Efficacy of methylprednisolone and urokinase pulse therapy combined with or without cyclophosphamide in severe Henoch-Schoenlein nephritis: a clinical and histopathological study

Yukihiko Kawasaki, Junzo Suzuki and Hitoshi Suzuki

Department of Pediatrics, Fukushima Medical University School of Medicine, Hikariga-oka, Fukushima City, Fukushima, Japan

Correspondence and offprint requests to: Department of Pediatrics, Fukushima Medical University School of Medicine, 1 Hikariga-oka, Fukushima City, Fukushima 960-1295, Japan. Email: tomo{at}fmu.ac.jp



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. There have been few controlled studies of combined therapy with multiple drugs, including immunosuppressives, for severe Henoch-Schoenlein nephritis (HSPN). We evaluated the efficacy of methylprednisolone and urokinase pulse therapy combined with cyclophosphamide for patients with HSPN of at least grade IVb.

Methods. We studied 37 patients who had been diagnosed with HSPN of at least grade IVb. Of them, 20 (Group A) were treated with methylprednisolone and urokinase pulse therapy, and 17 (Group B) were treated with methylprednisolone and urokinase pulse therapy combined with cyclophophamide. We analysed the clinical features, laboratory and pathological findings of the two groups retrospectively.

Results. After 6 months of treatment, mean urinary protein excretion in Group B had significantly decreased compared with Group A, and the activity index of both groups at the second biopsy was lower than that at the first. Furthermore, at the second biopsy, the chronicity index of Group B was lower than that of Group A. Four patients of Group A but none of Group B had persistent nephropathy (P<0.05).

Conclusions. Our study suggests that methylprednisolone and urokinase pulse therapy combined with cyclophosphamide is useful for patients with severe HSPN.

Keywords: cyclophosphamide; HSPN; methylprednisolone pulse therapy; urokinase pulse therapy



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Henoch-Schoenlein purpura (HSP) is an immunoglobulin (Ig) A-mediated immune-complex vasculitis that affects predominantly the skin, joints, gastrointestinal tract and kidneys. It occurs most frequently in childhood, and its prognosis is mainly predicted by the severity of renal involvement.

The proportion of patients reported to have renal involvement varies between 20 and 80%. The majority of children with Henoch-Schoenlein nephritis (HSPN) present only with haematuria and/or low-grade proteinuria, or both, and have a good chance to recover. However, patients with massive proteinuria at onset frequently have a progressive course [1,2]. In specialized centres, the proportion of children with HSPN who progress to renal failure or end-stage renal disease varies from 12 to 19% [36].

As for the treatment of severe HSPN, there are a few reports dealing with the use of multiple combined agents, including immunosuppressive drugs [710]. However, those studies were not controlled studies. We have recently reported that methylprednisolone and urokinase pulse therapy (MUT) was effective in HSPN patients of least type IIIb, though some of them nevertheless progressed and had increased sclerotic lesions [11].

In this study, we evaluate the efficacy of cyclophosphamide in severe HSPN, comparing clinical and histological outcomes in patients treated with methylprednisolone, warfarin, dipyridamole and urokinase pulse therapy (MUT) between 1988 and 1994 and with MUT plus cyclophosphamide (MUTC) between 1994 and 1998.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Patients
We collected data on 37 patients, who had been diagnosed to have HSPN of at least grade IVb, in the Department of Pediatrics of Fukushima Medical University School of Medicine between January 1988 and December 1998. Entry criteria included: (i) a diagnosis of HSP, made if the major manifestations of the illness consisted of a purpuric rash and abdominal pain without thrombocytopaenia, with additional features, arthritis and nephritis, accepted as being consistent with the diagnosis; (ii) age at the initiation of therapy and follow-up under 15 years; (iii) no previous treatment with corticosteroids or immunosuppressive drugs. These patients were retrospectively divided into two groups without randomization. Group A consisted of 20 patients treated with MUT, and Group B consisted of 17 patients treated with MUTC. The clinical features, laboratory data and pathological findings of both groups were retrospectively studied and comparisons were made between data obtained before and after therapy.

