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. Author manuscript; available in PMC: 2023 Jan 20.
Published in final edited form as: Arthritis Rheumatol. 2021 Jun 8;73(8):1478–1488. doi: 10.1002/art.41685

Camptothecin and topotecan, inhibitors of transcription factor Fli-1 and topoisomerase, markedly ameliorate lupus nephritis in NZBWF1 mice and reduce the production of inflammatory mediators in human renal cells

Xuan Wang 1,2, Jim C Oates 2,3, Kristi L Helke 4, Gary S Gilkeson 2,3, Xian K Zhang 2
PMCID: PMC9853877  NIHMSID: NIHMS1674780  PMID: 33559345

Abstract

Objective.

To examine the therapeutic effects of camptothecin (CPT) and topotecan (TPT), inhibitors of transcription factor Fli-1 and topoisomerase, on lupus nephritis in NZBWF1 mice and their effects on inflammatory mediators in human renal cells.

Methods.

NZBWF1 female mice were treated with vehicle, cyclophosphamide (CYC), CPT (1 or 2mg/kg), and TPT (0.03, 0.1, or 0.3mg/kg), beginning at the age of 25 weeks twice a week by intraperitoneal injection (8–10 mice/group). Blood and urine were collected for monitoring autoantibodies and proteinuria. Mice were sacrificed at 40 weeks, and renal pathological scores were examined. Human renal endothelial and mesangial cells were treated with CPT or TPT, expression of cytokines was measured.

Results.

None of NZBWF1 mice treated with 1 or 2mg/kg of CPT, 0.3mg/kg of TPT had proteinuria higher than 100mg/dl at the age of 40 weeks. One of eight mice treated with 0.1mg/kg of TPT and one of ten mice treated with CYC had proteinuria higher than 300mg/dl, whereas 90% of the mice treated with the vehicle had proteinuria higher than 300mg/dl. Compared to vehicle control, mice treated with 1 or 2mg/kg of CPT, 0.1 or 0.3mg/kg of TPT and CYC had significantly prolonged survival, attenuated renal injury, diminished splenomegaly, reduced anti-dsDNA autoantibody, and reduced IgG and C3 deposits in the glomeruli. Human renal cells treated with CPT or TPT had reduced expression of Fli-1 and decreased MCP1 following stimulation with IFN-α or IFN-γ.

Conclusion.

The results indicate that low dose CPT and TPT could be repurposed to treat lupus nephritis.

Introduction

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease and is characterized by the production of autoantibodies, immune-mediated inflammation in a variety of organs, and the accrual of organ damage over time (1–3). Lupus nephritis is one of the major causes of morbidity and mortality in SLE patients (4–6). Up to 60% of adult SLE patients and 80% of juvenile SLE patients develop clinical lupus nephritis during the course of their illnesses (6). Ten to thirty percent of patients with lupus nephritis progress to end-stage renal disease (ESRD) within 15 years of diagnosis (7). Despite the severe outcome of many patients with lupus nephritis, treatment options are limited by toxicity. The most severe side effects of medication for treating lupus nephritis are increased risk of infections and malignancies, and amenorrhea (8, 9). In more than 50 years, only one new drug, belimumab, has been approved for the treatment of SLE (10–13). Thus, novel approaches to the treatment of lupus nephritis is urgently needed (10, 14).

Fli-1 transcription factor belongs to the Ets gene family, and abnormal expression of Fli-1 has been associated with the pathogenesis of SLE in both human patients and murine models of lupus (15–18). Transgenic mice with over two-fold expression of Fli-1 developed a progressive immune complex-mediated lupus-like renal disease and ultimately died of renal failure (19). Reduced expression of Fli-1 in both MRL/lpr and NZM2410 mice resulted in profound, prolonged survival with significantly reduced severity of lupus nephritis (20, 21). In humans, increased expression of Fli-1 is significantly associated with new or recurrent lupus nephritis (22). We have demonstrated that Fli-1 is a key regulator in modifying the expression of several inflammatory cytokines, including MCP1, CCL5, IL-6, and CXCL-10, which are implicated in lupus development (23–25).

A previous report demonstrated that several groups of compounds, including camptothecin (CPT) and topotecan (TPT), can inhibit the expression and activity of Fli-1 (26). CPT is a natural product of a topoisomerase inhibitor discovered in the 1960s that has been used to treat leukemia in several countries (27). TPT is a semi-synthetic derivative of CPT with increased solubility and stability and is currently used to treat ovarian and lung cancers in the United States (28). In this report, we investigated whether low doses of these chemotherapeutic drugs could have therapeutic effects on lupus nephritis using NZBWF1 female mice and reduce the production of Fli-1-regulated cytokines in human renal cells. We have found that low doses of CPT and TPT significantly reduced autoantibody titers, improved renal function, and prolonged survival in NZBWF1 mice, and reduced production of inflammatory mediators in human renal cells following stimulation with IFN-α or IFN-γ.

