RESUMO
It is well-established that dysfunction of megalin-mediated albumin endocytosis by proximal tubule epithelial cells (PTECs) and the activation of the Renin-Angiotensin System (RAS) play significant roles in the development of Diabetic Kidney Disease (DKD). However, the precise correlation between these factors still requires further investigation. In this study, we aimed to elucidate the potential role of angiotensin II (Ang II), a known effector of RAS, as the mediator of albumin endocytosis dysfunction induced by high glucose (HG) in PTECs. To achieve this, we utilized LLC-PK1 and HK-2 cells, which are well-established in vitro models of PTECs. Using albumin-FITC or DQ-albumin as tracers, we observed that incubation of LLC-PK1 and HK-2 cells with HG (25 mM for 48 h) significantly reduced canonical receptor-mediated albumin endocytosis, primarily due to the decrease in megalin expression. HG increased the concentration of Ang II in the LLC-PK1 cell supernatant, a phenomenon associated with an increase in angiotensin-converting enzyme (ACE) expression and a decrease in prolyl carboxypeptidase (PRCP) expression. ACE type 2 (ACE2) expression remained unchanged. To investigate the potential impact of Ang II on HG effects, the cells were co-incubated with angiotensin receptor inhibitors. Only co-incubation with 10-7 M losartan (an antagonist for type 1 angiotensin receptor, AT1R) attenuated the inhibitory effect of HG on albumin endocytosis, as well as megalin expression. Our findings contribute to understanding the genesis of tubular albuminuria observed in the early stages of DKD, which involves the activation of the Ang II/AT1R axis by HG.
Assuntos
Albuminas , Angiotensina II , Endocitose , Células Epiteliais , Glucose , Túbulos Renais Proximais , Receptor Tipo 1 de Angiotensina , Endocitose/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Túbulos Renais Proximais/efeitos dos fármacos , Angiotensina II/farmacologia , Glucose/metabolismo , Glucose/farmacologia , Receptor Tipo 1 de Angiotensina/metabolismo , Animais , Células Epiteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Humanos , Albuminas/metabolismo , Suínos , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Sistema Renina-Angiotensina/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular , Losartan/farmacologiaRESUMO
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.
Assuntos
Endocitose , Túbulos Renais Proximais , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Receptor 4 Toll-Like , Receptor 4 Toll-Like/metabolismo , Endocitose/efeitos dos fármacos , Humanos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/virologia , Animais , Glicoproteína da Espícula de Coronavírus/metabolismo , SARS-CoV-2/metabolismo , Células HEK293 , Suínos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosforilação , COVID-19/metabolismo , COVID-19/virologia , COVID-19/patologia , Albuminas/metabolismo , Células LLC-PK1 , Células Epiteliais/metabolismo , Células Epiteliais/virologiaRESUMO
Sodium-glucose cotransporter-2 inhibitors (SGLT2i) reduce blood pressure (BP) in patients with hypertension, yet the precise molecular mechanisms remain elusive. SGLT2i inhibits proximal tubule (PT) NHE3-mediated sodium reabsorption in normotensive rodents, yet no hypotensive effect is observed under this scenario. This study examined the effect of empagliflozin (EMPA) on renal tubular sodium transport in normotensive and spontaneously hypertensive rats (SHRs). It also tested the hypothesis that EMPA-mediated PT NHE3 inhibition in normotensive rats is associated with upregulation of distal nephron apical sodium transporters. EMPA administration for 14 days reduced BP in 12-wk-old SHRs but not in age-matched Wistar rats. PT NHE3 activity was inhibited by EMPA treatment in both Wistar and SHRs. In Wistar rats, EMPA increased NCC activity, mRNA expression, protein abundance, and phosphorylation levels, but not in SHRs. SHRs showed higher NKCC2 activity and an abundance of cleaved ENaC α and γ subunits compared with Wistar rats, none of which were affected by EMPA. Another set of male Wistar rats was treated with EMPA, the NCC inhibitor hydrochlorothiazide (HCTZ), and EMPA combined with HCTZ or vehicle for 14 days. In these rats, BP reduction was observed only with combined EMPA and HCTZ treatment, not with either drug alone. These findings suggest that NCC upregulation counteracts EMPA-mediated inhibition of PT NHE3 in male normotensive rats, maintaining their baseline BP. Moreover, the reduction of NHE3 activity without further upregulation of major apical sodium transporters beyond the PT may contribute to the BP-lowering effect of SGLT2i in experimental models and patients with hypertension.NEW & NOTEWORTHY This study suggests that reduced NHE3-mediated sodium reabsorption in the renal proximal tubule may account, at least in part, for the BP-lowering effect of SGLT2 inhibitors in the setting of hypertension. It also demonstrates that chronic treatment with SGLT2 inhibitors upregulates NCC activity, phosphorylation, and expression in the distal tubule of normotensive but not hypertensive rats. SGLT2 inhibitor-mediated upregulation of NCC seems crucial to counteract proximal tubule natriuresis in subjects with normal BP.
