Immunoglobulin Light Chains Activate Tubular Epithelial Cells through Redox Signaling

Abstract

The renal proximal tubule metabolizes circulating low-molecular-weight proteins such as Ig free light chains. In the setting of plasma cell dyscrasias, the burden of filtered protein can be very high. Endocytosis of certain nephrotoxic light chains induces H 2 O 2 production and monocyte chemoattractant protein-1 (MCP-1) release, leading to recruitment of inflammatory cells and interstitial fibrosis, but how these processes are linked mechanistically is not well understood. This study investigated the relationship between H 2 O 2 generated after light chain endocytosis by human proximal tubular (HK-2) cells and activation of c-Src, a redox-sensitive tyrosine kinase. HK-2 cells exposed to two different light chains upregulated c-Src activity, which increased the production of MCP-1. In parallel, we observed a time-dependent oxidation of c-Src. Inhibition of c-Src activity and silencing c-Src expression abrogated the light chain-induced MCP-1 response, but had no effect on H 2 O 2 , indicating that production of H 2 O 2 is upstream of c-Src in the signaling cascade. Silencing megalin and cubilin expression inhibited the MCP-1 response, whereas extracellular catalase did not, indicating that endocytosis is required and that intracellular generation of reactive oxygen species activates c-Src. These data show that intracellular H 2 O 2 induced by endocytosis of monoclonal free light chains oxidizes and activates c-Src, which promotes release of MCP-1.

Receptor-Mediated Endocytosis Is a Trojan Horse in Light-Chain Nephrotoxicity

“…4 In the study by Basnayake et al, 6 the addition of catalase, a scavenger of H 2 O 2 , to the light-chain-containing medium did not reduce the cellular production of MCP-1. These findings led to the plausible conclusion that H 2 O 2 , accounting for the induction of MCP-1, originates in the intracellular, not the extracellular, compartment.…”

“…In view of these broad effects of c-Src on cell signaling, it is not surprising that c-Src is increasingly implicated in renal processes, including those that are physiologic (e.g., acid-base homeostasis, 8 renal hypertrophy 9 ) and those that are pathophysiologic (e.g., podocyte proliferation and dedifferentiation, 10 cystic kidney disease, 11 albumin-induced cytokine expression 12 ). The findings of Basnayake et al 6 are important for several reasons: First, they identify another disease instigated by the activation of c-Src; second, they provide an explanation for the recruitment of mitogen-activated protein kinase-dependent signaling and activation of NF-B described in previous studies of light-chain-exposed renal epithelial cells 2,3 ; and third, they direct attention to c-Src as an inducer of MCP-1, a chemokine strongly upregulated and broadly implicated in kidney injury.…”

“…Clearance of light chains by the proximal tubule would reduce their downstream delivery and their interaction with Tamm-Horsfall protein, thereby decreasing the risk for cast nephropathy; however, as made clear by a number of studies, 2-5 the uptake of light chains by the proximal tubule triggers a vigorous inflammatory response. The studies of Basnayake et al 6 in this issue of JASN examine the basis for this inflammatory behavior by exposing human proximal tubular epithelial cells to pathophysiologically relevant concentrations of light chains derived from patients with myeloma and kidney disease. Their findings demonstrate that light-chain uptake by megalin-cubilin receptors increases cellular generation of hydrogen peroxide, the latter activating c-Src, which in turn induces the chemokine monocyte chemoattractant protein 1 (MCP-1).…”

“…These findings led to the plausible conclusion that H 2 O 2 , accounting for the induction of MCP-1, originates in the intracellular, not the extracellular, compartment. 6 It would be of interest to determine the specific intracellular site and biochemical mechanism accounting for increased production of H 2 O 2 , because such oxidant generation links cellular uptake of light chains and activation of c-Src. The role of the hydroxyl radical and labile cellular iron would also be of interest: DMTU, used as a trap for H 2 O 2 in the study by Basnayake et al, 6 is also a hydroxyl radical scavenger, and H 2 O 2 can generate the hydroxyl radical in the presence of iron.…”

“…6 It would be of interest to determine the specific intracellular site and biochemical mechanism accounting for increased production of H 2 O 2 , because such oxidant generation links cellular uptake of light chains and activation of c-Src. The role of the hydroxyl radical and labile cellular iron would also be of interest: DMTU, used as a trap for H 2 O 2 in the study by Basnayake et al, 6 is also a hydroxyl radical scavenger, and H 2 O 2 can generate the hydroxyl radical in the presence of iron. 13 Defining the role of labile cellular iron is clinically relevant, because such involvement can be blocked by iron chelators.…”