Functional characterization of a novel missense CLCN5 mutation causing alterations in proximal tubular endocytic machinery in Dent's disease.

BACKGROUND/AIMS: Mutations of the endosomal chloride/proton exchanger gene, CLCN5, cause Dent's disease, an X-linked recessive proximal tubular disorder. The renal endocytic system was found to be affected in clcn5 knockout mice. However, the impaired endocytic machinery of Dent's disease patients has not been thoroughly investigated. METHODS: The CLCN5 gene was sequenced in a Japanese patient with Dent's disease and his family. The loss-of-function phenotype of the missense CLCN5 mutation was investigated by gene expression in Xenopus oocytes and CHO cells. Immunohistochemical analysis was performed on kidney biopsy specimens for endocytic machinery proteins, megalin, cubilin, and disabled-2 (Dab2) in proximal tubules. RESULTS: Genomic analysis revealed a novel G-to-A transition at the first nucleotide of the 333rd codon of CLCN5, causing a substitution of glycine with arginine. Inefficient expression of the mutant gene in Xenopus oocytes resulted in abolished chloride currents. Impaired N-glycosylation of the mutant protein was evident in the DNA-transfected CHO cells. Proximal tubular expression of megalin, cubilin, and Dab2 was markedly reduced and irregular staining in some portions was observed in the patient compared with controls. CONCLUSIONS: A novel G333R CLCN5 mutation caused defective expression of megalin, cubilin, and Dab2 in a patient with Dent's disease.

Role of megalin, a proximal tubular endocytic receptor, in the pathogenesis of diabetic and metabolic syndrome-related nephropathies: protein metabolic overload hypothesis.

Megalin is an endocytic receptor on the apical membranes of proximal tubule cells (PTC) in the kidney, and is involved in the reabsorption and metabolism of various proteins that have been filtered by glomeruli. Patients with diabetes, especially type 2 diabetes, or metabolic syndrome are likely to have elevated serum levels of advanced glycation end products, liver-type fatty acid binding protein, angiotensin II, insulin and leptin, and renal metabolism of these proteins is potentially overloaded. Some of these proteins are themselves nephrotoxic, while others are carriers of nephrotoxic molecules. Megalin is involved in the proximal tubular uptake of these proteins. We hypothesize that megalin-mediated metabolic overload in PTC leads to compensatory cellular hypertrophy and sustained Na+ reabsorption, causing systemic hypertension and glomerular hyperfiltration via tubuloglomerular feedback, and named this as 'protein metabolic overload hypothesis'. Impaired metabolism of bioactive proteins such as angiotensin II and insulin in PTC may enhance hypertrophy of PTC and/or Na+ reabsorption. Sleep apnoea syndrome, a frequent complication of diabetes and metabolic syndrome, may cause renal hypoxia and result in relative overload of protein metabolism in the kidneys. The development of strategies to identify patients with diabetes or metabolic syndrome who are at high risk for renal metabolic overload would allow intensive treatment of these patients in an effort to prevent the development of nephropathy. Further studies on the intracellular molecular signalling associated with megalin-mediated metabolic pathways may lead to the development of novel strategies for the treatment of nephropathies related to diabetes and metabolic syndrome.

Significance of proximal tubular metabolism of advanced glycation end products in kidney diseases.

Advanced glycation end products (AGEs) are formed by the nonenzymatic Maillard reaction between sugars and proteins. Low-molecular weight AGEs are filtered by renal glomeruli and then reabsorbed and metabolized by proximal tubule cells (PTCs). High-molecular weight AGEs are also delivered to PTCs in proteinuric states. In patients with diabetes, AGE generation is increased, and the actions of AGEs on PTCs are likely involved in the pathogenesis of diabetic nephropathy. In patients with chronic renal failure (CRF), reduced renal metabolism of AGEs likely accounts for the accumulation of AGEs in serum, leading to uremic complications including dialysis-related amyloidosis. AGE precursors such as reactive carbonyl compounds also accumulate in the sera of patients with CRF. It is likely that PTCs take up AGEs and AGE precursors via specific endocytotic receptors or transporters. Megalin is a multiligand endocytotic receptor that is abundantly expressed on PTCs. There is evidence that megalin is involved in the cellular uptake and degradation of AGEs. We previously reported a cell therapy model involving implantation of megalin-expressing cells into experimental mice with renal failure for elimination of uremic toxin proteins. Further studies are needed to clarify the molecular mechanisms of the metabolism of AGEs and their precursors to develop a strategy for the treatment of diabetic nephropathy and uremic complications of CRF.

Evidence for megalin-mediated proximal tubular uptake of L-FABP, a carrier of potentially nephrotoxic molecules.