Therapeutic intervention
A timetable of treatment for both groups is shown in Figure 1. Following diagnostic renal biopsy, the patients were treated with MUT before 1994 and were designated Group A. MUT was a combination of ‘pulse’ methylprednisolone, at 30 mg/kg/day i.v. bolus (maximum 1 g) for 3 consecutive days, and pulsed UK, at 5000 units/kg/day i.v. bolus (maximum 180 000 units) for 7 consecutive days, followed by daily oral prednisolone (1 mg/kg/day) for 6 months, along with anti-platelet agents (dipyridamole 5 mg/kg/day), and anti-coagulant (warfarin) for 24 months. Warfarin was given orally at a single morning dose of 1 mg (for <7-year-old patients) or 2 mg (for >7-year-old patients) to maintain the thrombotest at 30–50%. The corticosteroid was reduced subsequently over 3 months and dipyridamole and warfarin reduction was individualized according to individual improvement in 24 h creatinine clearance (24h Ccr), urinary sediment and urinary protein.



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Fig. 1. A timetable of treatment in both groups.

 
After 1994, the patients were treated with MUTC, and were designated Group B. MUTC involved the addition to the above described regimen of cyclophosphamide 2.5 mg/kg/day for 12 weeks. Cyclophosphamide was given orally at a single morning dose to maintain the pseudocholinesterase (ChE) level at more than half of the pre-therapy ChE or lymphocyte counts at >1500/µl, or both.

Definitions
‘Haematuria’ was considered a positive finding if urinary microscopic examination showed five or more red blood cells per high power field, and macrohaematuria if blood was visible to the naked eye [12]. Proteinuria was evaluated by 24 h quantitative measurements. Nephrotic syndrome was defined as the presence of proteinuria (>=40 mg/m2/h) and a serum albumin level <25 g/l, with or without oedema [12]. Hypertension was defined as a systolic or diastolic blood pressure greater than the 95th percentile for the patient's age, based on the recommendations of Pediatric Task Force [13].

The clinical status of each patient was classified as follows: Stage 0, normal: the patient was normal on physical examination, with normal urine and renal function; Stage 1, minor urinary abnormalities: the patient was normal on physical examination, and had microscopic haematuria or proteinuria <20 mg/m2/h; Stage 2, persistent nephropathy: the patient had proteinuria of 20 mg/m2/h or greater, and 24h Ccr of 40 ml/min/1.73 m2 or greater; Stage 3, renal insufficiency: the patient had 24h Ccr <40 ml/min/1.73 m2 (this category included patients who were on dialysis, were transplanted or who had died).

Pathology
First renal first biopsies were performed initially in all patients; second biopsies were performed in the recovery phase (5.3±2.5 months after onset) in 15 of 20 patients (75%) in Group A and 13 of 17 patients (76%) in group B in order to assess the efficacy of the therapy. Material for histological studies was fixed in 20% neutral formalin, embedded in paraffin, sliced at 2–3 µm in thickness, and stained with haematoxylin and eosin or periodic acid-Schiff reagent. The specimens were assessed by light microscopy (LM) and immunofluorescence (IF). The mean (±SD) number of glomeruli found in the biopsy specimens was 17.3±8.7 (range 10–46).

The glomerular changes were graded according to the classification devised by the pathologists of the International Society of Kidney Disease in Children [12], as follows: I, minor glomerular abnormalities; II, pure mesangial proliferation [(a) focal, (b) diffuse]; III, minor glomerular abnormalities or mesangial proliferation, with crescents in <50% of glomeruli [(a) focal, (b) diffuse mesangial proliferation]; IV, same as III but with crescents in 50–75% of glomeruli [(a) focal, (b) diffuse mesangial proliferation]; V, same as III but with crescents in >75% glomeruli [(a) focal, (b) diffuse mesangial proliferation]; VI, membrano-proliferative-like lesions.

