Glomerular Ultrafiltration Research Articles

Design and simulation of a microfluidics-based artificial glomerular ultrafiltration unit to reduce cell-induced fouling.

The microfluidic-based Glomerulus-on-Chips (GoC) are mostly cell based, that is, 3D cell culture techniques are used to culture glomerular cells in order to mimic glomerular ultrafiltration. These chips require high maintenance to keep cell viability intact. There have been some approaches to build non-cell-based GoCs but many of these approaches have the drawback of membrane fouling. This article presents a structural design and simulation study of a dialysate free microfluidic channel for replicating the function of the human glomerular filtration barrier. The key advancement of the current work is addressing the fouling issue by combining a pre-filter to eliminate cellular components and performing filtration on the blood plasma. The Laminar Flow Mixture Model in COMSOL Multiphysics 5.6 has been utilized to simulate the behavior of blood flow in the microchannels. The geometrical effect of microchannels on the separation of the filtrate was investigated. The velocity at the inlet of the microchannel and pore size of the filtration membrane are varied to see the change in outflow and filtration fraction. The efficiency of the device is calculated in terms of the filtration fraction (FF%) formed. Simulation results show that the filtrate obtained is ~20% of the plasma flow rate in the channel, which resembles the glomerular filtration fraction. Given that it is not dependent on the functionality of grown cells, the proposed device is anticipated to have a longer lifespan due to its non-cell-based design. The device's cost can be reduced by avoiding cell cultivation inside of it. It can be integrated as a glomerular functional unit with other units of kidney model to build a fully developed artificial kidney.

Demystifying Renal Tubular Disorders in children : A simplified approach

Renal tubular disorders essentially include all disorders of the highly specialized channels of the renal tubular system, from the proximal convoluted tubule to the cortical and medullary collecting ducts. This tubular system is responsible for reabsorption of 99% of glomerular ultrafiltrate, which contains a large amount of fluid (approximately 180 L/day) as well as most electrolytes and many metabolites such as glucose (G), amino acids, bicarbonate (HCO3-), phosphate (PO4 3-), and low-molecular-weight proteins and is essential for maintaining fluid, electrolyte, and acid–base balance. In this review, we will briefly discuss when to suspect a renal tubular disorder, a simplified algorithm for evaluation, and specific mutations of a few common disorders.

Receptor-associated protein impairs ligand binding to megalin and megalin-dependent endocytic flux in proximal tubule cells.

Proximal tubule (PT) cells retrieve albumin and a broad array of other ligands from the glomerular ultrafiltrate. Efficient uptake of albumin requires PT expression of both megalin and cubilin receptors. Although most proteins engage cubilin selectively, megalin is required to maintain robust flux through the apical endocytic pathway. Receptor-associated protein (RAP) is a chaperone that directs megalin to the cell surface, and recombinant RAP dramatically inhibits the uptake of numerous megalin and cubilin ligands. The mechanism by which this occurs has been suggested to involve competitive inhibition of ligand binding and/or conformational changes in megalin that prevent interaction with ligands and/or with cubilin. To discriminate between these possibilities, we determined the effect of RAP on endocytosis of albumin, which binds to cubilin and megalin receptors with high and low affinity, respectively. Uptake was quantified in opossum kidney (OK) cells and in megalin or cubilin (Cubn) knockout (KO) clones. Surprisingly, RAP inhibited fluid-phase uptake in addition to receptor-mediated uptake in OK cells and Cubn KO cells but had no effect on endocytosis when megalin was absent. The apparent Ki for RAP inhibition of albumin uptake was 10-fold higher in Cubn KO cells compared with parental OK cells. We conclude that in addition to its predicted high-affinity competition for ligand binding to megalin, the primary effect of RAP on PT cell endocytosis is to globally dampen megalin-dependent endocytic flux. Our data explain the complex effects of RAP on binding and uptake of filtered proteins and reveal a novel role in modulating endocytosis in PT cells.NEW & NOTEWORTHY Receptor-associated protein inhibits binding and uptake of all known endogenous ligands by megalin and cubilin receptors via unknown mechanism(s). Here, we took advantage of recently generated knockout cell lines to dissect the effect of this protein on megalin- and cubilin-mediated endocytosis. Our study reveals a novel role for receptor-associated protein in blocking megalin-stimulated endocytic uptake of fluid-phase markers and receptor-bound ligands in proximal tubule cells in addition to its direct effect on ligand binding to megalin receptors.

