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Am J Physiol Renal Physiol 295: F82-F90, 2008. First published April 30, 2008; doi:10.1152/ajprenal.00091.2008
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Silencing megalin and cubilin genes inhibits myeloma light chain endocytosis and ameliorates toxicity in human renal proximal tubule epithelial cells

Min Li, Saravanan Balamuthusamy, Eric E. Simon, and Vecihi Batuman

Section of Nephrology and Hypertension, Department of Medicine, Tulane University School of Medicine, New Orleans, Louisiana

Submitted 21 February 2008 ; accepted in final form 26 April 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Using target-specific short interfering (si) RNAs, we silenced the tandem endocytic receptors megalin and cubilin genes in cultured human renal proximal tubule epithelial cells. Transfection by siRNA resulted in up to 90% suppression of both megalin and cubilin protein and mRNA expression. In HK-2 cells exposed to {kappa}-light chain for up to 24 h, light chain endocytosis was reduced in either megalin- or cubilin-silenced cells markedly but incompletely. Simultaneous silencing of both the cubilin and megalin genes, however, resulted in near-complete inhibition of light chain endocytosis, as determined by measuring {kappa}-light chain protein concentration in cell cytoplasm and by flow cytometry using FITC-labeled {kappa}-light chain. In these cells, light chain-induced cytokine responses (interleukin-6 and monocyte chemoattractant protein-1) and epithelial-to-mesenchymal transition as well as the associated cellular and morphological alterations were also markedly suppressed. The results demonstrate that light chain endocytosis is predominantly mediated by the megalin-cubilin tandem endocytic receptor and identify endocytosis as a key step in light chain cytotoxicity. Blocking light chain endocytosis prevents its nephrotoxic effects on human kidney proximal tubule cells.

myeloma kidney; RNA interference; nephrotoxicity; flow cytometry; receptor-mediated endocytosis

IMMUNOGLOBULIN LIGHT CHAINS (LCs) are normally filtered relatively unhindered in the glomerulus, are endocytosed in proximal tubule cells, and enter the endosomal-lysosomal path for catabolism (1). We previously demonstrated that LC endocytosis in renal proximal tubule cells is a receptor-mediated process (2, 3, 11) and that the recently characterized multiligand endocytic receptor system megalin/cubilin plays a key role in this process (6, 16, 20, 28).

We have previously observed that excessive myeloma LC endocytosis induces inflammatory and proinflammatory cytokines in human renal proximal tubule epithelial cells (PTECs), identifying novel mechanisms of kidney injury in multiple myeloma. These responses were mediated by activation of nuclear factor-{kappa}B (NF-{kappa}B) and signaled through MAPKs, particularly p38 MAPK (1, 24, 25). The inhibitors of NF-{kappa}B, and MAPKs, especially p38 MAPK, markedly prevented LC-induced inflammatory responses in PTECs, confirming the pivotal role of these transcription factors and signaling pathways. We also noted in earlier studies that blocking LC endocytosis by maneuvers that interfere either with endosomal acidification (i.e., bafilomycin) or by clathrin lattice formation (i.e., by using hypertonic sucrose) had prevented LC-induced cytokine responses (1, 24, 25).

To further elucidate the role of endocytosis in LC-mediated cytotoxic phenomena, we evaluated the effect of silencing the megalin and cubilin genes, separately and simultaneously, on LC endocytosis and LC-induced cytotoxicity. In the experiments presented here, we used the immortalized human HK-2 proximal tubular cell line to test the hypothesis that silencing endocytic receptor genes inhibits LC endocytosis and that this in turn ameliorates LC cytotoxicity, LC-induced cytokines, and epithelial-mesenchymal transformation of LC-exposed human PTECs (18). The studies presented here demonstrate that there is a significant but less than complete inhibitory effect of individually silencing the megalin or cubilin gene on both the endocytosis of LC and its toxic effects, while simultaneous suppression of both megalin and cubilin has a near-total inhibitory effect.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
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Cell cultures. HK-2 immortalized human renal PTECs were purchased from the American Type Culture Collection (Manassas, VA) and were maintained in GIBCO Keratinocyte-Serum Free Medium supplemented with 5 ng/ml recombinant epidermal growth factor and 0.05 mg/ml bovine pituitary extract (Invitrogen, Carlsbad, CA). Grown as a monolayer, they showed the marker brush-border enzymes and biochemical and morphological characteristics similar to other widely used proximal tubule cells in stable culture. The cells were routinely cultured at 37°C in a humidified atmosphere of 95% air-5% CO2 and nourished at intervals of 3–4 days and were not passaged forward beyond about 20–25 passages.

