Kidney Disease and Proteomics: A Recent Overview of a Useful Tool for Improving Early Diagnosis.

Kidney disease is a critical and potentially life-threatening degenerative condition that poses a significant global public health challenge due to its elevated rates of morbidity and mortality. It manifests primarily in two distinct clinical forms: acute kidney injury (AKI) and chronic kidney disease (CKD). The development of these conditions hinges on a multitude of factors, including the etiological agents and the presence of coexisting medical conditions. Despite disparities in their underlying pathogenic mechanisms, both AKI and CKD can progress to end-stage kidney disease (ESKD). This advanced stage is characterized by organ failure and its associated complications, greatly increasing the risk of mortality. There is an urgent need to delve into the pathogenic mechanisms underlying these diseases and to identify novel biomarkers that can facilitate earlier diagnosis. Such early detection is crucial for enhancing the efficacy of therapy and impeding disease progression. In this context, proteomic approaches have emerged as invaluable tools for uncovering potential new markers of different pathological conditions, including kidney diseases. In this chapter, we overview the recent discoveries achieved through diverse proteomic techniques aimed at identifying novel molecules that may play a pivotal role in kidney diseases such as diabetic kidney disease (DKD), IgA nephropathy (IgAN), CKD of unknown origin (CKDu), autosomal dominant polycystic kidney disease (ADPKD), lupus nephritis (LN), hypertensive nephropathy (HN), and COVID-19-associated acute kidney injury (COVID-AKI).

O-GlcNAcylation in Renal (Patho)Physiology.

Kidneys maintain internal milieu homeostasis through a well-regulated manipulation of body fluid composition. This task is performed by the correlation between structure and function in the nephron. Kidney diseases are chronic conditions impacting healthcare programs globally, and despite efforts, therapeutic options for its treatment are limited. The development of chronic degenerative diseases is associated with changes in protein O-GlcNAcylation, a post-translation modification involved in the regulation of diverse cell function. O-GlcNAcylation is regulated by the enzymatic balance between O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) which add and remove GlcNAc residues on target proteins, respectively. Furthermore, the hexosamine biosynthetic pathway provides the substrate for protein O-GlcNAcylation. Beyond its physiological role, several reports indicate the participation of protein O-GlcNAcylation in cardiovascular, neurodegenerative, and metabolic diseases. In this review, we discuss the impact of protein O-GlcNAcylation on physiological renal function, disease conditions, and possible future directions in the field.

Subclinical Acute Kidney Injury in COVID-19: Possible Mechanisms and Future Perspectives.

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.

Albumin Expands Albumin Reabsorption Capacity in Proximal Tubule Epithelial Cells through a Positive Feedback Loop between AKT and Megalin.

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.

PKB is a central molecule in the modulation of Na+-ATPase activity by albumin in renal proximal tubule cells.

Evidence points to a possible role of tubular sodium reabsorption in worsening renal injury. Proximal tubule (PT) albumin overload is a critical process in the development of tubule-interstitial injury (TII), and consequently in progression of renal disease. We studied the possible correlation between changes in albumin concentration in the lumen of PT with modification of Na+-ATPase activity. An albumin overload animal model and LLC-PK1 cells as a model of PT cells were used. Albumin overload was induced by intraperitoneal injection of BSA in 14-week-old male Wistar rats. An increase in sodium clearance, fractional excretion of sodium, proteinuria, ratio between urinary protein and creatinine, and albuminuria were observed. These observations indicate that there could be a correlation between an increase in albumin in the lumen of PTs and renal sodium excretion. We observed that the activity of both Na+-ATPase and (Na++K+)ATPase decreased in the renal cortex of an albumin overload animal model. Using LLC-PK1 cells as a model of PT cells, inhibition of Na+-ATPase activity was observed with higher albumin concentrations, similar to that observed in the animal model. The inhibition of protein kinase B by higher albumin concentration was found to be a critical step in the inhibition of Na+-ATPase activity. Interestingly, activation of the ERK1/2/mTORC1/S6K pathway was required for protein kinase B inhibition. This mechanism leads to a decrease in protein kinase C activity and, consequently to inhibition of Na+-ATPase activity. Taken together, our results indicate that the molecular mechanism underlying the modulation of PT Na+-ATPase activity by albumin overload involves activation of the ERK1/2/mTORC1/S6K pathway, which leads to inhibition of the mTORC2/PKB/PKC pathway. Our findings contribute to better understanding regarding handing of renal Na+ induced by albumin overload in the lumen of PTs and, consequently, in the progression of renal disease.