Materials and Methods

Animals.

Twenty-week-old female NZBWF1 mice (stock No. 100008) were purchased from the Jackson Laboratory (Bar Harbor, ME), and mice were maintained at the animal facility at the Medical University of South Carolina (MUSC). All the animal experiments were approved by the Institutional Animal Care and Use Committee at MUSC.

Chemicals.

CPT, TPT and cyclophosphamide (CYC) were purchased from Selleckchem (Houston, TX), and dissolved in sterile PBS solution containing 1% DMSO. CYC was used as positive treatment control.

Treatment of mice with chemotherapeutic drugs.

Mice were randomized to the 7 groups with 8–10 mice per group. Mice were treated with vehicle (PBS solution containing 1% DMSO), CPT (1 or 2mg/kg), TPT (0.03, 0.1, or 0.3mg/kg), or CYC (25mg/kg) twice a week beginning at the age of 25 weeks by intraperitoneal (i.p.) injection. Doses of CPT and TPT used in this study were based on the doses used in mouse models for treating tumors and in the clinical treatment of human patients. The higher dose of CPT used in this study was about 1/4th to 1/17th the amount used in mouse tumor models (29, 30). The higher dose of TPT used in this study is about 1/6th to 1/20th of the dose used in the mouse tumor model (2.5mg/kg to 12.5mg/kg per week for tumor) (31), and the CYC dose was used as previously reported in murine lupus models (32, 33).

Proteinuria measurement.

Urine was collected as previously reported (20). Proteinuria was measured with Albustix (Siemens Healthcare Diagnostics) and quantified as grade 0 (negative), grade 1+ (≥ 30 mg/dl), grade 2+ (≥100 mg/dl), grade 3+ (≥300 mg/dl), or grade 4+ (≥2,000 mg/dl) according to the manufacturer’s recommendations.

Measurement of blood urea nitrogen (BUN), serum creatinine (sCr) and alanine aminotransferase (ALT).

Serum from NZBWF1 mice at 40 weeks was used for measurement. BUN, sCr and ALT were determined by the respective kits from BioAssay Systems (Hayward, CA) following the manufacturer’s instructions.

Measurement of serum immunoglobulin concentrations.

Total IgG, IgM, IgG1, IgG2a, IgG2b, and IgG3 levels in serum were determined by ELISA using a standard curve of known concentration of the same mouse Ig isotype as previously described (20).

Anti-ds-DNA autoantibody measurement.

Anti-dsDNA antibodies were measured by ELISA, as previously described (20).

Kidney pathology.

Kidneys were removed when the mice were sacrificed at 40 weeks. One kidney was fixed with 10% buffered formalin, embedded in paraffin, and then sectioned for H&E staining. The other kidney was frozen immediately in liquid nitrogen. Frozen sections (4 µm each) were stained with fluorescein-conjugated anti-mouse IgG or complement C3 (Jackson ImmunoResearch Lab, West Grove, PA). The intensity of IgG and C3 fluorescence staining was quantified by Image J after 10 high-power random fields taken from each section using a Keyence BZ-X700 microscope. The pathological scores of kidneys were examined in a blinded fashion as described previously (20).

Complement blood count (CBC) test.

20 µl whole blood from NZBWF1 mice at 23 or 39 weeks was collected in tubes with lithium heparin for the CBC test. The blood hematology profile was measured by the facility at the Department of Comparative Medicine, MUSC, using the Veterinary Multi-species Hematology System (Hemavet 950 FS, Drew Scientific, Miami Lakes, FL).

Cell culture.

Primary human renal glomerular endothelial cells (HRGECs) and human renal mesangial cells (HRMCs) were purchased from ScienCell Research Laboratories (Carlsbad, CA) and maintained in cell medium at 37oC with 5% CO2.

Measurement of cytokines.

MCP1, CXCL10, TNF-α, IL-10 and IL-6 concentrations in supernatants were determined by ELISA kit (R&D system, Minneapolis, MN).

Western blotting.

HRGECs or HRMCs were lysed with RIPA buffer, and the protein concentrations determined by the Pierce BCA protein assay. Expression of Fli-1 was detected by immunoblotting as previously described (20) .

DNA Transfection.

