Toll like receptor 4 mediates the inhibitory effect of SARS-CoV-2 spike protein on proximal tubule albumin endocytosis.

Tubular proteinuria is a common feature in COVID-19 patients, even in the absence of established acute kidney injury. SARS-CoV-2 spike protein (S protein) was shown to inhibit megalin-mediated albumin endocytosis in proximal tubule epithelial cells (PTECs). Angiotensin-converting enzyme type 2 (ACE2) was not directly involved. Since Toll-like receptor 4 (TLR4) mediates S protein effects in various cell types, we hypothesized that TLR4 could be participating in the inhibition of PTECs albumin endocytosis elicited by S protein. Two different models of PTECs were used: porcine proximal tubule cells (LLC-PK1) and human embryonic kidney cells (HEK-293). S protein reduced Akt activity by specifically inhibiting of threonine 308 (Thr308) phosphorylation, a process mediated by phosphoinositide-dependent kinase 1 (PDK1). GSK2334470, a PDK1 inhibitor, decreased albumin endocytosis and megalin expression mimicking S protein effect. S protein did not change total TLR4 expression but decreased its surface expression. LPS-RS, a TLR4 antagonist, also counteracted the effects of the S protein on Akt phosphorylation at Thr308, albumin endocytosis, and megalin expression. Conversely, these effects of the S protein were replicated by LPS, an agonist of TLR4. Incubation of PTECs with a pseudovirus containing S protein inhibited albumin endocytosis. Null or VSV-G pseudovirus, used as control, had no effect. LPS-RS prevented the inhibitory impact of pseudovirus containing the S protein on albumin endocytosis but had no influence on virus internalization. Our findings demonstrate that the inhibitory effect of the S protein on albumin endocytosis in PTECs is mediated through TLR4, resulting from a reduction in megalin expression.

Bradykinin produced during Plasmodium falciparum erythrocytic cycle drives monocyte adhesion to human brain microvascular endothelial cells.

Cerebral malaria (CM) pathogenesis is described as a multistep mechanism. In this context, monocytes have been implicated in CM pathogenesis by increasing the sequestration of infected red blood cells to the brain microvasculature. In disease, endothelial activation is followed by reduced monocyte rolling and increased adhesion. Nowadays, an important challenge is to identify potential pro-inflammatory stimuli that can modulate monocytes behavior. Our group have demonstrated that bradykinin (BK), a pro-inflammatory peptide involved in CM, is generated during the erythrocytic cycle of P. falciparum and is detected in culture supernatant (conditioned medium). Herein we investigated the role of BK in the adhesion of monocytes to endothelial cells of blood brain barrier (BBB). To address this issue human monocytic cell line (THP-1) and human brain microvascular endothelial cells (hBMECs) were used. It was observed that 20% conditioned medium from P. falciparum infected erythrocytes (Pf-iRBC sup) increased the adhesion of THP-1 cells to hBMECs. This effect was mediated by BK through the activation of B2 and B1 receptors and involves the increase in ICAM-1 expression in THP-1 cells. Additionally, it was observed that angiotensin-converting enzyme (ACE) inhibitor, captopril, enhanced the effect of both BK and Pf-iRBC sup on THP-1 adhesion. Together these data show that BK, generated during the erythrocytic cycle of P. falciparum, could play an important role in adhesion of monocytes in endothelial cells lining the BBB.

O-Linked GlcNAcylation mediates the inhibition of proximal tubule (Na++K+)ATPase activity in the early stage of diabetes mellitus.

