Hypertensive individuals are at greater risk for developing chronic kidney disease (CKD). Reducing proteinuria has been suggested as a possible therapeutic approach to treat CKD. However, the mechanisms underlying the development of proteinuria in hypertensive conditions are incompletely understood. Cardiac and vascular dysfunction is associated with changes in the -GlcNAcylation pathway in hypertensive models. We hypothesized that-GlcNAcylation is also involved in renal damage, especially development of proteinuria, associated with hypertension. Using the spontaneously hypertensive rat (SHR) model, we observed higher renal cortex -GlcNAcylation, glutamine-fructose aminotransferase (GFAT), and-GlcNAc transferase (OGT) protein expression, which positively correlated with proteinuria. Interestingly, this was observed in hypertensive, but not pre-hypertensive, rats. Pharmacological inhibition of GFAT decreased renal cortex -GlcNAcylation, proteinuria, and albuminuria in SHR. Using a proximal tubule cell line, we observed that increased-GlcNAcylation reduced megalin surface expression and albumin endocytosis , and the effects were correlated Moreover, megalin is -GlcNAcylated both and In conclusion, our results demonstrate a new mechanism involved in hypertension-associated proteinuria.

The role of albumin reabsorption in proximal tubule (PT) cells has emerged as an important factor in the genesis of albuminuria observed in the early stages of diabetes. Evidence has shown that a decrease in megalin expression could be the key mechanism in this process. In the present work, we investigated the molecular mechanism underlying the modulation of albumin endocytosis in LLC-PK1 cells, a model of PT cells. High glucose concentrations (HG) inhibited megalin expression and albumin endocytosis after 48 h of incubation. This inhibitory effect involves the entrance of glucose into PT cells through SGLT located at the luminal membrane. Once inside PT cells, glucose is diverted to the hexosamine biosynthetic pathway (HBP) increasing -GlcNAcylation of several intracellular proteins, including PKB. This process promotes the inhibition of PKB activity measured by its phosphorylation at Thr-308 and Ser-473 and phosphorylation of specific substrates, glycogen synthase kinase 3β (GSK3β) and tuberous sclerosis complex 2. The decrease in PKB activity led to a decrease in megalin expression and, consequently, reducing albumin endocytosis in LLC-PK1 cells. HG did not change mammalian target of rapamycin (mTOR) C2 activity, responsible for phosphorylated PKB at Ser-473. In addition, HG activated the mTORC1/S6K pathway, but this effect was not correlated to the decrease in megalin expression or albumin endocytosis. Taken together, our data help to clarify the current understanding underlying the genesis of tubular albuminuria induced by hyperglycemia in the early stage of diabetes pathogenesis.

Tubule-interstitial injury (TII) is a critical step in the progression of renal disease. It has been proposed that changes in proximal tubule (PT) albumin endocytosis plays an important role in the development of TII. Some reports have shown protective effects of lithium on kidney injury animal models that was correlated to proteinuria. We tested the hypothesis that lithium treatment ameliorates the development of TII due to changes in albumin endocytosis. Two experimental models were used: (1) TII induced by albumin overload in an animal model; (2) LLC-PK1 cells, a PT cell line. Lithium treatment ameliorates TII induced by albumin overload measured by (1) proteinuria; (2) collagen deposition; (3) area of tubule-interstitial space, and (4) macrophage infiltration. Lithium treatment increased mTORC2 activity leading to the phosphorylation of protein kinase B (PKB) at Ser473 and its activation. This mechanism enhanced albumin endocytosis in PT cells, which decreased the proteinuria observed in TII induced by albumin overload. This effect did not involve changes in the expression of megalin, a PT albumin receptor. In addition, activation of this pathway decreased apoptosis in LLC-PK1 cells, a PT cell line, induced by higher albumin concentration, similar to that found in pathophysiologic conditions. Our results indicate that the protective role of lithium treatment on TII induced by albumin overload involves an increase in PT albumin endocytosis due to activation of the mTORC2/PKB pathway. These results open new possibilities in understanding the effects of lithium on the progression of renal disease.

Increasing evidence has highlighted the role of tubule-interstitial injury (TII) as a vital step in the pathogenesis of acute kidney injury (AKI). Incomplete repair of TII during AKI could lead to the development of chronic kidney disease. Changes in albumin endocytosis in proximal tubule epithelial cells (PTECs) is linked to the development of TII. In this context, interleukin (IL)-4 has been shown to be an important factor in modulating recovery of TII. We have studied the possible role of IL-4 in TII induced by albumin overload. A subclinical AKI model characterized by albumin overload in the proximal tubule was used, without changing glomerular function. Four groups were generated: (1) CONT, wild-type mice treated with saline; (2) BSA, wild-type mice treated with 10 g/kg/day bovine serum albumin (BSA); (3) KO, IL4Rα −/− mice treated with saline; and (4) KO + BSA, IL4Rα −/− mice treated with BSA. As reported previously, mice in the BSA group developed TII without changes in glomerular function. The following parameters were increased in the KO + BSA group compared with the BSA group: (1) tubular injury score; (2) urinary γ-glutamyltransferase; (3) CD4 + T cells, dendritic cells, macrophages, and neutrophils are associated with increases in renal IL-6, IL-17, and transforming growth factor β. A decrease in M2-subtype macrophages associated with a decrease in collagen deposition was observed. Using LLC-PK1 cells, a model of PTECs, we observed that (1) these cells express IL-4 receptor α chain associated with activation of the JAK3/STAT6 pathway; (2) IL-4 alone did not change albumin endocytosis but did reverse the inhibitory effect of higher albumin concentration. This effect was abolished by JAK3 inhibitor. A further increase in urinary protein and creatinine levels was observed in the KO + BSA group compared with the BSA group, but not compared with the CONT group. These observations indicate that IL-4 has a protective role in the development of TII induced by albumin overload that is correlated with modulation of the proinflammatory response. We propose that megalin-mediated albumin endocytosis in PTECs could work as a sensor, transducer, and target during the genesis of TII.

Renal proximal tubule cells (PTECs) act as urine gatekeepers, constantly and efficiently avoiding urinary protein waste through receptor-mediated endocytosis. Despite its importance, little is known about how this process is modulated in physiologic conditions. Data suggest that the phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) pathway regulates PTEC protein reabsorption. Here, we worked on the hypothesis that the physiologic albumin concentration and PI3K/AKT pathway form a positive feedback loop to expand endocytic capacity. Using LLC-PK1 cells, a model of PTECs, we showed that the PI3K/AKT pathway is required for megalin recycling and surface expression, affecting albumin uptake. Inhibition of this pathway stalls megalin at EEA1+ endosomes. Physiologic albumin concentration (0.01 mg/mL) activated AKT; this depends on megalin-mediated albumin endocytosis and requires previous activation of PI3K/mTORC2. This effect is correlated to the increase in albumin endocytosis, a phenomenon that we refer to as “albumin-induced albumin endocytosis”. Mice treated with L-lysine present decreased albumin endocytosis leading to proteinuria and albuminuria associated with inhibition of AKT activity. Renal cortex explants obtained from control mice treated with MK-2206 decreased albumin uptake and promoted megalin internalization. Our data highlight the mechanism behind the capacity of PTECs to adapt albumin reabsorption to physiologic fluctuations in its filtration, avoiding urinary excretion.