Loss of S1P Lyase Expression in Human Podocytes Causes a Reduction in Nephrin Expression That Involves PKCδ Activation.

Sphingosine 1-phosphate (S1P) lyase (SPL, Sgpl1) is an ER-associated enzyme that irreversibly degrades the bioactive lipid, S1P, and thereby regulates multiple cellular functions attributed to S1P. Biallelic mutations in the human Sglp1 gene lead to a severe form of a particular steroid-resistant nephrotic syndrome, suggesting that the SPL is critically involved in maintaining the glomerular ultrafiltration barrier, which is mainly built by glomerular podocytes. In this study, we have investigated the molecular effects of SPL knockdown (kd) in human podocytes to better understand the mechanism underlying nephrotic syndrome in patients. A stable SPL-kd cell line of human podocytes was generated by the lentiviral shRNA transduction method and was characterized for reduced SPL mRNA and protein levels and increased S1P levels. This cell line was further studied for changes in those podocyte-specific proteins that are known to regulate the ultrafiltration barrier. We show here that SPL-kd leads to the downregulation of the nephrin protein and mRNA expression, as well as the Wilms tumor suppressor gene 1 (WT1), which is a key transcription factor regulating nephrin expression. Mechanistically, SPL-kd resulted in increased total cellular protein kinase C (PKC) activity, while the stable downregulation of PKCδ revealed increased nephrin expression. Furthermore, the pro-inflammatory cytokine, interleukin 6 (IL-6), also reduced WT1 and nephrin expression. In addition, IL-6 caused increased PKCδ Thr505 phosphorylation, suggesting enzyme activation. Altogether, these data demonstrate that nephrin is a critical factor downregulated by the loss of SPL, which may directly cause podocyte foot process effacement as observed in mice and humans, leading to albuminuria, a hallmark of nephrotic syndrome. Furthermore, our in vitro data suggest that PKCδ could represent a new possible pharmacological target for the treatment of a nephrotic syndrome induced by SPL mutations.

Cadmium-Induced Proteinuria: Mechanistic Insights from Dose-Effect Analyses.

Cadmium (Cd) is a toxic metal that accumulates in kidneys, especially in the proximal tubular epithelial cells, where virtually all proteins in the glomerular ultrafiltrate are reabsorbed. Here, we analyzed archived data on the estimated glomerular filtration rate (eGFR) and excretion rates of Cd (ECd), total protein (EProt), albumin (Ealb), β2-microglobulin (Eβ2M), and α1-microglobulin (Eα1M), which were recorded for residents of a Cd contamination area and a low-exposure control area of Thailand. Excretion of Cd and all proteins were normalized to creatinine clearance (Ccr) as ECd/Ccr and EProt/Ccr to correct for differences among subjects in the number of surviving nephrons. Low eGFR was defined as eGFR ≤ 60 mL/min/1.73 m2, while proteinuria was indicted by EPro/Ccr ≥ 20 mg/L of filtrate. EProt/Ccr varied directly with ECd/Ccr (β = 0.263, p < 0.001) and age (β = 0.252, p < 0.001). In contrast, eGFR values were inversely associated with ECd/Ccr (β = -0.266, p < 0.001) and age (β = -0.558, p < 0.001). At ECd/Ccr > 8.28 ng/L of filtrate, the prevalence odds ratios for proteinuria and low eGFR were increased 4.6- and 5.1-fold, respectively (p < 0.001 for both parameters). Thus, the eGFR and tubular protein retrieval were both simultaneously diminished by Cd exposure. Of interest, ECd/Ccr was more closely correlated with EProt/Ccr (r = 0.507), Eβ2M (r = 0.430), and Eα1M/Ccr (r = 0.364) than with EAlb/Ccr (r = 0.152). These data suggest that Cd may differentially reduce the ability of tubular epithelial cells to reclaim proteins, resulting in preferential reabsorption of albumin.

