[Anti-NEP and anti-PLA2R antibodies in membranous nephropathy: an update]. / Anticorps anti-EPN et anti-PLA2R dans les glomerulopathies extramembraneuses: le point en 2014.

Membranous nephropathy (MN) is the most common cause for nephrotic syndrome in adults and occurs as an idiopathic (primary) or secondary disease. Since the early 2000's, substantial advances have been made in the understanding of the molecular bases of MN. The neutral endopeptidase (NEP) and the receptor for secretory phospholipase A2 (PLA2R) have been identified as target antigens for circulating and deposited antibodies in allo-immune neonatal and adult " idiopathic " MN, respectively. These antibodies recognize specific antigens of podocytes, precipitate as subepithelial immune complexes and activate complement leading to proteinuria. Anti-PLA2R antibodies are of particular clinical importance. Indeed, they are detected in approximately 70% of primary MN in adults, demonstrating that MN actually is an autoimmune condition specific to the kidney. In Europeans, genome-wide studies have shown an association between alleles of PLA2R1 and HLA DQA1 (class II genes of tissue histocompatibility complex) genes and idiopathic MN. Newly developed diagnostic tests detecting circulating anti-PLA2R antibody and PLA2R antigen in glomerular deposits have induced a change in paradigm in the diagnostic approach of idiopathic MN. Measurement of circulating anti-PLA2R antibody is also very useful for the monitoring of MN activity. However, the mechanisms responsible for the formation of anti-PLA2R antibodies as well as those involved in the progression of MN to end-stage renal disease remain to be defined.

Autoantibodies specific for the phospholipase A2 receptor in recurrent and De Novo membranous nephropathy.

Recent findings in idiopathic membranous nephropathy (MN) suggest that in most patients, the disease is because of anti-phospholipase A(2) receptor (PLA(2) R1) autoantibodies. Our aim was to analyze the prevalence and significance of anti-PLA(2) R1 antibodies in recurrent and de novo MN after transplantation. We assessed circulating PLA(2) R1 autoantibodies by a direct immunofluorescence assay based on human embryonic kidney cells transfected with a PLA(2) R1 cDNA, and the presence of PLA(2) R1 antigen in immune deposits. We showed that PLA(2) R1 was involved in 5 of 10 patients with recurrent MN, but in none of the 9 patients with de novo MN. We also showed a marked heterogeneity in the kinetics and titers of anti-PLA(2) R1, which may relate to different pathogenic potential. We provide evidence that some patients with PLA(2) R1-related idiopathic MN and anti-PLA(2) R1 antibodies at the time of transplantation will not develop recurrence. Because PLA(2) R1 autoantibody was not always associated with recurrence, its predictive value should be carefully analyzed in prospective studies.

The L1 cell adhesion molecule is a potential biomarker of human distal nephron injury in acute tubular necrosis.

The L1 cell adhesion molecule (CD171) is a multidomain membrane glycoprotein of the immunoglobulin superfamily. We evaluated its expression in human acute kidney injury and assessed its use as a tissue and urinary marker of acute tubular injury. Using immunohistochemical studies with antibodies to the extracellular or cytoplasmic domains, we compared L1 expression in normal kidneys in 24 biopsies taken from patients with acute tubular necrosis. L1 was found at the basolateral and the lateral membrane in all epithelial cells of the collecting duct in the normal kidney except for intercalated cells. In acute tubular necrosis, L1 lost its polarized distribution being found in both the basolateral and apical domains of the collecting duct. Further, it was induced in thick ascending limb and distal tubule cells. Apically expressed L1 found only when the cytoplasmic domain antibody was used in biopsy specimens of patients with acute tubular necrosis. The levels of urinary L1, normalized for creatinine, were significantly higher in all 24 patients with acute tubular necrosis compared to five patients with prerenal azotemia or to six patients with other causes of acute kidney injury. Our study shows that a soluble form of human L1 can be detected in the urine of patients with acute tubular necrosis and that this may be a marker of distal nephron injury.

Target antigens and nephritogenic antibodies in membranous nephropathy: of rats and men.

