Effects of co-administration of urokinase and benazepril on severe IgA nephropathy

Xiangmei Chen, Qiang Qiu, Li Tang, Shuwen Liu, Guangyan Cai, Hongtao Liu and Yuansheng Xie

Kidney Center of PLA, Department of Nephrology, Chinese General Hospital of PLA, Beijing, China

Correspondence and offprint requests to: Professor Xiangmei Chen, Kidney Center of PLA, Department of Nephrology, Chinese General Hospital of PLA, Fuxing Road 28, Beijing 100853, China. Email: xmchen{at}public.bta.net.cn



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. The availability of treatment for IgA nephropathy (IgAN) is limited.

Method. A prospective randomized controlled clinical trial was performed to evaluate the effects of therapy with urokinase (UK) and benazepril (BZ, an angiotensin-converting enzyme inhibitor) or BZ alone on severe IgAN. We divided 71 cases of IgAN, Lee's grade >=III and with fibrinogen deposits, into two groups to be treated for 12 months with either UK + BZ or BZ alone.

Results. There was no significant difference between the two groups in baseline clinical and histopathological data. After 12 months of treatment, 25 of 35 patients (71.4%) in the UK + BZ group and 16 of 36 (44.4%) in the BZ-alone group had a >=50% decrease in 24-h urinary protein excretion compared with the baseline ({chi}2 test, P<0.05). Proteinuria significantly decreased at 6 and 12 months of treatment in both groups compared with baseline (P<0.01 in the UK + BZ group, P<0.05 in the BZ group), and the therapeutic efficiency of UK + BZ was better than that of BZ alone (P<0.05 at 6 and 12 months). The endogenous creatinine clearance rate (Ccr) was stable in the UK + BZ group, while Ccr declined significantly at 6 and 12 months in the BZ-alone group compared with baseline (P<0.05, respectively). The Ccrs of the two groups at 12 months of treatment were statistically different (P<0.05).

Conclusions. Combined therapy with UK and BZ was more effective than with BZ alone in reducing proteinuria and protecting renal function in patients with severe IgAN.

Keywords: angiotensin-converting enzyme inhibitor; benazepril; IgA nephropathy; randomized controlled trial; treatment; urokinase



   Introduction
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Immunoglobulin A nephropathy (IgAN) is the most common glomerulonephritis worldwide [1]. The impairment of renal function, severe proteinuria and arterial hypertension are the strongest and the most reliable predictors of an unfavourable clinical outcome [2]. Since the pathogenesis of IgAN is obscure, a specific treatment is not yet available. Although there remains no cure, some treatments that slow disease progression are becoming available. Previous approaches have included tonsillectomy, prednisolone, immunosuppressants, anti-hypertensive drugs (including angiotensin-converting enzyme inhibitors; ACEIs), anti-coagulants (including urokinase; UK), fish oils and others [3].

ACEIs can reduce glomerular hypertension and proteinuria, modifying capillary pressure and glomerular permselectivity [4]. In reported clinical trials, ACEIs have been shown to slow the progression of chronic renal insufficiency in patients with various renal diseases [5]. In IgAN, some randomized clinical trials and retrospective cohort studies have found that a variety of ACEIs moderately lowered urinary protein (UP) excretion [6,7]. However, no study has shown that ACEIs preserve renal function in patients with IgAN [8].

Disordered coagulation and fibrinolysis promote extravascular fibrin deposition in some diseases, in which anticoagulant or fibrinolytic strategies may be used to protect against acute inflammation or accelerated fibrosis [9]. Fibrin deposition in kidney is a common event in some forms of human and experimental glomerulonephritis, and is thought to result from local activation of blood coagulation, impaired removal by the fibrinolytic system or both [10]. Some patients have had marked improvement of proteinuria after UK therapy. ‘Consecutive’ UK administration might be useful for treatment of IgAN with moderate to advanced renal injuries [11].

Since both UK and ACEIs have reduced proteinuria in IgAN, we wondered if the combination of UK and an ACEI in the setting of severe IgAN could produce a better anti-proteinuric effect and preserve better renal function than ACEIs alone. We therefore designed a study to investigate the effects of administering UK and an ACEI (benazepril, BZ) together vs BZ alone on severe IgAN.



   Subjects and methods
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 Abstract
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 Subjects and methods
 Results
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The study was a prospective, randomized controlled clinical trial. The protocol was in accordance with the standards of the Ethics Committee of Chinese General Hospital of PLA, and all patients gave informed consent.

Patients
Between 1999 and 2001, 71 patients were enrolled in this study. They met the following criteria: (i) they underwent renal biopsy and were diagnosed as having IgAN; (ii) they had a renal pathological grade >=III as determined according to Lee's grading; (iii) investigation by immunofluorescence showed glomerular fibrinogen deposits; (iv) their serum creatinine levels were <354 µmol/l; and (v) they were not prone to haemorrhage. Therefore, the enrolled patients had moderate to advanced renal injuries with histologies positive for fibrinogen. The subjects had no systemic disease, such as Henoch–Schonlein syndrome or systemic lupus erythematosus. The patients had not received any ACEIs or UK treatment before the biopsy.

