Le Berre L, Godfrin Y, Lafond-Puyet L, Perretto S, Le Carrer D, Bouhours J F, Soulillou J P, Dantal J
Institut National de la Santé Et de la Recherche Médicale (INSERM), Unité de Recherche U437 "Immunointervention dans les allo et xenotransplantations," Institut de Transplantation et Recherche en Transplantation (ITERT), Nantes, France.
Kidney Int. 2000 Dec;58(6):2502-11. doi: 10.1046/j.1523-1755.2000.00434.x.
Patients suffering from focal and segmental glomerulosclerosis (FSGS) and in whom this disease recurs after transplantation are likely to have an active form of the disease and to have a factor(s) (such as, albuminuric factor) present in their blood that alters glomerular permeability for albumin.
We used a sequential 50 and 70% ammonium sulfate (AS) precipitation of plasma from patients with relapsing FSGS and non-FSGS nephrotic syndrome (NS), in addition to plasma from healthy individuals, to obtain both an immunoglobulin (Ig)-rich fraction (50% AS precipitate) and a non-Ig fraction (70% AS supernatant). These fractions were injected intra-arterially or intravenously/intraperitoneally into Sprague-Dawley rats, and proteinuria (g protein/mmol creatinine) was measured for 24 hours. Ig fractions eluted from immunoadsorption onto protein A were also tested. A biochemical characterization was then carried out on the 70% AS supernatants by ultrafiltration on 30 and 50 kD cut-off membranes and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Differentially stained bands were sequenced.
The 70% AS supernatants from FSGS patients induced proteinuria when injected intra-arterially into normal rats. This effect was significantly different (P < 0.05) from that observed when similar fractions were prepared from the plasma of patients suffering from non-FSGS NS, but was not different from that observed with fractions from healthy individuals and even with an injection of saline solution. Injections of other plasma fractions did not induce a significant proteinuria in the FSGS group versus the non-FSGS NS group. SDS-PAGE of 70% AS supernatants revealed a protein of 23 kD that was more concentrated in AS supernatants from FSGS plasma than the other plasma samples and that was identified by microsequencing as apolipoprotein A1. After sequential ultrafiltration of 70% AS supernatants on 30 and 50 kD cut-off membranes, a second band of 43 kD was found at a much higher concentration in the FSGS samples than in non-FSGS NS and healthy individuals samples. This band is likely to correspond to a candidate albuminuric factor recently reported by another group [1], and was identified by microsequencing as alpha1 acid glycoprotein or orosomucoid. Consequently, purified orosomucoid from the plasma of FSGS, non-FSGS NS patients, or healthy individuals was injected intra-arterially into rats. No differences were found between the proteinuria induced in each group.
These data strongly suggest that in vivo injection of material into the rat is not a reliable model for testing plasma fraction activity and that the 43 kD orosomucoid is not likely to be the albuminuric factor.
患有局灶节段性肾小球硬化症（FSGS）且移植后疾病复发的患者，其疾病可能处于活动期，血液中可能存在某种（如蛋白尿因子）改变肾小球对白蛋白通透性的因素。
我们对复发性FSGS患者、非FSGS肾病综合征（NS）患者以及健康个体的血浆依次进行50%和70%硫酸铵（AS）沉淀，以获得富含免疫球蛋白（Ig）的部分（50% AS沉淀）和非Ig部分（70% AS上清液）。将这些部分经动脉内或静脉内/腹腔内注射到Sprague-Dawley大鼠体内，并测量24小时蛋白尿（g蛋白/mmol肌酐）。还对从蛋白A免疫吸附洗脱的Ig部分进行了测试。然后通过在30和50 kD截留膜上超滤以及十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）对70% AS上清液进行生化表征。对差异染色的条带进行测序。
FSGS患者的70% AS上清液经动脉内注射到正常大鼠体内可诱导蛋白尿。与从非FSGS NS患者血浆制备的类似部分相比，这种效应有显著差异（P < 0.05），但与从健康个体血浆获得的部分以及注射生理盐水溶液时观察到的效应无差异。在FSGS组与非FSGS NS组中，注射其他血浆部分未诱导出显著蛋白尿。70% AS上清液的SDS-PAGE显示一条23 kD的蛋白条带，其在FSGS血浆的AS上清液中比其他血浆样本中更浓缩，经微量测序鉴定为载脂蛋白A1。在30和50 kD截留膜上对70% AS上清液依次超滤后，发现一条43 kD的第二条带，其在FSGS样本中的浓度远高于非FSGS NS和健康个体样本。这条带可能对应于另一组最近报道的候选蛋白尿因子[1]，经微量测序鉴定为α1酸性糖蛋白或血清类黏蛋白。因此，将从FSGS、非FSGS NS患者或健康个体血浆中纯化的血清类黏蛋白经动脉内注射到大鼠体内。各组诱导的蛋白尿之间未发现差异。
这些数据强烈表明，在大鼠体内注射物质并非测试血浆部分活性的可靠模型，且43 kD血清类黏蛋白不太可能是蛋白尿因子。
