Laboratory Investigation (2004) 84, 440–451 & 2004 USCAP, Inc All rights reserved 0023-6837/04 $25.00 www.laboratoryinvestigation.org Different types of glomerulopathic light chains interact with mesangial cells using a common receptor but exhibit different intracellular trafficking patterns Jiamin Teng1, William J. Russell1, Xin Gu1, James Cardelli2, M Lamar Jones1 and Guillermo A. Herrera1,3,4 1Department of Pathology; 2Department of Microbiology; 3Department of Cell Biology and Anatomy and 4Department of Medicine, Louisiana State University Health Sciences Center, Shreveport, LA, USA Patients with plasma cell dyscrasias may have circulating light chains (LCs), some of which are nephrotoxic. Nephrotoxic LCs can affect the various renal compartments. Some of these LCs may produce predominantly proximal tubular damage, while others are associated with distal nephron obstruction (the so-called ‘myeloma kidney’). Both these are considered tubulopathic (T) LCs. A receptor has been found in proximal tubular cells (cubilin/megalin complex), which mediates the absorption of LCs and is involved in the pathogenesis of tubulopathies that occurs in these patients. Another group of nephrotoxic LCs is associated with glomerular damage and these are considered as glomerulopathic (G). These patients with G-LCs may develop AL- amyloidosis (AL-Am) or LC deposition disease (LCDD). Recent evidence indicates that the physicochemical characteristics (amino-acid composition and conformation of the variable region) of a given nephrotoxic LC may be the most significant factor in determining the type and location of renal damage within the nephron. Other factors may also be involved, including yet uncharacterized host factors that may include genetic polymorphism, among others. Interestingly, the amount of LC production by the clone of plasma cells does not correlate directly with the severity of the renal alterations. Understanding the nature of the interactions between G-LCs and mesangial cells (MCs) is crucial to define key steps that may be targeted for therapeutic purposes. Experimental studies have delineated important aspects pertaining to interactions between G-LCs and MCs, indicating that these interactions are receptor mediated. The data presented in the current study support a single receptor present on MCs for both LCDD and AL-LCs, as clearly demonstrated with competition and colocalization immunofluorescence (IF) studies. This receptor resides in caveolae present on the plasma membrane of HMCs and is overexpressed when HMCs are incubated with G-LCs but not TLCs. Caveolae play a fundamental role in receptor-mediated endocytosis, a crucial process in the internalization of AL-LCs and amyloidogenesis. LC internalization is clathrin mediated. The data also indicate that intracellular trafficking in MCs is different for AL-LCs and LCDD-LCs. AL-LCs are delivered to the mature lysosomal compartment where amyloid formation occurs. LCDD-LCs alter mesangial function and phenotype by interacting with the MC surface membranes through similar receptors as the AL-LCs. The data also demonstrated that cubilin and megalin were absent on MCs, so the receptor involved is different from the one already characterized in the proximal tubules. Laboratory Investigation (2004) 84, 440–451, advance online publication, 1 March 2004; doi:10.1038/labinvest.3700069 Keywords: myeloma; light chains; plasma cell dyscrasia; mesangial cells; light-chain deposition disease; AL-amyloidosis; receptor While the majority of the nephrotoxic light chains 30% are primarily associated with glomerular (LCs) produce tubular damage, approximately pathology. Binding data relating to the tubular receptor for LCs have suggested that cubilin first binds to megalin on cell membranes, where Correspondence: GA Herrera, Department of Pathology, Louisiana the cubilin–megalin complex is internalized State University Health Sciences Center, 1501 Kings Highway, simultaneously for endosomal trafficking in Shreveport, LA, 71130, USA. the proximal tubules.1 The pathogenic effects of E-mail: [email protected] Received 17 June 2003; accepted 05 November 2003; published glomerulopathic (G)-LCs are centered in the online 1 March 2004 mesangium. Trafficking of light chains in mesangial cells J Teng et al 441 G-LCs alter mesangial homeostasis. Their effects proven by renal biopsy. The LCs were obtained and on mesangial cells (MCs) are variable depending on purified from the urine of these patients using the biochemical composition of the variable region ammonium sulfate precipitation and ion exchange of the LCs resulting in two well-characterized chromatography, as described previously.6 disease processes: Al-amyloidosis (AL-Am) and LC Briefly, affinity chromatography consisted of depostion disease (LCDD).2–4 The LCs obtained from passing urine over sepharose CL-4B columns to the urine of patients with these disorders are which either goat anti-human l or k antibodies have referred as AL and LCDD-LCs, respectively. Nephro- been conjugated. Free