Administration of UK and BZ
Combined therapy with UK + BZ was given to 35 patients with IgAN, and 36 IgAN patients received BZ alone. In the first group, 100 000 IU of UK in 250 ml of 5% glucose was injected intravenously daily for 10 days per month. Patients in both groups received BZ continuously at 10 mg/day throughout the study. All patients completed 12 months of therapy. The interval between the biopsy and the start of treatment was 2 months in all patients in order to avoid the haemorrhagic side effect of UK. Patients with hypertension (16 patients in the UK + BZ group and 19 patients in the BZ-alone group) were controlled below 140/90 mmHg by administrating non-ACEI anti-hypertensives, such as calcium antagonists and ß-blockers.

Patient monitoring and judgment of outcome
When the study began, complete medical histories were taken and physical examinations were performed on all patients to obtain baseline data. All patients were seen monthly during the study. At each follow-up visit, the patients were questioned about their symptoms and examined for side effects. Serum creatinine (Scr), blood urea nitrogen (BUN), serum cholesterol (CH), serum triglycerides (TG), serum albumin (Alb) and total protein (TP), amount of 24-h UP excretion and endogenous creatinine clearance rate (Ccr) were measured at the start, and at 6 and 12 months of treatment. Fibrinogen and prothrombin time were monitored monthly.

In view of the principal aim of the study, the primary outcome was the decrease of proteinuria, and its end point was a decrease of >=50% in UP excretion compared with the baseline. The secondary outcome was progression of renal disease, and its end points were an increase by >=50% in Scr, end-stage renal failure or death.

Pathological examination
Renal biopsy specimens were examined by light microscopy immunofluorescence, including fibrin-related antigen (FRA) and electric microscopy. The immunofluorescence findings were classified between none (-) and 3+ according to the intensity of fluorescence of each specimen. Pathological diagnosis of all patients with IgAN included the grades I–V of pathological damage determined in our department by light microscopy according to the grading system of Lee [12]. Lee’ s grades consist of: grade I, mostly normal glomeruli; grade II, less than half of the glomeruli show localized mesangial proliferation and sclerosis; grade III, diffuse mesangial proliferation and thickening with focal and segmental variation and focal interstitial oedema, and infiltrate occasionally present; grade IV, marked diffuse mesangial proliferation and sclerosis with tubular atrophy and interstitial inflammation; grade V, similar to grade IV, and but more severe.

The specific pathological features of the patients in this study were scored by light microscopy according to the scoring system of Katafuchi et al. [13]. The glomerular lesions (total score, 0–12) included glomerular hypercellularity, glomerular segmental lesions and glomerular sclerosis. The score for each glomerular lesion was fixed as follows: 0, no lesion; 1, <10% of glomeruli involved; 2, >=10% and <25%; 3, >=25% and <50%; 4, >=50% of glomeruli involved. The severity of interstitial cell infiltration, interstitial fibrosis and tubular atrophy in each case was scored (total score, 0–9) semi-quantitatively from 0 to 3 according to the percentage of injured tissue: 0, no lesion; 1, <25%; 2, >=25% and <50%; 3, >=50%.

In the UK + BZ group, 10 patients had repeat renal biopsies. Along with routine pathological examinations, the martius–scarlet–blue (MSB) trichrome stain method and the computer image analysis system (TIPAS/88) were used to quantify fibrin deposition in specimens from the first and second renal biopsies. The percentage of MSB-positive areas in the glomeruli was calculated.

Statistical analyses
The sample size of the study was calculated based on the change in proteinuria, to a decrease of >=50% of the UP excretion compared with baseline. The estimated sample size for two-sample comparison of proportions was calculated by STATA 7.0 statistic software and was based on the results of our preliminary investigation—a decrease of >=50% in UP excretion compared with the baseline in 75% of UK + BZ patients and 33% of BZ patients at 6 months follow-up. Ho test (null hypothesis): P1 = P2, where P1 was the proportion in the UK + BZ population and P2 was the proportion in the BZ population. Assumptions: {alpha} = 0.05, power = 0.90, P1 = 0.75, P2 = 0.33, n1/n1 = 1.00. The required estimated sample sizes were n1 = 33, n2 = 33, i.e. the calculated sample size of 33 subjects was based on a two-sided significance level of 0.05, and a statistical power of 90% at 6 months follow-up.