Membranous nephropathy, a disease characterized by an accumulation of immune deposits on the outer aspect of the glomerular basement membrane, is the most common cause of idiopathic nephrotic syndrome in white adults. In the rat model of Heymann nephritis, the target antigen of antibodies is megalin, a multiligand receptor expressed at the podocyte cell surface. This review summarizes key findings provided by this experimental model and by our discovery of neutral endopeptidase being the alloantigen involved in neonatal cases of membranous nephropathy. We discuss the role of alloimmunization as a new mechanism of renal disease and the approach that we use to identify new podocyte antigens. We also summarize current knowledge on the mechanism of proteinuria, with special emphasis on the role of complement. In conclusion, substantial progresses have been made in understanding molecular mechanisms of membranous nephropathy, which should lead to novel therapeutic approaches.

Renal cortical regulation of COX-1 and functionally related products in early renovascular hypertension (rat).

Renal volume regulation is modulated by the action of cyclooxygenases (COX) and the resulting generation of prostanoids. Epithelial expression of COX isoforms in the cortex directs COX-1 to the distal convolutions and cortical collecting duct, and COX-2 to the thick ascending limb. Partly colocalized are prostaglandin E synthase (PGES), the downstream enzyme for renal prostaglandin E(2) (PGE(2)) generation, and the EP receptors type 1 and 3. COX-1 and related components were studied in two kidney-one clip (2K1C) Goldblatt hypertensive rats with combined chronic ANG II or bradykinin B(2) receptor blockade using candesartan (cand) or the B(2) antagonist Hoechst 140 (Hoe). Rats (untreated sham, 2K1C, sham + cand, 2K1C + cand, sham + Hoe, 2K1C + Hoe) were treated to map expression of parameters controlling PGE(2) synthesis. In 2K1C, cortical COX isoforms did not change uniformly. COX-2 changed in parallel with NO synthase 1 (NOS1) expression with a raise in the clipped, but a decrease in the nonclipped side. By contrast, COX-1 and PGES were uniformly downregulated in both kidneys, along with reduced urinary PGE(2) levels, and showed no clear relations with the NO status. ANG II receptor blockade confirmed negative regulation of COX-2 by ANG II but blunted the decrease in COX-1 selectively in nonclipped kidneys. B(2) receptor blockade reduced COX-2 induction in 2K1C but had no clear effect on COX-1. We suggest that in 2K1C, COX-1 and PGES expression may fail to oppose the effects of renovascular hypertension through reduced prostaglandin signaling in late distal tubule and cortical collecting duct.

The cell adhesion molecule L1 is developmentally regulated in the renal epithelium and is involved in kidney branching morphogenesis.

We immunopurified a surface antigen specific for the collecting duct (CD) epithelium. Microsequencing of three polypeptides identified the antigen as the neuronal cell adhesion molecule L1, a member of the immunoglobulin superfamily. The kidney isoform showed a deletion of exon 3. L1 was expressed in the mesonephric duct and the metanephros throughout CD development. In the adult CD examined by electron microscopy, L1 was not expressed on intercalated cells but was restricted to CD principal cells and to the papilla tall cells. By contrast, L1 appeared late in the distal portion of the elongating nephron in the mesenchymally derived epithelium and decreased during postnatal development. Immunoblot analysis showed that expression, proteolytic cleavage, and the glycosylation pattern of L1 protein were regulated during renal development. L1 was not detected in epithelia of other organs developing by branching morphogenesis. Addition of anti-L1 antibody to kidney or lung organotypic cultures induced dysmorphogenesis of the ureteric bud epithelium but not of the lung. These results suggest a functional role for L1 in CD development in vitro. We further postulate that L1 may be involved in the guidance of developing distal tubule and in generation and maintenance of specialized cell phenotypes in CD.

Identification and epitope analysis of the renal Na+/Pi cotransport protein using monoclonal antibodies.