Assuntos
Compostos Benzidrílicos , Glucosídeos , Hipertensão , Ratos Endogâmicos SHR , Ratos Wistar , Inibidores do Transportador 2 de Sódio-Glicose , Trocador 3 de Sódio-Hidrogênio , Regulação para Cima , Animais , Masculino , Trocador 3 de Sódio-Hidrogênio/metabolismo , Trocador 3 de Sódio-Hidrogênio/genética , Trocador 3 de Sódio-Hidrogênio/antagonistas & inibidores , Hipertensão/tratamento farmacológico , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Glucosídeos/farmacologia , Compostos Benzidrílicos/farmacologia , Regulação para Cima/efeitos dos fármacos , Ratos , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/genética , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Rim/metabolismo , Rim/efeitos dos fármacosRESUMO
'On-a-chip' technology advances the development of physiologically relevant organ-mimicking architecture by integrating human cells into three-dimensional microfluidic devices. This method also establishes discrete functional units, faciliting focused research on specific organ components. In this study, we detail the development and assessment of a convoluted renal proximal tubule-on-a-chip (PT-on-a-chip). This platform involves co-culturing Renal Proximal Tubule Epithelial Cells (RPTEC) and Human Umbilical Vein Endothelial Cells (HUVEC) within a polydimethylsiloxane microfluidic device, crafted through a combination of 3D printing and molding techniques. Our PT-on-a-chip significantly reduced high glucose level, exhibited albumin uptake, and simulated tubulopathy induced by amphotericin B. Remarkably, the RPTEC:HUVEC co-culture exhibited efficient cell adhesion within 30 min on microchannels functionalized with plasma, 3-aminopropyltriethoxysilane, and type-I collagen. This approach significantly reduced the required incubation time for medium perfusion. In comparison, alternative methods such as plasma and plasma plus polyvinyl alcohol were only effective in promoting cell attachment to flat surfaces. The PT-on-a-chip holds great promise as a valuable tool for assessing the nephrotoxic potential of new drug candidates, enhancing our understanding of drug interactions with co-cultured renal cells, and reducing the need for animal experimentation, promoting the safe and ethical development of new pharmaceuticals.
Assuntos
Células Epiteliais , Túbulos Renais Proximais , Animais , Humanos , Células Endoteliais da Veia Umbilical Humana , Técnicas de Cocultura , Túbulos Renais Proximais/metabolismo , Dispositivos Lab-On-A-ChipRESUMO
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.
Assuntos
Diabetes Mellitus , Nefropatias Diabéticas , Camundongos , Suínos , Animais , Humanos , Túbulos Renais Proximais/metabolismo , Rim/metabolismo , Nefropatias Diabéticas/metabolismo , Sódio/metabolismo , Adenosina Trifosfatases/metabolismo , Diabetes Mellitus/metabolismoRESUMO
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.
