Characterization of proteinuria and tubular protein uptake in a new model of oral L-lysine administration in rats
2006; Elsevier BV; Volume: 69; Issue: 8 Linguagem: Inglês
10.1038/sj.ki.5000272
ISSN1523-1755
AutoresK. Thelle, Erik I. Christensen, Henrik Vorum, Hans Ørskov, Henrik Birn,
Tópico(s)Biomedical Research and Pathophysiology
ResumoIntravenous infusion of basic amino acids is used experimentally and pharmacologically to prevent renal proximal tubular uptake of filtered proteins. Intravenously injected L-lysine is rapidly cleared from plasma and the effect on tubular protein reabsorption is transient. To obtain a more sustained effect, we developed a model of oral L-lysine administration and characterized this model by analyzing urinary protein excretion and proximal tubule uptake of filtered proteins. Rats placed in metabolic cages were treated with 20 mmol/kg/6 h of L-lysine, glycine, or water. Urines were analyzed for proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and radioimmunoassay. Proximal tubule uptake of proteins and expression of apical membrane receptors were investigated by immunocytochemistry. In vitro uptake and receptor expression were studied using a yolk sac cell line. L-lysine administration produced increased urinary excretion of a large number of proteins while the effect on tubular accumulation of selected proteins was variable. L-lysine treatment induced changes in the localization of two receptors responsible for tubular endocytosis of filtered proteins. In conclusion, oral L-lysine treatment induced proteinuria, in particular albuminuria, as efficiently as previous reports on intravenous infusion. The effect on tubular protein accumulation was variable suggesting differential effects on tubular reabsorption and degradation of filtered proteins. Changes in tubular protein handling were accompanied by changes in the localization of the endocytic receptors, megalin, and cubilin. In vitro experiments supported the in vivo observations. The findings suggest that L-lysine may affect receptor trafficking in addition to possible effects on the direct binding of ligands to the receptors. Intravenous infusion of basic amino acids is used experimentally and pharmacologically to prevent renal proximal tubular uptake of filtered proteins. Intravenously injected L-lysine is rapidly cleared from plasma and the effect on tubular protein reabsorption is transient. To obtain a more sustained effect, we developed a model of oral L-lysine administration and characterized this model by analyzing urinary protein excretion and proximal tubule uptake of filtered proteins. Rats placed in metabolic cages were treated with 20 mmol/kg/6 h of L-lysine, glycine, or water. Urines were analyzed for proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and radioimmunoassay. Proximal tubule uptake of proteins and expression of apical membrane receptors were investigated by immunocytochemistry. In vitro uptake and receptor expression were studied using a yolk sac cell line. L-lysine administration produced increased urinary excretion of a large number of proteins while the effect on tubular accumulation of selected proteins was variable. L-lysine treatment induced changes in the localization of two receptors responsible for tubular endocytosis of filtered proteins. In conclusion, oral L-lysine treatment induced proteinuria, in particular albuminuria, as efficiently as previous reports on intravenous infusion. The effect on tubular protein accumulation was variable suggesting differential effects on tubular reabsorption and degradation of filtered proteins. Changes in tubular protein handling were accompanied by changes in the localization of the endocytic receptors, megalin, and cubilin. In vitro experiments supported the in vivo observations. The findings suggest that L-lysine may affect receptor trafficking in addition to possible effects on the direct binding of ligands to the receptors. Tubular uptake of filtered protein is mediated by proximal tubule endocytosis. This process involves the initial binding to endocytic receptors at the luminal surface of the proximal tubule cells. Previous studies have established two multiligand receptors, megalin and cubilin, as responsible for the endocytic uptake of a large number of different proteins and peptides, including lipoproteins, hormones, nutrients, and enzymes.1.Christensen E.I. Birn H. Megalin and cubilin: synergistic endocytic receptors in renal proximal tubule.Am J Physiol Renal Physiol. 