Interleukin-22 mediated renal metabolic reprogramming via PFKFB3 to treat kidney injury

Kidney damage initiates the deteriorating metabolic states in tubule cells that lead to the development of end-stage renal disease (ESTD). Interleukin 22 (IL-22) is an effective therapeutic antidote for kidney injury via promoting kidney recovery, but little is known about the underlying molecular mechanisms. Here we first provide evidence that IL-22 attenuates kidney injury via metabolic reprogramming of renal tubular epithelial cells (TECs). Specifically, our data suggest that IL-22 regulates mitochondrial function and glycolysis in damaged TECs. Further observations indicate that IL-22 alleviates the accumulation of mitochondrial reactive oxygen species (ROS) and dysfunctional mitochondria via the induction of AMPK/AKT signaling and PFBFK3 activities. In mice, amelioration of kidney injury and necrosis and improvement of kidney functions via regulation of these metabolism relevant signaling and mitochondrial fitness of recombinant IL-22 are certificated in cisplatin induced kidney damage and diabetic nephropathy (DN) animal models. Taken together, our findings unravel new mechanistic insights into protective effects of IL-22 on kidney and highlight the therapeutic opportunities of IL-22 and the involved metabolic regulators in various kidney diseases.


Introduction
Acute kidney injury (AKI), a common public health concern associated with high mortality and morbidity, affects millions of hospitalized patients worldwide and shows a fast-increasing incidence [1][2][3]. The underlying pathophysiology of AKI is not fully understood but involves the damage and apoptosis of renal tubular epithelial cells (TECs), especially in the proximal tubule [4][5][6][7]. Multiple literatures support that their severe and sustained damage often causes incomplete and maladaptive tissue repair, leading to inflammatory response, tubular degeneration, kidney fibrosis and eventually progression to end-stage renal disease (ESTD) [8][9][10]. Unfortunately, other than dialysis-based supportive care, no well-established therapeutics for kidney injury are available. Therefore, proactive treatment is much needed to reduce the suffering it causes to patients and the significant financial burdens of kidney injury to individuals and society.
Interleukin-22 , an important member of the IL-10 family cytokines, has recently attracted tremendous attention as a survival agent in numerous disorders driven by epithelial damage [11][12][13]. IL-22 elicits tissue protection and homeostasis primarily via activation of STAT3 signaling pathway and the promotion of epithelial proliferation [14][15]. Importantly, studies have demonstrated that the expression of IL-22R1 is limited to renal proximal TECs, and treatment with IL-22 can protect against renal epithelial injury and accelerate tubular regeneration [16][17]. However, although accumulating evidence indicates IL-22 is an effective therapeutic antidote for kidney injury, little is known about the underlying mechanisms of IL-22 induced TECs recovery. Understanding the molecular basis of IL-22 is significant both for exploring how IL-22 acts to inhibit AKI or ESTD and for discovering molecular modifiers as well as crucial processes involved in preventing kidney damage.
Kidney repair after damage is a metabolically-dependent and complicated process [18]. Recent evidence indicates that cell metabolic signatures can regulate renal cell survival and plasticity and thus provide useful mechanistic insights into how the metabolic processes control tissue repair [19][20]. Specifically, these findings show that metabolic reprogramming by PGC1α and the S-nitroso-CoA reductase system protects against kidney injury. Mechanistically, increased glycolysis and oxidative phosphorylation (OXPHOS) can promote growth and defensive signaling pathways or can supply the higher energetic demands of mitosis and anabolic biosynthesis during organ repair [19][20][21]. Recently, our studies have demonstrated that IL-22 alleviates mitochondrial dysfunction in liver, which involves the cellular metabolic processes [22][23][24]. Thus, we asked whether IL-22 protects against TECs damage and apoptosis via regulating their metabolic states. To determine the mechanisms of IL-22 mediated kidney repair, we treated human proximal tubular epithelial cells (PTECs) with stress stimulation. We found that IL-22 drives a metabolic reprogramming to enhance glycolysis and OXPHOS, which might prevent against TECs dysfunction. We further demonstrated that IL-22 induced this program via activating of AMPK/AKT signaling and its molecular modifiers to alleviate mitochondrial dysfunction. Our observations shed light on regulating metabolic states for treating and preventing kidney diseases, and provide new insights for developing efficient novel interventions for organ injury in general.