Equimolar concentrations of the Fli-1 expression construct and empty vector (pcDNA3.0, and pcDNA/Fli1) were transfected into the cells as described previously (34).

Results

NZBWF1 mice treated with CPT or TPT had reduced proteinuria, decreased serum levels of BUN and creatinine, and prolonged survival

NZBWF1 mice were initially treated with PBS control, CPT, TPT or CYC at the age of 25 weeks (Figure 1A), when around 40 to 50% of mice in each group had grade 1 proteinuria (data not shown). Proteinuria in the control group mice started to increase as mice aged (Fig. 1B). At the age of 34 weeks, 50% of control group mice and 60% of mice treated with 0.03mg/kg of TPT had proteinuria with grade 3 (≥300 mg/dl) (Fig. 1B). None of the mice treated with 1mg/kg of CPT., 2mg/kg of CPT, 0.1mg/kg of TPT, 0.3mg/kg of TPT had proteinuria with grade 2 (≥100mg/dl) at the age of 34 weeks (data not shown). One of 10 mice treated with 25mg/kg of CYC had grade 3 (≥300 mg/dl) proteinuria starting at the age of 34 weeks, and one of 8 mice treated with 0.1mg/kg of TPT had grade 3 proteinuria at the age of 40 weeks (Fig. 1B). Mice treated with 0.03mg/kg of TPT had similar proteinuria as mice treated with PBS. Ninety percent of mice that received PBS control had grade 3 proteinuria with (≥300 mg/dl), and seventy percent of these mice had grade 4 proteinuria (≥2000mg/dl) at 40 weeks (data not shown). Next, we measured the serum BUN and creatinine concentrations from all the mice at 40 weeks. Mice treated with 1mg/kg of CPT, 2mg/kg of CPT, 0.1mg/kg of TPT, or 0.3mg/kg of TPT, as well as 25mg/kg of CYC, had significantly lower BUN concentration compared to the mice treated with vehicle (Fig 1C). Mice treated with 1mg/kg of CPT, 0.1mg/kg of TPT, 0.3mg/kg of TPT, and 25mg/kg of CYC had significantly lower creatinine compared to the mice treated with vehicle (Fig. 1D). Mice treated with 2mg/kg of CPT had more than 50% lower creatinine compared to the control mice, though the difference was not statistically significant (Fig. 1D). All of the mice treated with CPT, the higher two doses of TPT (0.1 and 0.3mg/kg), or CYC survived to the age of 40 weeks, whereas only 60% of mice that received vehicle survived (p<0.05) (Fig. 1E). Fifty percent of mice treated with 0.03mg/kg of TPT survived to the age of 40 weeks. These data clearly demonstrate that low doses of CPT and TPT can keep NZBWF1 female mice in remission and increase survival rates. Mice treated with CPT or CYC developed slightly darken claws during the treatment, a known of side effects of CYC. Other than that, all the mice treated with 1mg/kg of CPT, 2mg/kg of CPT, 0.1mg/kg of TPT, or 0.3mg/kg of TPT, as well as 25mg/kg of CYC, did not show any clinical illness or abnormalities during the treatment.

Figure 1. Treatment with chemo drugs inhibiting Fli-1 prolonged survival and improved kidney function in NZBWF1 mice.

Figure 1.

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A. Timeline of drug treatment and sample collection in NZBWF1 mice. NZBWF1 mice were treated with CPT, TPT, CYC or vehicle at the age of 25 weeks twice a week (n=8–10 per group). B. Mice treated with CPT or TPT had significantly reduced proteinuria. The percentage of mice that have proteinuria with high grade of 3+ (≥ 300mg/dl). *p<0.05 compared with vehicle group by two-way analysis of variance (ANOVA). C. Reduced blood urea nitrogen (BUN) from the mice treated with CPT or TPT. D. Reduced serum creatinine (sCr) from the mice treated with CPT or TPT. BUN or sCr in serum from animals at the age of 40 weeks were measured. *p<0.05 compared with vehicle group by one-way ANOVA. n=8–10 mice each group. E. NZBWF1 mice treated with CPT or TPT had significantly improved survival. All mice (n = 8–10/per groups) treated with 1mg/kg and 2mg/kg of CPT, 0.1mg/kg and 0.3mg/kg of TPT and 25mg/kg of CYC survived to the age of 40 weeks compared with only 60% (6 of 10) of control groups. *p<0.05 compared to vehicle group via Kaplan-Meier log-rank test.