2001; 280: F562-F573PubMed Google Scholar, 2.Christensen E.I. Birn H. Megalin and cubilin: multifunctional endocytic receptors.Nat Rev Mol Cell Biol. 2002; 3: 256-266Crossref PubMed Scopus (603) Google Scholar Both receptors are heavily expressed at the luminal surface of proximal tubule cells and other absorptive epithelia, including yolk sac epithelial cells. Inhibition of proximal tubule reabsorption of proteins is an important experimental approach for the study of glomerular permeability and tubular function. Also inhibition of renal uptake has been introduced to protect the kidney from filtered, nephrotoxic agents, for example radiolabeled peptides and antibody fragments used experimentally as targeted anti-cancer drugs.3.de Jong M. Breeman W.A. Bernard B.F. et al.[177Lu-DOTA(0),Tyr3] octreotate for somatostatin receptor-targeted radionuclide therapy.Int J Cancer. 2001; 92: 628-633Crossref PubMed Scopus (167) Google Scholar, 4.Rosch F. Herzog H. Stolz B. et al.Uptake kinetics of the somatostatin receptor ligand [86Y]DOTA-DPhe1-Tyr3-octreotide ([86Y]SMT487) using positron emission tomography in non-human primates and calculation of radiation doses of the 90Y-labelled analogue.Eur J Nucl Med. 1999; 26: 358-366Crossref PubMed Scopus (100) Google Scholar, 5.Behr T.M. Sharkey R.M. Sgouros G. et al.Overcoming the nephrotoxicity of radiometal-labeled immunoconjugates: improved cancer therapy administered to a nude mouse model in relation to the internal radiation dosimetry.Cancer. 1997; 80: 2591-2610Crossref PubMed Google Scholar, 6.Behr T.M. Sharkey R.M. Juweid M.E. et al.Reduction of the renal uptake of radiolabeled monoclonal antibody fragments by cationic amino acids and their derivatives.Cancer Res. 1995; 55: 3825-3834PubMed Google Scholar, 7.Rutherford R.A. Smith A. Waibel R. Schubiger P.A. Differential inhibitory effect of L-lysine on renal accumulation of 67Cu-labelled F(ab')2 fragments in mice.Int J Cancer. 1997; 72: 522-529Crossref PubMed Scopus (11) Google Scholar, 8.Miao Y. Owen N.K. Whitener D. et al.In vivo evaluation of 188Re-labeled alpha-melanocyte stimulating hormone peptide analogs for melanoma therapy.Int J Cancer. 2002; 101: 480-487Crossref PubMed Scopus (87) Google Scholar, 9.Chen J. Cheng Z. Hoffman T.J. et al.Melanoma-targeting properties of (99 m)technetium-labeled cyclic alpha-melanocyte-stimulating hormone peptide analogues.Cancer Res. 2000; 60: 5649-5658PubMed Google Scholar Basic amino acids, notably L-lysine, inhibit the reabsorption of proteins in the proximal tubule whereas neutral or acidic amino acids do not.10.Mogensen C.E. Solling K. Vittinghus E. Studies on mechanisms of proteinuria using amino-acid-induced inhibition of tubular reabsorption in normal and diabetic man.Contrib Nephrol. 1981; 26: 50-65Crossref PubMed Google Scholar, 11.Mogensen C.E. Solling K. Studies on renal tubular protein reabsorption: partial and near complete inhibition by certain amino acids.Scand J Clin Lab Invest. 1977; 37: 477-486Crossref PubMed Scopus (212) Google Scholar Inhibition of proximal tubule endocytosis of plasma proteins and peptides by L-lysine infusion has been evaluated in several experimental studies12.Tencer J. Frick I.M. Oquist B.W. et al.Size-selectivity of the glomerular barrier to high molecular weight proteins: upper size limitations of shunt pathways.Kidney Int. 1998; 53: 709-715Abstract Full Text PDF PubMed Scopus (114) Google Scholar, 13.Ottosen P.D. Madsen K.M. Bode F. et al.Inhibition of protein reabsorption in the renal proximal tubule by basic amino acids.Ren Physiol. 1985; 8: 90-99PubMed Google Scholar, 14.Nielsen A.H. Hermann K.L. Mazanti I. Poulsen K. Urinary excretion of inactive renin during blockade of the renal tubular protein reabsorption with lysine.J Hypertens. 