IL-22 preserves cellular metabolism in TECs
Because mitochondria have been suggested to be important organelles that integrate cellular metabolism and apoptotic processes, we assessed mitochondrial function in TECs that were stimulated with kidney injury factors and treated with IL-22 [25]. As shown in Figure 1A, 1C, 1D, mitochondrial basal oxygen consumption rate (OCR), maximal respiratory capacity (MRC) and respiration were blocked substantially in the TECs under stress situations. Notably, these abnormalities were largely prevented by IL-22 treatment. Glycolysis in damaged TECs, evidenced by extracellular acidification rate (ECAR), was also markedly restrained, compared with IL-22 plus stimuli-challenged TECs (Figure 1B, 1E). Because multiple evidences have suggested that Glut1 plays a principal role in glycolysis and glucose homeostasis [26], we therefore ask whether IL-22 affects Glut1 translocation or expression. As anticipated, we demonstrated that IL-22 not only promoted the translocated Glut1 to the cell membrane surface (PKH26) and Glut1 expression, but also enhanced glucose uptake ( Figure 1F, and 1G). Overall, these observations indicated that IL-22 could enhance OXPHOS and glycolysis in injured TECs.

IL-22 ameliorates the accumulation of dysfunctional mitochondria in TECs via the activation of mitophagy
We next investigated whether the improvement of cellular metabolism by IL-22 in TECs is attributed to the prevention of mitochondrial dysfunction. As detected by MitoTracker Green and MitoSOX dye, we found that TECs had increased mitochondrial mass and mitochondrial ROS after exposure to stimuli (Figure 2A and   2B). To distinguish respiring and dysfunctional mitochondria, the TECs were then stained with MitoTracker Red to detect the mitochondrial membrane potential. Our results revealed that kidney injurious stimuli, such as cisplatin, significantly increased the mitochondria dysfunction (with higher MitoTracker Green and lower MitoTracker Red) ( Figure 2C). Importantly, IL-22 could preserve mitochondrial fitness in injured TECs, which was showed by the reduction of mitochondrial mass, mitochondrial ROS and mitochondrial dysfunction (Figure 2A, 2B

IL-22 maintains mitochondrial integrity of TECs via activation of AMPK/AKT signaling
The AMPK signaling, as a key signaling hub linking cell survival and metabolism, controls glucose metabolism, lipid synthesis and mitochondrial function [28].

IL-22 preserves cellular metabolism and mitochondrial integrity of TECs via induction of PFKFB3
Moreover, to explore how TECs mitochondrial fitness and metabolism were improved, we used RNA sequencing analysis (RNA-seq) to assess gene expression following stimuli injury and IL-22 treatment. We demonstrated that multiple genes were significantly changed in TECs after IL-22 incubation ( Figure 4A). Using the available databases, the gene set enrichment analysis of those up regulated genes was performed, which suggested a remarkable enrichment of genes in AMPK signaling pathway and MYC targets signaling pathway ( Figure S2). Notably, PFKFB3 was significantly induced by IL-22 from among the genes during kidney injury factors stimulation ( Figure 4A). These observations were also indicated by confocal images, Q-PCR and western bolt, and the high expression of PFKFB3 was AMPK/AKT signaling dependent (Figure 4B, 4C and S3).
Previous studies had suggested that PFKFB3 could be regulated by numerous signaling transductions to accelerate epithelial regeneration and repair following injury [29][30]. Thus, we speculated that IL-22 induces PFKFB3 to preserve cellular metabolism of TECs and maintain its mitochondrial fitness. We observed that the inhibition of AMPK/AKT signaling reduced PFKFB3 expression ( Figure 4D). So, we silenced PFKFB3 to further study its functions in TECs after IL-22 incubation. Our findings indicated that silenced PFKFB3 alleviated the metabolic reprogramming effects of IL-22 on restoring ECAR and OCR ( Figure 4E and 4F), and prevented glucose uptake as well as dysfunctional mitochondria (Figure 4G and 4H). Taken together, these findings indicated that the activation of PFKFB3 plays an important role in mitochondrial fitness and dysfunctional mitochondria elimination after kidney injury.