plus bound l or k LCs were toxic LCs may either affect both the tubular inter- eluted from the columns with 0.1 glycine, pH 2.2. stitial and glomerular compartments in a given case The purity of the LC was checked on immunodiffu- or may restrict their damage to one compartment, or sion plates and anti-IgG and anti-IgA affinity the two glomerulopathies may coexist. As such, columns were used to remove contaminating IgG some LCs may elicit their pathologic effects produ- and IgA. When purified, the LC migrated as a single cing a combination of lesions (ie proximal tubular band when resolved by zone electrophoresis on damage and distal nephron obstruction in the agarose gels using a 50 mM barbital buffer, pH 8.6, tubular interstitial compartment or LCDD and AL- and consisted entirely of either free l or k LC as Am in the glomerular compartment). The majority of determined by immunofixation. Purified LCs were the nephrotoxic LCs, however, produce a specific concentrated, dialyzed against 0.9 NaCl, filtered pattern of renal damage confined to one renal through a 0.2-m membrane and stored aseptically at compartment and restricted to a specific lesion. 1 mg/ml at 41C. The extracted LCs had the same Vascular alterations also occur in a significant composition as those circulating in the serum of the number of cases associated with AL-Am and in corresponding patients. some cases with LCDD. The present paper focuses on the interactions between G-LCs and MCs and how these produce Human Mesangial Cell (HMC) Culture pathologic alterations. The study aimed at elucidat- HMCs were cultured following the standard method ing whether a specific receptor was shared by all described by Harper et al.7 Briefly, the cortex of G-LCs or more than one receptor was involved, leading surgically removed human kidneys was separated to AL-Am and LCDD. Immunogold labeling studies and glomerular MCs were isolated by sieving the at the ultrastructural level, coimmunolocalization cortex through stainless-steel screens and a series of using IF, binding competition and crosslinking different pore size nylon sieves. The cells were studies demonstrated the presence of a single plated for culture in RPMI 1640 containing 20% receptor for both types of G-LCs. This receptor must fetal calf serum and penicillin, streptomycin and act differently when involved in LC–MC interac- fungizone. For some experiments, the HMCs were tions associated with AL-Am and LCDD, as the final grown on coverslips placed on 20 mm culture plates. result of the pathogenetic effects is entirely differ- Ultrastructural examination and immunocyto- ent. In the case of AL-LCs, endocytosis is a crucial chemistry staining with immunoreactivity for muscle- step in the formation of amyloid and resultant specific antigen (HHF-35 antigen) and vimentin and pathologic changes leading to mesangial matrix lack of staining for keratin and factor VIII were used degradation and replacement of normal matrix by to determine that these cells were a homogeneous amyloid fibrils. In contrast, in LCDD-LC interactions population of HMCs. Passage 5–6 HMCs, but not with MCs, internalization is not an important initial older, were grown on RPMI 1640 medium contain- process and the alterations in mesangial home- ing 15% fetal bovine serum (FBS) until confluent. ostasis dictated by the G-LCs appear to occur Once confluent, HMCs were cultured in low serum primarily at the MC surface.5 The receptor modulat- (0.5% FBS) RPMI 1640 medium for 48 h to render ing and regulating LC–MC interactions is different the cells quiescent prior to the experiments. from the receptor identified for LCs in the proximal tubular cells. The main objectives of the work reported in this paper are to confirm the existence Immunofluorescence (IF) of such receptor-mediating G-LC-MC pathologic events and to define whether AL and LCDD-LCs HMCs grown on coverslips incubated with one or exhibit similar or different intracellular trafficking two different G-LCs at the same time (LCDD and AL- pathways in MCs. Further studies to characterize LCs) and T-LCs were fluorescence tagged for k and this receptor fully are currently being conducted. or l LCs in order to label the various LCs and caveolin-1 to determine whether the G-LCs coloca- lized to the same surface MC areas and whether Materials and methods there was any attraction for the TLC to the MCs Protein purification membranes or competition with the G-LCs. A direct immunofluorescence stain procedure was per- G-LCs were purified from the urine of patients who formed.8 A polyclonal antibody to caveolin-1 (Santa had AL-Am, LCDD and myeloma cast nephropathy Cruz Biotechnology, Santa Cruz, CA, USA) was used Laboratory Investigation (2004) 84, 440–451 Trafficking of light chains in mesangial cells J Teng et al 442 at a dilution of 1/50 and those to k and l at a dilution to the slides for 1 min, rinsed with distilled water, of 1/200. LCs were labeled with either fluorescein, two changes and placed in Dako automation buffer rhodamine or Texas red.