Methods

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Patient Selection

To be eligible for the study, patients were required to have a nephrotic syndrome with biopsy-proven membranous nephropathy and deteriorating renal function. Exclusion criteria were as follows: age younger than 18 years, serum creatinine levels lower than 150 µmol/L, evidence for secondary types of membranous nephropathy (such as malignancy, hepatitis infection, positive test results for anti-DNA antibodies, or the use of drugs that could induce membranous nephropathy), child wish, diabetes mellitus, or clinical evidence of renal vein thrombosis. Renal biopsy specimens were taken from all patients at presentation and were re-evaluated by one investigator. Histologic staging was done according to the criteria of Ehrenreich and Churg [20]. All patients gave informed consent.

Study Design and Intervention

Patients were randomly assigned to one of two treatment protocols. Patients assigned to receive chlorambucil were given three cycles of treatment with steroids (intravenous pulses of methylprednisolone, 1 g on 3 consecutive days, followed by oral prednisone, 0.5 mg/kg of body weight per day for 27 days), and each cycle was followed by 1 month of treatment with oral chlorambucil (0.15 mg/kg per day). We used a lower dose of chlorambucil than that used by Ponticelli and colleagues [9] because in our previous study using chlorambucil at 0.2 mg/kg per day, leukopenia developed in more than half of the patients [18]. Patients assigned to receive cyclophosphamide were given intravenous pulses of cyclophosphamide, 750 mg/m², at monthly intervals for 6 months. They also received three intravenous pulses of methylprednisolone of 1 g for 3 consecutive days every 2 months. Cyclophosphamide was administered under forced saline diuresis. All patients received intravenous metoclopramide or ondansetron to avoid or reduce symptoms of nausea and vomiting. The patients received diuretics and antihypertensive drugs if required. Clinical examinations, biochemical profiles, and full blood counts were done at weekly intervals during immunosuppressive therapy and at regular intervals thereafter.

The trial was stopped when the serum creatinine levels doubled. For calculations of renal survival, renal death was defined as a doubling of the serum creatinine level or the start of renal replacement therapy. We have used the reciprocal of serum creatinine (1000/serum creatinine level) to assess the effect of the therapeutic interventions on the progression of renal insufficiency. Changes in this ratio parallel changes in endogenous creatinine clearance ([urinary creatinine concentration x V]/serum creatinine, where V = 24-h urine volume) because in a given patient, creatinine excretion (urinary creatinine concentration x V) is generally constant over time. For calculations, 1000 µmol/L (8.85 mg/dL) was taken as the creatinine level in patients who received hemodialysis. Complete remission was defined as a reduction of proteinuria to less than 0.3 g/24 h, and partial remission was defined as when proteinuria ranged between 0.3 and 3.0 g/24 h. To correct for inappropriate 24-hour urine collections, the amount of urinary protein per 10 mmol of creatinine (protein-creatinine index) was used for calculation.

At the start of the therapy, an interim analysis was planned after 20 patients were enrolled. By the end of 1992, we became aware of the study of Falk and colleagues [21], which showed no additional benefit of intravenous pulses of cyclophosphamide plus steroid treatment. Analysis of our patients' data also showed no effect of treatment with pulses of cyclophosphamide. It therefore seemed unjustified to continue the study protocol, and no more patients were enrolled.

Statistical Analysis

Results are given as the mean ±SD or as the median with range. Average changes in time from baseline values are given with 95% confidence intervals. Changes in serum creatinine levels were compared using repeated measures of analysis of variance. The Gehan log-rank test was used for survival analysis. The Wilcoxon test was used for all other statistical comparisons. A probability value of less than 0.05 was considered statistically significant.