Reduced splenomegaly in the NZBWF1 mice treated with CPT or TPT

Splenomegaly is closely associated with lupus disease activity in both human patients and murine models of lupus (3, 35). We collected and measured spleen weights when the mice were sacrificed at 40 weeks. Spleen index (spleen/body weight ratio) and spleen size were significantly smaller in mice treated with 1mg/kg, 2mg/kg of CPT, 0.1mg/kg, 0.3mg/kg of TPT and CYC compared to the control group (Fig. 2A, 2C). Total spleen cell number was significantly lower from the mice treated with 1mg/kg, 2mg/kg of CPT, 0.3mg/kg of TPT and CYC compared to the control mice (Fig. 2B).

Figure 2. NZBWF1 mice treated with CPT or TPT exhibited reduced splenomegaly, reduced number of splenocytes, and reduced expression of Fli-1 from spleen cells.

Figure 2.

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A. Reduced splenomegaly in the mice treated with CPT or TPT. The spleen/body weight ratio, which represents the ratios of the spleen weight to the mouse body weight were analyzed at the age of 40 weeks (group, n=8–10). B. Reduced total spleen cell number in the mice treated with CPT or TPT. The total number of spleen cells was calculated at the age of 40 weeks (N=8–10/per group). C. Representative spleen size was shown from each group of mice. Spleen size was measured at the age of 40 weeks when the mice were sacrificed. D. Representative Fli-1 levels from spleen cells from each group mice were shown. E. Reduced expression of Fli-1 from spleen cells treated CPT or TPT. Expression of Fli-1 proteins in spleen cells from NZBWF1 mice were evaluated by Western blot at the age of 40 weeks when the mice were sacrificed. β-actin was used as the protein loading control. Expression of Fli-1 was quantified by normalized with β-actin level from each mouse. *p<0.05 compared with vehicle group by one-way ANOVA.

Spleen cells from mice treated with CPT or TPT had reduced expression of Fli-1

To determine if the CPT or TPT treatment affects expression of Fli-1, spleen cells from 40-week-old mice were lysed, and Fli-1 expression was determined by immunoblotting. The expression of Fli-1 in spleen cells from mice treated with 2mg/kg of CPT or 0.3mg/kg of TPT was significantly lower compared to mice treated with vehicle or CYC. The Fli-1 protein levels in spleen cells from mice treated with 1mg/kg of CPT or 0.1mg/kg of TPT were decreased compared to the mice treated with vehicle though the difference is not statistically significant (Fig. 2D, 2E).

NZBWF1 mice treated with CPT or TPT had significantly lower autoantibodies and reduced total IgG in serum

Anti-dsDNA autoantibodies are hallmarks of lupus, and their role in pathogenesis is well demonstrated (36, 37). Next, we measured anti-dsDNA autoantibody levels in the sera from mice treated with CPT or TPT. Low levels of anti-dsDNA autoantibodies were detected from all mice at the age of 23 weeks (Fig. 3A). The anti-dsDNA antibody level gradually increased in the control group with age, whereas the mice treated with 1mg/kg, 2mg/kg of CPT, 0.1mg/kg, 0.3mg/kg of TPT, or CYC had significantly lower anti-dsDNA antibody levels at the ages of 30 and 38 weeks. Anti-dsDNA antibody levels in the mice treated with 1mg/kg, 2mg/kg of CPT, 0.1mg/kg, 0.3mg/kg of TPT and CYC were still significantly lower at 40 weeks. Mice treated with 0.03mg/kg of TPT had significantly lower anti-dsDNA antibody levels at 38 weeks compared to the mice treated with the vehicle. Next, we measured serum total IgG, IgM, IgG1, IgG2a, IgG2b, and IgG3 in all groups of mice at 40 weeks. Mice treated with 1mg/kg, 2mg/kg of CPT, 0.3mg/kg of TPT or CYC had significantly lower serum IgG compared to the control group (Fig. 3B). Only the groups of mice treated with 2mg/kg of CPT or CYC had significantly lower serum IgM compared to mice treated with vehicle (Fig. 3C). Mice treated with 1mg/kg, 2mg/kg of CPT, 0.3mg/kg of TPT, and 0.1mg/kg of TPT had statistically lower serum IgG1 compared to mice treated with vehicle (Fig. 3D). Mice treated with 1mg/kg, 2mg/kg of CPT, 0.03mg/kg, 0.1mg/kg or 0.3mg/kg of TPT had significantly lower serum IgG2a compared to mice treated with vehicle (Fig. 3D). All mice treated with CPT, TPT and CYC had lower serum IgG2b compared to mice treated with vehicle, but only mice treated with 1mg/kg, 2mg/kg of CPT, and 0.03mg/kg of TPT had statistically lower IgG2b. Mice treated with either dose of CPT or CYC had significantly lower serum IgG3, whereas mice treated with 0.1mg/kg of TPT had slightly higher serum IgG3 concentrations compared to mice treated with vehicle (Fig. 3D).

