Kidney Injury Molecule-1 Mediated Autophagy Pathway Participates in Ischemic Preconditioning Evoked Renal Protection


 Background

Ischemia-reperfusion injury (IRI) is one of the most vital pathogenesis causing kidney injury, especially during the perioperative periods of partial nephrectomy and renal transplantation, but lacking of effective prevention and treatment strategies. This study was conducted to investigate the influences of ischemic preconditioning (IPC) on the pathological process of mouse bilateral IRI, and to figure out the role of kidney injury molecule-1 (KIM-1) mediated autophagy pathway in this process.
Methods

Nephron major histocompatibility complex class II (MHC II) conditional knockout (cKO) mice (Six2-Cre+/−; MHC II flox/flox) and the age- and sex-matched littermates from the Six2-Cre−/−; MHC II flox/flox colony (Ctrl) were established to investigate these issues. A 15-minute period of IPC was performed 4 days before the 30-minute of bilateral renal vessel occlusion. Severity of renal IRI in cKO and Ctrl mice with or without IPC were analyzed respectively and correspondingly.
Results

MHC II cKO mice presented severer kidney injury in both acute and chronic phase of renal IRI. IPC could significantly attenuate ischemia/reperfusion-induced serum creatinine (sCr) and blood urea nitrogen (BUN) increasing, as well as histological KIM-1 expression. However, MHC II cKO mice undergoing IPC showed more deteriorated kidney injury when compared with Ctrl mice, with higher levels of sCr, BUN, KIM-1 expression in the acute phase, and aggravated interstitial fibrosis in the chronic phase.
Conclusions

IPC could attenuate renal IRI functionally and histologically. KIM-1 mediated autophagy pathway plays a vital role in the IPC induced renal IRI protection.


Background
Ischemia-reperfusion injury (IRI) is one of the most vital pathogenesis leading to kidney injury, especially during the perioperative periods of partial nephrectomy and renal transplantation [1]. Multiple strategies, including renoprotective drugs (diuretics, sodium bicarbonate, etc.) and renal replacement therapy have been brought up to prevent or treat renal IRI. However, most of these efforts have yielded limited success [2]. Ischemic preconditioning (IPC) is a kind of endogenous phenomenon that promotes tissue tolerance to IRI by a brief episode of ischemia and subsequent reperfusion before the index ischemic event, and may be a highly appealing, nonpharmacological and practical approach to attenuate renal IRI [3].
Since the concept of IPC was rstly proposed by Murry et al. in 1986[4], several studies have assessed the organ protective effect of IPC in various tissues and organs as well as across several species [5][6][7][8]. Over the past three decades, both experimental and clinical researches have demonstrated that IPC could enhance kidney tolerance to ischemic injury and has therapeutic potential for the prevention and/or reversal of the pathological sequelae associated with acute kidney injury (AKI) [9][10][11][12][13]. Nitric oxide [14], protein kinase C [15], MAP kinase and MAPKAP kinase 2 [16], NF-κB [17], mitochondria [18], microRNA [19], and decreased capacity of immune cells [20], etc. have all been implicated in mediating the protective effects of IPC. But the pivotal mechanisms of IPC still remain unclear.
Kidney injury molecule-1 (KIM-1) mediated epithelial cell phagocytosis of apoptotic or necrotic cells belongs to autophagy pathway which involves the process of KIM-1 mediated phagocytosis to autophagy induction, major histocompatibility complex (MHC) presentation and maintenance of peripheral T-cell tolerance [21], and is reported playing an important role in the process of kidney injury and epithelia renovation in both animal models and human diseases [22]. However, the role of this pathway in IPC evoked renal IRI protection is still unknown.
Thus, this study was conducted to investigate the in uences of IPC on the pathological process of mouse bilateral IRI, and to gure out the role of KIM-1 mediated autophagy pathway in this process by establishing a nephron MHC II conditional knockout (cKO) mouse model. [8][9][10][11][12] week old male nephron MHC II cKO (Six2-Cre +/− ; MHC II ox/ ox ) mice on a C57BL/6J background and the age-and sex-matched littermates from the Six2-Cre −/− ; MHC II ox/ ox colony (used as control (Ctrl)) weighing 20-25 g obtained from animal experiment center (Super-B&K Laboratory Animal Corp.