Results

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Clinical Findings

Between June 1989 and November 1992, 20 patients met the entrance criteria of the study and were randomly assigned to either treatment regimen. Two of the 20 patients (1 from each treatment group) immediately withdrew after assignment. One had to receive regular dialysis before treatment with methylprednisolone and cyclophosphamide had begun, and the other became psychotic 2 weeks after starting prednisone treatment. Because these 2 patients received neither chlorambucil nor cyclophosphamide, their data are not used for analysis. Of the remaining 18 patients, 9 were assigned to receive chlorambucil and 9 to receive cyclophosphamide. The clinical details are given in Table 1. All patients but 1 were men, and all had a nephrotic syndrome (proteinuria, 3.5 g/d) and renal insufficiency. In the 6 months before the start of treatment, the serum creatinine levels of patients in the chlorambucil group increased from a mean of 146 ±69 µmol/L to 260 ±112 µmol/L; in the cyclophosphamide group, the levels increased from 141 ±87 µmol/L to 218 ±85 µmol/L (P > 0.2 between the two groups). Six patients in the chlorambucil group and 5 patients in the cyclophosphamide group had been treated previously with short-term, high-dose prednisone according to a previously published schedule [5]. In the group assigned to receive chlorambucil, 7 of the 9 patients received antihypertensive treatment consisting of one drug (3 patients received an angiotensin-converting enzyme inhibitor and 4 patients, a ß-blocker), and in the group assigned to receive cyclophosphamide, 5 of the 9 patients received an antihypertensive drug, an angiotensin-converting enzyme inhibitor. Patients were followed for 6 to 36 months after the start of immunosuppressive therapy.

In both treatment groups, six patients had stage I or II membranous nephropathy. In the chlorambucil group, two patients had stage III nephropathy. The biopsy specimen of one patient could not be re-evaluated. Three of the patients treated with pulse cyclophosphamide had stage III nephropathy.

Response to Immunosuppressive Therapy

The course of renal function as reflected by 1000/serum creatinine for the individual patients is shown in Figures 1 and 2. During the 6 months of treatment, the serum creatinine level had decreased in all patients treated with chlorambucil by an average of 74 µmol/L (range, –19 to –175µmol/L; P = 0.003). In contrast, we observed an uninterrupted increase of the serum creatinine level in seven of the nine patients treated with cyclophosphamide, with an average increase of 79 µmol/L (range, –67 to 204 µmol/L; P = 0.02; difference between both groups, P < 0.001). The difference in the course of the serum creatinine level between the treatment groups was already apparent 3 months after therapy was started (P = 0.02 compared with the baseline creatinine level), and it persisted during the subsequent years of follow-up. Renal survival differed significantly between the two treatment groups (P = 0.03) in favor of the patients treated with chlorambucil. Both groups had similar increases of serum albumin levels and decreases of proteinuria and serum cholesterol levels (Table 2).

View larger version (16K): [in this window] [in a new window]   Figure 1. Renal function as shown by 1000/serum creatinine level in individual patients treated with chlorambucil, methylprednisolone, and prednisone. Treatment was started at t = 0 months. * = start of renal replacement therapy; Triangle = start of retreatment with immunosuppressive therapy. The Y-axis represents the reciprocal of serum creatinine ÷ 1000. This ratio reflects renal function.

View larger version (14K): [in this window] [in a new window]   Figure 2. Renal function as shown by 1000/serum creatinine level in individual patients treated with intravenous pulses of cyclophosphamide and methylprednisolone. Treatment was started at t = 0 months. * = start of renal replacement therapy in a single patient. = patient died. Triangle = start of retreatment with immunosuppressive therapy.

Follow-up

Follow-up data for each patient are given in Table 3. In the chlorambucil group, one patient had a complete remission of proteinuria, and three had a partial remission of proteinuria after 1 year. After the first year, a gradual decrease of renal function occurred in several patients. One patient reached end-stage renal failure 36 months after starting treatment and has recently received a renal transplant. Three years after initial treatment, serum creatinine levels had doubled in three other patients compared with the lowest serum creatinine levels after treatment (an increase of almost 40% compared with the value before treatment). These patients are currently being treated successfully with a new course of immunosuppressive therapy. In the cyclophosphamide group, two patients had a complete remission of proteinuria 9 months after therapy started. In these two patients, serum creatinine levels before the start of treatment were 170 and 152 µmol/L, respectively. One patient with deteriorating renal function died 6 months after start of treatment after receiving a coronary bypass graft. The serum creatinine level of one patient (patient 18 in Table 3) doubled after 7 months of treatment; the patient is currently receiving immunosuppressive therapy. Four patients reached end-stage renal disease at 12 months (n = 2) and 18 months (n = 2), respectively.

Infectious complications only occurred in patients treated with chlorambucil (Table 3). One patient had a subcutaneous abscess in the axilla 3 months after starting therapy, after which the patient decided to discontinue the immunosuppressive treatment. At that time, his serum creatinine level had decreased from 176 to 125 µmol/L. After discontinuing treatment, his serum creatinine level increased to 192 µmol/L at latest follow-up (18 months). In addition, leukopenia was seen only in patients treated with chlorambucil, and it necessitated dose reduction in three patients. Three patients reported nausea and anorexia during chlorambucil treatment, whereas seven patients in the cyclophosphamide group reported nausea and anorexia on the day of infusion and the 2 consecutive days.