Figure 3. CPT or TPT reduced anti-dsDNA antibody and total serum IgG levels in NZBWF1 mice.

Figure 3.

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A. Reduced anti-dsDNA antibody from mice treated with CPT or TPT. Serum levels of anti-dsDNA antibody were measured at the indicated ages. Data presented of anti-dsDNA Ab are the mean OD 450 ± S.E.M. at a 1:100 dilution in each group at the age of 23 to 40 weeks. Anti-dsDNA Ab were no different in each group at age of 23 weeks. *, # and & indicate p<0.05 compared with vehicle group at age of 30, 38 and 40 weeks, respectively, via two-way ANOVA. B-D. Comparison of serum levels of IgG, IgM, IgG1, IgG2a, IgG2b and IgG3 in NZBWF1 mice at the age of 40 weeks., *p<0.05 compared with vehicle group through one-way ANOVA. N=8–10 mice per group.

Mice treated with CPT or TPT had significantly reduced kidney weight index, decreased IgG and C3 deposits in glomeruli, and attenuated renal injury.

When mice were sacrificed at 40 weeks, kidney and body weights were measured, and kidney weight index (kidney/body weight ratio) was calculated. As shown in Fig. 4A, mice treated with 1mg/kg, 2mg/kg of CPT, 0.1mg/kg, 0.3mg/kg of TPT, or CYC had significantly decreased kidney weight index compared to control mice. The deposition of IgG and C3 in glomeruli was analyzed by immunofluorescence staining. Frozen sections of kidneys from all seven groups of mice were stained with fluorescein-conjugated anti-mouse IgG or C3. A random selection of 10 high-power field images was taken from each section, and the fluorescence intensity was analyzed using ImageJ software. The results showed significantly reduced IgG and C3 deposition in the glomeruli of mice treated with 1mg/kg or 2mg/kg of CPT, 0.1mg/kg or 0.3mg/kg of TPT, or CYC, compared to the control mice (Fig. 4B, 4C and 4E) . Renal pathological scores were assessed on the other kidney from each mouse. As shown in Fig. 4D and 4F, mice treated with 1mg/kg or 2mg/kg of CPT, 0.1mg/kg, or 0.3mg/kg of TPT or CYC had significantly reduced renal pathology scores compared to control.

Figure 4. CPT or TPT treatment reduced renal deposition of IgG and C3 and pathological renal scores in NZBWF1 mice.

Figure 4.

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A. Reduced kidney/body weight ratio from mice treated with CPT or TPT. Average kidney/body weight index by groups was measured at the age of 40 weeks. B-C. Reduced IgG deposits in the glomeruli from the mice treated with CPT or TPT. Assessment of glomerular IgG and C3 deposition in the kidney from the NZBWF1 mice treated with CPT, TPT, CYC or vehicle by immunofluorescence (IF) microscopy; sections were prepared from the kidneys of 40-week-old NZBWF1 mice. Quantified immunofluorescence density of IgG (B) and C3 (C) deposition. Each symbol represents an individual mouse. D. Reduced renal pathological scores from the mice treated with CPT or TPT. The renal pathology score of each kidney was measured according to glomerular proliferation, infiltration of inflammatory cells, crescents, and necrosis by a blinded observer. E. Representative images of IgG and C3 deposition from each group as indicated, white bar=200um. F. Representative images of H&E staining in kidneys (black bar=100um). Data are represented as mean±S.E.M. (n=8–10). *P<0.05 compared with vehicle group by one-way ANOVA.

Mice treated with CPT or TPT did not reveal liver toxicity or myelosuppression.