Animals
Ltd, Shanghai, China) were used in all experiments. Mice had free access to food and water, and were housed and maintained at Xinhua Hospital Laboratory Animal Resource Facility. Animals were assessed daily by veterinary staffs at our institution and by quali ed investigators in our group. All animal procedures were approved by the Animal Ethics Committee of Xinhua Hospital, and in conformity to the Guide for Care and Use of Laboratory Animals.
Bilateral renal IRI and IPC mouse model Brie y, experimental mice were anesthetized with iso urane (1.6-1.8%). Under aseptic conditions, two dorsal ank incisions were performed and the renal pedicles of both sides were identi ed and clamped using a small non-traumatic clamp (4*0.75 mm/16 mm, RWD Life Science, San Diego, CA, USA). After visual con rmation of ischemic changes, the kidneys were returned to the retroperitoneal cavity and vessel occlusion was maintained for 15 minutes [23]. The clamps were then released and blood ow restoration con rmed visually. Incisions were closed in two layers using 4 − 0 absorbable sutures and animals were volume resuscitated using normal saline (0.2 mL) injected subcutaneously. Subcutaneous buprenorphine (0.075 mg/kg) was given every 8-12 hours to ensure post-operative analgesia till 48 hours. 4 days later [23], the same procedure was performed again expect for the vessel occlusion maintaining for 30 minutes. Animals in sham-operated group were anesthetized and two ank incisions performed, renal pedicles dissected but not clamped. They were maintained for corresponding minutes mentioned above and the incisions were then closed in two layers.

Tissue harvest
To study acute phase of renal IRI, we allowed reperfusion for 2 days post-ischemia. To investigate postischemic progression to brosis, mice were followed for 42 days after injury. Following the reperfusion phase, animals were anesthetized, blood was obtained by mice tail at the time-point of 24 hours, 72 hours, and weekly from 1 week to 5 weeks after the surgery, and by cardiac puncture at the time-point of 6 weeks. Native kidneys were obtained from healthy anesthetized animals and processed immediately.
Mice were euthanized by high concentration of iso urane. Kidneys were surgically removed under anesthesia after reperfusion for 20 mL of phosphate-buffered saline (PBS), and then sectioned after removal of the renal capsule and extrarenal structures. Each kidney was sectioned along the transverse axis into two pieces: upper (3-to 4-mm length) and lower (3-to 4-mm length). The upper pieces were xed in 4% paraformaldehyde in 1X PBS for 24 hours at room temperature. Following xation, tissue samples were routinely processed and embedded in para n wax (Fisher Scienti c, Pittsburgh, PA, USA), and then for H&E staining, picrosirius red staining and immuno uorescence staining, etc. The medulla of the lower pieces were removed and the cortex were cut into small pieces and then ashed frozen in liquid nitrogen and then stored at -80 °C for further protein studies.

Detection of kidney injury related indicators
Western blot and immuno uorescence staining were used to detect the expression of KIM-1, cytokines including connective tissue growth factor (CTGF), alpha smooth muscle actin (α-SMA), transforming Picrosirius red staining and H&E staining, as well as sCr and BUN measurement were also conducted according to the manufacturer's protocols (abcam, Cambridge, MA, USA).

Statistical analysis
Statistical comparisons were performed using the statistical software GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA). The intergroup differences were tested using the Student's t test or analysis of variance (ANOVA). Statistical signi cance was de ned as a p < 0.05.

