Reduced Regime of Glucocorticoids and Cyclophosphamide for Mild ANCA-Associated Renal Vasculitis May Delay Deterioration of Renal Function

Objectives To compare the ecacy and safety of initial reduced-dose glucocorticoids combined with reduced-frequency cyclophosphamide and to determine risk predictors of end-stage renal disease in ANCA-associated vasculitis(cid:0)AAV(cid:0)patients with renal involvement which BVAS was less than 20. Methods This is a single-center retrospective cohort study that involved 58 patients who were newly diagnosed with ANCA-associated renal vasculitis. The ecacy and safety of reduced-frequency cyclophosphamide combined with initial reduced-dose glucocorticoids were compared using chi-square test. The cumulative probability to ESRD were estimated using the Kaplan–Meier method and compared using the log rank test. Potential variates were examined using multivariate Cox proportional hazard models to determine the risk predictors of end-stage renal disease.


Introduction
Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a group of necrotizing small vasculitis, includes granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA). 1 It also can be divided into PR3-ANCA positive and MPO-ANCA positive AAV according to the serological classi cation. Renal involvement occurs in 70% of AAV patients, 2 especially MPO-ANCA positive AAV 3 . ANCA-associated renal vasculitis is mainly manifested as rapidly progressive glomerulonephritis, and there are pauci-immune complex deposition and crescentic glomeruli in most kidney pathological biopsy.
Cyclophosphamide(CYC) combined with high-dose glucocorticoids has been standard regime in AAV as remission induction therapy for nearly ve decades. [4][5][6][7] However, considering the side effects of the longterm use of cyclophosphamide and glucocorticoids, on the one hand, new immunosuppressive regimen was tried by clinical researches, such as rituximab, whose e cacy was proved that not inferior to daily cyclophosphamide treatment for induction of remission in severe ANCA-associated vasculitis and may be superior in relapsing disease. 2,8 But due to economic reasons, rituximab is still di cult to conventionally apply to clinical practice. On the other hand, there is still controversy regarding the effective and relatively safe rate at which glucocorticoid doses can be tapered in patients with ANCA-associated vasculitis.
Although PEXIVAS trial was conducted to show that a rapid reduction of regimen of glucocorticoids was noninferior to a standard-dose regimen with respect to death or end stage renal disease (ESRD), 9,10 for non-serious diseases, there is still no consensus whether initial reduced-dose of glucocorticoids has the similar effects.
Infection is still the most common adverse event in immunosuppressive therapy of AAV. Currently, both laboratory and clinical studies have demonstrate that infection may trigger the formation of ANCA and severe cases may eventually develop into ANCA-associated vasculitis [11][12][13][14][15] Therefore if the infection caused by immunosuppressants and glucocorticoids cannot be better tapered in the treatment of AAV patients, it may accelerate the progression of disease.
This retrospective cohort study compared the e cacy and safety of reduced-frequency cyclophosphamide combined with initial reduced-dose glucocorticoids as a remission induction regimen for AAV patients with renal involvement which BVAS score was less than 20. We also examined clinical variates for ESRD to determine the risk factors that can be used to recognize patients at higher risk of ESRD in the short term.

Ethical statements
The study was approved by the Ethics Committee of Renji Hospital, and all participants signed the written informed consent.

Patients
This was a single-center retrospective cohort study involving 82 patients who were newly diagnosed in Department of Nephrology, Renji Hospital, Shanghai Jiao Tong University School of Medicine from 2013 to 2020.Inclusion criteria include:1. Kidney damage caused by MPA or GPA, the conditions include at least any one of the following: Necrotizing glomerulonephritis on biopsy; Red cell casts or hematuria (>=30 red cells per high power eld) on urinalysis; 2. ANCA to be positive needs to meet the following: through enzyme-linked immunosorbent assay ( ELISA) positive for MPO-ANCA/PR3-ANCA or positive for p-ANCA/c-ANCA by indirect immuno uorescence (IIF).Exclusion criteria include: 1. Except kidney, other organ-threatening AAV 2. Patients who have used any immunosuppressive agents for more than 2 weeks; 3. With other active autoimmune disease at the same time, such as systemic lupus erythematosus (SLE), eosinophilic granulomatosis with polyangiitis (EGPA) or anti-glomerulus basement membrane antibody-positive, etc; 4. Hepatitis B e antigen-positive, hepatitis C antibody-positive, HIV-positive; 5. History of malignant tumors; 6. Pregnant or lactating women; 7. Patients who have experience of oliguria at the rst visit. Finally involved 58 patients. (Figure 1)