Nitric-Oxide-Mediated Signaling in Podocyte Pathophysiology.

Nitric oxide (NO) is a potent signaling molecule involved in many physiological and pathophysiological processes in the kidney. NO plays a complex role in glomerular ultrafiltration, vasodilation, and inflammation. Changes in NO bioavailability in pathophysiological conditions such as hypertension or diabetes may lead to podocyte damage, proteinuria, and rapid development of chronic kidney disease (CKD). Despite the extensive data highlighting essential functions of NO in health and pathology, related signaling in glomerular cells, particularly podocytes, is understudied. Several reports indicate that NO bioavailability in glomerular cells is decreased during the development of renal pathology, while restoring NO level can be beneficial for glomerular function. At the same time, the compromised activity of nitric oxide synthase (NOS) may provoke the formation of peroxynitrite and has been linked to autoimmune diseases such as systemic lupus erythematosus. It is known that the changes in the distribution of NO sources due to shifts in NOS subunits expression or modifications of NADPH oxidases activity may be linked to or promote the development of pathology. However, there is a lack of information about the detailed mechanisms describing the production and release of NO in the glomerular cells. The interaction of NO and other reactive oxygen species in podocytes and how NO-calcium crosstalk regulates glomerular cells’ function is still largely unknown. Here, we discuss recent reports describing signaling, synthesis, and known pathophysiological mechanisms mediated by the changes in NO homeostasis in the podocyte. The understanding and further investigation of these essential mechanisms in glomerular cells will facilitate the design of novel strategies to prevent or manage health conditions that cause glomerular and kidney damage.

Ceramide-1-Phosphate as a Potential Regulator of the Second Sodium Pump from Kidney Proximal Tubules by Triggering Distinct Protein Kinase Pathways in a Hierarchic Way.

Kidney proximal tubules are a key segment in the reabsorption of solutes and water from the glomerular ultrafiltrate, an essential process for maintaining homeostasis in body fluid compartments. The abundant content of Na+ in the extracellular fluid determines its importance in the regulation of extracellular fluid volume, which is particularly important for different physiological processes including blood pressure control. Basolateral membranes of proximal tubule cells have the classic Na+ + K+-ATPase and the ouabain-insensitive, K+-insensitive, and furosemide-sensitive Na+-ATPase, which participate in the active Na+ reabsorption. Here, we show that nanomolar concentrations of ceramide-1 phosphate (C1P), a bioactive sphingolipid derived in biological membranes from different metabolic pathways, promotes a strong inhibitory effect on the Na+-ATPase activity (C1P50 ≈ 10 nM), leading to a 72% inhibition of the second sodium pump in the basolateral membranes. Ceramide-1-phosphate directly modulates protein kinase A and protein kinase C, which are known to be involved in the modulation of ion transporters including the renal Na+-ATPase. Conversely, we did not observe any effect on the Na+ + K+-ATPase even at a broad C1P concentration range. The significant effect of ceramide-1-phosphate revealed a new potent physiological and pathophysiological modulator for the Na+-ATPase, participating in the regulatory network involving glycero- and sphingolipids present in the basolateral membranes of kidney tubule cells.

Identification of VEGF Signaling Inhibition-Induced Glomerular Injury in Rats through Site-Specific Urinary Biomarkers.

