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.

Synthetic angiotensin II peptide derivatives confer protection against cerebral and severe non-cerebral malaria in murine models.

Malaria can have severe long-term effects. Even after treatment with antimalarial drugs eliminates the parasite, survivors of cerebral malaria may suffer from irreversible brain damage, leading to cognitive deficits. Angiotensin II, a natural human peptide hormone that regulates blood pressure, has been shown to be active against Plasmodium spp., the etiologic agent of malaria. Here, we tested two Ang II derivatives that do not elicit vasoconstriction in mice: VIPF, a linear tetrapeptide, which constitutes part of the hydrophobic portion of Ang II; and Ang II-SS, a disulfide-bridged derivative. The antiplasmodial potential of both peptides was evaluated with two mouse models: an experimental cerebral malaria model and a mouse model of non-cerebral malaria. The latter consisted of BALB/c mice infected with Plasmodium berghei ANKA. The peptides had no effect on mean blood pressure and significantly reduced parasitemia in both mouse models. Both peptides reduced the SHIRPA score, an assay used to assess murine health and behavior. However, only the constrained derivative (Ang II-SS), which was also resistant to proteolytic degradation, significantly increased mouse survival. Here, we show that synthetic peptides derived from Ang II are capable of conferring protection against severe manifestations of malaria in mouse models while overcoming the vasoconstrictive side effects of the parent peptide.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Plasmodium falciparum maturation across the intra-erythrocytic cycle shifts the soft glassy viscoelastic properties of red blood cells from a liquid-like towards a solid-like behavior.

The mechanical properties of erythrocytes have been investigated by different techniques. However, there are few reports on how the viscoelasticity of these cells varies during malaria disease. Here, we quantitatively map the viscoelastic properties of Plasmodium falciparum-parasitized human erythrocytes. We apply new methodologies based on optical tweezers to measure the viscoelastic properties and defocusing microscopy to measure the erythrocyte height profile, the overall cell volume, and its form factor, a crucial parameter to convert the complex elastic constant into complex shear modulus. The storage and loss shear moduli are obtained for each stage of parasite maturation inside red blood cells, while the former increase, the latter decrease. Employing a soft glassy rheology model, we obtain the power-law exponent for the storage and loss shear moduli, characterizing the soft glassy features of red blood cells in each parasite maturation stage. Ring forms present a liquid-like behavior, with a slightly lower power-law exponent than healthy erythrocytes, whereas trophozoite and schizont stages exhibit increasingly solid-like behaviors. Finally, the surface elastic shear moduli, low-frequency surface viscosities, and shape recovery relaxation times all increase not only in a stage-dependent manner but also when compared to healthy red blood cells. Overall, the results call attention to the soft glassy characteristics of Plasmodium falciparum-parasitized erythrocyte membrane and may provide a basis for future studies to better understand malaria disease from a mechanobiological perspective.

CXCR4 and MIF are required for neutrophil extracellular trap release triggered by Plasmodium-infected erythrocytes.

Neutrophil extracellular traps (NETs) evolved as a unique effector mechanism contributing to resistance against infection that can also promote tissue damage in inflammatory conditions. Malaria infection can trigger NET release, but the mechanisms and consequences of NET formation in this context remain poorly characterized. Here we show that patients suffering from severe malaria had increased amounts of circulating DNA and increased neutrophil elastase (NE) levels in plasma. We used cultured erythrocytes and isolated human neutrophils to show that Plasmodium-infected red blood cells release macrophage migration inhibitory factor (MIF), which in turn caused NET formation by neutrophils in a mechanism dependent on the C-X-C chemokine receptor type 4 (CXCR4). NET production was dependent on histone citrullination by peptidyl arginine deiminase-4 (PAD4) and independent of reactive oxygen species (ROS), myeloperoxidase (MPO) or NE. In vitro, NETs functioned to restrain parasite dissemination in a mechanism dependent on MPO and NE activities. Finally, C57/B6 mice infected with P. berghei ANKA, a well-established model of cerebral malaria, presented high amounts of circulating DNA, while treatment with DNAse increased parasitemia and accelerated mortality, indicating a role for NETs in resistance against Plasmodium infection.

A high salt diet induces tubular damage associated with a pro-inflammatory and pro-fibrotic response in a hypertension-independent manner.

High salt diet (HSD), considered a public health problem worldwide, is associated with chronic degenerative diseases including renal diseases. However, little is known about the effects of HSD on renal function independently of the development of hypertension. To address the hypothesis that HSD induces renal injuries even without changes in blood pressure, BALB/c mice were fed for 7 days with chow with a high salt content (0.3-8%). Blood pressure did not change and there was a decrease in cortical (Na+ + K+)ATPase and NHE3 exchanger and an increase in renal fractional excretion of sodium. Positive correlations between Na+ intake or urinary sodium excretion with proteinuria were found. HSD did not change glomerular function and structure, but induced tubule-interstitial injury measured by an increase in collagen deposition, interstitial space and γ-GT activity, a marker of tubular injury. These effects were associated with a decrease in cortical albumin reabsorption and megalin expression. Similarly, the addition of NaCl 20 mM to the incubation medium of LLC-PK1 cells reduced megalin expression and albumin endocytosis indicating that HSD could have a direct effect on proximal tubule cells. Furthermore, tubule-interstitial injury was associated with pro-inflammatory and pro-fibrotic phenotypes with an increase in Th1 and Th17 phenotypes and a decrease in Tregs followed by increases in IL-6, -17, -10, TNF-α, IFN-γ and TGF-ß. Our results reveal a complex network involved in renal injuries induced by HSD independently of changes in blood pressure. These findings strengthen the importance of restriction of salt intake for the general population even for salt-resistant individuals.

Effect of cell geometry in the evaluation of erythrocyte viscoelastic properties.

The red blood cell membrane-cytoskeleton is a complex structure mainly responsible for giving the cell rigidity and shape. It also provides the erythrocyte with the ability to pass through narrow capillaries of the vertebrate blood circulatory system. Although the red blood cell viscoelastic properties have been extensively studied, reported experimental data differ by up to three orders of magnitude. This could be attributed to the natural cell variability, to the different techniques employed, and also to the models used for the cell response, which are highly dependent on cell geometry. Here, we use two methodologies based on optical tweezers to investigate the viscoelastic behavior of healthy human red blood cells, one applying small cell deformations (microrheology) and another imposing large deformations (tether extraction). We also establish a defocusing microscopy-based method to characterize the cell geometry and thus the erythrocyte form factor, an essential parameter that allows comparisons among the viscoelastic properties at different conditions. Moreover, for small deformations, a soft glassy rheology model is used to discuss the results, while for large deformations two surface shear moduli and one surface viscosity are determined, together with the surface tension and bending modulus of the erythrocyte membrane lipid component. We also show that F-actin is not detected in tethers, although the erythrocyte membrane has physical properties like those of other adherent cells, known to have tethers containing F-actin inside. Altogether, our results show good agreement with the reported literature and we argue that, to properly compare the viscoelastic properties of red blood cells in different situations, the task of cell geometry characterization must be accomplished. This may be especially important when the influence of agents, like the malaria parasite, induces changes in both the geometry and chemical constituents of the erythrocyte membrane. Together, the new methodologies and procedures used in this study would allow the erythrocyte community to better explore the mechanical behavior of red blood cells and may be useful to characterize erythrocyte viscoelasticity changes in several blood diseases.