or antiinflammatory response that develops in the shocked/ septic patient/ experimental animal (Poston and Koyner 2019). Exposure to pro-inflammatory cytokines are thought to lead to renal cell death and dysfunction (Gomez et al. 2014). The administration of antibiotics directed at curbing septic pathogens also has been shown not to reduce the mortality rate associated with the development of AKI during the clinic course of the critically ill patient (Kellum et al. 2019). We and others have revealed that immune co-inhibitory receptor molecules appear to be involved in the pathology of the shocked/septic experimental animal's inflammatory response (Patil et al. 2017, Biron et al. 2018). As co-inhibitory receptors of the programmed cell death receptor-1 (PD-1) family bear an intracellular tyrosine inhibitory motif (ITIM) and/or intracellular tyrosine switch motif (ITSM), it has been shown that the suppression of T-cell function is a result of the recruitment of the phosphatase protein tyrosine phosphatase (PTP) family members (Keir et al. 2008, Riley 2009).
Src homology 2 domain-containing phosphatase 2 (SHP2) is one of the major PTPs. As such; it plays a significant role in maintaining immunological homeostasis by regulating many facets of immune cell signaling. Chichger et al. found that SHP2 activation protects the endothelial barrier against injury in the lung (Chichger et al. 2015). We recently reported that SHP1, but not SHP2, negatively modulated PD-L1 dependent regulation of T regulatory lymphocyte (Treg) function and in so doing contributed to resolving shock/sepsis-induced lung injury (Tang et al. 2015 activation may play distinct roles in different organs. Activation of SHP2 serves the role of a positive modulator of extracellular-regulated kinase (Erk) activity, which is downstream of the induction of a number of cytokines (Maroun et al. 2000). In addition to its role in growth factor receptor-bound protein 2 (Grb-2)-associated binder 1 (Gab1)-mediated Erk activation, SHP2 attenuates epidermal growth factor (EGF)-dependent phosphatidylinositol (PI)3 kinase activation by dephosphorylating Gab1 at the p85 binding sites (Zhang et al. 2002). It is thought that SHP2 is involve in the aspects of the inflammatory response that depend on Erk signaling. Thus, our study sets out to examine the hypothesis that the development of AKI, as a result of the sequential insults of hemorrhagic shock followed by septic challenge, is mediated, in part, through dysregulated SHP2 activation acting in-turn on Erk1/2 and signal transducers and activators of transcription (STAT)3.

Materials And Methods Animals and groups
All experiments were performed in accordance with National Institutes of Health guidelines and approved by the Animal Use Committee of Rhode Island Hospital (AWC# 5064-18). A total of 18 male C57BL/6 mice (10 to 12 weeks old) were included in the experiment. The animals were maintained in a 12/12-hour light/dark cycle at ambient temperature (23-25 °C) and provided with standard laboratory rodent chow and water ad libitum.
Mice were randomly divided into three groups of 6 animals each: 1) the control group: mice underwent sham hemorrhage (Hem) and sham septic surgical procedure (S/S); 2) the Hem/cecal ligation and puncture (CLP) sepsis + vehicle group; and 3) the Hem/CLP + PHPS1 (inhibitor of SHP2) group.

Experimental protocol
Hemorrhage and sepsis were elicited as described previously in our laboratory (Biron et al. 2018). In brief, bilateral femoral arteries of mice were catheterized under anesthesia. The mean blood pressure of arteries was continuously monitored through one catheter attached to a blood pressure analyzer (MicroMed, Louisville, Ky). When recovered from anesthesia, the mice were bled over a 5 to 10 min period to a mean blood pressure of 35 ± 5 mmHg and kept stable for 90 min. Then, mice were resuscitated by infusion with four times drawn blood volume of Ringer's solution. Mice in the S/S group were anesthetized and restrained in a supine position, and blood vessels were ligated, but no blood was drawn.
Twenty four hours after Hem, mice were submitted to sepsis, as elicited by the CLP technique as described previously (Biron, et al. 2018). Briefly, mice were anesthetized by isoflurane and a midline abdominal incision was performed. The cecum was mobilized and ligated at its middle portion below the ileocecal valve, punctured twice using a 22-gauge needle, and a small stool sample was squeezed out of the cecum to induce polymicrobial peritonitis. The abdominal wall was closed in 2 layers. Mice in the S/S group underwent the same procedure, including opening of the peritoneum and exposing the bowel, but without ligation or needle perforation of the cecum. After surgery, the mice were resuscitated by a subcutaneous injection of pre-warmed (37 °C) 0.6 mL normal saline.
The SHP2 selective inhibitor, Phenylhydrazonopyrazolone sulfonate 1 (PHPS1) (Cayman Chemical, Ann Arbor, MI), at 3 mg (dissolved in DMSO/PBS = 1:1 solvent and)/kg body weight was administered by a subcutaneous injection once immediately after Hem and, once again, following the performance of CLP. Twenty four hours after CLP (48 hours after Hem), mice were killed, blood and kidneys were collected for analyses.