Cancer therapies targeting the vascular endothelial growth factor (VEGF) signaling pathway can lead to renal damage by disrupting the glomerular ultrafiltration apparatus. The objective of the current study was to identify sensitive biomarkers for VEGF inhibition-induced glomerular changes in rats. Male Sprague-Dawley rats were administered an experimental VEGF receptor (VEGFR) inhibitor, ABT-123, for seven days to investigate the correlation of several biomarkers with microscopic and ultrastructural changes. Glomeruli obtained by laser capture microdissection were also subjected to gene expression analysis to investigate the underlying molecular events of VEGFR inhibition in glomerulus. ABT-123 induced characteristic glomerular ultrastructural changes in rats, including fusion of podocyte foot processes, the presence of subendothelial electron-dense deposits, and swelling and loss of fenestrations in glomerular endothelium. The subtle morphological changes cannot be detected with light microscopy or by changes in standard clinical chemistry and urinalysis. However, urinary albumin increased 44-fold as early as Day three. Urinary β2-microglobulin levels were also increased. Other urinary biomarkers that are typically associated with tubular injury were not significantly impacted. Such patterns in urinary biomarkers can provide valuable diagnostic insight to VEGF inhibition therapy-induced glomeruli injuries.

Glomerular Size in Transplanted Kidneys: Does Size Matter?

Glomerular Size in Transplanted Kidneys: Does Size Matter?

The Role of Obesity in the Development of Kidney and Glomerular Ultrafiltration Barrier Injuries: Leptin Contributions

Introduction Obesity is associated with multiple risk factors to the development of comorbidities such as kidney injuries. Lesions in the glomerular ultrafiltration barrier may cause impairment of the kidney filtration process. Nephrin is one of the main components of the glomerular ultrafiltration barrier, so changes in the expression of this protein indicate impaired renal function. Thus, the aim of this study is to investigate the role of obesity in the development of kidney and glomerular ultrafiltration barrier injuries. The contribution of leptin to kidney function in this context was also evaluated. Methods The protocols are in agreement with CEUA (nº 13/2017). Twenty-four mice aged 18 weeks were used: Six C57BL / 6J were assigned to the lean control group (CTL) and 18 ob/ob (provided by Prof. Dr. José Donato Junior from ICB-USP) were randomly assigned to obese control group (ob/ob) and obese treated with leptin group (ob/ob - lep). Leptin treatment was performed through osmotic mini-pump (model 1004, Alzet) insertion. Mini-pumps were filled with a solution containing leptin at a dose of 3.5 μg/day for 42 days. During the treatment period food intake was monitored daily. Weight gain and blood glucose were monitored weekly. At the end of the treatment period, the animals were kept in metabolic cages for 24 hours to evaluate water intake and urine collection. Ultimately, the animals were submitted to euthanasia by exsanguination. Renal function was assessed by plasma concentration of creatinine and urea, and urinary albumin excretion. Renal tissue was used to evaluate gene expression by qPCR, histological and immunofluorescence analysis. Results Plasma leptin measurements confirm that ob/ob animals did not produce leptin and that introduction of exogenous leptin elevated the levels of this hormone to values close to control. Ob/ob - lep group had a reduction in food intake and body weight when compared to ob/ob group, this reduction led to values of food intake and body weight similar to CTL group. There were no changes in water intake, urine flow and blood glucose among groups. There were also no changes in plasma creatinine values among groups, however, in the ob/ob group, there was an increase in serum urea and leptin treatment partially reversed this parameter to values closer to CTL. In ob/ob animals, there was an increase in urinary albumin excretion and leptin treatment lowered this parameter to values closer to CTL group. Inflammation of the renal tissue in obese animals was confirmed by increased IL-6 mRNA expression, leptin had no effect at this parameter. There was no significant difference in the expression of mRNA to MCP1 between groups. However, there is a tendency to increasing that gene's expression in obese animals and leptin treatment reduces that parameter back to control. In 42 days of protocol, there was no glomerular hypertrophy in the ob/ob group, however, there was a reduction in the expression of nephrin and leptin treatment did not reverse this parameter. Conclusion: Obesity impairs renal function, increases expression of inflammatory molecules and decreases nephrin expression. Leptin therapy was partially renoprotective since it reversed some of the observed parameters.