To measure liver toxicity of CPT or TPT at the doses used in the experiments, we measured the ALT in serum from mice at 40 weeks. As shown in Fig. 5A, mice treated with 1mg/kg or 2mg/kg of CPT, 0.03mg/kg, 0.1mg/kg or 0.3mg/kg of TPT had similar ALT compared to vehicle mice. To determine the potential impact of CPT and TPT on myelosuppression, we measured the CBC from mice at 23 and 39 weeks. Mice treated with vehicle had increased total white blood cells (WBCs), neutrophils, and lymphocytes at 39 weeks compared to the mice at 23 weeks, which reflects disease progression (Fig. 5B). Mice treated with 0.1mg/kg, 0.3mg/kg of TPT, or CYC at the age of 39 weeks had a similar number of white blood cells, neutrophils, and lymphocytes compared to the mice without treatment at the age of 23 weeks and significantly lower numbers of WBCs, neutrophils, and lymphocytes compared to the mice treated with vehicle at 39 weeks. Mice treated with 1mg/kg of CPT had a significantly higher number of WBCs, neutrophils and lymphocytes compared to the mice without treatment at 23 weeks. Counts of blood monocytes had similar effects as WBCs by group upon drug treatment (Fig. 5C). Mice treated with vehicle at 39 weeks had decreased red blood cells compared to mice without treatment at 23 weeks. Mice treated with 1mg/kg, 2mg/kg of CPT, 0.03mg/kg of TPT or CYC had reduced red blood cells compared to mice without treatment at 23 weeks. Mice treated with 0.1mg/kg, 0.3mg/kg of TPT had a similar number of red blood cells compared to the 23-week-old control mice, but significantly higher numbers compared to the 39-week-old control mice (Fig. 5D). All the mice had a similar number of platelets, except mice treated with 2mg/kg of CPT, which had a significantly higher number of platelets (Fig. 5E). Since it was reported Fli-1 deficiency is associated with systemic sclerosis (38), we examined mice treated with CPT or TPT regularly and did not find fibrotic manifestations of the skin in NZBWF1 mice during the entire treatment.

Figure 5. CPT or TPT had no significant side effect on liver function or hematopoietic system.

Figure 5.

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A. Comparison of ALT levels among the groups of mice. Serum levels of ALT from NZBWF1 mice at the age of 40 weeks were measured. B-E. Comparison of white blood cells, neutrophil, lymphocytes, monocytes, red blood cells and platelets among the groups of mice. Blood was collected from mice at 23 weeks (pre-treatment) and 39 weeks, and 20 hematology parameters were tested. Representative parameters of the hematopoietic system were presented including counts of WBCs (white blood cell), NE (neutrophil), LY (lymphocyte), MO (monocyte), EO (eosinophil), BA (basophil), RBC (red blood cell), Hb (hemoglobin), PLT (platelet) and MPV (mean platelet volume). The dotted line represents the mean of 23 weeks before onset of disease. ^P<0.05 compared with mice at 23 weeks by one-way ANOVA; *P<0.05 compared with vehicle control (PBS) mice at 39 weeks by one-way ANOVA. Data are represented as mean±S.E.M. (n=8–10).

HRGECs and HRMCs treated with CPT and TPT had reduced production of inflammatory mediators.

The involvement of HRGECs and HRMCs in lupus nephritis through the production of inflammatory cytokines and the expression of type I IFN in the pathogenesis of lupus development are well documented (39, 40). To test if CPT and TPT can inhibit Fli-1 in human cells, HRGECs and HRMCs were incubated with different concentrations of CPT (0.05µM to 0.5µM) for 12 hours. As low as 0.05 µM of CPT decreased Fli-1 protein expression (Fig. 6A). HRGECs treated with CPT and stimulated with IFN-α produced significantly less MCP1 and CXCL10 at 4 and 24 hours after stimulation with IFN-α (Fig. 6B). We are interested in MCP1 and CXCL10 levels given their involvement in lupus nephritis development, and we have shown that Fli-1 regulates expression of these two cytokines (23, 41). CPT and TPT also reduced Fli-1 expression in HRMCs (Fig. 6C). HRMCs treated with CPT or TPT significantly reduced the production of MCP1 following IFN-γ stimulation (Fig. 6D). Production of MCP1 and CXCL10 in HRMCs inhibited by CPT was dose-dependent (Supplemental Fig. 1). The levels of TNF-α and IL-10 in HRMCs and HRGECs treated with CPT were similar when compared to cells without treatment, whereas IL-6 that was regulated by Fli-1 was significantly reduced (25) (Supplemental Fig. 2). To verify if CPT reduced MCP1 by inhibiting Fli-1, HRMCs were treated with 0.25 µM of CPT for 12 hours and then transfected with 1μg plasmid pcDNA3.0 empty vector or pcDNA/Fli1 and stimulated with IFN-γ, and the MCP1 was measured after stimulation. Fli-1 protein levels and production of MCP1 were restored to similar levels in the cells transfected with plasmid pcDNA/Fli1 compared to the cells without CPT treatment (Fig. 6E, 6F). These data indicate that CPT reduced MCP1 largely through inhibition of Fli-1 expression.

Figure 6. CPT and TPT inhibited expression of Fli-1 and reduced inflammatory mediators in human renal cells.

Figure 6.