Phenotype of the nephron MHC II cKO mouse
To knock out MHC II gene from nephron, we crossed MHC II / mice (Six2-Cre −/− ; MHC II ox/ ox ) with transgenic mice expressing Six2-Cre. The Six2 gene has been linked to abnormalities of craniofacial and kidney development [24]. The ndings of our pilot experiments have con rmed that homozygous of Six2-Cre (Six2-Cre +/+ ) is embryolethal. Consequently, heterozygotes (Six2-Cre +/− ) were screened to express Cre recombinase in this study. To verify the e ciency of MHC II knockout, cortical primary proximal tubular cells (PTCs) were isolated from Ctrl and cKO mice respectively, and then cultured in Dulbecco's Modi ed Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) for 3 days before harvest. Isolated PTCs were used for western blot. As indicated in Fig. 1A, cKO mice were veri ed prominently lower MHC II expression.
Unless otherwise indicated, we con rmed our ndings in both strains (Six2-Cre +/− ; MHC II ox/ ox mice and Six2-Cre −/− ; MHC II ox/ ox mice). As showed in Fig. 1B-1G, cKO and Ctrl mice had similar appearance, behavior, body weight, kidney weight and baseline renal function (sCr and BUN). Intriguingly, cKO mice presented severer underlying kidney brosis (Fig. 1H), implying that MHC II cKO mice were more susceptible to factors inducing kidney injury in the environment, and furthermore a rmed that KIM-1 mediated autophagy pathway played an essential role in physiological kidney injury and epithelia renovation.
KIM-1 mediated autophagy pathway was a protective factor of renal IRI Bilateral 30 minutes of ischemia was formulated to establish the severe renal IRI model. The ideograph was shown in Fig. 2A. The time-point of day 2 post-ischemia was chosen to evaluate the acute phase and that of day 42 was optimized to investigate the chronic phase according to our research protocol. cKO mice showed dramatical differences in acute functional recovery from IRI, as indicated by diverse recovery patterns of both sCr and BUN, as well as different PTCs KIM-1 expressions 48 hours after kidney ischemia. However, from 1 week after the index ischemic injury, there were similar patterns of both sCr and BUN variations (Fig. 2B-2E). As for the chronic phase, nephron MHC II cKO led to deteriorated interstitial brosis, which was indicated by the results of picrosirius red staining (Fig. 2F). Summing up the above, all these results con rmed that KIM-1 mediated autophagy pathway played a protective role in the pathological process of renal IRI.

IPC attenuated renal IRI functionally and histologically
The delayed kidney ischemic preconditioning (DIPC) strategy was performed as shown in Fig. 3A. As expected, DIPC signi cantly attenuated ischemia/reperfusion-induced sCr and BUN increasing and KIM-1 expression in the early phase when compared to those without DIPC. Furthermore, in the groups of undergoing DIPC, cKO mice showed deteriorated kidney injury when compared to corresponding Ctrl mice. Nevertheless, consistent with the results above, from 1 week after the index ischemic injury, there were similar patterns of both sCr and BUN variations (Fig. 3B-3E). 48 hours after the index ischemia, renal protein levels of the pro brotic and brotic markers were detected by western blot. As shown in Fig. 3F, α-SMA, CTGF and TGF-β, etc. all presented a marked increase in the cKO mice.
In the chronic phase of renal IRI, DIPC also played a protective role. cKO mice suffered from an extremely serious atrophic kidney manifesting the lowest left kidney weight / body weight (Fig. 4A). Consistent with this, H&E staining of the kidney tissues showed that cKO mice exhibited severer histological kidney injury, as indicated by tubular dilation and distal tubular protein casts, nephrons loss, immunocytes in ltration and deposition of extracellular matrix, and these parameters were all diminished in the kidneys of Ctrl mice (Fig. 4B). Meanwhile, MHC II cKO led to increased renal interstitial brosis which was con rmed by picrosirius red staining and immuno uorescence staining of collagens I and IV (Fig. 4C-4E).
In a nutshell, these results implied that DIPC played a protection role in the pathological process of renal IRI, in both acute and chronic phase. Besides, KIM-1 mediated autophagy pathway played a vital role in the IPC induced renal IRI protection.

