Ceftriaxone Calcium Crystals Induce Acute Kidney Injury by NLRP3-Mediated Inflammation and Oxidative Stress Injury

Objective To investigate the role of inflammatory reactions and oxidative stress injury in the mechanisms of ceftriaxone calcium crystal-induced acute kidney injury (AKI) both in vivo and in vitro. Methods Male Sprague Dawley rats were randomly divided into five groups of ten each according to different concentrations of ceftriaxone and calcium. Based on the levels of serum creatinine (Scr) and blood urea nitrogen (BUN), the AKI group was chosen for the subsequent experiments. Kidney histological examination and immunohistochemistry were performed. The expression of NLRP3 and IL-1β protein and the concentrations of oxidative stress markers such as ROS, MDA, and H2O2 in kidney tissues were estimated. In parallel, HK-2 human renal proximal tubule cells were exposed to ceftriaxone calcium crystals. The mRNA expression levels of NLRP3 and IL-1β and the concentrations of oxidative stress markers were evaluated. Finally, cell viability and rat survival were also assessed. Results The results showed that significantly increased Scr and BUN levels, consistent with morphological changes and kidney stones, were found in the rats that received the highest concentration of ceftriaxone (1000 mg/kg) combined with calcium (800 mg/kg). The activation of the NLRP3 inflammasome axis and the marked elevation of MDA, H2O2, and ROS levels were observed both in vivo and in vitro. High expression of Nrf2, HO-1, and NQO1 was also documented. In addition, cell apoptosis and rat mortality were promoted by ceftriaxone calcium crystals. Conclusions Notably, we found that ceftriaxone-induced urolithiasis was associated with a high risk of AKI and NLRP3-mediated inflammasome and oxidative stress injury were of major importance in the pathogenesis.


Introduction
Ceftriaxone, a potent, semisynthetic, third-generation cephalosporin has a wide spectrum of powerful antimicrobial activities. The intravenous administration of ceftriaxone has been widely used for the treatment of microbial infections, particularly organ infection and sepsis [1]. Ceftriaxone is highly soluble as a sodium salt. However, it can bind with calcium ions, producing a poorly soluble ceftriaxone-calcium salt that forms precipitates in the urinary tract, also known as urolithiasis [2]. Although its incidence is relatively rare, ceftriaxone-induced urolithiasis could lead to severe complications, such as acute kidney injury (AKI) [3]. Based on our previous systematic review, the proportion of ceftriaxone calculi-induced AKI was 72.7%, which was much higher than the proportion of AKI induced by other types of stones, including 9.8% for melamine stones and only a rare occurrence for calcium oxalate stones [4]. The traditional concept of crystal-induced kidney injury focuses on urinary tract obstruction. Undoubtedly, bilateral obstructive urolithiasis can cause acute kidney injury, but tubular crystal plugs and casts rarely obstructed enough nephrons at the same time to explain AKI [5]. Our previous study also found that in addition to urinary obstruction, which is well-known, crystalline nephropathy could contribute to ceftriaxone calculiinduced AKI [4].
The evolving evidence that toxic and postischemic AKIs are largely driven by the associated inflammatory response raised the question of whether inflammation was also the driving factor in crystal-induced AKI [6]. Recently, it has been shown that CaOx crystals activated the NLRP3 inflammasome, resulting in progressive renal failure [7]. Other studies illustrated that cystine crystals, similar to CaOx, were endogenous inflammasome-activating stimuli [8]. These discoveries on the molecular mechanisms of crystal-induced inflammation now enforce a new view on crystal-related kidney injury. The most thoroughly described inflammasome is the nucleotide-binding domain and leucine-rich repeat protein-3 (NLRP3) [9][10][11]. NLRP3 assembles a multiprotein complex termed inflammasome, which comprises a caspase recruitment domain (Asc), and induces caspase-1 activation and the maturation of proinflammatory cytokines such as IL-1β and IL-18 [9]. The NLRP3 inflammasome is the best studied among all inflammasomes [10]; however, in ceftriaxone calcium crystal-induced AKI, the role of NLRP3 has yet not been researched.
Numerous studies have indicated that oxidative stress can play a significant role in the development of kidney stones [12,13]. In spite of urolithiasis, acute kidney injury was also associated with ROS production and impaired antioxidant activity [14]. In murine-accelerated, severe lupus nephritis, the production of the inflammatory cytokine IL-18 could be reduced by enhancing antioxidant activation [15]. So based on analogy with other forms of crystal-induced kidney injury, we speculated that oxidative stress and NLRP3mediated inflammation could both contribute to ceftriaxone calcium crystal-induced AKI. The purpose of this trial was to examine the role of NLRP3-mediated inflammation and oxidative stress injury in promoting the progressive renal failure observed in a model of ceftriaxone calcium crystal nephropathy.