1989; 7: 77-82PubMed Google Scholar primarily based on analysis of the excretion of specific proteins in the urine. The present study characterizes a new model of orally administered L-lysine to inhibit tubular protein uptake. We have characterized the effect of L-lysine treatment on tubular protein handling by comparing, qualitatively and quantitatively, the urinary protein excretion and cellular uptake of selected, endogenous proteins, including albumin, vitamin D-binding protein (DBP), retinol-binding protein (RBP), transferrin, and β2-microglobulin. Urinary protein excretion was characterized by one- and two-dimensional gel electrophoresis, immunoblotting, and radioimunoassay whereas tubular uptake of proteins and receptor expression were analyzed by immunocytochemistry. Futhermore, megalin- and cubilin-expressing yolk sac BN16 cells were exposed to L-lysine and the effects on albumin endocytosis and the expression of endocytic receptors were analyzed. Rats generally tolerated oral L-lysine treatment although one L-lysine-treated rat was excluded after 24 h of L-lysine treatment because of diarrhea. One rat exhibited heavy proteinuria prior to the administration of glycine and was excluded. Oral L-lysine treatment resulted in a significant, sixfold increase in plasma L-lysine concentration compared to water treated rats (Table 1) and caused a significant, 46- and 33-fold increase in urinary L-lysine excretion after 24 and 30 h of treatment, respectively, compared to baseline levels (Table 2). Plasma levels of glycine were increased fivefold in the glycine-treated rats (Table 1) resulting in a significant 12-fold increase in urinary glycine excretion after 24 h of treatment (Table 2). No significant changes in the plasma levels of sodium, potassium, glucose, or creatinine were observed, either in L-lysine- or glycine-treated rats. Glycine treatment caused a significant increase in plasma urea (Table 1) and urinary urea excretion after 24 h of treatment compared to baseline levels (Table 2). L-Lysine treatment induced an increase in urine output from 12.5 to 25 μl/min after 24 h treatment (Table 2). Oral L-lysine treatment did not result in any significant changes in the urinary excretion of sodium, creatinine, or urea (Table 2). A urine glucose dip-stick test was negative in all rats (Table 2). Electron microscopy of the glomerulus and proximal tubule cells showed no ultrastructural changes after L-lysine treatment (Figure 1a and b).Table 1Plasma analysis in rats treated orally for 30 h with L-lysine (n=4), glycine (n=4), or water (n=5)L-Lysine treatmentGlycine treatmentWater treatmentL-Lysine (mM)5.92±1.69aP=0.001 vs glycine treatment and P=0.0003 vs water treatment.1.16±0.230.99±0.21Glycine (mM)0.35±0.032.42±0.50bP=0.0002 vs L-lysine treatment and P=0.00006 vs water treatment.0.49±0.09Creatinine (μM)42.2±3.5937.8±2.3636.8±3.42Urea (mM)7.98±2.6010.20±0.43cP=0.0001 vs water treatment.7.02±0.76Sodium (mM)133±19155±20143±11Potassium (mM)3.83±0.403.71±0.333.68±0.34Glucose (mM)6.94±0.959.24±2.308.63±0.76Analyzed by one-way analysis of variance with post hoc t-test and Bonferroni correction. P<0.05 is considered significant.Values are expressed as mean±s.d.a P=0.001 vs glycine treatment and P=0.0003 vs water treatment.b P=0.0002 vs L-lysine treatment and P=0.00006 vs water treatment.c P=0.0001 vs water treatment. Open table in a new tab Table 2Results of urine analysis in rats before, after 24, and 30 h of oral treatment with L-lysine (n=4) or glycine (n=4)L-lysine treatmentBefore0–24 h24–30 hUrine output (μl/min)12.5±1.625.0±4.2a18.9±2.2L-Lysine (μmol/min)0.07±0.033.23±1.23b2.31±0.88bGlycine (μmol/min)0.50±0.210.26±0.100.20±0.09Albumin (μg/min)0.30±0.181.08±0.15c1.74±0.26dCreatinine (nmol/min)46.0±2.731.4±7.841.1±±11.9Urea (μmol/min)7.8±0.77.0±1.27.9±1.9Sodium (μmol/min)0.80±0.131.41±0.341.04±0.34GlucoseNegativeNegativeNegativeGlycine treatmentBefore0–24 h24–30 hUrine output (μl/min)12.2±4.214.7±4.613.5±5.3L-Lysine (μmol/min)0.06±0.030.08±0.060.03±0.02Glycine (μmol/min)0.45±0.145.61±1.20e2.14±1.37Albumin (μg/min)0.49±0.250.51±0.240.43±0.26Creatinine (nmol/min)48.9±6.344.3±5.558.7±12.3Urea (μmol/min)9.0±1.511.9±2.1f13.2±3.5Sodium (μmol/min)0.94±0.190.84±0.240.96±0.33GlucoseNegativeNegativeNegativeValues are expressed as mean±s.d.P-values vs before treatment: aP=0.009; bP=0.014; cP=0.0001; dP=0.005; eP=0.004; fP=0.006.Analyzed by one-way ANOVA with post hoc paired t-test and Bonferroni's correction. P<0.05 is considered