Uroguanylin modulates (Na++K+)ATPase in a proximal tubule cell line: Interactions among the cGMP/protein kinase G, cAMP/protein kinase A, and mTOR pathways.

BACKGROUND: The natriuretic effect of uroguanylin (UGN) involves reduction of proximal tubule (PT) sodium reabsorption. However, the target sodium transporters as well as the molecular mechanisms involved in these processes remain poorly understood. METHODS: To address the effects of UGN on PT (Na(+)+K(+))ATPase and the signal transduction pathways involved in this effect, we used LLC-PK1 cells. The effects of UGN were determined through ouabain-sensitive ATP hydrolysis and immunoblotting assays during different experimental conditions. RESULTS: We observed that UGN triggers cGMP/PKG and cAMP/PKA pathways in a sequential way. The activation of PKA leads to the inhibition of mTORC2 activity, PKB phosphorylation at S473, PKB activity and, consequently, a decrease in the mTORC1/S6K pathway. The final effects are decreased expression of the α1 subunit of (Na(+)+K(+))ATPase and inhibition of enzyme activity. CONCLUSIONS: These results suggest that the molecular mechanism of action of UGN on sodium reabsorption in PT cells is more complex than previously thought. We propose that PKG-dependent activation of PKA leads to the inhibition of the mTORC2/PKB/mTORC1/S6K pathway, an important signaling pathway involved in the maintenance of the PT sodium pump expression and activity. GENERAL SIGNIFICANCE: The current results expand our understanding of the signal transduction pathways involved in the overall effect of UGN on renal sodium excretion.

Mis-regulation of mammalian target of rapamycin (mTOR) complexes induced by albuminuria in proximal tubules.

High albumin concentrations in the proximal tubule of the kidney causes tubulointerstitial injury, but how this process occurs is not completely known. To address the signal transduction pathways mis-regulated in renal injury, we studied the modulation of mammalian target of rapamycin (mTOR) complexes by physiologic and pathophysiologic albumin concentrations in proximal tubule cells. Physiologic albumin concentrations activated the PI3K/mTORC2/PKB/mTORC1/S6 kinase (S6K) pathway, but pathophysiologically high albumin concentrations overactivated mTORC1 and inhibited mTORC2 activity. This control process involved the activation of ERK1/2, which promoted the inhibition of TSC2 and activation of S6K. Furthermore, S6K was crucial to promoting the over activation of mTORC1 and inhibition of mTORC2. Megalin expression at the luminal membrane is reduced by high concentrations of albumin. In addition, knockdown of megalin mimicked all the effects of pathophysiologic albumin concentrations, which disrupt normal signal transduction pathways and lead to an overactivation of mTORC1 and inhibition of mTORC2. These data provide new perspectives for understanding the molecular mechanisms behind the effects of albumin on the progression of renal disease.

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.

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.

Inhibition of SGLT2 co-transporter by dapagliflozin ameliorates tubular proteinuria and tubule-interstitial injury at the early stage of diabetic kidney disease.