Isolation and purification of LCs. The LCs used in these experiments were isolated and purified from the urine of multiple myeloma patients with myeloma kidney, as described previously (6, 22). Only urine with minimal or no albuminuria was selected, excluding patients with significant glomerulopathy. Thus, because they are obtained from patients without albuminuria, all the LCs used here must be considered "tubulopathic." The LCs used in the present study are previously shown to undergo endocytosis by PTECs, bind to cubilin and megalin, and induce epithelial-mesenchymal transition (EMT) and cytokine production through phosphorylation of MAPKs and activation of NF-{kappa}B (1–3, 6, 16, 18, 24, 25). Furthermore, the LC preparations were endotoxin free and did not contain detectable quantities of IL-1, IL-6, IL-8, MCP-1, or TNF-{alpha} (24, 25).

RNA interference of megalin and cubilin and LC treatment. Human megalin siRNA and cubilin siRNA consisted of three target-specific 20- to 25-nt siRNAs, and the siRNA negative control that contained a scrambled sequence were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). HK-2 cells, 2 x 105, in log phase were plated into 12-well tissue culture plates with normal growth medium ~24 h before transfection or when the cells reached 60–80% confluence. For each transfection well, the siRNA-transfection reagent complexes were overlaid onto the washed cells. Cells were incubated in normal cell culture conditions for 6 h, and then fresh normal growth medium was added to each well to maximize cell growth and prevent potential cytotoxicity. After 24 h of transfection, assays for target gene expression by Western blot and RT-PCR were performed. LC concentration (50 µM) was prepared from a stock solution (1 mM) as described previously (24, 25). Cell viability was measured by CellTiter 96 Aqueous One solution Cell Proliferation Assay (Promega, Madison, WI). To evaluate the role of LC endocytosis and to determine whether silencing the tandem scavenger receptor system cubilin/megalin by RNA interference inhibits the expression of cytokines, and protect cells from undergoing EMT, we used HK-2 cells cultured in serum-free fresh media, in the presence or absence of megalin and/or cubilin siRNA transfection. After exposure to {kappa}-LC for 4–6 or 24 h, culture supernatants were harvested and cells were lysed and stored at –70°C for cytokine assays and for determination of markers of EMT.

Western blot analysis. Protein extracts (20 µg) were boiled for 3 min in Laemmli sample buffer, then separated by 4–15% SDS-PAGE for megalin and cubilin, and 12.5% SDS-PAGE for E-cadherin and {alpha}-smooth muscle actin (SMA), and the separated proteins were electrophoretically transferred onto polyvinylidene difluoride or nitrocellulose membranes, respectively. After blotting, the membranes were blocked by 5% skim milk and subsequently were incubated with goat anti-human megalin (C-19) or cubilin (Y-20) antibodies (Santa Cruz Biotechnology), diluted at 1:1,000 overnight at 4°C or for 1 h at room temperature with an appropriate dilution of the primary antibody (1:500, monoclonal anti-E-cadherin, G-10, Santa Cruz Biotechnology), 4.2 µg/ml monoclonal anti-actin ({alpha}- SMA, Clone 1A4, Sigma, St. Louis, MO), or 0.9 µg/ml monoclonal anti-actin (clone AC-40, Sigma). After four washes in Tris-buffered saline-0.1% Tween 20 buffer, the membrane was incubated for 1 h at room temperature with horseradish peroxidase-conjugated donkey anti-goat immunoglobulin (Santa Cruz Biotechnology) or sheep anti-mouse immunoglobulin (GE Healthcare, Piscataway, NJ), respectively, and the antibody complexes were visualized by the Amersham ECL Plus Detection Reagents (GE Healthcare) as directed by the manufacturer. The protein expression levels were semiquantified using a Kodak Digital Molecular Image Station 4000MM with MI analysis program (Carestream Health, Rochester, NY).