significant. Open table in a new tab Analyzed by one-way analysis of variance with post hoc t-test and Bonferroni correction. P<0.05 is considered significant. Values are expressed as mean±s.d. Values are expressed as mean±s.d. P-values vs before treatment: aP=0.009; bP=0.014; cP=0.0001; dP=0.005; eP=0.004; fP=0.006. Analyzed by one-way ANOVA with post hoc paired t-test and Bonferroni's correction. P<0.05 is considered significant. One-dimensional electrophoresis and silver staining of urine from rats receiving water, glycine, or L-lysine revealed extensive proteinuria in L-lysine-treated rats and several protein bands were identified exclusively in urine from L-lysine-treated rats (Figure 2, lanes 9–12). There was no difference in urinary protein content following glycine treatment (Figure 2, lanes 5–8) compared to water treatment (Figure 2, lanes 1–4). Thus, in the following rats treated with glycine will be referred to as controls. Urine pooled from four control or four L-lysine-treated rats was analyzed by two-dimensional gel electrophoresis (Figure 3). Oral L-lysine treatment induced excretion of a large variety of primarily acidic proteins (Figure 3b) not identified in the control urine (Figure 3a). Immunoblotting allowed the identification of several of these spots as immunoreactive for albumin or DBP (Figure 3b) and thus representing these proteins or fragments thereof. Haptoglobin, hemopexin, and angiotensinogen were identified by mass spectrometry (Figure 3b). None of the latter proteins have previously been identified in L-lysine-induced proteinuria and the presence of haptoglobin was confirmed by non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting showing two bands of approximately 124 and 100 kDa (Figure 4a, lanes 5–8), corresponding to the two forms of this protein secreted by the rat liver.15.Hanley J.M. Haugen T.H. Heath E.C. Biosynthesis and processing of rat haptoglobin.J Biol Chem. 1983; 258: 7858-7869Abstract Full Text PDF PubMed Google Scholar In addition, one-dimensional gel electrophoresis and immunoblotting showed an increased excretion of specific proteins including transferrin (Figure 4b), DBP (Figure 4c), RBP (Figure 4d), and β2-microglobulin (data not shown).Figure 4Non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting of urines from (lanes 1–4) glycine and (lanes 5-8) L-lysine-treated rats. The urine volume loaded from each rat was corrected proportionally to the differences in urinary output from each rat. Increased excretion of (a) haptoglobin, (b) transferrin, (c) vitamin D-binding protein (DBP), and retinol-binding protein (RBP) (d) was observed in L-lysine-treated rats.View Large Image Figure ViewerDownload (PPT) Urinary albumin excretion, quantified by radioimmunoassay, revealed a four- and sixfold increase after 24 and 30 h of L-lysine treatment, respectively (Table 2). No increase in the albumin excretion rate was seen in the control rats when compared to water treated rats. Additional acidic, low molecular weight proteins were found in both control and L-lysine-treated rat urine (Figure 3a and b). These were identified by mass spectrometry as rat urinary protein 1 and 2. The tubular accumulation of albumin, β2-microglobulin, RBP, DBP, and transferrin was examined by immunocytochemistry. While intense proximal tubule vesicular labeling reflecting albumin endocytosis was observed in control rats (Figure 5a), virtually no intracellular albumin labeling was identified in the L-lysine-treated rat (Figure 5b) indicating a strong inhibition of albumin uptake. Only a partial inhibition of proximal tubule accumulation of β2-microglobulin (Figure 5c and d), DBP, and transferrin (data not shown), and no obvious changes in labeling of RBP (Figure 5e and f) were observed despite the documented increase in the urinary excretion of these proteins. Uptake of radiolabeled rat albumin in BN16 cells incubated with L-lysine was significantly reduced by 80% while glycine did not affect albumin uptake (Figure 6a). To test whether the inhibitory effect of L-lysine was reversible cells were preincubated with L-lysine and allowed to recover for 2 h before incubation with 125I-albumin resulting in a complete normalization of albumin endocytosis (Figure 6b). Immunocytochemistry