NDT Advance Access published online on October 7, 2008
Nephrology Dialysis Transplantation, doi:10.1093/ndt/gfn555
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Successful treatment of patients with lipoprotein glomerulopathy by protein A immunoadsorption: a pilot study
Nanjing University Clinical School of Medicine, Research Institute of Nephrology, Jinling Hospital, Nanjing, People's Republic of China
Correspondence and offprint requests to: Li Leishi, Professor of Medicine, Research Institute of Nephrology, Jinling Hospital, Nanjing University School of Medicine, Nanjing 210002, People's Republic of China. Tel: +86-25-80860210; Fax: +86-25-84801992; E-mail: lilsh{at}cae.cn
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Abstract |
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Background. No established therapy is available for patients with lipoprotein glomerulopathy (LPG). Protein A immunoadsorption has been proved to be effective in reducing proteinuria in patients with nephrotic syndrome. In this uncontrolled pilot study, we investigated the efficiency of immunoadsorption onto staphylococcal protein A as treatment for LPG.
Methods. Thirteen patients with renal biopsy-proven LPG were treated with staphylococcal protein A immunoadsorption. Immunoadsorption was administered for 10 cycles per session and 10 sessions as a course. A total of 30 l of plasma was regenerated in each course.
Results. Single immunoadsorption course led to a rapid decline in proteinuria from 4.01 ± 3.09 g/24 h to 1.21 ± 0.97 g/24 h (mean ± SD) (n = 13, P = 0.001), along with a dramatic decline in apolipoprotein E (apo E) from 9.79 ± 5.04 mg/dl to 6.20 ± 2.22 mg/dl (P = 0.004). A repeated renal biopsy (n = 12) showed that intraglomerular lipoprotein thrombi almost disappeared. Six patients were enrolled in the investigation of long-term outcome, and proteinuria returned to baseline levels within 12 months. Four recurrent patients received repeat immunoadsorption treatment; proteinuria decreased from 5.02 ± 1.85 g/24 h to 1.64 ± 0.55 g/24 h at the end of the treatment, serum apo E decreased from 14.65 ± 11.17 mg/dl to 7.90 ± 1.72 mg/dl. No patients suffered from severe complications.
Conclusion. Our observations suggest that immunoadsorption onto protein A might be an effective treatment for resolving intraglomerular thrombi and improving nephrotic syndrome in patients with LPG. Further studies are required to define the influence of immunoadsorption on long-term effects in LPG patients.
Keywords: apolipoprotein E; immunoadsorption; lipoprotein glomerulopathy
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Introduction |
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Lipoprotein glomerulopathy (LPG) is a newly identified kidney disease that is clinically characterized by increased apolipoprotein (apo) E and refractory nephrotic syndrome. In pathology, LPG is characterized by intraglomerular lipoprotein thrombi and a variable degree of mesangial proliferation. Because apo E is usually elevated and several apo E mutants have been reported in LPG patients, it has been speculated that abnormal apo Es may play a pathogenic role in the development of this disease. However, LPG does not appear to cause atherosclerosis, and lipoprotein thrombi have not been found in organs or tissues other than the kidneys. These findings indicate that there may be some specific factors involved in the glomeruli besides apo E abnormality.
Because the pathogenesis of the disease is unclear, therapy is focused on the symptomatic treatment. Many therapeutic trials including immunosuppression, glucocorticoids, hydroxymethyl-glutaryl coenzyme A reductase inhibitors, anti-platelet drugs, fibrinolytics, low-density lipoprotein apheresis and renal transplantation have all been performed. However, few trials have shown favourable effects of the disease, and no statistically supported clinical trial has been published [1,2]. Initially, immunoadsorption onto staphylococcal protein A was used for selective elimination of immunoglobulins and other circulating agents that might be involved in the pathogenesis of different diseases. Later, immunoadsorption was found to be effective in ameliorating proteinuria caused by non-immunologically mediated disease [3]. Here, we conducted an initial uncontrolled pilot study with the purpose of ascertaining the effects of immunoadsorption in patients with LPG.
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Subjects and methods |
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Patients
The diagnosis of LPG was based on the renal histology and abnormal lipid profiles of the peripheral blood. Thirteen patients (5 female, 8 male; aged 36.0 ± 10.7 years, range 14–56 years) diagnosed with LPG between 2000 and 2007 at the Research Institute of Nephrology, Jinling Hospital, Nanjing University School of Medicine underwent immunoadsorption treatment.