To compare the biopsies, a histological scoring system was modified to evaluate acute and chronic changes. Acute changes included mesangial proliferation (grades 0–3, 0 = normal, 1 = slight, 2 = moderate, 3 = severe), segmental necrosis with cellular crescent formation (scored according to the percentage of glomeruli involved, 0 = 0%, 1 = 1–20%, 2 = 20–50%, 3 = >50%), and interstitial oedema with mononuclear cell infiltration (0–3). The acuity index (AI) is the sum of the scores of those changes. Chronic renal injury was estimated by determining the number of glomeruli with fibrous crescents and segmental or global sclerosis. Each abnormality was scored 0–3 according to the number of glomeruli involved, as for the scoring of acute crescent formation. In addition, the combination of tubular atrophy and interstitial fibrosis was graded 0–3. The chronicity index (CI) is the sum of the scores of those chronic renal changes. Scoring was performed in a blinded fashion on specimens identified only by codes.

Tissue for IF was immediately fixed in OCT compound and frozen at –80°C until use for IF. IF searched for IgG, IgA, IgM, C1q, C3, C4 and fibrinogen. The intensity of immunofluorescence was graded on a scale where negative = 0, mild = 1, moderate = 2 and severe = 3.

Statistics
Data are expressed as mean values±SEM. Statistical analysis was performed on a Macintosh computer with a software package for statistical analysis (Stat View, Abacus Concepts, Berkeley, CA, USA). Several variables, that are clearly not normal in their distribution, were compared using non-parametric statistics such as the Mann–Whitney’ test or Wilcoxon signed-rank test. The renal survival rates were calculated using the life-table method (Kaplan-Meier). P<0.05 was considered significant.



   Results
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 Abstract
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 Subjects and methods
 Results
 Discussion
 References
 
Comparison of baseline characteristics and pathological findings between two groups at the time of the first renal biopsy (Table 1)
Age at onset and duration since onset were 8.0±2.8 and 7.4±3.1 years in Group A, and 7.9±3.1 and 6.2±1.7 years in Group B; male:female ratios were 9:11 and 8:9, respectively, in the two groups.


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Table 1. Comparison of laboratory findings between both groups at first renal biopsy and most recent follow-up

 
The glomerular abnormalities in the first biopsies of both groups were as follows: in Group A, grade IV in 18 patients, grade V in two patients; in Group B, grade IV in 15 patients, grade V in two patients. The percentage of crescentic formations was 55.5±6.5% in Group A and 60.7±12.3% in Group B.

Comparison of clinical and laboratory findings between two groups at the time of the first renal biopsy (Table 1)
In the pre-biopsy period, haematuria and proteinuria were present in all patients in either group. Proteinuria varied from 78 to 375 mg/m2/h (mean 154±73 mg/m2/h) in Group A and from 105 to 458 mg/m2/h (mean 181±85 mg/m2/h) in Group B. Nephrotic syndrome was present in 12 patients of Group A and in 14 patients of Group B (P = 0.15). 24h Ccr was decreased in eight patients of Group A and in eight patients of Group B, ranging from 16 to 67 ml/min/1.73 m2. Mean urinary protein excretions, incidence of haematuria, serum creatinine and creatinine clearances did not differ between the two groups.

Comparison of renal symptoms and laboratory data between two groups and the clinical stage at most recent follow-up (Figure 2 and Table 2)
We compared the renal symptoms and laboratory data before and after therapy. The mean urinary protein excretions of Group A and Group B were reduced from 154±73 and 181±85 mg/m2/h to 29±19 (P<0.001) and 10±5 mg/m2/h (P<0.001), respectively, at 6 months of follow-up. From 2 months after the initiation of treatment to the most recent follow-up, the mean urinary protein excretion in Group B was lower than that in Group A. At the most recent follow-up, the incidence of haematuria in Group A was higher than in Group B (P<0.05); however, serum albumin, serum creatinine and the mean blood pressure did not differ between the groups.



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Fig. 2. Comparison of proteinuria between Group A and Group B up to the most recent follow-up.