The STATA 7.0 statistic software was used to analyse the data. The {chi}2 test was employed to analyse enumeration data, and the t-test for measurement data. Continuous factors were summarized using means±SD. A two-tailed P-value of <0.05 was taken as the limit of statistical significance.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Baseline data
Thirty-five patients received UK and BZ together and 36 patients received BZ alone for 12 months. There was no significant difference between the two groups in age, gender, blood pressure, Scr, 24-h UP and Lee's grades of renal lesions at baseline (Table 1).


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Table 1. Comparison of baseline characteristics between the two groups

 
Comparison of efficiency of therapy in the two groups
After 12 months of treatment, there were 25 of 35 patients (71.4%) in the UK + BZ group and 16 of 36 (44.4%) in the BZ group whose amount of 24-h UP excretion decreased by >=50% compared with the baseline ({chi}2 test, P<0.05). The Scr of three patients in the BZ group increased by >=50%.

Clinical follow-up data (BUN, Scr, Ccr, UP, Alb, TP, TG and CH) for the two groups are shown in Table 2.


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Table 2. Comparison of clinical follow-up data between the two groups

 
After 12 months of treatment, the BUN of the BZ group increased significantly (P<0.05), while it remained unchanged in the UK + BZ group. The level of Scr remained unchanged in the UK + BZ group, but it significantly increased in the BZ group at 6 and 12 months of treatment compared with baseline (P<0.01, respectively). Ccr fell significantly at 6 and 12 months of treatment in the BZ group compared with the baseline (P<0.01, respectively) while it remained unchanged in the UK + BZ group. Ccr in the BZ group at 12 months of treatment was lower than that of UK + BZ (P<0.05) (Figure 1). Proteinuria significantly decreased at 6 and 12 months of treatment in both groups compared with the baseline (P<0.01 in the UK + BZ group, P<0.05 in the BZ group, respectively), and the efficiency of UK + BZ treatment was better than that of BZ alone at 6 and 12 months (P<0.05, respectively) (Figure 2). Alb and TP levels significantly increased compared with the baseline at 6 and 12 months of treatment in both groups, without a measurable difference between the two groups. The levels of TG and CH remained unchanged in both groups.



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Fig. 1. Creatinine clearance rate (Ccr) levels between the two groups. **Compared with baseline, P<0.01. #Comparison between the UK + BZ group and BZ-alone group, P<0.05.

 


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Fig. 2. Proteinuria levels between the two groups. *Compared with baseline, P<0.05. **Compared with baseline, P<0.01. #Comparison between the UK + BZ group and BZ-alone group, P<0.05.

 
Comparison of therapeutic efficiency vs different pathological score
ptIn order to clarify the relationship between severity of renal pathology and efficiency of therapy, we analysed the changes of UP and Scr after UK + BZ or BZ-alone treatment against different pathological scores. The results showed that UK + BZ or BZ alone decreased UP at 6 and 12 months of administration in all patients; but, in patients with glomerular scores >7 and tubular scores >5, the efficiency of decreasing proteinuria was better with UK + BZ than with BZ alone, and there was a significant difference between the two groups at 12 months of treatment (P<0.05). The combined therapy with UK + BZ kept Scr stable, whereas BZ alone did not. In the BZ group, Scr increased at 6 and 12 months of treatment (Table 3).


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Table 3. Comparison of therapeutic efficiency in patients with different pathological scores

 
Comparison between first and second renal biopsies
In the UK + BZ group, 10 patients had repeat renal biopsies during or after the treatment. The glomerular hypercellularity, segmental lesions and interstitial cell infiltration seen in most patients were decreased after UK + BZ treatment at the time of the second renal biopsy compared with the first.

The percentage of fibrin MSB-positive areas of the total of glomerular areas in the 10 patients was decreased significantly from 12.49±1.82 to 9.96±1.23% (P<0.05). The total proteinuria level of the 10 patients also declined (2.04±0.62 vs 0.81±0.40 g/24 h, P<0.01).

Safety and side effects
Distinct abnormalities of plasma fibrinogen and prothrombin time were not found during the study. The side effects of treatment included mild hyperkalaemia (three patients) due to BZ, and bleeding (mild nosebleed, two patients) due to UK, which did not influence the planned therapy, because they were mild and temporary.



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
In this study, we compared the efficiency of combined therapy with UK + BZ and BZ therapy alone in severe IgAN. Our results demonstrate that although BZ alone decreases proteinuria, the efficiency of combined treatment with UK + BZ is better. Furthermore, combined therapy kept renal function stable, while BZ alone did not. These results indicate that combined therapy with UK + BZ is more effective than BZ montherapy in reducing proteinuria and protecting renal function in severe IgAN (Lee's grade >=III).

