Cisplatin is a widely used chemotherapeutic agent known for its efficacy against various solid tumors, including lung, ovarian, bladder, and testicular cancers (Tang et al., 2023). However, its clinical use is often limited due to its nephrotoxicity, which can lead to renal injury in 3-48% of patients (Grillo-Marín et al., 2024). Cisplatin-induced nephrotoxicity is characterized by the accumulation of cisplatin in the kidneys, resulting in a progressive decline in renal function (Curry and McCormick, 2022). Key features of this nephrotoxicity include tubular cell death, oxidative stress, and inflammation (Volarevic et al., 2019). In particular, inflammation plays a crucial role in the pathogenesis of cisplatin-induced acute kidney injury (AKI), with the release of pro-inflammatory cytokines such as TNF-α, IL-6, IL-18, chemokines, and immune cell infiltration exacerbating the damage.

Lipopolysaccharide, a component of the outer membrane of Gram-negative bacteria, is known for its strong inflammatory response through toll-like receptor 4. LPS can induce sepsis, and approximately 60% of septic patients progress to septic AKI (Bagshaw et al., 2009). Therefore, the co-administration of LPS with cisplatin could potentially amplify the inflammatory cascade and worsen renal injury, providing a more powerful model production for studying nephrotoxicity.

To generate renal injury models, cisplatin is administered either at low doses (2.5-4 mg/kg) repeatedly or at higher doses (7-10 mg/kg) weekly for 1 to 4 weeks (Elbrolosy et al., 2024; Li et al., 2024; Santos et al., 2024). LPS administration is typically observed for up to 7 days post-injection to create an AKI model (Xiu et al., 2018, Yang et al., 2024). However, administering cisplatin alone presents challenges: at low doses, the model often recovers naturally, making it difficult to consistently induce renal injury, while at high doses, nephrotoxicity leads to significant mortality in mice. Additionally, LPS administration alone provides only short-term effects, limiting its utility for chronic kidney disease (CKD) modeling.

To address these limitations, we investigated the effects of co-administering cisplatin and LPS by analyzing survival rates, body weight, renal weight and area, and BUN levels to assess renal damage according to dosage and timing.

Chemical induced-renal injury model

Male C57BL/6 mice (8 weeks of age) were purchased from ORIENT BIO (Seongnam, Republic of Korea). Animal care followed the guidelines of the Laboratory Animal Research Center of CHA University. Cisplatin was administered intraperitoneally (IP) at doses of 8, 10, or 12 mg/kg once per week starting from day 0. LPS was administered IP at a dose of 5 mg/kg on day 1 and day 4 following each cisplatin injection, forming a weekly treatment set. This regimen was carried out for either 1, 2, or 4 weeks, depending on the experimental group. Mice were sacrificed on day 4, 18, or 32. In an in vivo experiment, the mice were divided into various treatment groups. The groups included control group (n = 5), cisplatin group (n = 5), and cisplatin + LPS group (n = 8). In experiments co-administering cisplatin and LPS, the groups included cisplatin 12 mg/kg + 2-wk group (n = 10), sham group (n = 5), cisplatin 10 mg/kg + 1-wk group (n = 3), cisplatin 10 mg/kg + 2-wk group (n = 10), cisplatin 8 mg/kg + 2-wk group (n = 8), cisplatin 8 mg/kg + 4-wk group (n = 5).

Body weight was measured using Precision Balance (HS-V Series, HANSUNG, Republic of Korea), and renal weight was measured using Analytical Balance (PX224KR/E, OHAUS, New Jersey), The renal area was measured using a ruler by taking multiple measurements of the long and short axes.

Immunohistochemistry

Kidney samples were fixed in 4% paraformaldehyde, gradually dehydrated, embedded in paraffin into 4-μm sections, and stained with H&E or MT. The slides were scanned and analyzed with ZEN 3.1 (Car zeiss, Germany). Images of the kidney cortex were captured at dimensions of 392.0 μm × 351.6 μm.