IL-22 alleviates kidney injury in cisplatin-induced AKI via suppression renal reactive oxygen species (ROS) accumulation and mitochondrial dysfunction
To test our hypothesis that IL-22 regulates renal metabolic profiles to suppress kidney tubule injury in vivo, we initially evaluated renal functions in IL-22 treated mice that had been administered with cisplatin ( Figure 5A). Four days after cisplatin injection, IL-22 not only substantially reduced tubular cellular damage and hemorrhage; their renal dysfunction also had been significantly attenuated, which were assessed by the serum levels of blood urea nitrogen (BUN) and creatinine (Cr) ( Figure 5B, 5C and 5D). We further suggested IL-22 prevents kidney injury via enhancing kidney regeneration and activating AMPK/AKT signaling and PFKFB3 ( Figure 5E, 5F and S4). Next, we employed MitoSOX (mitochondria-specific ROS dye) and JC-1 (mitochondrial membrane potential-dependent dye) to assess the mitochondrial dysfunction involved in cellular metabolism and apoptosis. In the kidney sections of mice that had been treated with cisplatin, there were comparable accumulations of ROS. Of note, at 4 days after IL-22 treatment, the levels of ROS were markedly reduced in the injured kidneys in comparison to ROS from mice after PBS treatment (Figure 5G). Taken together, these observations offer evidence that IL-22 plays a protective role in cisplatin-induced AKI via controlling mitochondrial fitness.

IL-22 ameliorates diabetes-induced renal injury via inhibition of mitochondrial dysfunction through the activation of AMPK/AKT signaling and PFKFB3
We and others have demonstrated that IL-22 could exert protective potency in diabetic nephropathy (DN), which is the main cause of ESTD [31-32]. To further study the underlying mechanisms, we fed db/db mice with high-fat diets (HFD) and then treated IL-22 (2.5 mg/kg, ip) for 8 weeks (Figure 6A). When mice were fed with HFD only, we found kidney tubular fibrosis and dilatation, in addition to atrophy. In contrast, when mice were treated with IL-22, renal fibrosis and histopathology were significantly improved, as well as kidney functions. Enhanced kidney functions were also demonstrated by decreased serum Cr, BUN levels and total urinary albumin/24 h (UAE) levels ( Figure 6B, 6C, 6D and 6E). To study whether these activities are associated with mitochondria, we assessed the mitochondrial function involved in cellular metabolism and apoptosis. The results indicated that the increased mitochondrial ROS levels and accumulated dysfunctional mitochondria were significantly alleviated by IL-22 treatment (Figure 6F and 6G). To further study whether the protective functions of IL-22 in kidney are mediated through PFKFB3, we injected mice with PFKFB3 shRNA adenovirus to knockdown PFKFB3. Our data indicated that the beneficial effects of IL-22 on HFD-induced renal injury, necrosis, steatosis, mitochondrial dysfunction and ROS accumulation and the activation of related signaling pathways were significantly blocked by PFKFB3 knockdown (Figure 6B, 6C, 6D, 6E, 6F and 6G). Additionally, the increased expression of AMPK/AKT signaling was also observed after treatment with IL-22 ( Figure 6H).
Along with the alleviation of kidney dysfunction, the expression of PFKFB3 and important enzymes in cellular metabolism were also improved in kidney of IL-22-treated mice (Figure 6H and 6I). Overall, our data suggested that IL-22 alleviated kidney damage via activation of AMPK/AKT signaling transduction, inhibition of ROS accumulation and mitochondrial function regulation through PFKFB3.