Treatments
The remission-induction period was 6 months. All of the patients have received intravenous pulses of cyclophosphamide, 15 mg/kg, given 4 weeks apart, the maximum dose per pulse was 1 g, adjusted for renal insu ciency. Initial prednisone or prednisolone was received at a dose of 1 mg per kilogram per day in standard-dose group and 0.3-0.7 mg per kilogram per day in reduced-dose group. Patients were allowed to undergo treatment plasma exchange or to receive one to three pulse of intravenous methylprednisolone (a maximum of 500mg) according to practice for managing severe disease.

De nitions
Renal remission was de ned as stable or improved renal function (stable or decreased serum creatinine), disappearance of hematuria (microscopic RBC <10/HP), and no other manifestations of AAV. Renal relapse was de ned as an increase in serum creatinine (Scr) of >30%, and/or new hematuria or proteinuria. Progressive disease was de ned as either the persistence of hematuria or proteinuria, and an elevation of serum creatinine. ESRD was de ned as the need for permanent dialysis or receipt of a renal transplant. Birmingham Vasculitis Activity Score (BVAS) range from 0 to 63 and higher scores indicating more active disease.

Outcomes
The primary end point was the occurrence of ESRD within the 24 months; the secondary end points were the remission rate and infection rate within the rst 3 and 6 months, the Scr drop rate at 3 month and persistent hematuria rate at 6 month.
Statistical analysis SPSS 22.0 statistical software was used for statistical analysis. If measurement data conforming to the normal distribution, they were expressed as mean ± standard deviation (x ± s), and the two-sample t test was used for comparison between the two groups. If it is not, they were expressed as the median (interquartile range) [M(P25, P75)], and the Mann-Whitney U test was used for comparison between the two groups. Categorical data was expressed as a number (proportion), and the comparison between two groups of unordered classi cation data uses the chi-square test. The cumulative probability of ESRD were estimated using the Kaplan-Meier method and compared using the log rank test. Include factors with P value <0.05 in Kaplan-Meier analysis into multivariate Cox proportional hazard models to identify the predictors of ESRD. Receiver operating characteristic (ROC) curve analysis was used to determine the predictive performance. The area under the curve (AUC) was calculated as the diagnostic measure of the test. P-value < 0.05 was considered to denote statistically signi cant differences.

Patient characteristics
A total of 82 patients with ANCA-associated renal vasculitis diagnosed in Department of Nephrology, Renji Hospital, Shanghai Jiaotong University School of Medicine from January 2013 to September 2020 were included. 24 people who did not meet the inclusion criteria were excluded, and nally 58 people were involved for this study. Among them, 35 were in the standard-dose glucocorticoids group and 23 were in the reduced-dose glucocorticoids group. The ratio of male to female among 58 people was 16/42, the average age was 62.45±12.70 years, the ratio of PR3-ANCA to MPO-ANCA was 5/53, and the baseline Scr  [12,17], 2 cases complicated with underlying pulmonary diseases, one was with pulmonary tuberculosis history, another was chronic obstructive pulmonary disease; 6 patients developed pulmonary infections within 3 months, and 4 patients had persistent hematuria for more than 6 months. The proportion of patients who developed ESRD in the standard-dose glucocorticoids group was signi cantly higher than that in the reduced-dose glucocorticoids group, but there is no signi cant statistical difference. (Table 1 Table 1 Risk predictors of ESRD Age, baseline Scr, infection, Scr drop rate and ANCA turned negative within rst 3 months, persistent hematuria for more than 6 months, baseline UACR, baseline albumin, baseline lymphocyte ratio, hypertension, diabetes, combined with chronic pulmonary disease were included in the Kaplan-Meier analysis, (Figure 3-7), and compared by Log rank test, ( Table 2). Among them, the baseline Scr, infection and Scr drop rate within rst 3 months, persistent hematuria for more than 6 months, and baseline UACR were signi cant correlated with endpoint event which P value were less than 0.05 and were included in the multivariate COX proportional hazard model. The results showed that baseline Scr (Figure 8).

