Cyclophosphamide treatment for hypertension and renal injury in an experimental model of systemic lupus erythematosus

Abstract

Cardiovascular disease is the major cause of mortality among patients with the autoimmune disorder systemic lupus erythematosus (SLE). Our laboratory previously reported that immunosuppression with mycophenolate mofetil, a common therapy in patients with SLE, attenuates the development of hypertension in an experimental model of SLE. Cyclophosphamide (CYC) is another common therapy for patients with SLE that has contributed to improved disease management; however, its impact on the development of hypertension associated with SLE is not clear. We tested whether treatment with CYC (25 mg/kg, once/week, IP injection) for 4 weeks would attenuate hypertension in an established female mouse model of SLE with hypertension (30-week-old NZBWF1 females). Plasma anti-dsDNA IgG levels, pathogenic for the disease, were lower in CYC-treated SLE mice compared to vehicle-treated SLE mice, suggesting efficacy of the therapy to suppress aberrant immune system function. Mean arterial pressure (MAP) was assessed by carotid artery catheters in conscious mice. Treatment did not attenuate the development of hypertension when compared to vehicle-treated SLE mice; however, urinary albumin excretion was lower in CYC-treated animals. Corresponding with the reduction in autoantibodies, data suggest that CYC treatment lowered circulating CD45R⁺ B cells. Paradoxically, circulating CD11b⁺ Ly6G⁺ neutrophils were increased in CYC-treated SLE mice compared to vehicle treated. Estrus cycling data also suggest that CYC treatment had an impact on ovarian function that may be consistent with reduced circulating estrogen levels. Taken together, these data suggest that CYC treatment attenuates autoantibody production and renal disease during SLE, but that the potential to affect MAP may be blunted by the increase in circulating neutrophils and CYC's impact on ovarian function.

Keywords: Autoimmunity; hypertension; immunosuppression; sex hormones.

Figure 1

Impact of cyclophosphamide (CYC) on mean arterial pressure (MAP). MAP was measured by indwelling, carotid artery catheter. Vehicle‐treated systemic lupus erythematosus (SLE) mice had increased MAP compared to vehicle‐treated control mice (128 ± 6 vs. 113 ± 3 mmHg, *P < 0.02). Results suggest that blood pressure was not altered by CYC treatment in SLE mice compared to vehicle‐treated SLE mice (128 ± 6 vs. 136 ± 4 mmHg, P = 0.6). □ Control Vehicle (n = 13), ■ Control CYC (n = 11), ○ SLE Vehicle (n = 7), and ● SLE CYC (n = 10).

Figure 2

(A) Data suggest that urinary albumin excretion (94.68 ± 90.07 vs. 0.07 ± 0.02 mg/day, P = 0.45) is reduced in cyclophosphamide (CYC)‐treated systemic lupus erythematosus (SLE) mice compared to vehicle‐treated SLE mice. No statistical differences were found. □ Control Vehicle (n = 21), ■ Control CYC (n = 18), ○ SLE Vehicle (n = 16), and ● SLE CYC (n = 14). (B). Neutrophil gelatinase‐associated lipocalin (NGAL) excretion (pg/day) was not significantly different among vehicle‐ and CYC‐treated control and SLE mice. No statistical differences were found. □ Control Vehicle (n = 19), ■ Control CYC (n = 17), ○ SLE Vehicle (n = 13), and ● SLE CYC (n = 14). (C) kidney injury molecule‐1 (KIM‐1) excretion was significantly lower in CYC‐treated SLE mice compared to vehicle‐treated control mice (265.90 ± 30.67 vs. 790.30 ± 139.00, *P < 0.02). □ Control Vehicle (n = 21), ■ Control CYC (n = 19), ○ SLE Vehicle (n = 16), and ● SLE CYC (n = 14). (D) Glomerular scoring index was not significantly altered by CYC treatment in control or SLE mice. □ Control Vehicle (n = 8), ■ Control CYC (n = 8), ○ SLE Vehicle (n = 8), and ● SLE CYC (n = 8). (E) Renal histological sections were stained with H&E. Representative glomeruli (×40) are from vehicle‐ and CYC‐treated Control and SLE mice.

Figure 3

Impact of cyclophosphamide (CYC) on circulating immune cells. Flow cytometry was performed on cryopreserved peripheral blood leukocytes to determine the impact of CYC treatment on lymphoid and myeloid cell populations. (A) Representative scatter plot of peripheral blood leukocytes. (B) Data suggest that CYC treatment reduced CD45R⁺ B cells compared to vehicle treatment in systemic lupus erythematosus (SLE) mice (15.87 ± 8.49% vs. 26.96 ± 4.72%, P = 0.06). (C) There was no significant difference in the percentage of circulating CD3⁺CD4⁺T cells between groups or (D) the percentage of CD3⁺CD8⁺T cells.(E) The percentage of circulating neutrophils was significantly increased in CYC‐treated SLE mice compared to vehicle‐treated mice (39.26 ± 4.92 vs. 20.58 ± 6.01, *P < 0.05). (F) There was no significant different in the percentage of circulating monocytes between groups. ○ SLE Vehicle (n = 8), and ● SLE CYC (n = 10).

Figure 4

Impact of cyclophosphamide (CYC) on circulating autoantibodies. Systemic lupus erythematosus (SLE) mice treated with CYC had reduced circulating anti‐dsDNA autoantibodies compared to vehicle‐treated SLE mice (180.50 ± 64.68 vs. 581.60 ± 148.3 units/mL, *P < 0.05). Vehicle‐treated SLE mice also had significantly increased plasma anti‐dsDNA IgG (*P < 0.05) compared to vehicle‐ (138.70 ± 60.37 units/mL) and CYC‐treated (149.60 ± 63.86 units/mL) control mice. □ Control Vehicle (n = 18), ■ Control CYC (n = 17), ○ SLE Vehicle (n = 15), and ● SLE CYC (n = 12).

Figure 5

Impact of cyclophosphamide (CYC) on renal lymphocyte infiltration. (A) Renal CD45R⁺ B cells were significantly increased in systemic lupus erythematosus (SLE) vehicle‐treated mice compared to all other treatment groups (*P < 0.05) (B) Renal CD3⁺CD4⁺ T cells were significantly increased in SLE vehicle‐treated mice compared to all other treatment groups (*P < 0.05). (C) Renal CD3⁺CD8⁺ T cells were not significantly different in response to CYC treatment in control or SLE mice. □ Control Vehicle (n = 5) ■ Control CYC (n = 5), ○ SLE Vehicle (n = 5), and ● SLE CYC (n = 5).

Figure 6

Impact of cyclophosphamide (CYC) on estrus cycling. Data suggest that CYC‐treated systemic lupus erythematosus (SLE) mice (n = 15) spent a greater amount of time in the estrus phase of the ovarian cycle compared to vehicle‐treated SLE mice (n = 17).