Liver-type fatty acid binding protein (L-FABP) binds with high affinity to hydrophobic molecules including free fatty acid, bile acid and bilirubin, which are potentially nephrotoxic, and is involved in their metabolism mainly in hepatocytes. L-FABP is released into the circulation, and patients with liver damage have an elevated plasma L-FABP level. L-FABP is also present in renal tubules; however, the precise localization of L-FABP and its potential role in the renal tubules are not known. In this study, we examined the cellular and subcellular localization of L-FABP in the rat kidney and tried to determine from where the L-FABP in kidney tissues had originated. Immunohistochemical studies of kidney sections localized L-FABP in the lysosomes of proximal tubule cells (PTC). In rats with carbon tetrachloride (CCl4)-induced acute liver injury, we detected high levels of L-FABP in the circulation and in the kidney compared with those in the control rat by immunoblotting, while reverse transcription-polymerase chain reaction showed that the level of L-FABP mRNA expression in the kidney of CCl4-treated rats was low and did not differ from that in the control rat. When 35S-L-FABP was intravenously administered to rats, the kidneys took up 35S-L-FABP more preferentially than the liver and heart, and histoautoradiography of kidney sections revealed that 35S-L-FABP was internalized via the apical domains of PTC. Quartz-crystal microbalance analysis revealed that L-FABP bound to megalin, a multiligand endocytotic receptor on PTC, in a Ca2+-dependent manner. Degradation assays using megalin-expressing rat yolk sac tumor-derived L2 cells demonstrated that megalin mediated the cellular uptake and catabolism of 125I-L-FABP. In conclusion, circulatory L-FABP was found to be filtered by glomeruli and internalized by PTC probably via megalin-mediated endocytosis. These results suggest a novel renal uptake pathway for L-FABP, a carrier of hydrophobic molecules, some of which may exert nephrotoxic effects.

Evidence indicating that renal tubular metabolism of leptin is mediated by megalin but not by the leptin receptors.

Leptin is secreted by adipocytes and is a circulating factor that regulates food intake and energy expenditure. Its serum level is elevated in patients with renal failure and has been suggested to be associated with malnutritional factors in these patients. Leptin has been suggested to be primarily metabolized by the kidneys, although the precise molecular mechanisms are not known. The purpose of this study was to determine the nephron segments and potential receptors involved in renal leptin metabolism. To determine the segment involved in leptin uptake, we performed histoautoradiography of kidney sections obtained from rats that had been injected iv with (125)I-leptin. The ability of megalin, a multiligand endocytic receptor in the proximal tubules, to bind and endocytose leptin was examined by ligand blotting analysis, quartz-crystal microbalance, and degradation assays using megalin-expressing rat yolk sac L2 cells. Immunohistochemistry was performed to localize leptin receptors (LEP-R) in the rat kidney using two antibodies that recognize different epitopes on the LEP-R proteins. Circulating (125)I-leptin was filtered by glomeruli and internalized by proximal convoluted tubules. Megalin bound leptin in the presence of Ca(2+) and mediated its cellular internalization and degradation. On immunohistochemistry, LEP-R were localized in the proximal straight tubules, loops of Henle, distal tubules, and collecting ducts. In conclusion, circulating leptin was filtered by glomeruli and taken up by proximal convoluted tubules, where megalin likely mediates its binding and uptake. The localization of LEP-R suggests that they are not primarily involved in leptin metabolism in the proximal tubules.

Role of megalin in endocytosis of advanced glycation end products: implications for a novel protein binding to both megalin and advanced glycation end products.

Advanced glycation end products (AGE) are filtered by glomeruli and reabsorbed and metabolized by proximal tubule cells (PTC). In renal failure, decreased renal AGE metabolism likely accounts for the accumulation in serum that is related to uremic complications. In diabetes, AGE generation is increased, and the handling mechanisms in PTC are likely associated with the pathogenesis of tubulointerstitial injury. It is therefore important to clarify the mechanisms of the AGE metabolism to develop a strategy for removing AGE in uremia and to elucidate the pathogenesis of diabetic nephropathy. To this end, this study focused on the molecular analysis of megalin, a multi-ligand endocytic receptor, in PTC. AGE uptake analysis was performed using the rat yolk sac-derived L2 cell line system established for the analysis of megalin's endocytic functions. The cells mediated specific internalization and degradation of AGE, which were significantly blocked by anti-megalin IgG, indicating that megalin is involved in the cellular processes. However, cell surface AGE-binding assays and ligand blot analysis revealed no evidence that megalin is a direct AGE receptor. Affinity chromatography and ligand blot analysis originally revealed that 200-kD and 400-kD proteins in the cells bind to AGE and the 200-kD protein to megalin in a Ca(2+)-dependent manner. The binding of megalin with the 200-kD protein was suppressed by receptor-associated protein (RAP), a ligand for megalin. In conclusion, megalin functions for endocytosis of AGE via an indirect mechanism. L2 cells express novel AGE-binding proteins, one of which may interact with megalin.