Discussion
The AAV patients in our study were those with BVAS less than 20, all of which no oliguria and no other organ-threating except kidney. Since most of the current studies are focused on severe cases of AAV, the optimal treatment to mild patients were not clear.
Cyclophosphamide has decades history as a classical and standard treatment agent for remission induction of ANCA-associated vasculitis, but considering the side effects of cumulative exposure to cyclophosphamide, including cancer, bladder injury, infection, gonadal damage, etc, 5,16-22 the exploration of different dose regime of cyclophosphamide has never stopped. Cyclophosphamide used to be given orally 2 mg/kg per day, or intravenously 15 mg/kg, every 2 to 3 weeks and treatment is continued for 3 to 6 months until remission. Long-term follow-up of the CYCLOPS trial showed that although pulse cyclophosphamide is associated with a higher relapse risk than oral cyclophosphamide, it was not associated with increased mortality or long-term morbidity. 23 Compared to CYCLOPS trial, all patients we studied were milder which BVAS score was less than 20 and most of which with only renal involvement. Therefore, the initial dose and frequency of administration of cyclophosphamide were both reduced on basis of standard protocol. The cumulative cyclophosphamide dose of our study was 2.4 [1.6, 3.7] g at 6 months in total included patients which was much lower than 8g in CYCLOPS trial. 24 After 6 months therapy, the remission rate was 86.21%, and the infection incidence rate was 25.86%, which were not inferior to CYCLOPS4 trial whose remission rate was 79.9%. This result suggested that for AAV patients with renal involvement and BVAS score<20, appropriately reducing the initial dose and frequency of administration of cyclophosphamide, while reducing the cumulative amount of cyclophosphamide, may still achieve effective clinical remission.
High-dose glucocorticoids used to be another cornerstone of remission induction treatment in AAV, [25][26][27] however due to numerous dose-dependent adverse effects, reducing dose of glucocorticoid and shortening using time of glucocorticoid have always been the key to nd optimal dose glucocorticoids which can keep a balance between e cacy and safety. The PEXIVAS trial compared two regimens of oral glucocorticoids in ANCA-associated vasculitis, and it did show that reduced-dose regimen of oral glucocorticoids was noninferior to standard-dose regimen. However, in PEXIVAS trial. 10 , the initial dose of glucocorticoid in both groups were the same, the dose was reduced rapidly by approximately 50% at the start of the second week in the reduced-dose group and the included patients were much severe. There is still absence of consensus whether initial glucocorticoids could be reduced in mild cases. In our retrospective study, all patients with a BVAS score less than 20 and were divided into standard-dose (1mg/kg) and reduced-dose group (0.3-0.7mg/kg) based on the initial dose glucocorticoids. After following up for 24 months, we did not get statistically signi cant difference in the incidence of ESRD between the two groups since the included number of patients were limited. But within the rst 3 months, there was signi cant difference of infection rate between two groups (reduced-dose 4.35% vs standarddose 25.72%, P=0.04). The result suggested that initial reduced-dose regimen maybe a better choice in treatment of mild AAV patients.
It is well known and quite important that infection may trigger the formation of ANCA and aggravate the course of AAV disease 28 . In our study, the data showed that infection rate within the rst 3 months after treatment was signi cantly different between two groups. Furthermore, the Cox proportional hazard models demonstrate that AAV patients who has infection within the rst 3 months are at higher risk of developing into ESRD [HR 9.835 have shown the relationship between infection and the development of AAV. high incidence of infection during induction therapy of Chinese AAV patients was been analyzed in a recent retrospective study, whose result showed that bacterial pneumonia was the main type of infection encountered. 29 In addition, the Staphylococcus was used of the greatest concern in a lot of number studies, which have shown that chronic nasal Staphylococcus aureus carriage in both GPA and MPA patients have a higher risk of relapse, especially GPA patients have an 8-fold increase in the risk of relapse and there are also evidences suggest that antibacterial treatment may reduce the risk of relapse in patients with sinusitis. [30][31][32][33][34][35] Besides, another focus was shown in some other prior studies that the persistent infection of Chlamydia pneumoniae contributes to the pathogenesis of MPO-ANCA associated renal vasculitis. They describe the process as that persistent infection of Chlamydia pneumoniae within macrophages and polymorphonuclear neutrophils would enhance the process of ANCA production, which elicits a hypersensitivity reaction of the host and nal resulting in strongly enhanced focal in ammatory reaction and necrotizing vasculitis. 36-38 From this point of view, control of infection has a pivotal effect during the therapeutic process which may affect the disease response and the course of AAV. Our study suggested that, for non-serious AAV patients with renal involvement, in order to reduce the incidence of infection and raise therapeutic effect, the regime of reduced-frequency cyclophosphamide combined with initial reduced-dose glucocorticoids may be more appropriate than standard-dose regimen and can be used as an option.