Discussion
AKI is a common critical illness with high risks of morbidity and mortality. Around 50% will eventually develop to chronic kidney disease, and 8.1% of these patients will progress to end-stage renal disease and require dialysis or renal transplantation [25,26]. Plenty of pathogenesis etiologies have been reported causing AKI, and IRI is one of the most important. Previous studies by Brooks et al. have discovered an important role of KIM-1 mediated autophagy pathway in recovery from renal IRI. As reported, KIM-1 mediated autophagy pathway involves the process of KIM-1 mediated phagocytosis to autophagy induction, MHC presentation and maintenance of peripheral T-cell tolerance, and is a protective factor in the process of kidney injury and epithelia renovation by regulating the PTCs immune reaction [21]. In this study, the phenomenon that KIM-1 mediated phagocytosis played a protective role in the pathological process of renal IRI was veri ed again in mouse bilateral IRI model by the construction of nephron MHC II cKO technique. Meanwhile, KIM-1 mediated autophagy pathway also participated in the IPC induced renal IRI protection. This is supported by Zarbock et al.'s hypothesis that renoprotection is mediated mainly through release of damage-associated molecular patterns that interact with pattern recognition receptors on renal tubular epithelial cells [12].
Although the evidences that IPC could attenuate kidney injury in several species and even in humans are irrefutable, IPC is still not currently recommended for renal transplantation or partial nephrectomy in clinical settings. The primary reason is that though numerous studies have investigated IPC and its effects, there has been no established or veri ed standard protocol of how to conduct the procedure, which may be attributable to the wide variety of methods that used for various facets of IPC, including the time window of ischemia [3]. Time window is the time between IPC and the index ischemic event. According to the time window of ischemia, IPC has been divided into acute IPC (AIPC, occurring immediately and lasting for 1-2 hours) and delayed IPC (DIPC, occurring 24-48 hours after the IPC stimulus and lasting for several days even weeks), which are both reported protective [27,28]. Thought it is generally believed that DIPC is more effective than AIPC in reducing renal IRI [13], there is still no speci c time window of ischemia has been recommended to maximize the renal protective effect of IPC. Zhang et al. previously studied and demonstrated that IPC could reduce renal IRI when conducted 4 days before the index ischemia [23]. Thus, we chose the time window of 4 days in this research, and con rmed that DIPC could signi cantly attenuate renal IRI. However, whether is this the most ideal time window still needs more evidences and to be veri ed in other animal models or even in clinical settings. Secondly, it might be detrimental to apply local IPC to humans by repeatedly clamping and declamping renal vessels. As a result, some studies have studied the effect of remote IPC (RIPC), triggered by brief episodes of ischemia and reperfusion applied in distant tissues or organs before injury of the target organ [12].
Recently, experimental and clinical evidences have demonstrated that RIPC might be an effective, noninvasive and inexpensive strategy to protect kidneys from injury [12]. More intriguingly, no difference in e cacy between local and remote IPC has been observed. Nevertheless, a therapeutic index for RIPC and the appropriate intensity of RIPC have not been established, which limits its clinical application. Thirdly, the renal protective effects of IPC in renal transplantation or partial nephrectomy in clinical settings are still ambiguous. Huang et al. investigated the effect of IPC on renal function in patients undergoing laparoscopic partial nephrectomy, and found that IPC was associated with a lower incidence of glomerular ltration rate (GFR) reduction (measured by renal scintigraphy) at 1 month after surgery (8.8% versus 15%, p = 0.03). However, there were no differences in sCr level or estimated GFR (eGFR) at 1 and 6 months between IPC group and control group [29]. Besides, as reported by Chen at el., IPC did not improve early renal function in patients receiving living-donor renal transplantation [30]. Thus, the clinical application of IPC in patients undergoing partial nephrectomy or renal transplantation still needs more research supports. Further large multicenter trials are required to establish the clinical bene ts of IPC as well as to understand the optimal dose and patient selection.

Conclusions
The present study determines that IPC plays a protective role in the pathological process of renal IRI. Selective deletion of nephron MHC II deteriorates kidney injury and tubulointerstitial brosis resulting from renal IRI. KIM-1 mediated autophagy pathway plays a vital role in IPC evoked renal IRI protection. And clearly, more researches are desiderated to form a better evidentiary basis for the use of IPC, to understand the therapeutic potential and potential risks, to determine when and in whom the intervention works.

Availability of data and material
The datasets generated and analyzed during the current study are available from the corresponding authors, Haibo Shen and Zhengqin Gu, upon reasonable request.