Real-time quantitative RT-PCR. Total RNA was purified from HK-2 cells by extraction with the RNeasy Mini kit (Qiagen, Valencia, CA), and 50 ng from each sample was used for real-time quantitative RT-PCR analysis using Stratagene Brilliant II SYBR Green QRT-PCR reagents (Agilent Technologies, La Jolla, CA). The primers for megalin and cubilin were obtained from Santa Cruz Biotechnology, and the pairs of primers used for amplification of human E-Cadherin and {alpha}-SMA were designed with the MacVector program (MacVector, Cary, NC) based on reported sequences. The fluorescent product was detected during annealing/extension periods, and dissociation (melting curve) analysis was used to confirm the specificity of the amplification products. The quantification data were analyzed using Stratagene MxPro QPCR software and expressed as baseline subtracted fluorescent reading normalized to the reference dye (dRn).

ELISA for {kappa}-LC, IL-6, MCP-1, and soluble E-cadherin. The intracellular concentration of endocytosed {kappa}-LC in human renal PTECs was determined using a newly developed sandwich ELISA with monoclonal antibodies specific for human free {kappa}-LCs and Bence-Jones {kappa}-LCs; it is nonreactive with {lambda}-LCs. Before proceeding with the ELISA, the intracellular proteins, which were kept at –70°C after extensive washing and extraction from the HK-2 cells, were mixed thoroughly and protein concentrations were quantified by using Bio-Rad DC protein assay reagents (Bio-Rad, Hercules, CA). The levels of human IL-6, MCP-1, and soluble E-cadherin in the media of HK-2 cell cultures, in the presence or absence of siRNA transfections, were measured using sensitive immunometric human ELISA kits (Quantikine, R&D Systems, Minneapolis, MN).

Flow cytometric analysis of LC endocytosis. To confirm the efficacy of the megalin and cubilin siRNA-mediated gene silencing on inhibiting LC endocytosis in HK-2 cells, {kappa}-LCs were FITC-conjugated using a FluoroTag FITC Conjugation Kit (Sigma). From the absorbance readings of the conjugate samples, the ratio of fluorescein to LC protein between 0.85 and 1.0 was obtained, which proved satisfactory for flow cytometric studies. The confluent monolayers were washed with serum-free medium and allowed to equilibrate for 2 h at 37°C. The cells were then transfected with human megalin or cubilin siRNA for 1 day before exposure to 50 µM FITC-labeled {kappa}-LCs as described above. The cells were washed several times with PBS (–), trypsinized, and suspended in PBS. Fluorescence was detected by using a Beckman Coulter FC500 Cytomics with CXP software. The positive control was FITC-labeled {kappa}-LCs without siRNA transfection; the negative control was unlabeled {kappa}-LCs.

Materials. All drugs were in saline. Unless otherwise specified, all chemicals were obtained from Sigma.

Statistical analyses. All experiments were repeated at least three times, and the experimental condition was repeated in quadruplicate wells for each experiment. The density of each band for the protein or transcripts was digitized and defined in terms of pixels (PSL), which is the total pixel minus the background. For each sample, the signal strength of the protein or transcript was normalized for the corresponding actin signal. The results were expressed as means ± SE. Multiple comparisons were made with an ANOVA and Tukey-Kramer post hoc tests (InStat, GraphPad, San Diego, CA). Statistical analyses, curve fitting, and calculations were done using GraphPad Prism 4.0c. Statistical significance was defined as P < 0.05 for all analyses.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
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Megalin and cubilin gene-specific siRNA transfection and their expression. HK-2 cells were transfected with megalin siRNA, cubilin siRNA, or negative control siRNA. After 24 h, cells were exposed to 50 µM {kappa}-LCs for up to 24 h. No significant changes in megalin and cubilin mRNA expression were observed in cells transfected with scrambled (control) siRNA compared with the untransfected control cells. However, the scrambled siRNA diminished cubilin protein expression slightly (Figs. 1 and 2). Both the untreated control cells and the LC-exposed cells showed similar receptor expression level. Transfection by megalin or cubilin siRNA suppressed their corresponding protein expression (Fig. 1A). The silencing of the cubilin gene did not affect megalin expression. However, cubilin expression in cells transfected with megalin siRNA was reduced to the same level as the transfection of cubilin siRNA alone (Fig. 1A). The combination of megalin and cubilin siRNAs almost completely knocked down both megalin and cubilin expression (Figs. 1 and 2). Transfection of HK-2 cells with specific megalin siRNA reduced the levels of both megalin (to 22.8 ± 4.5%) and cubilin (to 17.2 ± 2.3%) of control levels (Fig. 1B). With cubilin siRNA, there was a significant decrease in cubilin to 14.6 ± 3.3% of control with no change in the levels of megalin (Fig. 1B). Consistent with this, the real-time quantitative RT-PCR results showed that megalin siRNA greatly reduced the mRNA of both megalin and cubilin (Fig. 2).


