Low molecular weight proteins in renal disease

Le travail porte sur les protéines de faible poids moléculaire ou microprotéines dans la pathologie rénale. La première partie de la thèse résume les données de la littérature sur le catabolisme rénal des protéines, sur le syndrome urémique et sur la physiopathologie de la protéinurie. La deuxième partie présente une série de cinq études réalisées chez les patients en dialyse (hémodialyse et dialyse péritonéale) et ceux présentant un dysfonctionnement tubulaire. Les principales microprotéines étudiées sont : la β2-microglobuline, la cystatine C, la protéine A ou la protéine des cellules de Clara (CC16), la protéine transporteuse de la vitamine A (RBP) et l’alpha-1-microglobuline.
L’objectif était de déterminer les concentrations sériques de ces protéines chez les patients en dialyse, leur clairance péritonéale chez les patient en dialyse péritonéale, et d’identifier les facteurs qui influencent leur élimination dans ces deux modes de dialyse. Par la dosage des protéines spécifiques dans l’urine, nous avons cherché à caractériser la protéinurie en cas d’atteinte tubulaire et surtout à déterminer la fiabilité des microprotéines comme marqueurs de diagnostic de dysfonctionnement tubulaire.
Les résultats ont montré que les concentrations sériques de base des microprotéines sont très élevées chez les patients en dialyse chronique et influencées essentiellement par des facteurs liés au patient (âge, sexe et diurèse résiduelle). Ce qui a trait au procédé de dialyse ne semble pas avoir d’influence significative sur ces concentrations. Chez les patients en hémodialyse, les concentrations post-dialyse sont déterminées par la taille de la protéine, la perméabilité de la membrane de dialyse et l’hémoconcentration. LA CC16 apparaît comme un nouveau marqueur potentiel de biocompatibilité des membranes d’hémodialyse et ses variations lors d’une séance de dialyse avec une membrane bioincompatible comme un moyen pour détecter les altérations pulmonaires chez les patients en dialyse chronique. La dialyse péritonéale s’accompagne de l’élimination tant de substances potentiellement néfastes que des protéines nécessaires à l’organisme telle que l’albumine.
Le transport des protéines à travers le membrane péritonéale est fonction de leur taille et se fait à travers au moins deux types de pores différents, l’un pour les microprotéines et l’autre pour les protéines plus larges. La néphropathie due aux herbes chinoises est caractérisée par une protéinurie tubulaire sélective, témoin d’une atteinte toxique du tubule proximal, principal site de réabsorption des microprotéines. L’infection à VIH quant à elle, est associée à une prévalence très élevée d’anomalies glomérulaires et/ou tubulaires infracliniques qui ne semblent pas liées à la sévérité de l’affection initiale. Pour la détection des anomalies tubulaires la CC16 et la RBP urinaires apparaissent comme des marqueurs fiables et très sensibles, tandis que le rapport microprotéine/albumine urinaire apparaît comme un bon marqueur pour distinguer une protéinurie tubulaire d’une protéinurie glomérulaire

The first part of the thesis reviews the literature on the renal catabolism of proteins, and especially on the renal handling of plasma five LMWP (β2-m, cyst C, CC16, RBP and α1-m), on the uremic syndrome and the pathophysiology of proteinuria. The second part presents a series of clinical studies on the fate of LMWP in renal diseases. Factors influencing the serum concentrations of these microproteins in long-term dialyzed patients have been investigated, and the characteristics of dialysis membranes have been examined in relation to protein removal during the dialysis procedure (Chap. 1). Tubular proteinuria is characterized in a new pathological entity, the Chinese herbs nephropathy and in analgesic nephropathy (Chap. 2.1). Tubular proteinuria in HIV-infection is reported (Chap. 2.2), and the reliability of urinary LMWP as markers of tubular dysfunction is also determined (Chap. 2.1 & 2.2).
These studies indicate that in various pathological states involving the kidney, the concentrations of LMWP in serum and/or in urine are invariably increased: slightly in case of mild lesions and more markedly in case if advanced disease states.