Assuntos
Injúria Renal Aguda , Nanopartículas Metálicas , Camundongos , Animais , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Ouro/farmacologia , Túbulos Renais Proximais , Modelos Animais de Doenças , Proteinúria/metabolismo , Proteinúria/patologia , Albuminas/metabolismo , Injúria Renal Aguda/metabolismoRESUMO
Since the outbreak of COVID-19 disease, a bidirectional interaction between kidney disease and the progression of COVID-19 has been demonstrated. Kidney disease is an independent risk factor for mortality of patients with COVID-19 as well as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leading to the development of acute kidney injury (AKI) and chronic kidney disease (CKD) in patients with COVID-19. However, the detection of kidney damage in patients with COVID-19 may not occur until an advanced stage based on the current clinical blood and urinary examinations. Some studies have pointed out the development of subclinical acute kidney injury (subAKI) syndrome with COVID-19. This syndrome is characterized by significant tubule interstitial injury without changes in the estimated glomerular filtration rate. Despite the complexity of the mechanism(s) underlying the development of subAKI, the involvement of changes in the protein endocytosis machinery in proximal tubule (PT) epithelial cells (PTECs) has been proposed. This paper focuses on the data relating to subAKI and COVID-19 and the role of PTECs and their protein endocytosis machinery in its pathogenesis.
Assuntos
Injúria Renal Aguda , COVID-19 , Insuficiência Renal Crônica , Humanos , COVID-19/complicações , SARS-CoV-2 , Injúria Renal Aguda/metabolismo , Insuficiência Renal Crônica/metabolismo , Túbulos Renais Proximais/metabolismoRESUMO
INTRODUCTION: Amphotericin B (AmB), used for systemic fungal infections, has a limited clinical application because of its high nephrotoxicity. Natural antioxidant and anti-inflammatory substances have been widely studied for protection against drug-induced nephrotoxicity. α-Bisabolol (BIS) has demonstrated a nephroprotective effect on both in vitro and in vivo models. AIMS: The aim of this work was to evaluate the effect of BIS against AmB-induced nephrotoxicity in vitro. MATERIAL AND METHODS: LLC-MK2 cells were pre- and post-treated with non-toxic BIS concentrations and/or AmB IC50 (13.97 µM). Cell viability was assessed by MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)] assay. Flow cytometry analyses were used to assess cell death mechanism, production of reactive oxidative stress (ROS) and mitochondrial transmembrane potential. Kidney Injury Molecule-1 (KIM-1) levels were measured via ELISA. KEY FINDINGS: The present work showed that BIS pretreatment (125; 62.5 and 31.25 µM) increased cell viability when compared to the group treated only with AmB IC50. AmB treatment induced both necrosis (7-AAD-labeled cells) and late apoptosis (AnxV-labeled). BIS was able to prevent the occurrence of these events. These effects were associated with a decrease of ROS accumulation, improving transmembrane mitochondrial potential and protecting against tubular cell damage, highlighted by the inhibition of KIM-1 release after BIS treatment. SIGNIFICANCE: BIS presented a potential effect on model of renal cytotoxicity induced by AmB, bringing perspectives for the research of new nephroprotective agents.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Túbulos Renais Proximais/efeitos dos fármacos , Sesquiterpenos Monocíclicos/farmacologia , Anfotericina B/farmacologia , Anfotericina B/toxicidade , Animais , Antifúngicos/farmacologia , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Receptor Celular 1 do Vírus da Hepatite A/metabolismo , Rim/metabolismo , Túbulos Renais Proximais/metabolismo , Macaca mulatta , Sesquiterpenos Monocíclicos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/farmacologiaRESUMO
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.
Assuntos
Células Epiteliais/metabolismo , Retroalimentação Fisiológica , Túbulos Renais Proximais/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Albuminas/metabolismo , Animais , Biomarcadores , Endocitose , Células Epiteliais/efeitos dos fármacos , Imunofluorescência , Expressão Gênica , Túbulos Renais Proximais/citologia , Masculino , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Transporte Proteico , Transdução de Sinais/efeitos dos fármacosRESUMO
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.