Six patients among the total 13 patients followed up for more than 24 months were enrolled in the investigation of the long-term effects. Immunoadsorption was not repeated for these six patients during the follow-up. Another four patients were treated with repeat immunoadsorption.
Treatment and study protocol
After completion of baseline measurements, immunoadsorption was performed on every other day. A double lumen single-needle catheter was inserted into the right jugular vein. A combination of citrate and low-molecular weight heparin was administered for anticoagulation. Blood circulation was set up by a Gambro AK10 blood pump; the blood flow rate was 100 ml/min. A plasma-separation device (Plasmaflux P1S/P2S, Fresenius) was used for conventional plasmapheresis. The plasma was separated at the rate of 30 ml/min. The plasma was adsorbed through a staphylococcal protein A adsorption column (Immunoadsorba, Fresenius). After circulating for 10 min, the column was infused with acid eluent (pH 2.2), and then rinsed with the buffer solution (pH 7.0), until the pH in the adsorption columns was restored to 7.0, then the next cycle started. The plasma flow rate was maintained at 30 ml/min. Each treatment session consisted of 10 cycles, and a total of 3 000 ml plasma was regenerated. One treatment course consisted of 10 treatment sessions, and a total of 30 l plasma was regenerated.
No lipid-lowering therapy, diuretics or angiotensin-receptor blockades (ARBs) were given to the patients during the immunoadsorption course. All the 13 patients were on maximal medical therapy after immunoadsorption, including hydroxymethyl-glutaryl coenzyme A reductase inhibitors for lipid-lowering therapy, ARBs, calcium-channel blocker and diuretics for blood pressure control. No immunosuppressive agents were used.
Laboratory investigations
Daily 24-h urine, venous blood samples were collected on the day before the first immunoadsorption and after a single immunoadsorption course as well as at Months 6, 12 and 24 after the last immunoadsorption treatment session. Serum creatinine and albumin levels, immunoglobulin (Ig) G, were determined according to standard laboratory procedures. As for lipid profiles measurement, the blood was drawn from the vein into the tubes containing 0.1% of ethylene-diaminetetraacetic acid (EDTA)-2Na, and was separated by centrifugation (2000 g*15 min). Total cholesterol and triglyceride values were determined enzymatically. High-density lipoprotein (HDL) and low-density lipoprotein (LDL) were isolated from plasma by sequential ultracentrifugation as described elsewhere [4]. Apo A, apo B and apo E were measured by the immunoturbidimetric assay (Apo A, Apo B: RANDOX Laboratories Ltd, United Kingdom; Apo E: Daiichi Pure Chemicals, Co., Japan).
Renal histological examination
Percutaneous renal biopsy was performed under ultrasonographic guidance in each patient before the immunoadsorption treatment (<10 days). In order to detect the effect of immunoadsorption treatment, a repeat renal biopsy was performed after immunoadsorption (<3 days, n = 12). Formalin-fixed tissue was embedded in paraffin using routine procedures. Sections of 2 µm in thickness were stained with haematoxylin and eosin (HE), periodic acid-Schiff (PAS), periodic acid-methenamine silver and Masson's trichrome-elastica for microscopic pathological diagnosis. Interstitial fibrosis was graded semiquantitatively on a scale of 0 to 3+ on the basis of the percentage of the cortical area affected (<5, 6–25, 26–50 and >50%, respectively). Immunofluorescence staining was performed on 3 µm cryostat sections by using fluorescein isothiocyanate- (FITC) labelled rabbit, anti-human IgG, IgA, IgM, C3, C4 and C1q antibodies. In snap-frozen sections, apo A, apo B and apo E were analysed with the indirect immunofluorescence technique using antisera monospecific to each (DAKO Denmark A/S. Denmark). The pattern of apos’ distribution was described as focal or diffuse, and segmental or global. The intensity of apo A, apo B and apo E staining was staged as negative (0), weak (1+), moderate (2+), or strong (3+). Electron microscopy was also used routinely in all cases. Two pathologists, who were blinded to the clinical data, performed histopathological evaluation.
This study was approved by the ethics committee of Jinling Hospital. Written informed consents were obtained from all patients for immunoadsorption treatment and renal biopsy.
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Statistical analysis |
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Statistical analyses were performed using SPSS software (version 13.0). Values were expressed as mean ± standard deviation. The paired t-test was used for the comparison of individual paired values. Statistical significance was assumed for P < 0.05, and a very high significance level was defined as P < 0.01.