using antibodies against megalin (Figure 7a and b) and cubilin (Figure 7c and d) revealed an increased apical immunoreactivity in proximal tubules from L-lysine-treated rats (Figure 7b and d) compared to controls (Figure 7a and c). In control rats labeling for megalin (Figure 7a) and cubilin (Figure 7c) was localized along the base of the brush border as normally observed in segment S1 rat proximal tubules while in the L-lysine-treated rats a consistent and intense receptor-labeling extending to the tip of the brush border was identified in all proximal tubule profiles (Figure 7b and d). Furthermore, immunoblotting revealed an increased urinary excretion of the cubilin receptor in L-lysine-treated rats (Figure 8a, lanes 5–8) compared to controls (Figure 8a, lanes 1–4). Megalin fragments, but not intact megalin, were identified in the urine of both control and L-lysine-treated rats (data not shown). BN16 cells incubated with L-lysine revealed no change in megalin or cubilin expression levels (Figure 8b) as well as no change in lactate dehydrogenase (LDH) activity in the medium (Figure 8c) when incubated for up to 24 h with low doses of L-lysine (10 mM) comparable to the concentration in plasma of L-lysine-treated rats. At higher concentrations a decrease in megalin and cubilin expression and an increase in LDH activity in the medium were observed after 24 h (Figure 8b and c).Figure 8Effect of L-lysine on receptor expression and urinary excretion. (a) Non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting of urine from (lanes 1–4) glycine and (lanes 5–8) L-lysine-treated rats using anti-cubilin antibodies. L-Lysine treatment resulted in an increased excretion of the cubilin receptor and fragmented cubilin in the urine whereas only weak bands could be detected in control urine. The urine volume loaded from each rat was corrected proportionally to the urinary output. (b) Non-reducing SDS-PAGE and immunoblotting for megalin or cubilin on 5 μg per lane of homogenized BN16 cells incubated for 8 or 24 h with glycine (lane 1: 10 mM, 2: 25 mM, 3: 50 mM) or L-lysine (lane 4: 10 mM, 5: 25 mM, 6: 50 mM). At 8 h no changes in total immunoreactive megalin or cubilin expression was observed in cells incubated with L-lysine as compared to glycine. At 24 h no change was observed at 10 mM L-lysine while at higher concentrations megalin and cubilin expression decreased compared to cells incubated with similar concentrations of glycine. (c) LDH activity in the incubation medium from BN16 cells incubated for 8 or 24 h with 10, 25, or 50 mM of glycine or L-lysine (n=1). Only the high concentrations of L-lysine for 24 h induced a marked increase in LDH-release to the incubation medium.View Large Image Figure ViewerDownload (PPT) The present study characterizes the induction of proteinuria in rats by oral L-lysine treatment. Two-dimensional gel electrophoresis revealed a large number of proteins and protein fragments excreted in excessive amounts in rats treated orally with L-lysine. A number of these proteins were identified by immunoblotting or mass spectrometry including albumin, transferrin, DBP, RBP, β2-microglobulin, haptoglobin, hemopexin, and angiotensinogen, several of which are known ligands to the proximal tubule endocytic receptors megalin or cubilin. Intravenously infused L-lysine has been frequently used in order to inhibit tubular uptake of proteins11.Mogensen C.E. Solling K. Studies on renal tubular protein reabsorption: partial and near complete inhibition by certain amino acids.Scand J Clin Lab Invest. 1977; 37: 477-486Crossref PubMed Scopus (212) Google Scholar, 12.Tencer J. Frick I.M. Oquist B.W. et al.Size-selectivity of the glomerular barrier to high molecular weight proteins: upper size limitations of shunt pathways.Kidney Int. 1998; 53: 709-715Abstract Full Text PDF PubMed Scopus (114) Google Scholar, 13.Ottosen P.D. Madsen K.M. Bode F. et al.Inhibition of protein reabsorption in the renal proximal tubule by basic amino acids.Ren Physiol. 