Analysis of the Ecacy and Renal Prognosis of a Reduced-Dose Immunosuppressive Regimen of Mild to Moderate ANCA-Associated Renal Vasculitis

Secondary infection caused by high-dose immunosuppressive therapy is one of the most common causes of death in patients with ANCA-associated vasculitis. This study aimed to explore whether reduced-dose immunosuppressive therapy can achieve the same ecacy while reducing secondary infection in mild to moderate ANCA-associated renal vasculitis and to determine whether early secondary infections will affect the deterioration of renal function. The ecacy and safety of the reduced-dose regimen and standard regimen were compared using the chi-square test. The renal survival rates 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. blood lymphocyte ratio, c-reaction protein(CRP),procalcitonin(PCT), Erythrocyte sedimentation rate(ESR), serum albumin, total bilirubin(TBil), glutamic-pyruvic transaminase(ALT), glutamic oxalacetic transaminase(AST), previous history(hypertension, diabetes, hyperlipemia, concomitant symptom, etc.

Although cyclophosphamide (CYC) combined with high-dose glucocorticoids has been the standard regimen in AAV as remission induction therapy for nearly ve decades [9][10][11][12] , new immunosuppressive agents have been tried in clinical trials considering the side effects of the long-term use of cyclophosphamide and glucocorticoids [13][14][15] . For instance, rituximab is not inferior to daily oral cyclophosphamide for the induction of remission in severe ANCA-associated vasculitis and may be superior in relapsing disease, which has been proven by both RAVE and RITUXVAS trials 3,16 . However, due to economic reasons, rituximab is still di cult to conventionally apply in clinical practice. Additionally, there is still controversy regarding the hunting of regimens allowing for glucocorticoid reduction, which can balance e cacy and safety in patients with ANCA-associated vasculitis. Although the PEXIVAS trial was conducted to show that a rapid reduction in glucocorticoid regimen was noninferior to a standard regimen with respect to death or end-stage renal disease (ESRD) 17,18 , there is still no consensus on whether an initial reduced dose of glucocorticoids has similar e cacy for nonsevere diseases.
Secondary infection is the most common adverse event and the primary cause of death in immunosuppressive therapy for AAV [19][20][21] . Currently, both elementary experiments and clinical studies have indicated that infection may trigger the formation of ANCAs and that severe cases may eventually develop into ANCA-associated vasculitis 1,22−25 . Therefore, the core of management of AAV is far more than achieving disease remission, and it is necessary to concentrate on reducing mortality, preventing deterioration of renal function and improving the survival quality.
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 mild to moderate AAV patients with renal involvement. 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.

Patients
This was a single-center retrospective cohort study collecting information on 82 patients who were newly diagnosed in the 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 positivity needs to be met 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. Alveolar hemorrhage or anuria 2. Patients who had used any immunosuppressive agents for more than 2 weeks; 3. With other active autoimmune diseases at the same time, such as systemic lupus erythematosus (SLE), eosinophilic granulomatosis with polyangiitis (EGPA) or anti-glomerulus basement membrane antibody positivity; 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 had experience with oliguria at the rst visit. Finally, 58 patients meet the research conditions. (Figure 1)

Treatments
The remission-induction period was 6 months. All of the patients received intravenous pulses of cyclophosphamide (15 mg/kg), and the frequency was extended from once every 2-3 weeks according to the recommendation 26 to once every 4 weeks. 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 the standard-dose group and 0.3-0.7 mg per kilogram per day in the reduced-dose group. Patients were allowed to undergo treatment plasma exchange according to practice according to the clinician's experience. During the remission maintenance period, the frequency of cyclophosphamide switched from once every 4 weeks to once every 3 months with the same dose as before, combined with prednisone or prednisolone at a dose of 5-15 mg per day.

De nitions
Mild to moderate was de ned as BVAS 18. Renal remission was de ned as 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 an elevation of serum creatinine and either the persistence of hematuria or proteinuria. ESRD was de ned as the need for permanent dialysis or receipt of a renal transplant. The Birmingham Vasculitis Activity Score (BVAS) ranges from 0 to 63, and higher scores indicate more active disease.