Rats Administered with High Concentration of Ceftriaxone
and Calcium Developed AKI. We previously successfully created a rat model of ceftraxone-induced urolithiasis [4]. In the present trial, the rats were randomly divided into two groups. Compared with the rats in the NC group, the rats (group 1) that were administered with ceftriaxone (1000 mg/kg)+CaCl 2 (800 mg/kg) revealed a severe increase in BUN (Figure 1(a)) and creatinine (Figure 1(b)), consistent with renal failure (P < 0:01). H&E staining also showed histopathological damage to the kidney, including severe interstitial edema, cellular infiltrate, tubular cell swelling, and necrosis (Figures 1(c) and 1(d)).

High Concentration of Ceftriaxone and Calcium Induced
Crystal Nephropathy and Inflammation. As shown in Figure 2, histological examination using von Kossa staining (Figures 2(a) and 2(b)) demonstrated profound crystal deposition in the kidneys of rats administered with ceftriaxone and calcium. The expansion of the kidney tubules was also observed. Transmission electron microscopy (TEM) showed that there were both intracellular and extracellular crystal precipitates (Figures 2(c) and 2(d)). To further define the inflammatory infiltrate, we stained for the presence of macrophages/monocytes with immunohistochemistry. As indicated in Figures 2(e) and 2(f), macrophage/monocyte staining was strongly positive in the group treated with ceftriaxone and calcium compared with the control group.

Ceftriaxone Calcium Crystals Upregulated the NLRP3
Inflammasome Axis. We measured the expression of NLRP3 and IL-1β in the kidney lysates, and the western blot results demonstrated that they were noticeably elevated in the AKI group (Figures 3(a) and 3(b)). In addition, immunohistochemical staining also showed that NLRP3 and IL-1β were upregulated in the tubules of rats in the AKI group compared with those in the control group ( The results showed that the H 2 O 2 and MDA concentrations in the kidney tissues were significantly increased in the AKI group compared with those in the control group. Next, we investigated whether ceftriaxone calcium crystals affected the Nrf2 pathway in the kidneys of rats with AKI. As shown in Figures 4(f)-4(h), the results of western blot analysis revealed that the expression of Nrf2 including its downstream enzymes such as HO-1 and NQO1 were enhanced. Consistent with the protein analyses, Nrf2, HO-1, and NQO1 mRNA levels were also found to be markedly elevated (Figures 4(i)-4(k)). 2.6. Ceftriaxone Calcium Crystals Induced Cell Apoptosis and Rat Mortality. As shown in Figure 6, scanning electron microscopy (SEM) images of the control group showed normal healthy morphology with minimal apoptosis (Figures 6(a), 6(c), and 6(e)). In contrast, ceftriaxone calcium crystal treatment for 24 h induced massive apoptosis with cellular and nuclear condensation and fragmentation ( Figure 6(f)). The dose-dependent effects of ceftriaxone calcium crystals (0-1000 mg/L) on HK-2 cell viability ( Figure 6(g)) showed that a ceftriaxone crystal concentration ≥ 500 mg/L was capable of significantly reducing cell viability (P < 0:05). To examine the effect of ceftriaxone calcium calculi-induced AKI on rat mortality, we continued to observe the NC group and AKI group for 30 days. The 30-day mortality of the AKI group was 100% (Figure 7). Death likely occurred following progressive renal insufficiency, but the contribution of inflammation 2 Oxidative Medicine and Cellular Longevity in other organ systems cannot be excluded. In contrast, the NC group showed 100% survival at 30 days.