 

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Table 2. Comparison of clinical stage between both groups at first renal biopsy and most recent follow-up

 
At the most recent follow-up, 24hCcr had returned to within the normal range in eight patients of Group A and in eight patients of Group B who had 24hCcr <70 ml/min/1.73 m2 at the initiation of therapy. Eight patients of Group A and 13 patients of Group B had recovered clinically, and eight patients of Group A and four patients of Group B had minimal urinary abnormalities. Four patients of Group A and none of Group B had persistent nephropathy (P<0.05) and no patient in either group had renal insufficiency.

Results of repeat renal biopsies
Comparison of ISKDC classification between both groups at first and second biopsies (Table 3). The number of patients with grade IV and grade V did not differ between the two groups. At the second biopsy, there were more patients with grade IV in Group A than in Group B.


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Table 3. Comparison of ISKDC classification between both groups at first and second renal biopsies

 
Comparison of AI and CI between both groups at first and second biopsies (Table 4). The AI of both groups at second biopsy was lower than that at first biopsy. At second biopsy, the AI of Group B was lower than that of Group A (P<0.05). The CI of Group A did not differ between the first and second biopsies, but the CI of Group B at second biopsy decreased when compared with the first biopsy. At second biopsy, the CI of Group B was lower than that of Group A (P<0.05).


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Table 4. Comparison of AI and CI between both groups at first and second biopsies

 
Side effect of treatment
Five patients of Group A and five patients of Group B showed Cushingoid changes, three patients in Group A and two patients in Group B developed mild glaucoma, and one patient in Group A and one patient in Group B presented with mild growth retardation. One patient in Group A (but none in Group B) developed mild hypertension, which was well controlled by treatment with nifedipine. The doses of urokinase and anticoagulants used in the treatment regimen did not result in bleeding tendencies such as epistaxis or gastrointestinal haemorrhage. One patient in Group B developed leucopaenia and discontinued the cyclophosphamide for 8 weeks. One patient in Group A and one patient in Group B developed infections—the former acute bronchitis, the latter acute pneumonia. Both recovered with antibiotic treatment. Most of the side effects were mild and well controlled; all were reversible.



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Our study suggests that MUTC significantly reduced urinary protein excretion and prevents any increase of crescentic and sclerosed glomeruli in HSPN patients with at least type IV, when compared with MUT. At their most recent follow-ups, there were no patients with persistent nephropathy and renal insufficiency among the patients treated with MUTC.

As for reports concerning the prognosis of HSPN, Counahan et al. [6] reviewed 88 patients with HSPN and found that 15 of 26 patients (58%) with at least grade IV and five of 38 (13%) without treatment had active renal disease, renal insufficiency or both. Yoshikawa et al. [14] reported that HSPN was a significant cause of childhood chronic renal failure, accounting for 16% of all children entering dialysis in Japan. Specifically, 52% (13 of 25) of patients studied by them, with at least grade IV, had renal insufficiency.

The pathological features that are valuable portents of prognosis are histological grade, especially the percentage of glomeruli with crescents, and higher chronicity indices [1,2,5,6]. Therefore, it is necessary to assure that therapy for severe HSPN is adequate. Niaudet and Habib [3] reported that methylprednisolone pulse therapy was effective in patients at risk of progression of their nephropathy, especially if it was started early during the course of the disease, before the crescents become fibrosed. Iijima et al. [7] mentioned that a multiple combined therapy with prednisolone, cyclophosphamide, heparin or warfarin, and dipyridamole could be effective in histologically severe HSPN. In addition, Flynn et al. [9] report that treating children with HSPN with high-dose corticosteroids plus oral cyclophosphamide is safe and, as in nephrotic syndrome, appears to significantly reduce proteinuria. This study, however, was not a controlled study. Therefore, we resorted to a controlled study to investigate the efficacy of that therapy combined with cyclophosphamide in patients with severe HSPN.