Outcomes
The primary end point was the occurrence of ESRD within the 24 months; the secondary end points were the rate of secondary infection and ANCA turned negative within the rst 3 months and the rate of adverse events and persistent hematuria within the rst 6 months.
Statistical analysis SPSS 22.0 statistical software was used for statistical analysis. If measurement data conformed to a normal distribution, they were expressed as the mean ± standard deviation (x ± s), and the two-sample t test was used for comparison between the two groups. If 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 were expressed as a number (proportion), and the comparison between two groups of unordered classi cation data used the chi-square test. The renal survival rate was estimated using the Kaplan-Meier method and compared using the log rank test. Factors with a P value <0.05 in Kaplan-Meier analysis were included in 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. A 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 the Department of Nephrology, Renji Hospital, Shanghai Jiaotong University School of Medicine from January 2013 to September 2020 were included. Twenty-four people who did not meet the inclusion criteria were excluded, and 58 people were involved in this study. Among them, 35 were in the standard-dose glucocorticoid group, and 23 were in the reduced-dose glucocorticoid group. The ratio of males to females 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, the baseline Scr was 251. 35 [12,17], 2/9 cases complicated with underlying pulmonary diseases (one had pulmonary tuberculosis history, another had chronic obstructive pulmonary disease); 6/9 patients developed pulmonary infections within 3 months, and 4/9 patients had persistent hematuria for more than 6 months. The proportion of patients who developed ESRD in the standard-dose glucocorticoid group was signi cantly higher than that in the reduced-dose glucocorticoid group, but there was no signi cant difference due to the limited sample size. (Table 1) Secondary end points  Table 1) Risk predictors of ESRD Age, baseline Scr, infection, Scr drop rate and ANCA became negative within the rst 3 months, persistent hematuria for more than 6 months, baseline UACR, baseline albumin, baseline lymphocyte ratio, hypertension, diabetes, and chronic pulmonary disease were included in the Kaplan-Meier analysis (Figure 3-7), and the renal survival rate was compared by the log rank test (Table 2). Among them, the baseline Scr, infection and Scr drop rate within the rst 3 months, persistent hematuria for more than 6 months, and baseline UACR were signi cantly correlated with endpoint events with P values less than 0.05 and were included in the multivariate Cox proportional hazard model.  (Table 3)