SRT1720 Administration Attenuates Albuminuria and Renal Fibrosis on Adriamycin‐Induced Glomerulosclerosis

The focal segmental glomerulosclerosis (FSGS) is the leading cause of progression of chronic kidney disease (CKD) and its responsible for the increasing number of cases of dialysis and kidney transplants worldwide. The lack of knowledge about the mechanisms underlying the progression of FSGS makes it difficult to develop therapeutic approaches with regarding the proteinuria and CKD progression. The aim of the study is to investigate the role of SIRT1 on FSGS induced by adriamycin, focusing on renoprotective responses. For this we used a synthetic SIRT1 agonist, the SRT1720. Male Balb/c mice aged 5 weeks were organized into two treatment groups: 48h and 14 days, being thus distributed in four experimental conditions: control (vehicle), adriamycin (ADR, 10 mg/kg), treated with SRT1720 (20 mg/kg/day) and treated with ADR+SRT1720. After the treatment, physiological parameters such as body weight, renal weight, weight gain, and plasma parameters such as creatinine, urea and osmolarity were evaluated. The glomerulosclerosis index was assessed by quantifying the renal sections stained with periodic acid-schiff (PAS). Urinary albumin was detected after staining the SDS-polyacrylamide gel. For the 48h group, the expression of mRNA was performed by quantitative real time PCR. For the 14-day group, GRP78, claudin-1, phospho eIF2αSer51 and phospho AMPKαThr172 protein expression were evaluated by immunoblotting. Type-IV collagen and nephrin staining were verified by immunofluorescence using confocal microscopy. The results indicate that for the ADR 48h group there was a decrease in final body weight, renal weight and an increase in plasma creatinine concentration compared to the control group. Administration of SRT1720 mitigates part of these effects, improving renal function. Our data indicate that although there is no positive stain for glomerulosclerosis in PAS or differences in the gene expression of Kim1, Tnf and Nphs1, the animals already have significant albuminuria within 48 hours after ADR administration in comparison to control, group, which suggests an initial damage on glomerular ultrafiltration barrier. Regarding the 14-day group, a decrease in final weight, an increase in renal weight, an increase in plasma creatinine, a significant increase in urinary albumin and the glomerulosclerosis index for ADR animals are observed, and this effect was clearly reversed by administration of SRT1720. There were no changes in creatinine clearance between groups after 14 days of treatment, despite an increase in urinary flow for the ADR + SRT1720 group. Although there were no significant differences on GRP78, phospho eIF2αSer51 or claudin-1 protein expression, the ADR+SRT1720 group showed an increase on phospho AMPKαThr172. Type IV collagen deposition was observed in ADR mice, as well as an important reduction in glomerular nephrin marking. These effects were partially reversed by the administration of the SRT1720. In conclusion, our data indicate that SRT1720 administration, a synthetic SIRT1 agonist, can improve renal function, mainly by partially preserving the glomerular ultrafiltration barrier and inhibiting renal fibrosis.

Metformin Protects against Podocyte Injury in Diabetic Kidney Disease.

Metformin is the most commonly prescribed drug for treating type 2 diabetes mellitus (T2D). Its mechanisms of action have been under extensive investigation, revealing that it has multiple cellular targets, either direct or indirect ones, via which it regulates numerous cellular pathways. Diabetic kidney disease (DKD), the serious complication of T2D, develops in up to 50% of the individuals with T2D. Various mechanisms contribute to the development of DKD, including hyperglycaemia, dyslipidemia, oxidative stress, chronic low-grade inflammation, altered autophagic activity and insulin resistance, among others. Metformin has been shown to affect these pathways, and thus, it could slow down or prevent the progression of DKD. Despite several animal studies demonstrating the renoprotective effects of metformin, there is no concrete evidence in clinical settings. This review summarizes the renoprotective effects of metformin in experimental settings. Special emphasis is on the effects of metformin on podocytes, the glomerular epithelial cells that are central in maintaining the glomerular ultrafiltration function.

Crosstalk between tubular epithelial cells and glomerular endothelial cells in diabetic kidney disease.