1985; 8: 90-99PubMed Google Scholar, 16.Zager R.A. Johannes G. Tuttle S.E. Sharma H.M. Acute amino acid nephrotoxicity.J Lab Clin Med. 1983; 101: 130-140PubMed Google Scholar and radiolabeled compounds used in experimental cancer therapy.6.Behr T.M. Sharkey R.M. Juweid M.E. et al.Reduction of the renal uptake of radiolabeled monoclonal antibody fragments by cationic amino acids and their derivatives.Cancer Res. 1995; 55: 3825-3834PubMed Google Scholar, 7.Rutherford R.A. Smith A. Waibel R. Schubiger P.A. Differential inhibitory effect of L-lysine on renal accumulation of 67Cu-labelled F(ab')2 fragments in mice.Int J Cancer. 1997; 72: 522-529Crossref PubMed Scopus (11) Google Scholar, 8.Miao Y. Owen N.K. Whitener D. et al.In vivo evaluation of 188Re-labeled alpha-melanocyte stimulating hormone peptide analogs for melanoma therapy.Int J Cancer. 2002; 101: 480-487Crossref PubMed Scopus (87) Google Scholar The use of intravenous infusion requires venous catheterization limiting the time for which this can be applied. In addition, bladder catheterization is often used for urine collection. In our hands, as well as others, this procedure has caused difficulties in the analysis of urinary proteins since minimal damage to the urinary tract epithelial cells may cause leak of interstitial and/or plasma proteins.16.Zager R.A. Johannes G. Tuttle S.E. Sharma H.M. Acute amino acid nephrotoxicity.J Lab Clin Med. 1983; 101: 130-140PubMed Google Scholar In our preliminary studies, using i.v. L-lysine, anesthesia, and bladder catheterization, baseline albumin concentrations prior to treatment were 12-fold higher than in non-catheterized animals most likely due to epithelial damage caused by the catheter. In the present study, one rat was excluded due to diarrhea. Whether this was a result of L-lysine treatment is unresolved, however, there was no evidence of major electrolyte disturbances in the remaining L-lysine treated rats. Oral L-lysine was significantly absorbed to produce a sixfold increase in plasma-L-lysine concentration and proved as efficient as i.v. L-lysine in producing albuminuria causing a sixfold increase in albumin excretion rate after L-lysine treatment similar to previous findings (four- to sixfold increases) when L-lysine was given intravenously.12.Tencer J. Frick I.M. Oquist B.W. et al.Size-selectivity of the glomerular barrier to high molecular weight proteins: upper size limitations of shunt pathways.Kidney Int. 1998; 53: 709-715Abstract Full Text PDF PubMed Scopus (114) Google Scholar, 16.Zager R.A. Johannes G. Tuttle S.E. Sharma H.M. Acute amino acid nephrotoxicity.J Lab Clin Med. 1983; 101: 130-140PubMed Google Scholar No changes in the ultrastructure of the proximal tubule were observed as a result of L-lysine treatment. Also, no changes in renal excretion of sodium and glucose were identified suggesting that tubular function otherwise was unaffected. This was supported by the observation that LDH release into the medium was unchanged when BN16 cells were incubated with comparable concentrations (10 mM) of L-lysine. The effect of L-lysine treatment on urinary albumin excretion has previously been described,11.Mogensen C.E. Solling K. Studies on renal tubular protein reabsorption: partial and near complete inhibition by certain amino acids.Scand J Clin Lab Invest. 1977; 37: 477-486Crossref PubMed Scopus (212) Google Scholar, 12.Tencer J. Frick I.M. Oquist B.W. et al.Size-selectivity of the glomerular barrier to high molecular weight proteins: upper size limitations of shunt pathways.Kidney Int. 1998; 53: 709-715Abstract Full Text PDF PubMed Scopus (114) Google Scholar, 16.Zager R.A. Johannes G. Tuttle S.E. Sharma H.M. Acute amino acid nephrotoxicity.J Lab Clin Med. 1983; 101: 130-140PubMed Google Scholar however, the mechanism for the increased excretion is largely unknown. Based on urinary excretion patterns11.Mogensen C.E. Solling K. Studies on renal tubular protein reabsorption: partial and near complete inhibition by certain amino acids.Scand J Clin Lab Invest. 