The pre-dialysis serum levels of all LMWP studied are markedly increased and multiplied on the average by a factor of 20 in dialyzed patients. They are elevated to the same extent whether patients are on long-term hemodialysis or peritoneal dialysis. They are independent of the duration of dialysis treatment (except for RBP whose serum level declines with time in peritoneal dialysis) and the dialysis membrane characteristics. Only patient-related factors (residual diuresis, age and sex) significantly influence pre-dialysis serum concentrations of these proteins. The retention of LMWP in the serum of dialyzed patients and the lack of correlation between LMWP serum concentrations and duration of dialysis therapy lead to some considerations. Firstly, the possible implication of these proteins in the pathogenesis of the uremic syndrome. The role of β2-m in dialysis-related amyloidosis has been already established, that of other microproteins such as CC16, cyst C and α1-m remains to be delineated. Secondly, the existence of a feedback controls limiting the synthesis of these proteins when their serum levels are elevated. Such mechanism is described mainly for hormones, but it is not yet reported for the other studied LMWP. Thirdly, routes other than residual GFR, dialysis removal, deposition in bone joint (for β2-m) and liver uptake (for RBP), might contribute to the disposal of LMWP in long-term hemodialyzed patients, especially in anuric patients.
The perdialytic changes of LMWP in serum are determined by the size of the protein, the permeability of dialysis membranes, including the peritoneum, and hemoconcentration. In hemodialysis, only proteins of the size of β2-m or smaller are removed by high flux membranes (UFC ≥ 15 ml/h . m² . mmHg), whereas peritoneal dialysis results in a marked loss of all LMWP as well as HMWP. The use of a wide range (in size) of LMWP in addition of HMWP (in peritoneal dialysis) allows us to estimate the pore size of hemodialysis membranes and to determine the peritoneal membrane characteristics : the peritoneum behaves like a two-pore model in the handling of proteins. Long cycle exchanges, during peritoneal dialysis, is associated with a significant loss of albumin and other HMWP without significant change in the clearance of small and middle size molecules. A prospective study, analyzing the effect of dialysis membrane biocompatibility on serum levels of low molecular weight proteins allows us to describe a new markers of biocompatibility related to the activation of the complement cascade, i.e. a transient rise of the serum level of CC16 during cuprophane dialysis, whose interest stems from the fact that it reflects a transient alteration of the bronchoalveolar/blood barrier permeability. The magnitude of the transient rise of serum CC16 declines with duration of hemodialysis therapy, suggesting a progressive alteration of the bronchoalveolar/blood barrier during long-term hemodialysis.
The assessment of several LMWP, albumin and NAG in urine has allowed us to characterize the proteinuria in subjects exposed to toxic Chinese herbs included un a slimming cure, to report the first detailed analysis of tubular proteinuria in patients with analgesic nephropathy and to demonstrate the existence of preclinical renal defects in HIV-infected patients, before the onset of HIV associated nephropathy.
The pattern of tubular proteinuria following Chinese herbs consumption is variable. Mild dysfunction characterized by a raise of the urinary level of only 3 of the LMWP as a consequence of mild tubular injury, which proves reversible in some patients. A more severe tubular dysfunction is associated with increased urinary excretion of all LMWP, albumin and NAG. Total proteinuria is elevated and moderate renal failure is also present. These abnormalities are not reversible and worsen is some patients during the follow-up. This pattern is identical to that observed in analgesic and cadmium nephropathies. The largest increase in all urinary markers is associated with elevated serum levels of LMWP as a consequence of more reduced GFR. Total proteinuria is manifestly increased. Almost all of these patients reach ERSD during the follow-up.
In HIV-infected patients without clinical evidence of HIV nephropathy, an abnormal pattern of urinary proteins is present in 81% of the patients. It is characterized by a tubular as well as a glomerular proteinuria confirming the presence of proximal tubular and/or glomerular permeability defects. These defects do not depend on the severity of HIV-infection. Like serum β2-m and CD-4 lymphocyte count, urinary β2-m appears as a new marker of the severity of the disease.
As previously reported for β2-m, we confirm that the concentration ratio of LMWP over albumin in urine is the best indicator to discriminate tubular from glomerular proteinuria. CC16 and RBP in urine appear as more reliable markers than other LMXP for the detection of tubular dysfunction, with a sensitivity of 64% (RBP), 80%(CC16) and 94% (both two parameters). The sex-dependant of urinary CC16 should be taken into account for the determination of normal values