Discussion

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Our study shows that treatment with oral chlorambucil and prednisone can benefit patients with membranous nephropathy and deteriorating renal function. Although we did not include an untreated control group in our study, it seems likely that the reversal of deterioration in renal function observed in patients treated with oral chlorambucil is related to this treatment. First, the improvement in renal function was uniform and closely related to the time at which treatment was started. Second, in most untreated patients with membranous nephropathy and renal insufficiency, a continuous decrease of renal function is observed, whereas improvement of renal function in such patients has been reported rarely [22]. Third, the effects of oral chlorambucil treatment were clearly superior to the effects of cyclophosphamide. Monthly treatment with intravenous cyclophosphamide was ineffective, and the course of disease in our patients treated with cyclophosphamide closely resembles the course in untreated patients.

Our results on the beneficial effects of chlorambucil closely agree with those of Mathieson and colleagues [16], who observed an improvement in renal function in six of eight treated patients with membranous nephropathy and deteriorating renal function. It is clear from this and other studies that chlorambucil treatment poses major risks. Patients with renal insufficiency cannot tolerate a dose of 0.2 mg/kg per day as used by Ponticelli and colleagues [8]. Even at the reduced dose of 0.15 mg/kg per day that we used, bone marrow toxicity occurred in three patients. In addition, infectious complications, nausea, anorexia, and vomiting were frequent. The potential long-term risks of chlorambucil treatment (such as leukemia, lymphoid tumors, and skin cancer) must also be considered. On the other hand, the risks of treatment may be acceptable if the treatment prevents or delays the development of end-stage renal disease with its accompanying health hazards, such as infections and malignancies (as is the case with hemodialysis and kidney transplantation). In this respect, results obtained after long-term follow-up are important. In both our study and that of Mathieson and colleagues [16], beneficial effects of chlorambucil treatment were maintained during the first 18 months of treatment. However, in the few patients in our study who were followed for more than 3 years, deterioration of renal function recurred. Although three patients responded to re-treatment with immunosuppressive therapy, the numbers are too small to draw any firm conclusions. At this time we can conclude that chlorambucil treatment delays the development of end-stage renal disease for more than 18 months. However, treatment effects may disappear after 3 years.

Monthly intravenous pulses of cyclophosphamide were clearly ineffective. We chose this particular regimen to minimize side effects. It has been shown in patients with lupus nephritis that the use of intravenous pulses of cyclophosphamide is associated with fewer side effects than oral cyclophosphamide [19]. The concomitant use of oral steroids is debatable because response to treatment has been reported with the use of oral cyclophosphamide alone [13, 15]. Indeed, our treatment regimen with cyclophosphamide and methylprednisolone pulses had only minor side effects. However, the effects of the treatment were disappointing. Recently, Falk and colleagues [21] described similar results: The addition of monthly pulses of cyclophosphamide (although in a lower dose than used by us) to prednisone offered no additional benefit to prednisone alone [21]. Their results, as well as ours, differ from those of previous studies of West and coworkers [13, 15] and Bruns and colleagues [14]. These investigators observed improved renal survival in patients with membranous nephropathy and renal insufficiency after treatment with oral cyclophosphamide for at least 12 months. Most of those patients were concomitantly treated with oral steroids. Although the lack of effect of intravenous cyclophosphamide in our study might be explained by the omission of oral steroid treatment, an alternative explanation seems more likely. We tend to agree with Falk and colleagues [21], who argued that either difference in route of administration (intravenous compared with oral), in treatment duration (6 months compared with 12 months), or in the total dose of cyclophosphamide used could conceivably explain the differences in treatment effects. The possibility remains that cyclophosphamide pulses at shorter intervals might be effective.

In conclusion, intermittent monthly pulses of cyclophosphamide are ineffective in patients with membranous nephropathy and deteriorating renal function. Three therapies seem promising: 1) the original regimen reported by Ponticelli and colleagues [8] using chlorambucil at 0.2 mg/kg per day; 2) a modification of this regimen with lower-dose chlorambucil as used in our study; and 3) the use of oral cyclophosphamide. Long-term follow-up is needed to determine how long the treatment effect persists. Further randomized trials are needed to define the optimal timing of the initiation and duration of therapy and the role of steroids.