Seven monoclonal antibodies (mAbs) were raised against a rabbit renal brush-border glycoprotein (molecular mass, 63-66 kDa), presumably involved in Na+/Pi cotransport, which we had previously purified and reconstituted in active form in proteoliposomes (Debiec, H., Lorenc, R., and Ronco, P. M. (1992) Biochem. J. 286, 97-102). Antibody specificity for the 63-66-kDa protein was analyzed by enzyme-linked immunosorbent assay and confirmed by Western blotting and immunoaffinity chromatography of solubilized brush-border membranes (BBM), which both yielded a single 63-66-kDa band. Enzyme-linked immunosorbent assay and immunoblotting of renal cortical cell subfractions localized the immunoreactive protein to the brush-border membrane. This location was confirmed by indirect immunofluorescence of kidney cortex sections. Binding of two of the seven mAbs (63A20 and 206A126) to native BBM only occurred when the related epitope was exposed in the presence or absence of Na+, respectively; the other mAbs did not react with native BBM probably because of intramembranous orientation of the epitopes. mAb 63A20 inhibited dose-dependently Na+/Pi cotransport when preincubation of BBM was carried out in the presence of Na+ but did not affect Na+/D-glucose cotransport. Proteoliposomes formed from BBM proteins depleted of the 63-66-kDa protein by affinity chromatography with mAb 63A20 showed an 85% reduction in Na+/Pi cotransport, whereas Na+/D-glucose cotransport was not modified. These results thus establish that the 63-66-kDa BBM protein is the essential component of the Na+/Pi cotransport system. The present study also provides the first immunologic tools available for immunohistochemical localization of the Na+/Pi cotransporter. Finally, the identification of a functional epitope by mAb 63A20 opens up new ways to explore the molecular aspects of Pi uptake.

Reconstitution and characterization of a Na+/Pi co-transporter protein from rabbit kidney brush-border membranes.

A protein with Na+/Pi co-transporter activity has been extracted from rabbit brush-border membranes with chloroform/methanol and purified by hydroxyapatite chromatography. The protein has been incorporated by the dilution method into liposomes formed from different types and ratios of lipids. The greatest reconstitution has been achieved into liposomes prepared from cholesterol (20%), phosphatidylcholine (20%), phosphatidylethanolamine (30%) and phosphatidylserine (30%) (CH/PC/PE/PS). Pi uptake by these proteoliposomes had the following characteristics: (i) the initial rate was markedly greater in the presence of an inwardly directed Na+ gradient (600 pmol/10 s per mg) than with a K+ gradient (65 pmol/10 s per mg); (ii) maximal uptake was increased 8-fold above the equilibrium value ('overshoot') when a Na+ gradient was applied; (iii) Pi was not merely bound to proteoliposomes but was transported intravesicularly; and (iv) Na(+)-dependent Pi uptake was sensitive to the known phosphate transport inhibitors. This first successful attempt of reconstitution of Na+/Pi transport activity into proteoliposomes led us to isolate and characterize physico-chemically the protein responsible. Its isoelectric point was about 5.8, and urea/SDS gel electrophoresis revealed a broad band of molecular mass ranging from 63 to 66 kDa under both reducing and non-reducing conditions. In the native form, the molecular mass analysed by gel filtration was estimated to be 170 +/- 10 kDa, suggesting that the protein is a polymer, probably stabilized by hydrophobic bonds. Endoglycosidase F treatment decreased the molecular mass to approx. 50 kDa. It is postulated that this acidic glycoprotein might represent a subunit of the intact Na+/Pi co-transporter from rabbit kidney brush-border membranes.

1,25-Dihydroxycholecalciferol-related Na+/D-glucose transport in brush-border membrane vesicles from embryonic chick jejunum. Modulation by triiodothyronine.

1,25-Dihydroxycholecalciferol, when present at and above 10 nM in an organ-culture system of embryonic chick jejunum, approximately doubled the rate of Na(+)-gradient-driven D-glucose uptake by brush-border membrane vesicles, but had no effect on Na(+)-independent D-glucose transfer. The sterol also had no effect on Na+ influx along an outside/inside Na+ gradient ([Na+]o = 100 mM; [Na+]i = 0 mM). This renders it unlikely that in embryonic intestine, calcitriol raises Na(+)-dependent D-glucose transport through changes in the electrochemical Na+ gradient. D-[U-14C]Glucose tracer exchange, measured under voltage-clamp condition at Na+/D-glucose equilibrium, revealed that addition of calcitriol to the culture medium approximately doubled the activity of the Na+/D-glucose transporter in the brush-border membrane. This was also reflected by an corresponding increase in the maximal velocity of the transfer process. Increased [3H]phlorizin binding after calcitriol treatment suggests that the steroid hormone activates Na+/D-glucose transport through increasing the number of carrier molecules in the brush-border membrane. 10 nM triiodothyronine, which by itself has no effect on Na(+)-dependent D-glucose transport, potentiated the effect of 1,25-dihydroxycholecalciferol such that in the presence of both hormones, Na+/D-glucose-carrier activity was increased fourfold above control levels.