Assessments of BUN

The mouse blood was collected in a heparin tube on ice and centrifuged at 3,000 rpm for 15 minutes. The supernatant was used for the assay. BUN levels in mouse serum were measured using an automated analyzer (Accute TBA-40FR, TOSHIBA, Japan), following the manufacturer’s instructions.

Statistical analysis

All data are presented as the mean ± standard error of the mean. Independent-group t-tests were used for comparisons between two groups, or ANOVA with Tukey’s post-hoc analysis for multiple group comparisons. All statistical analyses were conducted using GraphPad Prism software (version 8), and p < 0.05 was considered statistically significant.

Effect of cisplatin and LPS co-administration to generate renal injury model

We initially confirmed the effectiveness of using cisplatin (12 mg/kg) combined with LPS (5 mg/kg) compared to cisplatin alone inducing a renal injury model. The cisplatin group received a single administration on day 0, while the cisplatin + LPS group received cisplatin on day 0, followed by LPS on days 1 and 4 (Fig. 1A). Our data revealed that the co-administration of cisplatin and LPS resulted in significantly lower body weight, renal weight, area and BUN levels compared to the control group (Fig. 1B). Based on this observation, we decided to apply the co-administration of cisplatin and LPS in our experiments to further optimize and investigate the dose- and time-dependent effects in a mouse model.

Figure 1.Effects of administration of cisplatin and cisplatin + LPS to generate renal injury mouse model. (A) Experimental schedule of cisplatin and LPS administration. Schematic representation of the treatment groups and dosing schedule. (B) Physiological and biochemical effects of cisplatin and cisplatin + LPS treatment. The co-administration of cisplatin and LPS was associated with significant reductions in body weight and renal weight, area, and BUN (5-8 mice per group) suggesting enhanced renal injury compared to the control group. Mean ± SEM, 5-8 mice per group. *p < 0.05 by ANOVA with Tukey’s analysis and t-test. Cis, cisplatin; LPs, lipopolysaccharide; Cis + LPS, cisplatin + lipopolysaccharide; BUN, blood urea nitrogen.

Dose and duration-dependent effects of cisplatin and LPS co-administration

To assess the dose- and duration-dependent effects to induce real injury in mice, we treated mice with varying doses of cisplatin (8, 10, or 12 mg/kg) in combination with LPS (5 mg/kg) for durations of 1, 2, and 4 weeks (Fig. 2A). When cisplatin was treated for 2 weeks, in the cisplatin (12 mg/kg) group, 50% of the mice did not survive, indicating the high toxicity and potential lethality of this dose (Fig. 2B). In contrast, all mice in the 8 and 10 mg/kg groups survived, suggesting no lethality at these doses.

Figure 2.Dose and duration-dependent effects of cisplatin to generate renal injury mouse model. (A) Schematic representation of the experimental schedules. All groups received cisplatin on days 0 and 7, and LPS treated on days 1 and 4 following each cisplatin injection. (B) Survival rate of mice treated with varying doses of cisplatin with LPS over a two-week period were monitored until day 18. Mice treated with 8 mg/kg (black dotted line) and 10 mg/kg (green solid line) cisplatin with LPS 5 mg/kg, exhibited a 100% survival rate. In contrast, the group treated with 12 mg/kg cisplatin (red line) with LPS 5 mg/kg showed a 50% mortality rate. (C) Physiological and biochemical effects for 1 or 2 weeks of 10 mg/kg cisplatin with LPS 5 mg/kg treatment. Mice were sacrificed on day 18. Administering cisplatin at a dose of 10 mg/kg resulted in significant alterations in body weight, renal weight, renal area, and serum BUN (3-10 mice per group) levels when compared to the sham group. (D) Physiological and biochemical effects for 2 or 4 weeks of 8 mg/kg cisplatin treatment. Mice were sacrificed on days 18 or 32 for 2 weeks or 4 weeks treatment, respectively. Compared to 2 weeks treatment group, the 4 weeks cisplatin with LPS 5 mg/kg treatment group exhibited lower body weight and renal weight, with elevated BUN (8-10 mice per group) levels that reflect the induction of renal injury. Mean ± SEM, *p < 0.05 by (C) ANOVA with Tukey’s analysis, 3-10 mice per group or (D) t-test, 8-10 mice per group. Sham group received saline injections. Cis, cisplatin; LPS, lipopolysaccharide; Cis 12 + 2-wk, cisplatin 12 mg/kg + LPS 5 mg/kg treatment for 2 weeks; Cis 10 + 1-wk, cisplatin 10 mg/kg + LPS 5 mg/kg treatment for 1 week; Cis 10 + 2-wk, cisplatin 10 mg/kg + LPS 5 mg/kg treatment for 2 weeks; Cis 8 + 2-wk, cisplatin 8 mg/kg + LPS 5 mg/kg treatment for 2 weeks; Cis 8 + 4-wk, cisplatin 8 mg/kg + LPS 5 mg/kg treatment for 4 weeks; BUN, blood urea nitrogen.