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A. Reduced Fli-1 by CPT in HRGECs. HRGECs treated with CPT for 12 hours and Fli-1 protein was evaluated by immunoblotting. B. Reduced MCP1 and CXCL10 production in HRGECs by CPT. HRGECs treated with 0.25 µM CPT or DMSO for 12 hours and stimulated with IFN-α. MCP1 or CXCL10 were measured by ELISA. C. Reduced Fli-1 in HRMCs by CPT or TPT. HRMCs were treated with 0.25 µM of CPT, 0.1µM of TPT, or control for 12 hours, and Fli-1 protein was evaluated by immunoblotting. D. Reduced MCP1 production in HRMCs by CPT or TPT. MCP1 in supernatants from HRMCs treated with 0.25 µM CPT, 0.1 µM TPT or control were measured by ELISA. E. Fli-1 protein levels in HRMCs was restored following the transfection with plasmid pcDNA/Fli1. F. MCP1 levels in HRMCs was restored following the transfection with plasmid pcDNA/Fli1. The cells were treated with 0.25 µM of CPT or vehicle for 12 hours and transfected with 1μg plasmid pcDNA3.0 empty vector or pcDNA/Fli1 and stimulated with FN-γ, and the MCP1 was measured 24 hours after stimulation. *P<0.05 treated vs control group.

Discussion

In this report, our data clearly demonstrated that chemotherapeutic drugs CPT and TPT, at least in part through inhibiting expression of transcription factor Fli-1, markedly ameliorated lupus nephritis in NZBWF1 mice. The NZBWF1 mice treated with 1mg/kg, 2mg/kg of CPT, 0.1mg/kg, 0.3mg/kg of TPT had completely eliminated splenomegaly and significantly decreased total serum IgG, serum anti-dsDNA antibodies, proliferative glomerulonephritis, renal inflammation, and proteinuria, and significantly prolonged survival.

The impact of treatment with CPT or TPT on splenomegaly, anti-dsDNA autoantibodies, and total serum IgG was profound. Mice treated with 1mg/kg, 2mg/kg of CPT and 0.3mg/kg of TPT had similar or lower anti-dsDNA autoantibodies at the age of 40 weeks compared to the titers at the age of 23 weeks, before the treatment started (Fig. 3). One of the possible mechanisms that mice treated with CPT or TPT had lower anti-dsDNA autoantibodies is through their immunosuppressive function; this is supported by our data showing the total serum IgG was significantly lower in the mice treated with CPT or 0.3mg/kg of TPT compared to the mice treated with vehicle (Fig. 3). CPT treatment had a more profound impact on total serum IgG and anti-dsDNA antibodies compared to the TPT treatment. The total serum IgG concentration in the mice treated with 2mg/kg of CPT was reduced more than 80% compared to the mice without treatment, despite the fact that there was no leukopenia in these mice (Fig. 3B, 5B). We have demonstrated that NZM2410 mice and MRL/lpr mice, murine models of lupus, with reduced expression of Fli-1 had significantly lower anti-dsDNA autoantibody levels and lower total serum IgG (20, 21). Mice treated with 1mg/kg, 2mg/kg of CPT, or 0.1mg/kg, 0.3mg/kg of TPT had significantly lower expression of Fli-1 (Fig. 2D). Thus, reduced expression of Fli-1 likely contributed to the decreased expression of anti-dsDNA antibodies in the mice treated with CPT or TPT. It is interesting to note that mice treated with 0.03mg/kg of TPT had lower anti-dsDNA antibodies but had similar renal pathological scores and survival compared to the control mice, which indicates additional factors other than anti-ds-DNA antibodies affect renal scores and survival in the mice with TPT treatment. The mice treated with 0.1mg/kg, 0.3mg/kg of TPT had similar total WBC and lymphocyte counts at 39 weeks compared to 23-week-old control mice but significantly lower counts compared to the 39-week-old control mice, which suggests these treatments induced disease remission. Mice treated with 1mg/kg or 2mg/kg of CPT had significantly higher total WBCs and lymphocytes at 39 weeks compared to 23-week-old control mice, having significantly reduced splenomegaly and renal pathological scores and prolonged survival (Fig. 5). These data indicate that CPT and TPT likely have different effects on myelotoxicity or myelosuppression.