Diabetic kidney disease (DKD) is characterized by progressive impairment of kidney function. It has been postulated that tubule-interstitial injury, associated with tubular albuminuria, precedes glomerular damage in the early stage of DKD. Here, we wanted to determine if the development of tubule-interstitial injury at the early stage of DKD implies modulation of megalin-mediated protein reabsorption in proximal tubule epithelial cells (PTECs) by SGLT2-dependent high glucose influx. Rats with streptozotocin (STZ)-induced diabetes were treated or not with dapagliflozin (DAPA) for 8 weeks. Four experimental groups were generated: (1) CONT, control; (2) DAPA, rats treated with DAPA; (3) STZ, diabetic rats; (4) STZ + DAPA, diabetic rats treated with DAPA. No changes in glomerular structure and function were observed. The STZ group presented proteinuria and albuminuria associated with an increase in the fractional excretion of proteins. A positive correlation between glycemia and proteinuria was found. These phenomena were linked to a decrease in luminal and total megalin expression and, consequently, in albumin reabsorption in PTECs. We also observed tubule-interstitial injury characterized by an increase in urinary tubular injury biomarkers and changes in tubular histomorphometry parameters. In addition, inverse correlations were found between cortical albumin uptake and tubule-interstitial injury or glycemia. All these modifications were attenuated in the STZ + DAPA group. These results suggest that SGLT2-dependent high glucose influx into PTECs promotes a harmful effect on the PTECs, leading to the development of tubular albuminuria and tubule-interstitial injury preceding glomerular damage. These results expand current knowledge on the renoprotective effects of gliflozins.

Rapamycin treatment induces tubular proteinuria: role of megalin-mediated protein reabsorption.

Introduction: Rapamycin is an immunosuppressor that acts by inhibiting the serine/threonine kinase mechanistic target of rapamycin complex 1. Therapeutic use of rapamycin is limited by its adverse effects. Proteinuria is an important marker of kidney damage and a risk factor for kidney diseases progression and has been reported in patients and animal models treated with rapamycin. However, the mechanism underlying proteinuria induced by rapamycin is still an open matter. In this work, we investigated the effects of rapamycin on parameters of renal function and structure and on protein handling by proximal tubule epithelial cells (PTECs). Methods: Healthy BALB/c mice were treated with 1.5 mg/kg rapamycin by oral gavage for 1, 3, or 7 days. At the end of each treatment, the animals were kept in metabolic cages and renal function and structural parameters were analyzed. LLC-PK1 cell line was used as a model of PTECs to test specific effect of rapamycin. Results: Rapamycin treatment did not change parameters of glomerular structure and function. Conversely, there was a transient increase in 24-h proteinuria, urinary protein to creatinine ratio (UPCr), and albuminuria in the groups treated with rapamycin. In accordance with these findings, rapamycin treatment decreased albumin-fluorescein isothiocyanate uptake in the renal cortex. This effect was associated with reduced brush border expression and impaired subcellular distribution of megalin in PTECs. The effect of rapamycin seems to be specific for albumin endocytosis machinery because it did not modify renal sodium handling or (Na++K+)ATPase activity in BALB/c mice and in the LLC-PK1 cell line. A positive Pearson correlation was found between megalin expression and albumin uptake while an inverse correlation was shown between albumin uptake and UPCr or 24-h proteinuria. Despite its effect on albumin handling in PTECs, rapamycin treatment did not induce tubular injury measured by interstitial space and collagen deposition. Conclusion: These findings suggest that proteinuria induced by rapamycin could have a tubular rather than a glomerular origin. This effect involves a specific change in protein endocytosis machinery. Our results open new perspectives on understanding the undesired effect of proteinuria generated by rapamycin.

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.

(Na+ + K+)-ATPase is a target for phosphoinositide 3-kinase/protein kinase B and protein kinase C pathways triggered by albumin.

In recent decades, evidence has confirmed the crucial role of albumin in the progression of renal disease. However, the possible role of signaling pathways triggered by physiologic concentrations of albumin in the modulation of proximal tubule (PT) sodium reabsorption has not been considered. In the present work, we have shown that a physiologic concentration of albumin increases the expression of the α1 subunit of (Na(+) + K(+))-ATPase in LLC-PK1 cells leading to an increase in enzyme activity. This process involves the sequential activation of PI3K/protein kinase B and protein kinase C pathways promoting inhibition of protein kinase A. This integrative network is inhibited when albumin concentration is increased, similar to renal disease, leading to a decrease in the α1 subunit of (Na(+) + K(+))-ATPase expression. Together, the results indicate that variation in albumin concentration in PT cells has an important effect on PT sodium reabsorption and, consequently, on renal sodium excretion.