Figure 1
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  		Fig. 1. Western blots of megalin and cubilin in short interfering RNA (siRNA)-transfected human renal proximal tubule epithelial cells (PTECs). A: transfection with 50-pmol megalin siRNA or 100-pmol megalin plus cubilin siRNAs resulted in decreased expression of both megalin (600 kDa) and cubilin (460 kDa). Transfection with 50-pmol cubilin siRNA alone had significant suppressive effect (gene silencing) in cubilin expression at 24 h, but no change in megalin expression. B: graphic representation of Western blots demonstrates the effect of megalin and/or cubilin siRNA on their protein expression in HK-2 cells. Each value normalized to β-actin represents the mean ± SE of 4 separate experiments. **P < 0.01 and *P < 0.05 vs. corresponding "None" control.

 

Figure 2
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  		Fig. 2. Effect of megalin and/or cubilin siRNA transfection on the expression of megalin and cubilin transcripts in human renal PTECs. HK-2 cells were transfected with human megalin siRNA or cubilin siRNA for 24 h. Transfection with megalin siRNA (50 pmol) or megalin plus cubilin siRNAs (100 pmol) effectively silenced or successfully suppressed both megalin and cubilin mRNA expression. In contrast, 50 pmol of cubilin-specific siRNAs blocked cubilin mRNA expression only, and megalin levels were not different from untransfected control. **P < 0.01 vs. None (n = 4).

 
Effect of silencing megalin and cubilin genes on LC endocytosis. To determine whether silencing the megalin and cubilin genes impedes LC endocytosis, we tracked internalization of FITC-conjugated LC in human renal PTECs transfected with megalin and cubilin siRNAs individually and simultaneously. In control and mock-transfected cells after 4 h exposure, FITC-{kappa}-LC (50 µM) is taken up into endocytic vesicles that can be readily quantitated by a 530-nm laser beam by measuring fluorescence intensity in FITC-positive cells (Fig. 3A). In either megalin or cubilin knockdown cells, the uptake of FITC-{kappa}-LC was inhibited by ~80% (P < 0.001) compared with control cells (Fig. 3, A and B). In contrast, in cells depleted of megalin and cubilin simultaneously, almost no punctate FITC fluorescence was observed, indicative of virtually complete inhibition of endocytosis (Fig. 3, A and B). Figure 3C shows the effect of silencing megalin and cubilin individually on intracellular {kappa}-LC protein concentrations measured by ELISA in HK-2 cells exposed to 50 µM {kappa}-LCs. Uptake of {kappa}-LC by HK-2 cells is inhibited after transfection by either megalin siRNA or cubilin siRNA. When either megalin or cubilin was silenced, there were significant decreases in LC uptake by 54.6 or 42.8%, respectively, compared with untransfected LC-exposed cells (Fig. 3C). The largest inhibition of endocytosis of {kappa}-LC (84.5%) was caused by simultaneous knockdown of both megalin and cubilin. Thus both methodologies, i.e., flow cytometry and determination of intracellular LC protein, are in close agreement and confirm that silencing the megalin and cubilin genes inhibits LC endocytosis.