A high yield preparation of brush border membrane vesicles from organ-cultured embryonic chick jejunum: demonstration of insulin sensitivity of Na(+)-dependent D-glucose transport.

Although embryonic chick small intestinal segments provide a very limited amount of tissue for preparation of enterocyte brush border membrane vesicles (BBMV), we were able to develop a procedure for isolation of BBMV from cultured 20-d-old embryonic chick jejunum in high yield by modifying a divalent cation precipitation method. Total yield of the brush border marker enzyme alkaline phosphatase in the vesicle fraction as compared to the crude homogenate was approximately 40%, and the specific activity of the enzyme was increased 25-fold on the average. The brush border membrane vesicle fraction was only contaminated with other cellular organelles (basolateral membranes, mitochondria, lysosomes or endoplasmic reticulum) to a minor extent. Functional integrity of the brush border vesicles was indicated by Na+ gradient-driven electrogenic D-glucose transport leading to concentrative transfer (overshoot) of the sugar into an osmotically active intravesicular space. When jejuna were cultured for 48 h in the presence of 10(-6) mol/L insulin, the initial rate of Na(+)-dependent D-glucose uptake by brush border membrane vesicles as well as Na(+)-dependent [3H]phlorizin binding to brush border membranes was approximately twice as high as in vesicles from untreated controls. This strongly suggests that insulin could enhance intestinal absorption of D-glucose by increasing the intrinsic activity of the Na(+)-dependent D-glucose transport system at the luminal membrane of enterocytes.

Mechanism and regulation of intestinal phosphate absorption.

Proper absorption of inorganic phosphate (Pi) from the lumen of the small intestine is of great importance for the achievement of Pi homeostasis. Although due to intralumenal H+ and Pi concentrations, Pi probably can be absorbed as H2PO4- by passive means in the duodenum, transepithelial transport of HPO4(2)- requires uptake from the lumen by an active transport system. The latter has been identified in many species as a Na(+)-Pi cotransport system at the brush-border membrane of the enterocyte. Although it is still a matter of debate whether the intestinal Na+ gradient-driven Pi transport system is electrogenic or electroneutral, there is agreement that the transporter accepts H2PO4- and HPO4(2)- alike. Recently, two laboratories independently isolated a Na(+)-Pi-binding protein which has been tentatively identified as part of the Na(+)-Pi cotransport system. Movement of Pi from the cytosol across the basolateral membrane into the interstitial space has only been preliminarily characterized as transfer by facilitated diffusion. Na(+)-Pi cotransport across the brush-border membrane is under control by the active vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). The sterol increases the intrinsic activity, i.e. the number and/or mobility of Na(+)-Pi carriers through genomic and probably also nongenomic actions. In addition, the rate of Na(+)-gradient-driven Pi transport can be enhanced by the hormone also through reduction of transmembrane Na+ fluxes so that more energy for translocation becomes available from the transmembrane Na+ gradient. Evidence is accumulating that thyroid hormones as well as glucocorticoids, apart from stimulating vitamin D-independent Pi uptake, potentiate the effect of 1,25-(OH)2D3 on Na(+)-Pi cotransport across the brush-border membrane.

D-glucose uptake is increased in jejunal brush-border membrane vesicles from hyperthyroid chicks.

Jejunal brush-border membrane vesicles were harvested from 4-week old chicks whose thyroid status had been altered either by a daily injection of 20 micrograms T3 for 1 week or which through the preceding 4 weeks had received propylthiouracil and that had been repleted with either 20 or 80 micrograms T3 in divided doses within 48 h. T3 markedly stimulated D-glucose uptake in brush-border membrane vesicles in the presence of an outside/inside (100/0 mmol/l) Na+ gradient. T3 administration had no detectable influence on the Na+ permeability of the isolated vesicles. The effect of the thyroid hormone on Na+ gradient-driven D-glucose uptake was fully preserved at zero transmembrane potential difference. These findings exclude that T3 stimulates Na+-dependent D-glucose transport in the small intestine through changes of the electrochemical Na+ gradient or through alteration of the transmembrane potential difference. Tracer exchange experiments under equilibrium and voltage-clamp conditions revealed a significantly shorter half-time of D-glucose uptake in brush-border membrane vesicles from T3-treated chicks. Kinetic analysis showed that T3 administration significantly increases the apparent maximal velocity of D-glucose transport in brush-border membrane vesicles, whereas the apparent Km values were virtually unaltered. From these data we conclude that T3 increases the activity of Na+-dependent D-glucose carriers in the brush-border membrane. This is interpreted as consistent with a greater rate of D-glucose absorption from the intestinal lumen under conditions of hyperthyroidism.