1977; 37: 477-486Crossref PubMed Scopus (212) Google Scholar and analysis on glomerular structure12.Tencer J. Frick I.M. Oquist B.W. et al.Size-selectivity of the glomerular barrier to high molecular weight proteins: upper size limitations of shunt pathways.Kidney Int. 1998; 53: 709-715Abstract Full Text PDF PubMed Scopus (114) Google Scholar it is generally believed that L-lysine exerts an effect on tubular reabsorption. Analysis of the proximal tubule endocytic uptake of filtered proteins by immunocytochemistry following L-lysine treatment revealed an almost absent labeling for albumin, a reduced labeling for transferrin, DBP, and β2-microglobulin, however, little or no change in accumulation of RBP, suggesting a differential effect on the uptake and degradation of different filtered proteins. Except for angiotensinogen, haptoglobin, and hemopexin all proteins identified as excreted in increased amounts in the urine from L-lysine-treated rats are known ligands to megalin and/or cubilin.17.Christensen E.I. Moskaug J.O. Vorum H. et al.Evidence for an essential role of megalin in transepithelial transport of retinol.J Am Soc Nephrol. 1999; 10: 685-695Crossref PubMed Google Scholar, 18.Birn H. Fyfe J.C. Jacobsen C. et al.Cubilin is an albumin binding protein important for renal tubular albumin reabsorption.J Clin Invest. 2000; 105: 1353-1361Crossref PubMed Scopus (238) Google Scholar, 19.Nykjaer A. Dragun D. Walther D. et al.An endocytic pathway essential for renal uptake and activation of the steroid 25-(OH) vitamin D3.Cell. 1999; 96: 507-515Abstract Full Text Full Text PDF PubMed Scopus (769) Google Scholar, 20.Nykjaer A. Fyfe J.C. Kozyraki R. et al.Cubilin dysfunction causes abnormal metabolism of the steroid hormone 25(OH) vitamin D(3).Proc Natl Acad Sci USA. 2001; 98: 13895-13900Crossref PubMed Scopus (234) Google Scholar, 21.Orlando R.A. Rader K. Authier F. et al.Megalin is an endocytic receptor for insulin.J Am Soc Nephrol. 1998; 9: 1759-1766PubMed Google Scholar, 22.Kozyraki R. Fyfe J. Verroust P.J. et al.Megalin-dependent cubilin-mediated endocytosis is a major pathway for the apical uptake of transferrin in polarized epithelia.Proc Natl Acad Sci USA. 2001; 98: 12491-12496Crossref PubMed Scopus (213) Google Scholar The receptors colocalize in the apical endocytic apparatus of the proximal tubule cell and are responsible for the tubular reabsorption of a variety of filtered proteins.2.Christensen E.I. Birn H. Megalin and cubilin: multifunctional endocytic receptors.Nat Rev Mol Cell Biol. 2002; 3: 256-266Crossref PubMed Scopus (603) Google Scholar Interestingly, the L-lysine-induced proteinuria and decreased tubular accumulation of protein were accompanied by a redistribution of the receptors as indicated by an increase in the brush border receptor immunoreactivity as well as an increased excretion of cubilin to the urine. BN16 cells, known to internalize several of the proteins studied, that is, transferring,22.Kozyraki R. Fyfe J. Verroust P.J. et al.Megalin-dependent cubilin-mediated endocytosis is a major pathway for the apical uptake of transferrin in polarized epithelia.Proc Natl Acad Sci USA. 2001; 98: 12491-12496Crossref PubMed Scopus (213) Google Scholar DBP,20.Nykjaer A. Fyfe J.C. Kozyraki R. et al.Cubilin dysfunction causes abnormal metabolism of the steroid hormone 25(OH) vitamin D(3).Proc Natl Acad Sci USA. 2001; 98: 13895-13900Crossref PubMed Scopus (234) Google Scholar and RBP,17.Christensen E.I. Moskaug J.O. Vorum H. et al.Evidence for an essential role of megalin in transepithelial transport of retinol.J Am Soc Nephrol. 1999; 10: 685-695Crossref PubMed Google Scholar by a megalin and cubilin-mediated mechanism, revealed a very efficient L-lysine-induced inhibition of albumin endocytosis as in the in vivo model, but showed no change in total immunoreactive receptor protein levels. This suggests that the luminal accumulation of receptors observed in vivo is the result of altered trafficking rather than a change in total protein levels. Cubilin, having no transmembrane domain, is loosely associated with the plasma membrane and may be released by nonenzymatic and nonsolubilizing procedures.23.Moestrup S.K. Kozyraki R. Kristiansen M. et al.The intrinsic factor-vitamin B12 receptor and target of teratogenic antibodies is a megalin-binding peripheral membrane protein with homology to developmental proteins.J Biol Chem. 1998; 273: 5235-5242Crossref PubMed Scopus (210) Google Scholar It is possible that L-lysine causes a similar release of receptor protein. In addition to effects on receptor trafficking L-lysine may also directly inhibit binding of filtered proteins to ligand binding areas on the rece
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