In recent years, although the development of clinical therapy for diabetic kidney disease (DKD) has made great progress, the progression of DKD still cannot be controlled. Therefore, further study of the pathogenesis of DKD and improvements in DKD treatment are crucial for prognosis. Traditional studies have shown that podocyte injury plays an important role in this process. Recently, it has been found that glomerulotubular balance and tubuloglomerular feedback (TGF) may be involved in the progression of DKD. Glomerulotubular balance is the specific gravity absorption of the glomerular ultrafiltrate by the proximal tubules, which absorbs only 65% to 70% of the ultrafiltrate. This ensures that the urine volume will not change much regardless of whether the glomerular filtration rate (GFR) increases or decreases. TGF is one of the significant mechanisms of renal blood flow and self‐regulation of GFR, but how they participate in the development of DKD in the pathological state and the specific mechanism is not clear. Injury to tubular epithelial cells (TECs) is the key link in DKD. Additionally, injury to glomerular endothelial cells (GECs) plays a key role in the early occurrence and development of DKD. However, TECs and GECs are close to each other in anatomical position and can crosstalk with each other, which may affect the development of DKD. Therefore, the purpose of this review was to summarize the current knowledge on the crosstalk between TECs and GECs in the pathogenesis of DKD and to highlight specific clinical and potential therapeutic strategies.

Діуретична активність похідного метилксантину фуроксану за одноразового та тривалого застосування на функцію видільної системи щурів на тлі спонтанного діурезу

The study of the effect of furoxane on renal activity under conditions of spontaneous diuresis was performed by the method of Berkhin. Hydrochlorothiazide was chosen as a reference drug. Furoxane and hydrochlorothiazide were administered intragastrically at a dose of 25 mg/kg for 7 days. The content of Na+ and K+in the urine was determined by flame photometry. The concentration of creatinine in the urine was studied by the method of Folin in the modification of Berkhin. The obtained results were calculated using the methods of variation statistics. The analysis of the results shows that under one-time action of furoxane in rats the daily urination increased by 210.4 % (p &lt;0.001), the concentration of Na+ in the urine of rats increased by 51.9 % (p &lt;0.05), and the concentration of K+ in the urine tended to increase by 5.6 %. Natriuresis increased 1.7 times (p &lt;0.05), potassium - 1.23 times (p &lt;0.05). Excretion of endogenous creatinine increased 1.49 times (p &lt;0.05), indicating an improvement in glomerular ultrafiltration. After administration of hydrochlorothiazide there was an increase in diuresis by 92.2 % (p &lt;0.05), urinary Na+ concentration increased by 49% (p &lt;0.05) and increased Na+excretion from urine by 23.3 % (p &lt;0.05). K+ concentration in urine increased by 12.6 %, and K+excretion - by 27.5 % (p &lt;0.05). Thus, a single administration of furoxane at a dose of 25 mg/kg led to an increase in daily diuresis, Na+ and K+ diuresis, which indicates the potential diuretic and saluretic properties of the drug. Long-term (7 days) furoxane administration contributed to the increase of urination in animals. After 3 days diuresis increased by 226,1 % (p &lt;0.001), on 7th day – by 236,3 % (p &lt;0.001). Salures was increased: the excretion of Na+ after 3 days increased by 2.27 times (p &lt;0,05), after 7 days - by 2,38 times (p &lt;0,05), K+ - 1.83 times (p &lt;0.05). The gradual increase in creatinine excretion in the dynamics of the use of furoxane, indicated a possible prolongation of the effect of furoxane. Thus, furoxane, which by its chemical structure belongs to methylxanthine derivatives is a promising pharmacologically active substance for further study of its specific activity and safety in order to create a highly effective diuretic and implementation in clinical practice.