Identification of Na+,Pi-binding protein in kidney and intestinal brush-border membranes.

An Na+, Pi-binding protein has been extracted from kidney and intestinal brush-border membranes with an organic solvent and has been purified by Kieselghur and Sephadex LH-60 chromatography. The molecular mass of this protein has been estimated to be about 155 kDa as determined by gel-filtration chromatography on Sepharose 2B. Under denaturing conditions, polyacrylamide-gel electrophoresis revealed a monomer of molecular mass about 70 kDa. The protein has high specificity and high affinity for Pi [K0.5 (concentration at which half-maximal binding is observed) near 10 microM]. Na2+ binding also exhibits saturation behaviour, with a K0.5 near 7.5 mM. Pi binding is inhibited by known inhibitors of Pi transport in brush-border membrane vesicles. It appears that this protein could be involved in Na+/Pi co-transport across the renal and intestinal brush-border membranes.

Influence of lactose on phosphate metabolism in rats.

1. Lactose, sucrose or maltose were administered to rats by gavage together with 32PO4, and blood 32P was determined. 2. PO4 uptake into blood was increased only when lactose and PO4 were administered into the gut. 3. Weaned male rats were fed on a control diet or a diet containing 30, 60 or 120 g lactose/kg. After a 5 d period of adaptation, a 6 d P balance was carried out. After a further 24 d blood levels of PO4, and calcium and PO4 uptake by brush-border membrane vesicles prepared from the renal cortex, were determined. 4. The absorption and retention of P were higher in lactose-fed rats. Faecal P excretion decreased with increasing dietary lactose content. Urinary P excretion was lower in the rats fed on 30 and 60 g lactose/kg whereas the 120 g lactose/kg diet increased urinary P. 5. Membrane vesicles isolated from rats fed on the diets containing 30 and 60 g lactose/kg showed a higher uptake of PO4, but with the 120 g lactose/kg diet uptake was lower than with membrane vesicles isolated from rats fed on the control diet. 6. A statistically significant increase in the serum PO4 level was observed in the rats fed on the diet with 120 g lactose/kg. 7. The presence of lactose in the diet caused a stimulatory effect on PO4 absorption and retention.

Effect of lactose on phosphate transport into rat intestinal brush border membrane vesicles.

Brush border membrane vesicles (BBMV) were used to evaluate the influence of lactose on phosphate (Pi) transport in the intestine of the rat. BBMV were prepared from duodenum, jejunum and ileum of 3-, 4- and 7-wk-old rats. Na+-dependent active transport of Pi was investigated in the presence of an Na+ gradient; Na+-independent, diffusional transport was investigated by substitution of choline chloride for NaCl. In 3-wk-old rats active Pi transport dominated, whereas in 4-wk-old rats a decrease in active transport and an increase of the diffusional component of total Pi transport were observed. Furthermore, in 7-wk-old rats, Pi was transported by simple diffusion alone. Addition of lactose to the preincubation medium had no effect on Pi transport in duodenal and jejunal BBMV isolated from 3-wk-old rats. In the remaining BBMV preparations, lactose caused a twofold enhancement of diffusional Pi uptake. Glucose and galactose had no effect on diffusional Pi uptake into BBMV. These results indicate that lactose may directly interact with luminal membranes, thus leading to increased diffusional transport. The membranes with a decreased Na+-dependent Pi transport activity are most susceptible to the influence of lactose.

Biological time-related changes in tolerance of male rats to hypoxia--I. Survival rate and carbohydrate metabolism.

Investigations were carried out on male Wistar rats, synchronized in standard conditions to a light-dark regiment (LD 12:12 with L from 0600 to 1800). Rats exposed to hypoxia equivalent to 10,500m at a clock-hour of 1000 had a survival time twice as long as that of animals exposed at 2200. Data from this study indicate the ability to mobilize energy stores through the conversion of liver glycogen to glucose along with circadian differences in hormonal response (e.g. corticosterone and insulin) contributes to the tolerance to hypoxia being greater during diurnal rest than nocturnal activity in rats.