ACEIs can slow the progression of renal disease in patients with type 1 diabetic nephropathy, hypertensive nephrosclerosis and chronic non-diabetic glomerular and interstitial renal disease, by reducing glomerular hypertension and proteinuria [8]. A reduction in proteinuria is considered by many investigators to be the hallmark of effective treatment to preserve renal function in non-diabetic renal diseases [14]. However, some randomized clinical trials and retrospective cohort studies [6,7] found that a variety of ACEIs moderately lowered UP excretion without an accompanying improvement in renal function in IgAN. Our present study also demonstrates that BZ alone decreases proteinuria at 6 and 12 months of treatment without an accompanying improvement in renal function in severe IgAN (Lee's grade >=III).

In the anti-glomerular basement membrane glomerulonephritis model, fibrinogen-deficient mice with no fibrin(ogen)-related material detectable in their kidneys developed milder glomerulonephritis—with fewer necrotic glomeruli, diminished macrophage infiltration and reduction in the number of glomerular crescents and of Scr—when compared with control mice [15]. These results indicate that fibrin deposits in glomeruli play an important role in renal injuries. Glomerular fibrin deposits may occur within vessels or in extracapillary spaces. Studies suggest that intravascular thrombosis is promoted by endothelial cell activation or injury, resulting in the release of endothelial cell-derived tissue factor procoagulant, fibrinolytic inhibitors, platelet-activating factor and large multimers of von Willebrand factor. Glomerular fibrin may be removed by fibrinolytic or phagocytic mechanisms or may persist and lead to glomerular obsolescence. Suppression or elimination of factors that promote glomerular fibrin deposition and enhancement of mechanisms that remove glomerular fibrin may be important in the recovery from several forms of human kidney disease [16]. Our study demonstrated that fibrin deposition may be related to the plasminogen activator (PA)/plasminogen activator inhibitor (PAI-1) system in glomerular endothelial cells, and we found an abnormal expression of tissue-type PA, urokinase PA and PAI-1 in IgAN [17,18]. PAI-1 has been implicated in several renal pathogenetic processes, including thrombotic microangiopathies and proliferative or crescentic glomerulopathies. Most recently, it has become clear that PAI-1 also plays a pivotal role in progressive renal disease, both glomerulosclerosis and tubulointerstitial fibrosis [19].

Fibrinolysis studies by Colucci et al. [10] showed that both PA activity and UK antigen were significantly lower in patients with serum creatinine >1.5 mg/dl compared with those with normal creatinine and with non-inflamed kidneys. Reduced fibrinolytic activity in those patients’ urine was due to decreased excretion of UK [10]. Since the intra-glomerular hypercoagulable state is related to renal injury, it is thought that fibrinolytic agents, including UK, might be a beneficial to preserving the renal function in patients with IgAN. Miura et al. reported that long-term UK therapy in IgAN resulted in the marked improvement of proteinuria and the preservation of Scr levels. They suggested that UK administration might be useful for treating IgAN with moderate to advanced renal injuries [11].

In glomerulonephritis, UK is considered to exert its therapeutic effects by the removal of intra-glomerular FRA, or by fibrinolysis. Other actions of UK, such as mediating proteolysis in the mesangial extracellular matrix, have also been investigated [20]. We used repeat renal biopsy and quantitative methods to analyse the relationship between the decrease of fibrin deposits and the decrease of proteinuria in patients. Total fibrin deposits in the 10 patients from the UK + BZ group who had repeat renal biopsies decreased, as did their proteinuria. These results suggest that, although the mechanism of proteinuria reduction in combined UK + BZ therapy was unclear, it is related to the removal of intraglomerular FRA or fibrin. In addition, the decrease of glomerular hypercellularity, segmental lesions and interstitial cell infiltration after UK + BZ administration also indicates that the combined treatment might have an anti-inflammatory role, because there are distinct correlations between coagulation and inflammation.

Concerning the relationship of the severity of pathological injury and therapeutic efficiency, our results indicate that among the patients with glomerular scores >7 and tubular scores >5, the efficiency of UK + BZ in decreasing UP excretion was better than that of BZ alone, while there was no significant difference between the two therapies among patients with glomerular scores <=7 and tubular scores <=5. This result indicates that the UK + BZ treatment may be more effective in severe IgAN patients with distinct glomerular hypercellularity, segmental lesions and interstitial cell infiltration. In summary, the efficacy of combined treatment with UK and BZ in severe IgAN suggests that it is a potentially useful therapy given that presently there are no other effective treatments available for patients with IgAN.



   Acknowledgments
 
We are grateful to Mr Suozhu Shi, Dr Jie Wu, Dr Nobuaki Yamanaka and Dr Jianzhong Wang for their work or comments. This study was supported by a grant from the Creative Research Group Fund of the National Foundation Committee of Natural Science of the Peoples Republic of China (30121005), and a grant from the Key Technologies Research and Development Programme of the Tenth Five-year Plan of the Peoples Republic of China (2001BA701A14a).

Conflict of interest statement. None declared.



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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 3.12.02
Accepted in revised form: 18. 9.03





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