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Results |
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Short-term effects
After a single course of immunoadsorption, clinical data changed dramatically. All of the 13 patients presented with oedema at the onset of the treatment, after the immunoadsorption treatment, symptoms of nephrotic subsided completely. Proteinuria declined in all patients, and the mean level decreased from 4.01 ± 3.09 g/24 h at the beginning of immunoadsorption to 1.21 ± 0.97 g/24 h at the end of the immunoadsorption series (P = 0.001). The mean decrease was 65.71% (range 29.23–93.44%). Proteinuria <0.5 g/24 h was experienced in 3 (23.08%) of 13 patients after the treatment. Although four patients had a decrease of serum albumin, the mean serum albumin level increased from 29.82 ± 6.54 g/l to 31.90 ± 8.36 g/l at the end of the immunoadsorption series (P = 0.166). Apo E values decreased from 9.79 ± 5.04 mg/dl to 6.20 ± 2.22 mg/dl (P = 0.004); the mean decrease was 32.22% (range 4.34–60.32%). There was a 10.41% decrease in serum creatinine from 1.13 ± 0.53 mg/dl to 0.97 ± 0.33 mg/dl (P = 0.089). Changes in laboratory values before and after treatment were summarized in Table 1.
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Twelve patients had the renal biopsy before the immunoadsorption treatment and repeat biopsy after the immunoadsorption treatment. Before the treatment, specimens from all cases showed slightly lobular and enlarged glomeruli under light microscope. Capillary lumina in the affected glomeruli were markedly dilated, and stained a pale colour with PAS. In the non-dilated areas, the capillary loops appeared to be collapsed, with mesangial proliferation and small areas of segmental sclerosis (Figure 1A). Mild and limited interstitial fibrosis was observed in light microscopy specimens, and no foam cells were observed in the interstitium (Figure 2A). As for immunofluorescence microscopy, strong and massive depositions of apo E, apo A and apo B were found in the capillary lumina (Figure 3A). The mean level of the semiquantitative score of apo E was 1.83 ± 0.58. Under electron microscope, the glomerular capillary lumina were occupied by lipid granules and vacuoles, which formed striae resembling fingerprints (Figure 4). After the immunoadsorption treatment, most capillaries showed almost complete disappearance of lipoprotein thrombi. Mesangial cells and matrix proliferated compared with those seen at the first renal biopsy (Figure 1B). In terms of percentage of global sclerosis, segmental sclerosis, as well as the severity of the tubulo-interstitial lesions, no significant changes were observed between the first and second biopsies (Table 2, Figure 2B). The semiquantitative score of apo E decreased to 1.25 ± 0.75, with significant changes compared to the score before immunoadsorption treatment (Table 2). Not only the intensity of apo E, apo A and apo B dropped but also the pattern of immunofluorescence changed from global and diffuse to focal and segmental (Figure 3B).
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Long-term effects
Proteinuria fell significantly (P = 0.024) from a mean of 5.27 ± 4.12 g/24 h at baseline to 2.60 ± 2.45 g/24 h by 6 months, but the proteinuria tended to return to baseline levels within 12 months (Table 3). However, the increase in proteinuria between 6 and 12 months did not reach statistical significance (P = 0.49). During the initial 12 months of the follow-up, the mean serum albumin increased significantly (P = 0.015) from a baseline of 30.12 ± 7.16 g/l to 39.52 ± 6.70 g/l. After 24 months of immunoadsorption discontinuation, serum albumin also returned to baseline levels, which was consistent with an increase in proteinuria. No significant differences in serum creatinine were observed during the years of follow-up (Table 3).
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About repeat immunoadsorption
Patient 1, 2, 5 and 6 received the repeat immunoadsorption on the 6, 19, 19 and 38 months after the first immunoadsorption treatment course, respectively. After the treatment, the mean level of proteinuria decreased from 5.02 ± 1.85 g/ 24 h to 1.64 ± 0.55 g/24 h at the end of the treatment, and serum apo E decreased from 14.65 ± 11.17 mg/dl to 7.90 ± 1.72 mg/dl (Table 4). The mean decrease in proteinuria and serum apo E was 65.25%, 31.25%, respectively. The immediate effect of repeat immunoadsorption was just as same as the first treatment.
