= 5 experiments at each concentration in each group

= 5 experiments at each concentration in each group. in vitro. When used in a rodent model of cast nephropathy, this cyclized peptide construct served as an effective inhibitor of intraluminal cast formation and prevented the functional manifestations of acute kidney injury in vivo. These experiments provide proof of concept that intraluminal cast formation is integrally involved in the pathogenesis of acute kidney injury from cast nephropathy. Further, the data support a clinically relevant approach to the management of renal failure in the setting of multiple myeloma. Introduction One of the Paullinic acid functions of the kidney is to filter and metabolize low molecular weight proteins that include immunoglobulin free light chains (FLCs). Polyclonal FLCs are secreted normally in the circulation and appear in the glomerular ultrafiltrate. FLCs are reabsorbed into the proximal tubular epithelium and then hydrolyzed. In the setting of overproduction of SSH1 monoclonal FLCs, a wide variety of renal pathologies can develop, including glomerular diseases, such as Amyloid Light-chain (AL-type) amyloidosis and monoclonal light chain deposition disease, or tubular damage, known as proximal tubulopathy (1C5). In addition, FLCs that escape tubular reabsorption are presented to the distal nephron and, in the proper conditions, form intraluminal casts that obstruct tubular fluid flow (3, 6C8). Clinical manifestations of this phenomenon, known as cast nephropathy, include acute kidney injury (AKI) and progressive renal failure. Because this complication occurs in multiple myeloma, which constitutes 12%C13% of hematologic malignancies in the US (9), the term myeloma kidney has also been used. Cast nephropathy is a seminally important and common complication in myeloma, since reduced renal function contributes to morbidity and mortality and limits therapeutic options (10C12). At the time of presentation, nearly half of these patients have renal dysfunction, as defined by a serum creatinine concentration greater than or equal to 1.3 mg/dl (10). When kidney tissue was examined histologically, cast nephropathy was the major cause of renal failure (13). Prior studies determined an important role for Tamm-Horsfall glycoprotein (THP) in cast nephropathy (7). THP possesses a single FLC-binding domain, termed LCBD (14, 15), and the complementarity-determining region 3 (CDR3) of most FLCs tested specifically interacted with this site (16). The following experiments were designed to analyze the binding interaction between FLCs and THP and to test the hypothesis that a competitive inhibitor of the interaction between THP and monoclonal FLCs prevents AKI induced in cast nephropathy. Results The CDR3 of FLCs demonstrated varying binding affinities to THP. Previous publications demonstrated that FLCs bind to a specific domain on human THP, but possess variable affinities for Paullinic acid THP (14, 15). Initial experiments expanded the original studies by using the variable light chain (VL) domain of 20 unique human FLCs from the I, III, IV, V, VI, I, II, and IV families. The yeast 2-hybrid system originally designed by Fields and Song (17) was employed to determine the site on the light chain that interacted with THP (16). The binding interactions of these and FLCs with recombinant 26-residue and 263-residue fragments of THP, which contained the previously described LCBD, were quantified. The findings were similar when either the smaller or larger THP fragment was used, so the data presented in this paper are from experiments that used the larger fragment (Table ?(Table1).1). All tested families of FLCs bound to THP, with members of the V family demonstrating the lowest binding affinity. The relative strength of the interactions differed among the 20 different FLCs (Table ?(Table1).1). The variable domain of Paullinic acid the V FLCs, LKPBLL53, showed the lowest affinity interaction: yeast transformed with this construct did not grow in leucine-deficient medium and possessed low -gal activity. The intact VL of the IIIa FLCs, ITPBLL86, demonstrated the highest binding affinity among the FLCs tested. A series of truncation mutations performed on the FLCs again confirmed that the CDR3 of both and FLCs specifically interacted with the THP constructs. Reactivity with THP correlated weakly (= 0.23; = 0.02) with the number of amino acid residues in the CDR3. Table 1 Binding affinities of 20 different FLCs with THP Open in a separate window Key amino acid residues in the CDR3 of FLCs determined binding to THP. An artificial construct was designed using framework 2 and framework 3 of LKPBLL53, an FLC that did not interact significantly with THP (Table ?(Table1).1). Various CDR3 sequences were then inserted into this construct (Figure ?(Figure1).1). The construct that did not contain an insert did not interact.