Discussion
The AAV patients in our study were those with BVAS scores less than 18, all of whom had no alveolar hemorrhage or anuria. Since most of the current studies focused on severe cases of AAV, the optimal treatment of mild to moderate patients is worthy of further discussion.
Multiple studies demonstrated that the most common cause of death in patients with ANCA-associated vasculitis was secondary infection caused by high-dose immunosuppressive therapy, very few due to active vasculitis. The RITUXVAS trial indicated that most deaths occur in the early stages of treatment (within 3 months), 50% of which resulted from secondary infection 3 . A clinical study from China also found that secondary infection was the leading cause of death through a one-year follow-up of AAV patients [19][20][21] . The hazard factors associated with severe infection in AAV patients include the use of chronic immunosuppression, long-term placement of central intravenous catheters, uraemia/dialysis dependency, and gamma globulin depletion caused by plasma exchange 27 ; in particular, the use of highdose glucocorticoids and cyclophosphamide was an independent risk factor for infection in AAV patients 26,28,29 . Consequently, based on the presupposition of achieving remission, a reduced regimen of immunosuppressive therapy can be considered an option to lower the infection rate, especially in patients with mild to moderate ANCA-associated renal vasculitis. The frequency of intravenous pulses of cyclophosphamide and the initial dose of prednisone or prednisolone were both reduced in our study, considering some factors as follows: most patients with AAV have no or mild extrarenal manifestations, some concomitant infection at the rst visit and cyclophosphamide itself has certain nephrotoxicity.
The secondary infection of AAV patients is not only the main cause of death during the course of the disease but also may be an important factor in aggravating the condition. A large number of elementary experimental and clinical studies have explored the causal relationship between infection and vasculitis. Elementary experimental studies have summarized some proposed mechanisms of ANCA formation during the process of infection as follows: initiation of autoimmune response by microbial peptides that are complementary to auto antigens; epigenetic silencing and antigen complementarity leading to upregulation of autoantigen genes; molecular mimicry between bacterial and self-antigens; formation of neutrophil extracellular traps that stimulate immune processes including production of ANCA; and interaction of bacterial components with Toll-like receptors, which leads to formation of mediators affecting the immune responses to infections and can trigger ANCA production [22][23][24][25] . Clinical studies have shown that chronic nasal Staphylococcus aureus carriage in both GPA and MPA patients has a higher risk of relapse, especially GPA patients, who have an 8-fold increase in the risk of relapse, and there is also evidence suggesting that antibacterial treatment may reduce the risk of relapse in patients with sinusitis [30][31][32][33][34][35] ; simultaneously, persistent infection of Chlamydia pneumoniae has been proven to contribute to the pathogenesis of MPO-ANCA-associated renal vasculitis in a process that elicits a hypersensitivity reaction of the host and ultimately results in strongly enhanced focal in ammatory reaction and necrotizing vasculitis 36-38 . In our study, the data showed that the infection rate within the rst 3 months after treatment was signi cantly different between the two groups. Furthermore, the Cox proportional hazard models demonstrated that AAV patients who had infection within the rst 3 months had a lower renal survival rate, and the results of the area under the curve (AUC) analysis demonstrated the predictive value of this variable.
In addition to infection, the multivariate Cox regression model showed that patients with higher baseline creatinine and persistent hematuria for more than 6 months were also at higher risk of developing ESRD in the early stages of the disease. In AAV patients, 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 a median follow-up time of 17 months, AAV patients with persistent hematuria for more than 6 months were at higher risk of developing 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 histopathologic types, including other types of active glomerulus nephritis 43 , which indicates that patients with persistent hematuria may have new renal pathological manifestations on the basis of the original disease, which would affect the prognosis.
In summary, we believe that in the process of immunosuppressive treatment, 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 were mainly MPA patients; therefore, the results may not be applicable to GPA and EGPA patients. The median follow-up time in this study was 17 months, which could be longer in subsequent studies.
In conclusion, our study proved that a decrease in the secondary infection rate would lower mortality in the early stage and may prevent the progression or relapse of disease, thereby delaying the deterioration of kidney function. Hence, the regimen of reduced-frequency cyclophosphamide combined with initial reduced-dose glucocorticoids may be more appropriate than the standard-dose regimen in mild to moderate AAV and can be used as an option. In addition, we propose that patients with higher baseline serum creatinine, secondary infection within the rst 3 months, and persistent hematuria for more than 6 months of treatment were at higher risk of developing ESRD.

Declarations
Ethics approval and consent to participate The study have been performed in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Renji Hospital, and due to the retrospective nature of the study, the need for patients' informed consent was waived.

Consent for publication
Not applicable.

Availability of data and materials
All data generated or analysed during this study are included in this published article.

Competing interests
The authors report no con icts of interest. The authors alone are responsible for the content and writing of the article.

Funding
None.
Authors' contributions The Kaplan-Meier method was used to estimate the cumulative probability of renal survival rate, and the log rank test was used for comparison. A P-value < 0.05 was considered to denote statistically signi cant differences. Factors with a P value < 0.05 in Kaplan-Meier analysis were included in multivariate Cox proportional hazard models. A 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. Twenty-four patients who did not meet the inclusion criteria were excluded, and a total of 58 patients were involved in this study. All patients received intravenous pulses of cyclophosphamide, 15 mg/kg, given 4 weeks apart, and the maximum dose per pulse was 1 g, adjusted for renal insu ciency. Thirty-ve patients received prednisone or prednisolone at a dose of 1 mg per kilogram per day in the standard-dose group, and 23 patients received prednisone or prednisolone at a dose of 0.3-0.7 mg per kilogram per day in the reduced-dose group. A total of 9 patients developed ESRD, 7 patients in the standard-dose glucocorticoid group and 2 patients in the reduced-dose glucocorticoid group.