Figure 3
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  		Fig. 3. Effect of silencing megalin and/or cubilin on light chain (LC) endocytosis and cytotoxicity in human renal PTECs. A: flow cytometric analysis of megalin/cubilin gene silencing on uptake of fluorescently labeled {kappa}-LC by HK-2 cells. Histograms represent the distributions of events for FITC-LC (n = 6). B: FITC-{kappa}-LC endocytosis by HK-2 cells is significantly but partially inhibited in cells transfected with megalin or cubilin siRNA, whereas in cells cotransfected with megalin and cubilin siRNAs FITC-LC endocytosis is almost completely inhibited. C: after transfection with human megalin (50 pmol) and/or cubilin (50 pmol) siRNAs, the intracellular {kappa}-LC concentrations in LC-exposed HK-2 cells were determined by ELISA. **P < 0.01 vs. {kappa}-LC treatment alone (n = 6).

 
Effect of silencing megalin and cubilin genes on LC-induced cytokine production and cytotoxicity. As we have previously observed (1, 24, 25), upregulation of IL-6 and MCP-1 production was again observed in the HK-2 cells exposed to human {kappa}-LC for 6 h (Fig. 4, A and B). The cytokine levels were still elevated after nontargeting siRNA (negative control) transfection. In contrast, in the cells transfected with either megalin or cubilin siRNA the IL-6 and MCP-1 upregulations were inhibited significantly by 30–70% (Fig. 4, A and B), whereas IL-6 secretion appeared completely inhibited down to baseline levels after combined transfection with megalin siRNA and cubilin siRNA (Fig. 4A).


Figure 4
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  		Fig. 4. Silencing megalin and cubilin genes inhibits cytokine production and ameliorates LC cytotoxicity in human renal PTECs. Effect of {kappa}-LC on IL-6 (A) and MCP-1 (B) production in human megalin and/or cubilin siRNA-transfected HK-2 cells is shown. Megalin and cubilin siRNA transfections significantly blocked both IL-6 and MCP-1 secretion. Blockade of megalin/cubilin on the effect of cell morphology (C) and LC cytotoxicity assay (D) in HK-2 cells is also shown. The number of viable cells in culture exposed to {kappa}-LC after siRNA transfection was similar to the untreated "None" control. Cells were visualized using a charge-coupled Nikon Coolpix 995 digital CCD camera attached to a Nikon Diaphot inverted phase-contrast microscope with x10/0.30 numeric aperture objective. Each value represents the mean ± SE of 3–4 separate experiments. **P < 0.01 and *P < 0.05 vs. 50 µM {kappa}-LC-exposed control cells.

 
Exposure to 50 µM {kappa}-LC induced morphological alterations in cultured human renal HK-2 cells that included detachment of the cells from the culture plate and aggregation and necrosis of the cells (data not shown). The attached cells showed gross elongation, with filopodia formation (Fig. 4C). Silencing megalin and cubilin genes prevented these morphological changes with preservation of the appearance of a normal epithelial phenotype (Fig. 4C). We determined cell viability by MTS Cell Proliferation/Cytotoxicity Assay and noted preservation of cells in culture (significant reduction in the number of injured tubule cells) after transfection with megalin and cubilin siRNAs separately or in combination (Fig. 4D).

Effect of silencing megalin and cubilin genes on LC-induced EMT. We recently demonstrated that LC is a direct stimulus for EMT in human renal PTECs (18). We observed that the expression of E-cadherin (a marker for the epithelial phenotype) decreased, while the expression of {alpha}-SMA (a marker for the myofibroblast phenotype) increased in HK-2 cells exposed to {kappa}-LCs for 48 h, showing that these cells lost their epithelial characteristics and acquired mesenchymal cell properties (Fig. 5 A). Densitometric analyses of the Western blots revealed that the transfection by siRNAs abrogated the expected decrease in E-cadherin expression following exposure to LCs (Fig. 5B). Similarly, the expected rise in the levels of {alpha}-SMA after exposure to {kappa}-LC was also prevented and remained at basal levels after silencing of the megalin and/or cubilin genes. Furthermore, silencing the megalin and cubilin genes prevented release of human soluble E-cadherin into cell culture supernates, paralleling the preservation of E-cadherin at basal levels despite exposure to {kappa}-LC (Fig. 5C). At the transcriptional level, after {kappa}-LC-exposed cells were transfected with either megalin or cubilin siRNA to block receptor-mediated endocytosis, we observed that E-cadherin and {alpha}-SMA mRNA expression remained unchanged from basal levels (Fig. 6), compared with LC control, indicating that the cells preserved their epithelial phenotype despite exposure to LC.