Besides infection, the multivariate COX regression model showed that patients with higher baseline creatinine and persistent hematuria for more than 6 months are also at higher risk of development to ESRD in the early stages of the disease. In AAV patients, the microscopic hematuria is a marker of renal damage and disease activity, but the signi cance is still controversial. Although previous studies have indicated that persistent hematuria can be used as an important predictor of future renal relapse in AAV patients after induction therapy 39,40 , other studies showed that persistent hematuria only indicates renal damage degree and cannot predict the progression of the disease 41 . A retrospective study demonstrated that no disappearance of hematuria for more than 90 days following standard treatment after diagnosis of ANCA-associated renal vasculitis was not related to a lower eGFR at 1 year of follow-up. 42 However, in our study, with median follow up time of 17 months, the AAV patients with persistent hematuria for more than 6 months were at higher risk of development to ESRD. A small series clinical trial has given us some mentality. This trial involved nine AAV patients in clinical remission with either persistent or new hematuria, and given them repeat kidney biopsies, the results found various of histopathologic types including other types of active glomerulus nephritis, 43 which indicating that patients with persistent hematuria may have new renal pathological manifestations on basis of the original disease, which would affect the prognosis. In summary, we believe that in the process of immunosuppressive treatment, the persistent hematuria may represent the degree of renal damage, and may also have a certain predictive value for the prognosis of the disease. For patients with persistent hematuria, clinicians should promptly pay attention to and adjust the treatment strategy to prolong the occurrence of ESRD.
However, our study is a retrospective study with a limited sample size, and its results need to be further con rmed in a larger prospective cohort study. The patients included in this study are mainly MPA, so its results may not be applicable to GPA and EGPA patients. The median follow-up time in this study was 17months which could be longer in the following studies.
In conclusion, the current study demonstrated that the regime of initial reduced-dose glucocorticoids combined with reduced-frequency cyclophosphamide can get effectively remission in AAV patients with renal involvement which BVAS score was less than 20, while reducing the incidence of infection at the same time. We propose that patients with higher baseline serum creatinine, infection within rst 3 months, and persistent hematuria for more than 6 months of treatment were at higher risk in development of ESRD. To reduce the incidence of infection and thus delay deterioration of renal function, the reduced-dose regime may be more appropriate than standard-dose regimen and can be used as an option in mild patients.

Supporting information
Table1. The anonymized data set of the present study.

Con icts of Interest
The authors report no con icts of interest. The authors alone are responsible for the content and writing of the article. The Kaplan-Meier method was used to estimate the cumulative probability of ESRD, and log rank test was used to compare. P-value < 0.05 was considered to denote statistically signi cant differences. Include factors with P value < 0.05 in Kaplan-Meier analysis into multivariate Cox proportional hazard models. P-value < 0.05 was considered to denote statistically signi cant differences. Figure 1 A total of 82 patients with ANCA-related vasculitis were included. 24 patients who did not meet the inclusion criteria were excluded, and a total of 58 patients were involved for this study. All patients has received intravenous pulses of cyclophosphamide, 15 mg/kg, given 4 weeks apart, the maximum dose per pulse was 1 g, adjusted for renal insu ciency. 35 patients have received prednisone or prednisolone at a dose of 1 mg per kilogram per day in standard-dose group and 23 patients have received prednisone or prednisolone at a dose of 0.3-0.7 mg per kilogram per day in reduced-dose group. A total of 9 patients developed to ESRD, 7 patients in the standard-dose glucocorticoids group and 2 patients in the reduceddose glucocorticoids group.