Mechanism and prevention of acute kidney injury from cast nephropathy in a rodent model

Abstract

A common renal complication of multiple myeloma is "myeloma kidney," a condition also known as cast nephropathy. The renal lesions (casts) are directly related to the production of monoclonal immunoglobulin free light chains (FLCs), which coprecipitate with Tamm-Horsfall glycoprotein (THP) in the lumen of the distal nephron, obstructing tubular fluid flow. Here, we report that analysis of the binding interaction between FLCs and THP demonstrates that the secondary structure and key amino acid residues on the complementarity-determining region 3 (CDR3) of FLCs are critically important determinants of the molecular interaction with THP. The findings permitted development of a cyclized competitor peptide that demonstrated strong inhibitory capability in the binding of FLCs to THP 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.

Figure 1. Key amino acid residues in the CDR3 of the V_L domain determine binding to THP.

A construct that permitted ligation of the CDR3 of interest between portions of framework 2 and framework 3 from an FLC (LKPBLL53) that did not interact with THP was used to test the interaction of the CDR3 with THP (A). As predicted, the construct that did not contain a CDR3 insert (no insert) did not interact with THP in the yeast 2-hybrid assay (B). Also as expected, the CDR3 from LKPBLL53 did not interact with THP. Binding was strongest with CDR3 (LSADSSGSYLYV) from ITPBLL86. Binding was also demonstrated with the use of the CDR3 (QVWDSTSDHYV) from ITPBLL1, but the interaction was markedly reduced with the use of the CDR3 (QVWHSSSDHYV) from ITPBLL2. These regions differ in only 2 amino residues, which are underlined in the figure. *P < 0.05, compared with no insert; ^†P < 0.05, compared with ITPBLL2 CDR3 construct. n = 6–8 experiments in each group. Data are shown as mean ± SEM.

Figure 2. Mutational analysis of the CDR3 sequence demonstrated the importance of the primary sequence, in particular the fourth amino acid residue, of the CDR3 in binding to THP.

*P < 0.05, compared with no insert. n = 7 experiments in each group. Data are shown as mean ± SEM.

Figure 3. Graphs of the effect of competitor peptides and cyclized competitor peptides on binding of THP to FLCs.

While both CDR3 mimetic peptides inhibited binding of FLCs to THP, cyclization, which was accomplished through intramolecular disulfide bonding between cysteine residues added onto the amino and carboxyl termini, shifted the inhibition curves to the left. n = 5 experiments at each concentration in each group. Data are shown as mean ± SEM.

Figure 4. The cyclized peptide (AHX-CLSADSSGSYLYVCKK) (circles) was a highly effective inhibitor that completely prevented the binding of THP to 6 different human monoclonal FLCs.

The control peptide (AHX-CLSAFSFGSYLYVCKK) (white squares) did not effectively inhibit binding of THP to any of the 6 FLCs. n = 4 experiments in each group. Data are shown as mean ± SEM.

Figure 5. Overlay (far-Western) assays demonstrating the interaction between FLCs and THP.

(A) Coomassie-stained gel (left) depicts the monomeric and dimeric forms of a monoclonal FLC and the V_L domain, which had been enzymatically cleaved from the FLCs. The adjacent blot (right) demonstrates that human THP bound to monomers, dimers, and polymeric FLCs, as well as the V_L domain of the FLCs. (B) Results of an experiment that used cyclic peptide 1 (AHX-CLSADSSGSYLYVCKK) to compete with the biotinylated V_L domain of FLC ITPBLL86 for binding to THP immobilized onto PVDF membrane. The reversible protein stain demonstrated the presence of THP in each lane (top). Addition of increasing molar amounts, relative to THP, of the cyclic peptide prevented binding of the V_L domain to THP. (C) Results of an overlay assay that used cyclized peptide 1 (AHX-CLSADSSGSYLYVCKK) or a cyclized control peptide (AHX-CLSAHSSGSYLYVCKK) to compete with biotinylated THP for binding to κ2 FLC immobilized onto PVDF membrane. The reversible protein stain demonstrated the presence of κ2 FLC in each lane (top). Cyclized peptide 1, but not the cyclized control peptide, dose dependently inhibited binding of THP to the κ2 FLC (bottom).

Figure 6. Light and immunofluorescence micrographs depicting the beneficial effect of the synthetic, cyclized competitor peptide in preventing cast nephropathy in vivo.

Rats were injected intraperitoneally with the cyclic peptide (AHX-CLSADSSGSYLYVCKK), 8 mg, or vehicle 2 hours prior to injection with 1 of 2 unique monoclonal FLCs. Both were obtained from patients who had myeloma and renal dysfunction compatible with cast nephropathy. The patient who donated the λ2 FLC had biopsy-proven cast nephropathy. Addition of the cyclic peptide prevented cast formation and accumulation of the monoclonal FLCs in the kidney, as demonstrated by H&E staining and by immunofluorescent labeling of human κ or λ light chains, respectively, using specific primary antibodies with Texas red–labeled secondary antibody. Nuclei were visualized in the fluorescent images by DAPI staining. n = 3 rats in each group. Scale bars: 100 μm.

Figure 7. Experimental peptide 1 served as an effective inhibitor of cast nephropathy in vivo.

Changes in (A) mean serum creatinine, determined by tandem mass spectrometry, and (B) medullary cast formation of 3 groups of rats (n = 5–6 per group) were determined in a rescue experiment in which the competitor cyclized experimental (exp.) peptide 1 (AHX-CLSADSSGSYLYVCKK), a control cyclized peptide (AHX-CLSAHSSGSYLYVCKK), or vehicle alone was administered intraperitoneally 4 hours after rats received the nephrotoxic FLC (κ2). Mean serum creatinine increased in the vehicle-treated and control cyclized peptide–treated groups, but rats given competitor cyclized peptide 1 were protected from AKI. Animals that received experimental peptide 1 had fewer casts in the medulla compared with animals that received either vehicle or the control peptide. Rats that received the control peptide also had fewer casts (P < 0.05) than animals that received vehicle alone. *P < 0.05, compared with the other 2 groups. Data are shown as mean ± SEM.

Figure 8. Schematic depicting the different domains on THP as well as potential alternative splice sites (represented by vertical lines).

The FLC-binding domain (LCBD) lies between the EGF-like repeats and the zona pellucida domains.