Consequently, we explored the difference in renal injury development between 1- and 2-week treatments of cisplatin (10 mg/kg administered once per week) (Fig. 2A and 2C). The 2 weeks treatment group showed significant changes in body weight, renal weight, renal area, and serum BUN levels compared to the sham group, suggesting robust renal injury. However, the one-week treatment group only showed significant differences in renal weight, with other parameters being less affected, indicating that a single administration may not be sufficient to fully induce renal damage and emphasizing the need for prolonged exposure to observe more pronounced effects.

In the case of low-dose cisplatin (8 mg/kg administered once per week) administration, comparing 2 weeks and 4 weeks treatment groups (Fig. 2A and 2D), no significant injury was observed in the 2 weeks treatment group, as evidenced by the absence of alterations in physiological parameters, including body weight and renal weight, and the stability of serum BUN levels, suggesting insufficient model induction at this dose and time frame. However, the four-weeks treatment group resulted in a noticeable increase in renal injury, evidenced by significantly reduced body weight, renal weight, area, and increased BUN levels compared to the two-week treatment group. These results suggest that a longer duration is required for renal injury to manifest at lower doses of cisplatin.

Histological abnormality and fibrosis in renal injury mouse model

Histological analysis of mice with 10 mg/kg cisplatin for two weeks of treatment revealed renal injury characteristics, including tubule dilation (black arrow), cast formation (red arrow), and brush-border loss (blue arrow) (Fig. 3A). MT staining further confirmed an increase in fibrosis within the renal tissue, suggesting the onset of chronic damage after prolonged treatment (Fig. 3B).

Figure 3.Histological abnormality and fibrosis after inducing renal injury. Histological comparison between the sham group and 2 weeks treatment of 10 mg/kg of cisplatin with LPS 5 mg/kg group (Cis 10 + 2-wk) were performed using H&E (A) and MT (B) staining. Sham group received saline injections. Compared to the sham group, notable renal injury features, including tubule dilation (black arrow), cast formation (red arrow), and brush-border loss (blue arrow), were observed, along with evidence of fibrosis. Sham group received saline injections. H&E, hematoxylin and eosin staining; MT, Masson’s trichrome staining; Cis, cisplatin; Cis 10 + 2-wk, cisplatin 10 mg/kg + LPS 5 mg/kg treatment for 2 weeks. Scale bar represents 50 μm.

This study showed that the combination treatment of cisplatin and LPS was more effective at inducing the model compared to cisplatin treatment alone. These results support the increasing evidence that inflammatory components play a crucial role in the severity and progression of nephrotoxicity (Manohar and Leung, 2018).

In the high-dose cisplatin (12 mg/kg) with LPS (5 mg/kg) group, we observed a 50% mortality rate in the 2-week treatment cohort, indicating that while this dose effectively induces renal injury, it also poses significant toxic risks (Zheng et al., 2024). The observed lethality suggests that this dosage may not be ideal for long-term studies or applications where survival is critical. Therefore, we administered twice injections of LPS at 5 mg/kg per week to induce inflammation, effectively simulating renal injury while minimizing mortality in the mouse model. These results highlight the importance of balancing between dosage and duration of cisplatin treatment to generate an effective renal injury model.