Figure 5
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  		Fig. 5. Effect of megalin and/or cubilin interference on the expression of E-cadherin and {alpha}-smooth muscle actin (SMA) in LC-exposed human renal PTECs. A: Western blot analysis of E-cadherin and {alpha}-SMA expressions in LC-treated HK-2 cells transfected with megalin and/or cubilin siRNAs. Cells exposed to 50 µM {kappa}-LC for 48 h exhibited significant depression in the expression of the epithelial cell marker E-cadherin and a significant increase in the mesenchymal marker {alpha}-SMA. B: transfection by megalin and/or cubilin siRNA achieved preservation of the expression of E-cadherin or {alpha}-SMA (somewhat less on E-cadherin after cubilin siRNA transfection only). C: immunoassay of soluble E-cadherin released from HK-2 cells exposed to 50 µM {kappa}-LC at 48 h showed significant increase in the level of soluble E-cadherin in culture medium, a characteristic event during the loss of the epithelial cell phenotype. Transfection of HK-2 cells with megalin and cubilin siRNAs for 1 day before {kappa}-LC exposure and subsequent cotreatment prevented secretion of soluble E-cadherin to medium. Each value represents the mean ± SE of 3 replicates/group. **P < 0.01 and *P < 0.05 vs. corresponding 50 µM {kappa}-LC alone.

 

Figure 6
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  		Fig. 6. Real-time RT-PCR analysis of E-cadherin and {alpha}-SMA expression after megalin and/or cubilin siRNA transfection in LC-exposed human renal PTECs. Graphic presentation of real-time semiquantitative RT-PCR demonstrates suppression of E-cadherin and {alpha}-SMA induction in HK-2 cells exposed to 50 µM {kappa}-LC. The {alpha}-SMA induction at 24 h coincided with the loss of E-cadherin and was prevented by the blockage of megalin and/or cubilin expression. **P < 0.01 vs. 50 µM {kappa}-LC-exposed control cells (n = 4).

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
GRANTS
 REFERENCES
 
Kidney involvement in multiple myeloma is a serious complication affecting up to 50% of patients with myeloma and is associated with a much worse prognosis (1, 7, 17, 19). Direct toxicity by LCs on kidney cells, especially on proximal tubule cells, appears to be an important mechanism of kidney injury in multiple myeloma (1, 4, 5, 13, 22). Most of the kidney abnormalities seen with myeloma are associated with LC proteinuria and increased LC production, confirming previous studies from our laboratory and others that increased endocytosis and protein overloading might be important in LC nephrotoxicity (25, 30).

The tandem endocytic receptors megalin and cubilin play an important role in LC endocytosis. We have previously demonstrated that multiple species of LCs, including both the {kappa}- and {lambda}-subtypes, are ligands for either megalin or cubilin; some preferentially bind to megalin while others to cubilin (6, 16). These receptors are giant glycoproteins related to the low-density lipoprotein receptor (LDLR) superfamily, and only megalin (also designated GP330) expressed on the apical membrane domain of epithelial cells has a transmembrane domain capable of internalizing its ligand directly. In contrast, cubilin, although anchored in the plasma membrane, lacks a cytosolic domain to initiate endocytic signaling. Ligands that bind to cubilin are endocytosed in conjunction with megalin, which acts as a chaperone for cubilin while also internalizing its own ligands (8, 28). We previously have demonstrated that neutralizing antibodies directed against both megalin and cubilin were capable of blocking endocytosis of LCs, indicating the importance of this receptor system in LC endocytosis (6, 16). In the present study, we show that silencing megalin or cubilin individually has a lesser effect than silencing both. Megalin siRNA knockdown also had a dramatic effect on cubilin protein levels (to the same degree as knocking down cubilin itself). Because knockdown of either cubilin or megalin/cubilin had the same effect on LC endocytosis and cytokine production, this also suggested that megalin may only function to tether cubilin to membranes for endocytosis of cubilin-bound ligands, such as vitamin B12, apolipoprotein A1, albumin, and LC.