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Adverse effects
Two infections occurred in the total 17 immunoadsorption-treated courses (11.76%). The patients recovered soon after the treatment of antibiotic and the replacement of double lumen single-needle catheter. Because of the use of low-molecular weight heparin after the renal biopsy, one single episode of gross haematuria occurred. One anaphylactic episode was seen, which was relieved by intravenous administration of dexamethasone. No other adverse effects were observed.
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Discussion |
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LPG was first reported as a rare glomerular disease in 1987 [5], but it became more common in recent years [6]. In our clinical unit, 25 patients had been diagnosed as LPG from 1996 to 2007, accounting for 0.18% of all the renal biopsies. Although the lesions appear to be limited in the kidney [7], the patients with LPG face a dismal prognosis with a poor quality of life. We reported that the percentage of oedema, nephrotic syndrome and abnormal levels of serum creatinine were 100%, 87.5%, 37.5%, respectively [8].
No specific treatment for LPG has been established yet. LPG patients often show abnormal levels of serum lipids and apolipoproteins, so lipid-lowering therapy represents the generally accepted therapeutic approach. However, to the best of our knowledge, the reported experience of lipid-lowering therapy is to date restricted to case reports [9–11]. Probucol may be useful for early-stage LPG, but does not seem to be effective in patients with nephrotic LPG [12]. Bezafibrate and intensive lipid-lowering therapy consisting of four kinds of lipid-lowering agents (fenofibrate, niceritrol, ethyl-icosapentate and probucol) were reported to be effective in patients with nephrotic LPG, but the improvement in proteinuria and apolipoprotein level took several months to develop. Novel therapeutic options have, therefore, been investigated in order to ameliorate the clinical manifestation quickly and effectively.
In our investigation, the short-term effects of immunoadsorption in patients with LPG were dramatic. Only after one treatment course, proteinuria decreased by >50%, and apo E level nearly converted to normal range. Immunoadsorption not only improved proteinuria and apo E levels but also resolved lipoprotein thrombi in LPG. However, mesangial cell proliferation as well as expansion of the mesangial matrix became more obvious in the repeat renal biopsy. This finding was not surprising, just as patients with diabetic nephropathy [13] and postinfectious glomerulonephritis [14], mesangial matrix expansion or mesangial cell proliferation were often observed and sustained for a long duration after the termination of the initial pathogenic insult. Arai [10] and Ieiri [11] also reported this phenomenon in LPG patients after Bezafibrate and the intensive lipid-lowering therapy treatment. In our study, the differences between the percentage of global sclerosis and segmental sclerosis before and after the immunoadsorption treatment were not significant, the extent of tubulointerstitial fibrosis did not change, and serum creatinine remained stable, so we speculated that this phenomenon was due to glomerular healing after the removal of lipoprotein thrombi. As lipid-lowering therapy was not given to the patients before or during the immunoadsorption course, which suggested that the marked improvement of proteinuria, apo E level and histologic resolution of the lipoprotein thrombi were the effect of immunoadsorption.
As a relatively rare disease, controlled therapeutic trials are difficult to perform, so whether immunoadsorption would prolong the progression of LPG is unknown. But epidemiologic studies suggested that proteinuria was associated with more rapid kidney function loss [15], so we reasonably speculated that immunoadsorption treatment of LPG patients, especially the reduction of proteinuria excretion, might lead to improvement of renal prognosis. However, immunoadsorption was not a causative therapy option. In the previous immunoadsorption study of focal segmental glomerulosclerosis recurrence in kidney transplants and other disease with nephrotic syndrome, proteinuria tended to return to baseline levels within 1 month after immunoadsorption discontinuation [3,16,17]. As a genetic predisposition disease, in our study, although with the lipid-lowering therapy and tight blood pressure control, after immunoadsorption, proteinuria still tended to return to baseline levels within 12 months. We applied repeat immunoadsorption to four recurrent patients, which also had dramatic therapeutic effects. Repeat immunoadsorption might be an acceptable alternative treatment for recurrent patients, as no causative therapy has been established yet.
In conclusion, we demonstrated for the first time that immunoadsorption onto protein A was associated with a significant response shown by reduced proteinuria, decreased apo E and resolved intraglomerular thrombi in patients with LPG, and repeat immunoadsorption might also be effective in recurrent patients. Our observations suggest that immunoadsorption is an acceptable alternative treatment option in patients with LPG. This hypothesis should be investigated by randomized clinical trials including a control group, and further studies are also required to define the influence of immunoadsorption on long-term effects in LPG patients.
Conflict of interest statement. None declared.
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[Abstract/Free Full Text]
Accepted in revised form: 11. 9.08
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