BACKGROUND: Diabetic kidney disease (DKD) is a severe complication of diabetes mellitus (DM). It has been proposed that modifications in the function of proximal tubule epithelial cells (PTECs) precede glomerular damage during the onset of DKD. This study aimed to identify modifications in renal sodium handling in the early stage of DM and its molecular mechanism. METHODS: Streptozotocin (STZ)-induced diabetic BALB/c mice (STZ group) and LLC-PK1 cells, a model of PTECs, were used. All parameters were assessed in the 4th week after an initial injection of STZ. RESULTS: Early stage of DKD was characterized by hyperfiltration and PTEC dysfunction. STZ group exhibited increased urinary sodium excretion due to impairment of tubular sodium reabsorption. This was correlated to a decrease in cortical (Na++K+)ATPase (NKA) α1 subunit expression and enzyme activity and an increase in O-GlcNAcylation. RNAseq analysis of patients with DKD revealed an increase in expression of the glutamine-fructose aminotransferase (GFAT) gene, a rate-limiting step of hexosamine biosynthetic pathway, and a decrease in NKA expression. Incubation of LLC-PK1 cells with 10 µM thiamet G, an inhibitor of O-GlcNAcase, reduced the expression and activity of NKA and increased O-GlcNAcylation. Furthermore, 6-diazo-5-oxo-L-norleucine (DON), a GFAT inhibitor, or dapagliflozin, an SGLT2 inhibitor, avoided the inhibitory effect of HG on expression and activity of NKA associated with the decrease in O-GlcNAcylation. CONCLUSION: Our results show that the impairment of tubular sodium reabsorption, in the early stage of DM, is due to SGLT2-mediated HG influx in PTECs, increase in O-GlcNAcylation and reduction in NKA expression and activity.

Gold nanoparticles reduce tubule-interstitial injury and proteinuria in a murine model of subclinical acute kidney injury.

Subclinical acute kidney injury (subAKI) is characterized by tubule-interstitial injury without significant changes in glomerular function. SubAKI is associated with the pathogenesis and progression of acute and chronic kidney diseases. Currently, therapeutic strategies to treat subAKI are limited. The use of gold nanoparticles (AuNPs) has shown promising benefits in different models of diseases. However, their possible effects on subAKI are still unknown. Here, we investigated the effects of AuNPs on a mouse model of subAKI. Animals with subAKI showed increased functional and histopathologic markers of tubular injury. There were no changes in glomerular function and structure. The animals with subAKI also presented an inflammatory profile demonstrated by activation of Th1 and Th17 cells in the renal cortex. This phenotype was associated with decreased megalin-mediated albumin endocytosis and expression of proximal tubular megalin. AuNP treatment prevented tubule-interstitial injury induced by subAKI. This effect was associated with a shift to an anti-inflammatory Th2 response. Furthermore, AuNP treatment preserved megalin-mediated albumin endocytosis in vivo and in vitro. AuNPs were not nephrotoxic in healthy mice. These results suggest that AuNPs have a protective effect in the tubule-interstitial injury observed in subAKI, highlighting a promising strategy as a future antiproteinuric treatment.

Surface megalin expression is a target to the inhibitory effect of bradykinin on the renal albumin endocytosis.

Megalin-mediated albumin endocytosis plays a critical role in albumin reabsorption in proximal tubule (PT) epithelial cells (PTECs). Some studies have pointed out the modulatory effect of bradykinin (BK) on urinary protein excretion, but its role in PT protein endocytosis has not yet been determined. Here, we studied the possible correlation between BK and albumin endocytosis in PT. Using LLC-PK1 cells, a model of PTECs, we showed that BK specifically inhibited megalin-mediated albumin endocytosis. This inhibitory effect of BK was mediated by B2 receptor (B2R) because it was abolished by HOE140, an antagonist of B2R, but it was not affected by Lys-des-Arg9-BK, an antagonist of B1. BK induced the stall of megalin in EEA1+ endosomes, but not in LAMP1+ lysosomes, leading to a decrease in surface megalin expression. In addition, we showed that BK, through B2R, activated calphostin C-sensitive protein kinase C, which mediated its effect on the surface megalin expression and albumin endocytosis. These results reveal an important modulatory mechanism of PT albumin endocytosis by BK, which opens new possibilities to understanding the effect of BK on urinary albumin excretion.