Our previous observations that inhibiting LC endocytosis by either blocking endosomal acidification by bafilomycin or interfering with the clathrin-coated pathway by adding hypertonic sucrose in the medium markedly inhibited LC-induced inflammatory responses (1, 3, 24, 25) suggested that excessive endocytosis is a prerequisite for LC cytotoxicity. The current studies were designed to evaluate directly the role of megalin/cubilin in LC endocytosis and LC-induced cytotoxic phenomena in human PTECs. The combined knockdown of megalin/cubilin has a more prominent effect on E-cadherin levels than does cubilin alone. This further supports a connection between endocytosis and EMT, because compared with cubilin knockdown alone, combined megalin/cubilin knockdown was much more effective in blocking LC endocytosis. However, there may be additional consequences of combined megalin/cubilin knockdown, including a possible contribution to EMT beyond blocking LC endocytosis.

Before proceeding with endocytosis experiments, we first evaluated the effect of the target-specific megalin and cubilin siRNAs on suppressing the protein and mRNA levels for these glycoproteins by Western blotting and real-time quantitative RT-PCR. After 50-pmol siRNA treatment for 24 h, both megalin and cubilin protein and mRNA levels were markedly suppressed. Interestingly, our result also showed that megalin knockdown not only decreased cubilin protein levels but also had a dramatic effect on cubilin mRNA. Megalin, characterized by a cluster of cysteine-rich class A repeats, EGF-like repeats, and YMTD repeats, specifically binds to cubilin in the NH2-terminal region that contains the EGF-like repeats and CUB domains 1 and 2. Human megalin siRNA is a pool of three 20- to 25-nt siRNAs designed to knock down their gene expression that may contain target-nonspecific nucleotides (e.g., encoding for EGF repeats), which could be responsible for the inhibition of cubilin mRNA expression. Thus it is conceivable that there may be a feedback link between megalin and cubilin messages and that the knockdown of megalin also decreases cubilin expression levels. Equally possible is that megalin siRNA might have additional nonspecific effects influencing cubilin expression.

Next, we studied the endocytosis of LCs in the transfected cells and monitored the uptake of {kappa}-LC by determining the LC concentration in the cytosolic extracts of cells. The siRNA transfection studies show that the tandem endocytic receptor megalin/cubilin is responsible for LC endocytosis. In the megalin- and cubilin-specific, siRNA-transfected proximal tubule cells, LC uptake was inhibited by ~85%, underscoring the role of this receptor system in LC endocytosis. That the inhibition of endocytosis of LCs was <100% in megalin/cubilin-silenced cells also suggests that a small but significant amount of LC internalization can occur via a megalin/cubilin-independent pathway. This is consistent with our previous observations tracking LC endocytosis by flow cytometry in the presence and absence of neutralizing megalin and cubilin antibodies (6, 16).

The cubilin/megalin tandem endocytic receptor belongs to a unique class of giant glycoprotein receptors related to the LDL receptor superfamily and can bind multiple ligands (8, 29). Many of the low-molecular-weight proteins that are relatively unhindered in the glomerulus are endocytosed after binding to cubilin/megalin. Among these proteins are {alpha}1- and β2-microglobulin, cytochrome c, retinol binding protein, peptide hormones, such as insulin, parathyroid hormone, etc., and {kappa}- and {lambda}-LCs (1, 6, 8, 16, 29). Megalin deficiency, as seen in selective knockout models and in CLCN5-deficiency (Dent's disease), and cubilin deficiency, as seen in Imerslund-Grasbeck syndrome (12, 15), have generally been associated with low-molecular-weight proteinuria in addition to a variety of other disorders such as vitamin B12 deficiency, thyroid deficiency, etc. (9, 10, 12, 14, 21, 23, 26). All of these disorders confirm the role of the tandem megalin/cubilin endocytic receptor system in proximal tubule endocytosis of filtered proteins that include the relatively positively charged smaller than albumin proteins such as LCs, β2-microglobulin, retinol binding protein, but also significant quantities of albumin (9, 14).