Cyclophosphamide is a potent alkylating agent which inhibits lymphocyte proliferation, leading to repression of the function of B and T lymphocytes as well as to a reduction of their numbers [15]. The dose of cyclophosphamide we used was based on the classic approach to minimal-change nephrotic syndrome, where cyclophosphamide has been shown to have a potent immunomodulating effect, especially in steroid-sensitive patients [1618]. Cyclophosphamide has also been used with success in the treatment of several other forms of crescentic glomerulonephritis, particularly those characterized by altered autoimmunity, including Wegener's granulomatosis and systemic lupus erythematosus [15,19,20].

Some side effects of cyclophosphamide have been observed, such as myelosuppression, haemorrhagic cystitis and interstitial pneumonia [15]. In our hospital, we have used serum cholinesterase activity in addition to WBC and lymphocyte counts to monitor for the side effects of cyclophosphamide. Serum cholinesterase activity might be an index of cyclophosphamide therapy. Imai et al. [21] showed the importance of maintaining serum cholinesterase (ChE) levels at more than half of the pre-therapy ChE level for preventing the side effects. In our study, it is possible that the side effects of cyclophosphamide were minimized by careful regulation of serum cholinesterase activity. Therefore, we suggest that it would be useful to monitor serum cholinesterase activity during cyclophosphamide therapy.

Our cocktail therapy with cyclophosphamide included reduced urinary protein excretion and mesangial IgA deposition, and prevented the increase of sclerosed glomeruli in children with severe HSPN of at least grade IVb. The rationale for using prednisolone and cyclophosphamide in severe HSPN is that corticosteroids and immunosuppressive agents reduce IgA production and minimize the abnormal immune response and inflammatory events that follow IgA deposition in glomeruli. Urokinase, warfarin and dipyridamole are used to inhibit the mediators of glomerular damage [4,8,22].

UK, a plasminogen activator derived from fresh human urine, first attracted attention as a therapeutic agent for thrombotic diseases such as cardiovascular diseases or cerebral thrombosis. The molecular weight of UK is 54 000. UK has been used in patients with chronic glomerulonephritis, including IgA nephropathy, with fibrinogen or fibrin deposits detected by immunofluorescence. The rationale for such treatment was as follows: (i) the defibrinating activity of UK was stronger than of anti-coagulants; (ii) specific accumulations of UK were seen in the kidney and liver despite a very short turnover rate; (iii) adverse effects were very rare even if UK was administered for a long period [23,24].

Recently, Glass et al. [25] reported that UK may mediate proteolysis in the mesangial extracellular matrix, and Yasunaga et al. [26] showed in vitro that plasminogen enhanced the angiogenesis of bovine capillary endothelial cells and an anti-UK antibody inhibited this effect. We have reported that the improvement of hypercoagulable states and acute inflammatory responses, and the suppression of sclerotic changes in glomeruli, were found in patients on MUT. The studies mentioned suggest that UK and plasminogen may regulate the repair of affected capillaries and may digest the proliferated matrix in diseased glomeruli.

Dipyridamole is an anti-platelet agent, and it abrogates the mitogenic effects of platelet-derived growth factor [27]. Hillis et al. [27] reported that dipyridamole significantly inhibited the growth of human mesangial cells in vitro in a dose-dependent fashion. Nagai et al. [28] showed that warfarin ameliorated hyperfiltration and urinary albumin excretion in streptozotocin rats. Futhermore, Lee et al. [29] found that dipyridamole and low-dose warfarin were effective in patients with IgA nephropathy and renal impairment.

The beneficial effects of combined prednisolone, cyclophosphamide, urokinase, warfarin and dipyridamole treatment were accompanied by few serious side effects specifically attributable to those drugs. Most of the side effects were mild and well controlled, and all were reversible. The cocktail therapy was well tolerated in all patients, and its safety was monitored by using serum cholinesterase as the index of the side effects of cyclophosphamide.

In conclusion, our study suggests that cocktail therapy with MUTC is effective in patients with the risk of the progression of their severe HSPN. Long-term and additional observations are necessary to evaluate the long-term efficacy of this cocktail therapy.

Conflict of interest statement. None declared.



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 1. 4.03
Accepted in revised form: 1.10.03