SARS-CoV-2 spike protein inhibits megalin-mediated albumin endocytosis in proximal tubule epithelial cells.

Patients with COVID-19 have high prevalence of albuminuria which is used as a marker of progression of renal disease and is associated with severe COVID-19. We hypothesized that SARS-CoV-2 spike protein (S protein) could modulate albumin handling in proximal tubule epithelial cells (PTECs) and, consequently contribute to the albuminuria observed in patients with COVID-19. In this context, the possible effect of S protein on albumin endocytosis in PTECs was investigated. Two PTEC lines were used: HEK-293A and LLC-PK1. Incubation of both cell types with S protein for 16 h inhibited albumin uptake at the same magnitude. This effect was associated with canonical megalin-mediated albumin endocytosis because: (1) DQ-albumin uptake, a marker of the lysosomal degradation pathway, was reduced at a similar level compared with fluorescein isothiocyanate (FITC)-albumin uptake; (2) dextran-FITC uptake, a marker of fluid-phase endocytosis, was not changed; (3) cell viability and proliferation were not changed. The inhibitory effect of S protein on albumin uptake was only observed when it was added at the luminal membrane, and it did not involve the ACE2/Ang II/AT1R axis. Although both cells uptake S protein, it does not seem to be required for modulation of albumin endocytosis. The mechanism underlying the inhibition of albumin uptake by S protein encompasses a decrease in megalin expression without changes in megalin trafficking and stability. These results reveal a possible mechanism to explain the albuminuria observed in patients with COVID-19.

The monoterpene 1,8-cineole prevents cerebral edema in a murine model of severe malaria.

1,8-Cineole is a naturally occurring compound found in essential oils of different plants and has well-known anti-inflammatory and antimicrobial activities. In the present work, we aimed to investigate its potential antimalarial effect, using the following experimental models: (1) the erythrocytic cycle of Plasmodium falciparum; (2) an adhesion assay using brain microvascular endothelial cells; and (3) an experimental cerebral malaria animal model induced by Plasmodium berghei ANKA infection in susceptible mice. Using the erythrocytic cycle of Plasmodium falciparum, we characterized the schizonticidal effect of 1,8-cineole. This compound decreased parasitemia in a dose-dependent manner with a half maximal inhibitory concentration of 1045.53 ± 63.30 µM. The inhibitory effect of 972 µM 1,8-cineole was irreversible and independent of parasitemia. Moreover, 1,8-cineole reduced the progression of intracellular development of the parasite over 2 cycles, inducing important morphological changes. Ultrastructure analysis revealed a massive loss of integrity of endomembranes and hemozoin crystals in infected erythrocytes treated with 1,8-cineole. The monoterpene reduced the adhesion index of infected erythrocytes to brain microvascular endothelial cells by 60%. Using the experimental cerebral malaria model, treatment of infected mice for 6 consecutive days with 100 mg/kg/day 1,8-cineole reduced cerebral edema with a 50% reduction in parasitemia. Our data suggest a potential antimalarial effect of 1,8-cineole with an impact on the parasite erythrocytic cycle and severe disease.

Megalin-mediated albumin endocytosis in renal proximal tubules is involved in the antiproteinuric effect of angiotensin II type 1 receptor blocker in a subclinical acute kidney injury animal model.