Our results here clearly show that gene knockdown of megalin/cubilin protects kidney cells from toxic effects of excessive LCs and that LC toxicity may be directly related to endocytosis. When the cells are transfected with either megalin or cubilin siRNA, the production of IL-6 and MCP-1 induced by {kappa}-LC were inhibited significantly but incompletely, whereas the IL-6 secretion appeared completely inhibited down to baseline levels after combined transfection with megalin siRNA and cubilin siRNA. This suggested that the inflammatory and cell-toxic effects of excessive filtered LCs are predominantly, but not completely related to endocytosis. Some endocytosis-independent processes between the inflammatory response and LCs cannot be excluded. Diwakar et al. (10) found in studies of albumin that showed albumin-mediated TGF-β1 secretion in proximal tubule cells was independent of endocytosis. Similarly, Thelig et al. (27) did not find that abrogation of protein endocytosis in megalin-deficient mice protects from tubulointerstitial disease in a model of anti-glomerular basement membrane, serum-mediated glomerulonephritis characterized by heavy proteinuria. The apparent discrepancy can be explained by the differences between albumin and LCs, and the characteristics of the kidney disease model used in the study by Thelig et al. In head-to-head comparisons, we generally found albumin much less toxic than LCs on human PTEC, and the amount of albumin used in the studies showing inflammatory effects on PTEC is generally much higher (up to 5–10 vs. 0.25–1 mg/ml LC) (24, 25). Additionally, some intrinsic differences between albumin and LC cannot be excluded.

In the case of anti-glomerular basement membrane, serum-induced glomerulonephritis, the predominant pathophysiology is glomerular injury, inflammation, and release of multiple inflammatory factors from glomerular cells, factors independent of and beyond "protein overloading." This complex set of events is likely to cause and mediate tubulointerstitial injury independently of protein reabsorption in the kidney, and it is not surprising that megalin deficiency was not protective. In contrast, in multiple myeloma, the tubulointerstitial disease appears to be mediated predominantly through LC-PTEC interaction and excessive endocytosis. Our earlier studies with pharmacological inhibitors of endocytosis (1, 24, 25) and the current experiments in which endocytosis is abrogated through gene knockdown of megalin and cubilin resulting in near-complete abrogation of cytokine responses and EMT demonstrate that endocytosis is a required step in LC-mediated inflammatory effects.

The kidney abnormalities in myeloma are almost always associated with LC proteinuria as a result of "overflow proteinuria" because of excessive LC production by the malignant clone of plasma cells. This leads to presentation of large quantities of LC to the proximal tubule and forces excessive endocytosis, which could trigger cell stress responses, such as phosphorylation of MAPKs, activation of nuclear transcription factors, and induction of inflammatory and proinflammatory cytokines (24, 25). Although our earlier studies with maneuvers that interfere with endocytosis provided support for this sequence of events (24, 25), we had not directly demonstrated that decreased or blocked endocytosis counters these inflammatory responses. Near-total knockdown of megalin and cubilin using siRNA provided the opportunity to examine in detail the effect of blocking LC endocytosis on LC cytotoxicity.

In conclusion, the experiments presented here demonstrate that silencing the megalin and cubilin genes results in marked inhibition of LC endocytosis and this in turn prevents the LC-induced cytokine responses, as well as LC-induced EMT. Thus we present here the most direct evidence linking endocytosis to cytotoxicity. These studies identify inhibition of endocytosis as a possible therapeutic strategy against renal disorders mediated by protein overloading.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was funded in part by research grants from the Veterans Administration (Merit Review Award) and the Louisiana Cancer Research Consortium (Cancer Research Pilot Incentive Award).


   ACKNOWLEDGMENTS
 
The authors thank Wei Cai and Christina Primo for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: V. Batuman, Section of Nephrology and Hypertension, Dept. of Medicine, Tulane Univ. School of Medicine, 1430 Tulane Ave., SL-45, New Orleans, LA 70112-2632

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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