Lower doses of cisplatin (8 and 10 mg/kg) with LPS (5 mg/kg) provided a more sustainable approach, as all mice survived in these groups, and significant renal injury was observed during extended treatment periods. The results from the 10 mg/kg cisplatin with LPS 5 mg/kg group also suggest that mild renal injury can initially be recovered, but cumulative damage becomes more evident with continued administration. These results suggest that the progression of renal injury is time-dependent, and longer treatment duration may be required to observe more pronounced physiological changes.

Interestingly, the 8 mg/kg cisplatin with 5 mg/kg LPS group did not show significant renal injury at the 2-week time point. However, extending the treatment duration to 4 weeks resulted in clear signs of renal injury, including body weight loss, decreased renal weight, and increased BUN levels. This finding emphasizes the importance of treatment duration in low-dose nephrotoxicity models. While high-dose cisplatin may induce renal damage at earlier exposure time, lower doses require longer exposure times to achieve similar levels of injury, supporting the notion that cisplatin-induced nephrotoxicity is cumulative and increases gradually with repeated exposure (Fu et al., 2024).

Both the 10 mg/kg cisplatin with 5 mg/kg LPS treatment for 2 weeks and 8 mg/kg cisplatin with 5 mg/kg LPS treatment for 4 weeks effectively induced renal injury in our model, but each approach had distinct advantages and limitations regarding time and dosage. The 10 mg/kg cisplatin with 5 mg/kg LPS treatment for 2 weeks provided a faster onset of injury, demonstrating significant physiological and histological damage, making it a more efficient choice when a shorter treatment duration is required. On the other hand, 8 mg/kg cisplatin with 5 mg/kg LPS treatment for 4 weeks may be more suitable for experiments that require lower drug dosages to minimize toxicity.

We measured renal length as an indicator of renal injury. It is widely recognized that several pathological processes contribute to a reduction in renal area, including tissue damage and fibrosis (Sanz et al., 2023). Following the initial death of tubular epithelial cells changes such as tubular vacuolization and nephron loss occur, leading to a decrease in functional tissue, which is subsequently replaced by fibrotic tissue (Berru et al., 2019). Our results also demonstrate fibrosis, along with characteristic features of renal injury, such as brush-border loss and tubule dilation, further supporting the idea that these cellular and structural changes contribute to renal size reduction. Additionally, we relied on histological analysis (fibrosis) and serum BUN levels as markers of renal injury following LPS and cisplatin administration. In future studies, we aim to further investigate this renal injury model by exploring the expression of inflammatory cytokines and fibrosis markers, aiming to develop potential therapeutic agents for renal injury.

In conclusion, incorporating LPS, known to amplify inflammatory responses, alongside cisplatin administration has led to the development of a renal injury model that more effectively induces nephrotoxicity. When cisplatin is administered alone, there are challenges in creating a renal injury model, as mice either die due to severe nephrotoxicity or the model recovers naturally. However, by adding LPS, we were able to establish a CKD model that maintains mouse survival while effectively inducing renal damage. Based on these findings, we have successfully developed a renal injury model that not only facilitates the study of nephrotoxicity mechanisms but also supports the exploration of potential therapeutic interventions.

None.

Conceptualization, S-B.H., S-S.K., Y.L., and E.K.; methodology, S-B.H., S-S.K., Y.L., and E.K.; investigation, S-B.H., S-S.K., Y.L., and E.K.; data curation, S-B.H., S-S.K., Y.L., and E.K.; writing-original draft preparation, S-B.H., and E.K.; writing-review and editing, S-B.H., S-S.K., Y.L., and E.K.; supervision, S-S.K., Y.L., and E.K.; project administration, Y.L., and E.K.; funding acquisition, Y.L., and E.K.

This work was supported by an intramural research program from the CHA Advanced Research Institute, grant number CARI-RD-017. This research was supported by a grant of Korean Cell-Based Artificial Blood Project funded by the Korean government (The Ministry of Science and ICT, The Ministry of Trade, Industry and Energy, the Ministry of Health & Welfare, the Ministry of Food and Drug Safety) (grant number: RS-2023-KH140925).

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No potential conflict of interest relevant to this article was reported.