Determination of serum biochemical indicators
Blood urea nitrogen (BUN) and creatinine (Cre) concentrations were measured using corresponding kits (Abcam, Cambridge, MA). Blood inflammatory cytokine/chemokine levels were detected by commercial ELISA kit (BD Biosciences, San Jose, CA). All procedures were performed according to the manufacturer's instructions.

Calculation of kidney/body weight index (KI).
The left kidney was harvested, weighed, frozen rapidly in liquid nitrogen and stored at − 80 °C for subsequent analysis. The kidney/body weight index (KI) was calculated as: kidney wet weight (mg)/total body weight (g) × 100%. Under normal conditions, KI is relatively constant; when the kidneys suffering congestion, edema even hypertrophy, the ratio of kidney to body weight increased ).

Statistical analysis
Values are expressed as the mean ± standard deviation. The statistical analysis was performed with the SPSS version 24.0 statistical software package (IBM Inc., Armonk, NY, USA). Data were tested for normality and equality of variance. Comparisons among the three groups for each dependent variable were performed using an analysis of variance (ANOVA) with a post hoc Newman-Keuls multiple comparison test. The level of statistical significance was set at P < 0.05.

Results
Hem/CLP-induced SHP2 activation in the kidney is attenuated by PHPS1 treatment PHPS1, a cell-permeable highly selective inhibitor for SHP2, has been shown to inhibit SHP-2dependent cellular signaling and tumor cell colony formation (Hellmuth et al. 2008). In this study, subcutaneous injection of mice with PHPS1 immediately after Hem and CLP procedure significantly decreased the level of activated SHP2 detected when compared with vehicle-treated mice as shown in Here, we observed a marked increase in the KI of Hem/CLP mice. This is important since an increased KI reflects kidney damage such as edema, hypertrophy and organ congestion. The KI in Hem/CLP group mice increased compared with S/S group, however, PHPS1 treatment suppressed this increase as shown in Fig. 2. This implies that PHPS1 treatment directly or indirectly reduced the kidney edema and cell hypertrophy occurring here.
SHP2 inhibition provides a renal protective effect in Hem/CLP mice.
As expected, mice subjected to Hem/CLP procedure exhibited a significantly elevated BUN and Cre levels in serum when compared to that of S/S control mice. Here again, treatment with PHPS1 significantly improved renal function by reducing the levels of BUN and Cre ( Fig. 3A and 3B).
It has been reported that NGAL is a promising biomarker for diagnosing acute kidney injury (Kellum et al.). Here we attempted to detect changes in the expression of NGAL in the kidney by Western Blot.
As shown in Fig. 3C, NGAL was significantly upregulated in renal tissue of both Hem/CLP group mice compared with S/S mice. Interestingly, Hem/CLP group administered the SHP2 inhibitor PHPS1 exhibited reduced NGAL levels compare to vehicle-treated Hem/CLP mice. We also examined the change induced in cleaved (activated) caspase 3 in renal tissue by Western Blot. Hem/CLP increased the expression of cleaved caspase 3 compared to S/S mice, however, the injection of Hem/CLP with PHPS1 attenuated the rise in cleaved caspase 3 to levels seen is S/S mice, when compared to vehcle treated Hem/CLP mice (Fig. 3D).
The changes in kidney histopathologic induced by Hem/CLP are attenuated by PHPS1 treatment.
As shown by histology examination in Fig. 4, Hem/CLP caused an increase in pathological changes in the kidney; including tubular epithelial cell sloughing, edema, inflammatory cell infiltration, loss of brush border, tubular dilation, and tubular distortion, when compared with that of S/S mice. However, PHPS1 treatment markedly ameliorated these changes and preserved the renal architecture.
The Hem/CLP-induced increase in systemic blood inflammatory cytokine/ chemokine levels is suppressed by PHPS1 administration.
The levels of inflammatory factors assessed included IL-6, TNF-α, IL-10, as well as chemokines KC and MIP-2 in blood that were significantly increased in the Hem/CLP group mice when compared with S/S group. Alternatively, while PHPS1 injection dramatically reduced the levels of TNF-α, IL-10, and MIP-2.
Interestingly, there was no significant reduction/change observed in IL-6 and KC levels following Hem/CLP (Fig. 5). To further illuminate the nature of the protective effects of PHPS1 on the development of AKI here, we chose to examine the extent to which not only SHP2, but Erk1/2 and STAT3, were activated/phosphorylated in renal tissue. As shown in Fig. 7, compared to S/S mice, Hem/CLP induced marked activation of SHP2, Erk1/2 and STAT3, as shown by the significant elevation in the ratio of phosphprylated-SHP2:total-SHP2, phosphorylated-Erk1/2:total-Erk1/2 and phosphorylated-STAT3:total-STAT3. However, inhibition of SHP2 activation with PHPS1 treatment, markedly decreased this Hem/CLP-induced rise.