Conclusions
In conclusion, we demonstrated the therapeutic potential and underlying mechanisms of IL-22 in kidney damage. IL-22 regulated renal cell metabolism via a metabolic reprogramming to preserve mitochondrial integrity. Inhibition of their regulators (i.e., AMPK, AKT, PFKFB3) could lead to abnormal metabolic profiles and loss of mitochondrial fitness, as indicated in TECs with spontaneous IL-22 treatment, which had increased dysfunctional mitochondria and mitochondrial ROS accumulation. Most importantly, the kidney damage by mitochondrial dysfunction and ROS accumulation in mice models was substantially alleviated by IL-22 treatment through the activation of STAT3-AMPK-AKT-PFKFB3 signaling. Thus, our study indicated that targeting associated metabolic signaling could be directly favorable for numerous kidney damage diseases and IL-22 is a potential therapeutic agent for preventing and treating these diseases.

Seahorse experiments
We analyzed TECs using a Seahorse XF96 Extracellular-Flux-Analyzer for the changes in the extracellular acid rate (ECAR) and oxygen consumption rate (OCR) and. Briefly, HK-2 (1 × 10 5 ) was grown on 96-well Seahorse-plates for overnight. Committee of School of Pharmacy, Fudan University. Immunohistochemical staining, measurement of mitochondrial membrane potential, histological and ROS staining of kidney sections were performed as previously described [22][23][24].

Real-time PCR
We obtained the total-RNA by TRNzol reagents (Beyotime Biotechnology, China) from cell or tissue samples and then transcribed to cDNA using a MMLV-reverse transcriptase test kit (Beyotime Biotechnology, China). The expression level of mRNA was analyzed by the real-time PCR instruments (BioRad, USA) using SYBR green qPCR-mix assay kit (Beyotime Biotechnology, China) and standardized to GAPDH.

Flow cytometry
Kidney cells were grown and treated as described above. MitoSOX (mitochondrial ROS), MitoTracker Green (total mitochondrial mass) and MitoTracker Red (mitochondrial membrane potential) staining were carried out according to the manufacturer's instructions and previous researches [22][23][24]. We obtained the results by the Beckman Coulter Flow Cytometer and the CytExpert software (BD Biosciences, USA).

Glucose uptake
We measured the glucose concentrations in cell culture supernatant by a glucose uptake assay test according to the manufacturer's protocol and previous researches manufacturer's (GAHK20, Sigma, USA). Results normalization was carried out according to the number of cells.

Gene knockdown
We used the small interfering RNA (siRNA, RiboBio, China) to knockdown specific gene. Firstly, Lipofectamine RNAiMAX and siRNA (100 pmol) were gently mixed via pipetting and incubated at 37 o C for 0.5 hour. Then we changed the standard cell culture medium to transfection cocktail and incubated the cells for 6 hours for siRNA gene transfection. Next, we changed the transfection cocktail to fresh cell culture medium. After 48 hours, kidney cells were incubated with IL-22 for further studies.

Western blots
We extracted total proteins from cell or tissue by RIPA lysis buffer (Beyotime Biotechnology, China). Then, we collected the lysates by centrifugation at 4 °C at 12000 rpm for 10 min. Subsequently, the lysates were heated with the sample buffers at 100 °C for 15 min, subjected to sodium dodecyl sulfate polyacrylamide gel-electrophoresis (SDS-PAGE) and electro transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Germany). The membranes were then blocked with 5% bovine serum albumin (BSA) for 2 hours and co-incubated with primary antibodies at 4°C overnight. After washing three times, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (CST, USA). The protein blots were visualized by the enhanced chemiluminescence imaging system.

Immunofluorescence
The kidney cells were fixed by 3%-4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Then, we blocked the cells with 5% bovine serum albumin (BSA) for 2 hours. Cells were incubated overnight with anti-GLUT1 antibody at 4°C, washed four times, and then stained with PKH26. Subsequently, kidney cells were stained with Hoechst 33342 and mounted on slides. Data were obtained by the confocal microscopy.

Statistics
Data were expressed as means ± standard deviations (SD) and were shown using GraphPad Prism 5.0. For results comparing two or more groups, differences were calculated using the Student's t-test or the one-way of analysis (ANOVA). ***, **, and * suggested P < 0.001, P < 0.01 and P < 0.1, respectively.