Urinary A-megalin excretion is associated with the proximal tubular endocytosis activity in db/db mice, a process modulated by empagliflozin

Megalin, a type I transmembrane glycoprotein, is a multi-ligand endocytic receptor expressed primarily in the apical membrane of proximal tubules (PTs). Megalin is excreted into urine as ectodomain (A-megalin) and full-length (C-megalin) forms (Diabetes Care, 2012). In patients with type 2 diabetes, urinary A-megalin levels were elevated during the normoalbuminuric and microalbuminuric stages but declined at the macroalbuminuric stage in cross-sectional analyses. Longitudinal studies have linked higher urinary A-megalin levels to accelerated estimated glomerular filtration rate (eGFR) decline in these patients (J Diabetes Complications, 2022). The endo-lysosomal system is well developed in segment 1 of PTs, where megalin plays a central role in receptor-mediated endocytosis (RME). In contrast, fluid-phase endocytosis (FPE) predominates in segment 2, in which megalin is also likely to be involved. These processes can be assessed by fluorescent lysozyme and low-molecular-weight dextran uptake, respectively. Sodium–glucose cotransporter-2 (SGLT2) inhibitors provide renoprotection in diabetic kidney disease, but underlying mechanisms are not fully defined. Increased PT endocytosis is associated with metabolic overload, and its suppression may contribute to renoprotection. This study investigated the association of urinary A-megalin excretion with PT endocytosis in db/db mice, a model of type 2 diabetes, and examined the effect of empagliflozin, an SGLT2 inhibitor.

Urinary A-megalin was measured in male db/db mice and controls (db/m) at 6, 8, 10, and 12 weeks of age. At 6 weeks, PT uptake of fluorescent lysozyme and dextran was examined in both groups. In 6-week-old db/db mice, after 14-day treatment with empagliflozin or vehicle, urinary excretion of A-megalin and α1-microglobulin (an endocytic megalin ligand) was measured, and renal megalin protein and mRNA expression levels were evaluated using immunoblotting and qPCR, respectively. In addition, after 5-day treatment with empagliflozin or vehicle in 6-week-old db/db mice, RME and FPE in PTs were evaluated, and transdermal GFR measurement was conducted. The interaction of empagliflozin with megalin was assayed using surface plasmon resonance.

Urinary A-megalin excretion increased from 6 weeks of age in db/db mice and was accompanied by enhanced RME in PTs. Empagliflozin treatment for 14 days reduced urinary A-megalin but increased urinary α1-microglobulin in db/db mice. Megalin protein and mRNA expression levels were unchanged between empagliflozin- and vehicle-treated groups. Short-term (5-day) empagliflozin treatment suppressed both RME and FPE in PTs of db/db mice without affecting GFR. Surface plasmon resonance analysis revealed that empagliflozin has very low affinity for megalin (KD > 1 mM).

Urinary A-megalin excretion increases early in db/db mice, paralleling enhanced RME in PTs. Empagliflozin suppresses both RME and FPE independently of GFR, leading to reduced urinary A-megalin. Indirect mechanisms likely mediate the inhibitory effects of empagliflozin on megalin’s endocytic function. Urinary A-megalin may reflect the PT endocytic activity and may serve as a therapeutic biomarker for SGLT2 inhibitor treatment in diabetic kidney disease.