Effects of MCC950 on Cell Viability and Cytoprotection.
MCC950, the inhibitor of NLRP3, was added to HK-2 cell cultures containing ceftriaxone calcium crystals for 12 hours, after which they were subjected to MTT assays. The results ( Figure 8) showed that the ceftriaxone calcium crystaltreated cells had significantly increased MTT absorbance levels, which were prevented by the addition of MCC950.

Discussion
Recently, AKI was found secondary to ceftriaxone-induced urolithiasis [16,17]. Our previous study also demonstrated impaired renal function in 72.7% of the patients with ceftriaxone calculi [4]. Renal stone disease is widely considered to be a mechanical problem causing intrarenal or extrarenal urinary outflow obstruction, but crystal nephropathy is also associated with significant intrarenal inflammation and oxidative stress that can cause acute kidney injury [5]. In the present study, our findings suggested that the inflammasome and oxidative stress injury were of major importance in the loss of kidney function that occurred in ceftriaxone calcium crystal-induced kidney disease, an important example of crystal nephropathy. The inflammasome is thought to have the unique ability to integrate a myriad of signals from pathogen-and damage-associated molecular patterns into a proinflammatory response [18]. As the most extensively investigated of the inflammasomes identified, the activation of the NLRP3 inflammasome can be achieved by a wide range of structurally dissimilar agonists, including pathogens, pore-forming toxins, environmental irritants, and endogenous damageassociated molecular patterns [19][20][21]. Our study found that NLRP3 activation was involved in the pathogenesis of ceftriaxone calcium crystal-induced AKI and cell apoptosis. And the inhibitor of NLRP3 could effectively protect renal cells from ceftriaxone attack, suggesting that ceftriaxone crystalmediated cytotoxicity was likely associated with NLRP3 inflammasome. In fact, NLRP3 activation has been demonstrated to contribute to multiple diseases by increasing IL-1β and IL-18 secretion and amplifying the inflammatory response, including acute lung injury and acute liver failure [22,23]. Evolving data suggest that the inflammasome IL-1/18 axis contributes to acute and chronic inflammation and tissue remodeling in the kidney [5]. Our study defined the relative importance of the inflammasome in specific ceftriaxone calcium crystal-induced AKI and suggested further study to identify the therapeutic opportunities afforded by targeting the inflammasome response.
Oxidative stress has been implicated in the pathogenesis of AKI [24]. In the present study, markers of oxidative stress such as ROS, MDA, and H 2 O 2 were significantly increased in the animal model of ceftriaxone calculi-induced AKI, and similar changes were also observed in HK-2 cells. As reported, Nrf2 is a pivotal coordinator of the pathways that protects cells against oxidative and inflammatory damage, with HO-1 and NQO1 being two of the major downstream anti-inflammatory and antioxidative enzymes [25]. Nrf2 translocation from the cytoplasm to the nucleus was essential for its regulation of antioxidant/detoxification enzyme expression [26]. We showed here that ceftriaxone calcium crystal stimulation increased Nrf2 levels in the nuclei, as well as HO-1 and NQO1 protein and mRNA levels, which was indicative of a stress response. Our results were also  [27].
In addition to the indirect mechanisms involving inflammation and oxidative stress injury that we have discussed, ceftriaxone calcium crystals could also harm tubular cells by directly inducing tubular cell death. Crystals are known to be cytotoxic when they come in direct contact with the tubular epithelium [28]. For example, Thongboonkerd et al. have demonstrated that CaOx monohydrate crystals (COM) tightly adhere to MDCK cells with subsequent detrimental effects on the cells [29]. The internalization of COM crystals by MDCK cells resulted in the alteration of several proteins involved in energy production, which contributed to mitochondrial dysfunction and a subsequent increase in ROS production [30]. We have shown that upon exposure, ceftriaxone calcium crystals could directly induce massive apoptosis, consistent with the cellular and nuclear condensation and fragmentation of HK-2 cells. H&E staining also showed severe interstitial edema, cellular infiltrate, tubular cell swelling, and necrosis. Taken together, these results indicated that the pathological changes promoted the elevation of Cr and BUN levels and the subsequent progressive renal insufficiency, which eventually induced rat mortality.
There is limited information about ceftriaxone-induced urolithiasis and its association with AKI. This severe complication is often overlooked or misinterpreted, although its incidence is extremely high. To our knowledge, this is the 5 Oxidative Medicine and Cellular Longevity first study to investigate its pathogenesis, and the present study revealed several new findings. First, we developed an animal model of ceftriaxone calcium crystal nephropathy with development of renal failure and death. Second, we demonstrated that inflammation and oxidative stress played a critical role in the mechanism of ceftriaxone calcium crystal-induced AKI. The necroptosis of renal tubules and cell apoptosis were consistent with the increased expression of IL-1β and concentrations of ROS, MDA, and H 2 O 2 , suggesting that inflammation and oxidative stress could promote renal injury. Finally, we highlighted a novel role of NLRP3 and Nrf2 in these events. Based on our study, ceftriaxone calcium crystals significantly elevated the expression of the NLPR3-IL-1β axis and the transcription of Nrf2, including the downstream enzymes, such as HO-1 and NQO1. Collectively, these results suggested that ceftriaxone calcium crystals could induce acute kidney injury by NLRP3-mediated inflammation and oxidative stress injury. Further research is necessary to determine the therapeutic value of blocking of the inflammasome pathway consistent with antioxidants.
The current study still has several limitations. First, the present trial lacked positive and negative controls to further verify the role of NLRP3 and Nrf2 in the pathogenesis of ceftriaxone calcium crystal-induced AKI. For example, a series of measures such as NLRP3 inhibitor, si-NLRP3 cells, NLRP3 knockout rats, and si-Nrf2 cells could be used to make the results more convincing. Second, as reported, ROS production could also lead to the activation of the NLRP3 inflammasome [20]. Thus, the effect of oxidative stress on increasing the expression of the NLRP3 axis cannot be ruled out, and whether ceftriaxone calcium crystals could have a direct effect on NLRP3 inflammasome activation is not clear. Further studies should be performed to determine the in-depth mechanisms.