Previous studies have reported that another topoisomerase I (Top I) inhibitor, irinotecan, has improved lupus nephritis in NZBWF1 mice (42, 43). In their reports, the total serum IgG and anti-dsDNA autoantibodies were similar from the sera between the mice treated with irinotecan and vehicle, though glomerular IgG and C3 deposits were significantly reduced in the mice treated with irinotecan (42, 43). Frese-Schaper and colleagues suggested that irinotecan protected NZBWF1 mice mainly by changing DNA relaxation and reducing anti-dsDNA binding to the autoantibodies (43). We found that both total serum IgG and anti-dsDNA autoantibodies were significantly reduced in the mice treated with 1mg/kg, 2mg/kg of CPT or 0.1mg/kg, 0.3mg/kg of TPT (Fig. 3). The differences between these previous reports and our data are likely due to different topoisomerase inhibitors used in the studies. Though CPT, TPT, and irinotecan belong to the same class of CPT analogs, they have different properties and different targets. CPT is water-insoluble with a relatively low ability to inhibit Top I (IC50 of 0.68 μM) (44). TPT is water soluble with high efficacy to inhibit Top I (45). Irinotecan is an inactive prodrug that is activated in vivo (46). We have found that CPT and TPT had different effects on neutropenia and platelets in NZBWF1 mice (Fig 5). Mice treated with 2mg/kg of CPT had significantly higher numbers of WBCs, neutrophils, lymphocytes, and platelets compared to the mice treated with TPT. (Fig. 5).

We further demonstrated that HRGECs and HRMCs treated with CPT and TPT had reduced expression of Fli-1 and decreased production of inflammatory cytokines following IFN-α or IFN-γ stimulation. (Fig. 6). An increased expression of type I IFN-regulated genes has been known to be associated with patients with SLE (40). Human T cells and macrophages treated with CPT and TPT had significantly reduced expression of Fli-1 and decreased production of inflammatory mediators (Wang et. al, unpublished data). These findings suggest that CPT and TPT could have therapeutic effects on human patients with lupus nephritis too.

Currently, CYC is one of the most reliable and effective treatments for treating patients with severe lupus nephritis (2). The major side effects of CYC include infertility, urotoxicity, and oncogenicity, and a significant portion of patients do not response to CYC treatment or do not tolerate CYC well as induction or maintenance therapy (2, 47). A high dose of CYC is used for treating patients with severe lupus nephritis at 500–1000 mg/m2 IV monthly for 6 doses, whereas CYC is used for treated breast cancer at 400–1800 mg/m2 at intervals of 2–4 weeks (2, 47, 48). The dose of CYC used in this study equals 600mg/m2/4weeks, which is a similar dose used for chemotherapy. The dose of TPT used in this study for inducing remission in lupus nephritis is equal to 0.6mg/m2 per week for human (1.8 mg/m2 per three weeks, for conversion, see F.D.A. guidance, Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers; https://www.fda.gov/media/72309/download) whereas the 7.5mg/m2 per three-week dose of TPT is used for human ovarian cancer and small cell lung cancer (49, 50). Thus, the dose used in this study is about 1/4th the dose for the cancer chemotherapy that achieved similar or better effects compared to the CYC, which suggesting an effective dose of TPT could have similar efficacy with less toxicity for treating lupus nephritis.

Decreased expression of Fli-1 has been reported to be associated with systemic sclerosis (38) , we did not find any signs of skin fibrosis from mice treated with CPT or TPT. Additionally, there is no literature that reports cancer patients who utilize high doses of TPT to be at an elevated risk of developing systemic sclerosis. As a result, we believe the risk of systemic sclerosis is low for patients that utilize CPT and TPT in treating lupus nephritis. A limitation of this study is that only about half of mice had proteinuria when the treatment started. We predict CPT or TPT will have therapeutic effects on mice with established lupus nephritis. As shown in Fig. 1B, for one mouse with grade 3 proteinuria (≥300mg/dl) at the age of 26 weeks, upon receiving 1mg/kg of CPT treatment, proteinuria levels were suppressed for the entirety post-treatment. Another area to study is when lupus nephritis relapse after treatment is discontinued with CPT or TPT.

In Summary, our data indicate that chemotherapeutic drugs CPT and TPT, at least in part by inhibiting expression of Fli-1, significantly ameliorated lupus nephritis in NZBWF1 mice as effectively or better than CYC. TPT is an FDA approved drug; thus, it is possible that the drug can be repurposed to treat lupus nephritis after further clinical investigation.

Supplementary Material

FIG S1-S2

Acknowledgments

This study was supported in part by a fund from Lupus Research Alliance (No. 6670 to X.K.Z.) South Carolina Clinical & Translational Research Institute (SCTR) Voucher Pilot Program NIH/NCRR Grants UL1 TR001450. We would like to thank Ms. Brittany Henry for her technical supports and Mr. Davis Borucki for the critical reading of the manuscript.

Supported in part by a grant from Lupus Research Alliance (6670 to X.Z.)

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Supplementary Materials

FIG S1-S2
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