Comparative in vitro analyses of the effect of immunoglobulin \u1d740 light chain and fatty acid free albumin on proximal tubular epithelial cells-involvement of megalin phosphorylation

摘要

Kidney disease is a major challenge for health care systems, and the prevalence is increasing. Proteinuria is a hallmark of progressive renal dysfunction and describes the pathological excess of plasma proteins in urine, mainly albumin.\udMultiple Myeloma is a cancer of plasma cells that leads to excessive presence of free light chain protein (FLC) in blood. Renal failure due to overproduction of FLC and the associated light chain proteinuria occurs as a result of decreased renal function or as a direct toxic effect on the proximal tubular cells (PTCs) by excessive protein. Proteins are normally reabsorbed by endocytosis via megalin receptor that binds proteins and mediates their uptake. Exceeding the proximal tubular epithelial cells (PTECs) reabsorption capacity might trigger inflammation detrimental to the kidney. In proteinuric nephropathy the cytoplasmic tail of megalin (MegCT) is phosphorylated after interaction between proteins and megalin on the PTECs, which activates signalling cascades that regulate the phosphorylation.\udAn in vitro proteinuric model was established using HK2 cells (a proximal tubular epithelial cell line derived from normal human kidney) treated with high concentrations of essentially fatty acid free human serum albumin (FAF-HSA) or lambda light chain (\u1d706-LC) isolated and purified from the urine of a myeloma patient, to induce cellular damage. The potential pathogenic role for FAF-HSA and \u1d706-LC on HK2 cells was examined. Also, renal toxicity that comes from the intracellular signalling through phosphorylation of MegCT was addressed by utilising antibodies directed against specific phosphorylation site (PPPSP) of the intracellular portion of megalin in HK2 cells stimulated with different concentrations of FAF-HSA and \u1d706-LC, so-called pre-stimulated HK2.\udIn vitro analyses showed (i) a detrimental effect of FAF-HSA and \u1d706-LC on viability of HK2, (ii) phosphorylation of the cytoplasmic tail of megalin in pre-stimulated HK2 cells. (iii) Production of inflammatory cytokines and H2O2 generation, activation of autophagy process and increase in several kidney biomarkers/ injury mediators, which are involved in different pathways in response to protein overload. All these reasons are likely to contribute to direct PTECs injury and kidney failure in patients.\udPotentially these mechanisms may be attractive for drug development to benefit patients with kidney failure and help to inhibit the progression of proteinuric nephropathy and as such may save lives.