BACKGROUND: Tubule-interstitial injury (TII) is one of the mechanisms involved in the progression of renal diseases with progressive proteinuria. Angiotensin II (Ang II) type 1 receptor blockers (ARBs) have been successfully used to treat renal diseases. However, the mechanism correlating treatment with ARBs and proteinuria is not completely understood. The hypothesis that the anti-proteinuric effect of losartan is associated with the modulation of albumin endocytosis in PT epithelial cells (PTECs) was assessed. METHODS: We used a subclinical acute kidney injury animal model (subAKI) and LLC-PK1 cells, a model of PTECs. RESULTS: In subAKI, PT albumin overload induced TII development, measured by: (1) increase in urinary lactate dehydrogenase and γ-glutamyltranspeptidase activity; (2) proteinuria associated with impairment in megalin-mediated albumin reabsorption; (3) increase in luminal and interstitial space in tubular cortical segments. These effects were avoided by treating the animals with losartan, an ARB. Using LLC-PK1 cells, we observed that: (1) 20 mg/mL albumin increased the secretion of Ang II and decreased megalin-mediated albumin endocytosis; (2) the effects of Ang II and albumin were abolished by 10-8 M losartan; (3) MEK/ERK pathway is the molecular mechanism underlying the Ang II-mediated inhibitory effect of albumin on PT albumin endocytosis. CONCLUSION: Our results show that PT megalin-mediated albumin endocytosis is a possible target during the treatment of renal diseases patients with ARB. GENERAL SIGNIFICANCE: The findings obtained in the present work represents a step forward to the current knowledge on about the role of ARBs in the treatment of renal disease.

High Doses of Essential Oil of Croton Zehntneri Induces Renal Tubular Damage.

The essential oil of Croton zehntneri (EOCZ) and its major compounds are known to have several biological activities. However, some evidence shows potential toxic effects of high doses of EOCZ (>300 mg/kg) in amphibian and human kidneys. The aim of the present work was to investigate the effects on renal function of EOCZ at 300 mg/kg/day in healthy Swiss mice and a subclinical acute kidney injury (subAKI) animal model, which presents tubule-interstitial injury (TII). Four experimental groups were generated: (1) CONT group (control); (2) EOCZ, mice treated with EOCZ; (3) subAKI; (4) subAKI+EOCZ, subAKI treated simultaneously with EOCZ. EOCZ treatment induced TII measured by increases in (1) proteinuria; (2) cortical tubule-interstitial space; (3) macrophage infiltration; (4) collagen deposition. A decrease in tubular sodium reabsorption was also observed. These results were similar and nonadditive to those observed in the subAKI group. These data suggest that treatment with EOCZ at higher concentrations induces TII in mice, which could be mediated by protein overload in the proximal tubule.

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.

Eugenol disrupts Plasmodium falciparum intracellular development during the erythrocytic cycle and protects against cerebral malaria.

BACKGROUND: Malaria is a parasitic disease that compromises the human host. Currently, control of the Plasmodium falciparum burden is centered on artemisinin-based combination therapies. However, decreased sensitivity to artemisinin and derivatives has been reported, therefore it is important to identify new therapeutic strategies. METHOD: We used human erythrocytes infected with P. falciparum and experimental cerebral malaria (ECM) animal model to assess the potential antimalarial effect of eugenol, a component of clove bud essential oil. RESULTS: Plasmodium falciparum cultures treated with increasing concentrations of eugenol reduced parasitemia in a dose-dependent manner, with IC50 of 532.42 ± 29.55 µM. This effect seems to be irreversible and maintained even in the presence of high parasitemia. The prominent effect of eugenol was detected in the evolution from schizont to ring forms, inducing important morphological changes, indicating a disruption in the development of the erythrocytic cycle. Aberrant structural modification was observed by electron microscopy, showing the separation of the two nuclear membrane leaflets as well as other subcellular membranes, such as from the digestive vacuole. Importantly, in vivo studies using ECM revealed a reduction in blood parasitemia and cerebral edema when mice were treated for 6 consecutive days upon infection. CONCLUSIONS: These data suggest a potential effect of eugenol against Plasmodium sp. with an impact on cerebral malaria. GENERAL SIGNIFICANCE: Our results provide a rational basis for the use of eugenol in therapeutic strategies to the treatment of malaria.