Conclusion
In summary, we have developed an animal model of ceftriaxone calcium crystal nephropathy with development of renal failure and death. Using this model, we demonstrated that NLRP3-mediated inflammasome and oxidative stress injury are of major importance in the pathogenesis of ceftriaxone calcium crystal-induced AKI. This work emphasized the potential value of anti-inflammatory and antioxidant therapies in ceftriaxone calcium crystal nephropathy.

Animal Experiment.
The animal experiment procedure consisted of two steps.
Step1. A total of 20 male SD rats, initially weighing 100:0 ± 5:1 g, were housed under standard conditions (the animal experimental center of Nanjing Medical University, SCXK 2008-0004). All of the rats were given free access to deionized distilled water throughout the experiment. This experiment was permitted by the Veterinary Directorate of Nanjing Medical University, in accordance with the Chinese legislation and the Council Directive of the Asian Community.
The rats were randomly divided into two groups (ten rats each) as follows: (1) NC group, no treatment, and (2) group 1, ceftriaxone (1000 mg/kg)+CaCl 2 (800 mg/kg). Ceftriaxone was administered by intramuscular injections, while CaCl 2 was administered by gavage. All of the reagents were administered for 7 days.
On day 7, all the rats were anesthetized with ketamine (100 mg/kg), and blood samples (3 mL each) were collected by cardiac puncture to assay the Cr (creatinine) and BUN (blood urea nitrogen) concentrations. All of the collected samples were kept at -20°C until measurements were taken. Then, the rats were sacrificed, and their kidneys were removed for histological examination (the procedure was described in Section 5.2).
Step2. The AKI group was rebuilt for the next experiments. The amount of living rats was documented, and the 30-day survival rates were calculated. (e, f) Immunohistochemistry staining of IL-1β in kidney tissue ((e) NC group, (f) group 1, bars = 100 μm). (g, h) Semiquantitative analysis for NLRP3 (g) and IL-1β (h) using Image-Pro plus 6.0 software (the results are expressed as mean ± SD; * * P < 0:01 compared with the control group). 6 Oxidative Medicine and Cellular Longevity  Oxidative Medicine and Cellular Longevity and setting a pixel intensity threshold that identifies crystals separate from background tissue. Ultrastructural analysis was performed with a transmission electron microscope EM 410 (Philips; Eindhoven, Netherlands). An avidin-biotin immunoperoxidase method was used with a monoclonal antibody directed against macrophages/monocytes (F4/80; clone BM8, Abcam, no. 16911). The injury scores were graded according to the degree of damage based on the percentage of involvement of the kidney. The damage quantification from ten areas corresponding to the renal tissue was graded using the following parameters: tubular cell necrosis, apoptosis, cytoplasmic vacuole formation, hemorrhage, and tubular dilatation based on a five-score system (1, histopathological changes < 10%; 2, =10-25%; 3, =25-50%; 4, =50-75%; and 5, =75-100%) [31]. The pathological scores were given by two independent blinded pathologists.