Discussion
These findings demonstrate that, hemorrhagic shock followed by sepsis can induce significant kidney injury as well as lung injury as we have previously documented (Ayala et al. 2002). This study also provides some of the first evidence demonstrating the therapeutic potential of PHPS1 as a treatment directed against AKI induced by Hem/CLP, as our data revealed that PHPS1 could improve renal function. The mechanism we would propose to account for protective effect of PHPS1 involves However, once produced in a sequential fashion, Hem followed CLP, indices of lung injury were clearly induced (Ayala, et al. 2002) and that appears to also be the case for AKI based on this study. In this respect, patients suffering from trauma often have significant blood loss and receive fluid during recovery in emergency room. And while hemostatic supportive care is critical during this initial period, the impact of this initial event on patient's capacity to handle subsequent insults, infectious or otherwise, can be significant, culminating in multiple organ failure and death in the most severe cases. Using a rodent model of Hem in combination with a subsequent septic challenge, which approximates aspects of what is seen in traumatic shock patients, our laboratory has shown that injection of PHPS1, a specific inhibitor of SHP2, attenuates Hem/CLP-induced AKI in mice. The mechanism by which SHP2 inhibition protects against double hit induced AKI remains poorly understood. SHP2, encoded by the PTPN11, plays a significant role in maintaining homeostasis during inflammation by regulating many facets of cell signaling. Our data indicate that injection of SHP2 inhibitor PHPS1 attenuated the release of inflammatory cytokines and renal tissue HMGB1 levels.
Previous studies have revealed that select inflammatory signaling pathways are involved in the processes driving AKI. Teng et al. (Teng, et al. 2018) showed that the lentivirus-mediated silencing of SHP2 improved I/R-AKI via inhibition of the TLR4/NF-kB Pathway. Chen et al.  revealed that the SHP2 inhibitor PHPS1 exerted a protective effect against atherosclerosis via suppression of the SHP2:ERK pathway activation. Our current study indicates that SHP2 suppression significantly reduced the levels of phosphorylated Erk1/2 and STAT3 in kidney. This is important as Erk1/2 and STAT3 signaling pathways are thought to be widely involved in the course of inflammation.
That said; there are several limitations to the current study, which should be considered. First, we found an amelioration of systemic and local organ inflammation by SHP2 blockade, however, what the cellular target(s) are involved in the course of mediating the SHP2 to activation of Erk 1/2 and/or STAT3 signaling is still unclear. In previous studies, our laboratory found SHP1 negatively modulated PD-L1 dependent regulation of Treg cell function during the resoltion of shock/sepsis-induced lung injury (Tang, et al. 2015). As a component of co-inhibitory receptor signaling sequence of PD-1, how SHP2 recruitment actually suppresses T-cell function in Hem/CLP is incompletely understood. Further studies will be needed to confirm the phenotypic changes that occur at a cellular level in leukocytes from Hem, CLP or Hem/CLP mice. Secondly, while we chose to use PHPS1 to selectively inhibit SHP2 phosphorylation/activation here, it has been reported that when activation of SHP2 by many stimuli include IL-1 and IL-6, growth factors (insulin, EGF, FGF, etc.), they produced a deleterious effect in cancer (Zhang et al. 2015), which is supportive of our findings. Additionally, different species have different sensitivity to such agents, so it is unclear whether PHPS1 would necessarily have a similar positive effect in the clinical trauma patient. Inasmuch; we feel these data indicate that further clinical research should be done to establish if such issues exist. Finally, Erk1/2 and STAT3 signaling pathway are both involved in mediating IL-6 inflammatory signaling, however, IL-6 levels in our model were not effected by PHPS1 treatment. This suggests, there may be other cytokines involved in the course of this double hit induced kidney injury that are mediating the effects of PHPS1 inhibition in the kidney. Perhaps hemorrhagic shock induced noninfectious inflammation is a critical contributory factor in this model, however, its precise mechanism of action is still unclear. Despite this, our data also indicates that the inhibition of Erk1/2 and/or STAT3 signaling may be the key aspects of the protective actions of PHPS1 treatment against Hem/CLP induce AKI.

Conclusion
In conclusion, our data support the tenet that SHP2 inhibition attenuates AKI-induced by our doublehit/sequential insult model of Hem/CLP and that this protective action may be attributable to its antiinflammatory property in mitigating activation of the Erk1/2 and STAT3 signaling pathway. This Conflict of Interest: It should also be noted that all authors concur with the material submitted in this manuscript and that none of the authors have any financial interests or affiliations with commercial organizations whose products or services are related to the subject matter of this       Effects of PHPS1 on expression of HMGB1 in kidney after Hem/CLP. The kidneys were collected twenty four hours post Hem/CLP or S/S operation and tissue homogenates were obtained. The expression of HMGB1 was determined by western blot. The levels of HMGB1 from Hem/CLP mice was significantly increased compared to S/S. PHPS1 treated Hem/CLP mice showed reduced HMGB1 levels compared to Veh treated HEM/CLP mice. *, P<0.05, versus S/S; #, P<0.05, PHPS1 treated group (Hem/CLP + PHPS1) versus Hem/CLP+vehicle (Hem/CLP). One-way ANOVA and a Student-Newman-Keuls' test, Mean ± SD; n=6 mice/group.