Cell Experiments.
The human kidney-2 cells (HK-2) were purchased from HonorGene company (Changsha, China). They were cultured in Eagle's minimum essential medium supplemented with 10% fetal bovine serum (Huihe, Guangzhou), penicillin (100 U/mL, Rongbo, Shanghai), and streptomycin (100 μg/mL, Kairui, Beijing). For experiments, cells were seeded in six-well plates or T-75 flasks at the initial cell density of 2 × 10 5 cells/mL and divided into a control group and ceftraxone calcium crystal-treated group. The MDA (malondialdehyde) and H 2 O 2 concentrations were measured, and the mRNA expression levels of NLRP3 and IL-1β were examined (the methods were described in Sections 5.5 and 5.8). The specimens were sputter-coated with platinum and examined by JEOL JSM-6500 TFE-SEM at an accelerating voltage of 10 keV.
5.9. Cell Viability Test. After treatment with various concentrations of ceftriaxone calcium crystals (0-1000 mg/L), the cell viability was then determined at the specified times using the MTT-based toxicology assay, following the manufacturer's protocol (Sigma, Shanghai, China). Then, HK-2 cells were divided into 2 groups as follows: ceftriaxone calcium crystals (1000 mg/L) and ceftriaxone calcium crystals (1000 mg/L)+MCC950 (10 μM, Jinhui Biotechnology, Shanghai). After 12 h treatment, cell viability was determined using the MTT-based toxicology assay, following the manufacturer's protocol (Sigma, Shanghai, China).

Statistical Analysis.
All of the statistical analyses were conducted by SPSS (version 19.0). Discrete variables were represented as frequencies, and the differences were evaluated by the Pearson χ 2 test. Continuous variables were summarized as the mean ± standard deviation (SD), and the tests of significance were performed by t-tests for two groups or by an analysis of variance for more than two groups. A P value of less than 0.05 was considered significant.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
We wish to confirm that there are no known conflicts of interest associated with this publication.

Authors' Contributions
Z.Y.F. and N.B.X. were responsible for the conception and design, collection and/or assembly of data, data analysis and interpretation, financial support, manuscript writing, and final approval of the manuscript. X.Z. and X.L.W. were responsible for the data analysis, manuscript writing, and final approval of the manuscript. Z.L.H. and G.Y.Z. performed the experiments and data collection. J.R.P. was responsible for the conception and design, manuscript preparation, financial and administrative support, and final approval of the manuscript. Zhang Yifan and Ning Benxiang are co-first authors.  Ceftriaxone calcium crystals Ceftriaxone calcium crystals + MCC950 ⁎ Figure 8: Effects of MCC950 on ceftriaxone calcium crystalinduced cytotoxicity. HK-2 cells were treated with ceftriaxone calcium crystals alone and in combination with MCC950 (10 μM) for 3, 6, 9, and 12 hours, and MTT absorbance was examined (the results are expressed as mean ± SD; * P < 0:05 compared with the control group).