INTERLEUKIN 6 METABOLISM AND FUNCTIONAL KIDNEY STATUS IN EARLY RHEUMATOID ARTHRITIS

Much attention paid to the problem of early rheumatoid arthritis (eRA) is due to its socio-economic significance. Kidney damage in RA occurs is reported as highly prevalent ranging from 36 to 73% and of great clinical significance, since it contributes much to the course of the underlying disease and increases mortality. The aim of the study was to evaluate the interrelation between interleukin-6 metabolism (IL-6) and impaired kidneys functional status at the debut of rheumatoid arthritis. Material and methods. This study included 35 patients aged over 18 with eRA. The average age of patients was 50.71 ± 2.25 years (from 18 to 76 years), there were 80% of women; the average duration of the disease at the time of the initial examination was 9.21 ± 0.43 months. Results. The index of microalbuminuria was found to increase by 154.57% with an elevation of IL-6 values ​​above 50 percentile. Also, microglobulinuria values ​​increased by 207.14% in patients of the subgroup with IL-6 values ​​above 50 percentile compared to the patients below 50 percentile. The glomerular permeability index, reflecting the average concentration of albumin in the glomerular ultrafiltrate, increased by 87.93% along with the increase in pro-inflammatory marker IL-6 in patients of the subgroup above 50 percentile relative to patients with low IL-6 values. Progressively with an increase in IL-6 values, a renal functional reserve dropped by 69.42% between subgroups below and above 50 percentile. Conclusion. Elevation of pro-inflammatory cytokine IL-6 is associated with changes in key markers of renal function, reflecting the presence of a direct connection between the degree of inflammatory activity in RA and the degree of renal dysfunction as the disease progresses.

A New Estimate of the Glomerular Sieving Coefficient for Retinol-Binding Protein 4 Suggests It Is Not Freely Filtered

A New Estimate of the Glomerular Sieving Coefficient for Retinol-Binding Protein 4 Suggests It Is Not Freely Filtered

The Effect of Albumin on Podocyte Apoptosis: the Role of PKC δ, p38 MAPK and Endoplasmic Reticulum Stress

Chronic kidney disease (CKD) is a public health problem characterized by high morbidity and mortality and with enormous social and economic impact on the health system. The high prevalence of patients maintained in dialysis programs, and the perspective of increasing the number of cases of CKD is worrisome for health professionals worldwide. One of the most serious consequences of CKD progression is albuminuria, which is mainly due to severe lesions in the glomerular basement membrane and tubular injury. Increased permeability of the glomerular ultrafiltration barrier to albumin is directly related to podocyte injury, mainly due to loss of foot processes, oxidative stress and apoptotic events. However, the cellular mechanisms involved in such processes have not yet been elucidated. Thus, the aim of this study is to investigate the mechanisms by which albumin participates in podocyte injury, considering the contribution of cubilin receptors, PKC δ (delta), p38 MAPK and the endoplasmic reticulum stress.MethodsDifferentiated mouse podocytes were distributed in four experimental groups: control and treated with albumin at the concentrations of 1, 5 and 10 mg/ml, in serum‐free RPMI 1640, for 24 hours. After the treatment, apoptosis was assessed by flow cytometry. For immunofluorescence experiments, cells were treated with FITC albumin 1 mg/mL for 30 min, 1h, 3h and 24h. The images were obtained by confocal microscopy, objective of 63x. Protein expression was analyzed by immunoblotting; GRP78, PKC δ, p38 MAPK and Caspase‐12 were evaluated 30 min, 1h, 3h and 24h after the treatment of the cells with albumin 1 mg/ml. GAPDH was used as endogenous control. All results are presented as the mean ± standard error. Statistical analysis was assessed by one‐way ANOVA followed by the Bonferroni's test. Significance was assumed at p&lt;0.05.ResultsAlbumin treatment at the concentration of 1 mg/ml for 24h resulted in a significant increase on the total apoptosis rate compared to the control. In immunofluorescence assay, colocalizations between FITC albumin and cubilin were observed in almost all the time periods tested and mainly after 1 hour of the treatment. Regarding protein expression, a significant increase on GRP78 was observed 1 hour after the albumin treatment. In addition, a significant increase on the expression of phosphorylated PKC δ, p38 MAPK and cleaved Caspase‐12 were also observed in the periods of 1h, 3h and 24h after the albumin treatment. These last two parameters were reduced by SB203580, a p38 MAPK specific inhibitor (100 nM)ConclusionsThese results suggest that the protein cubilin has an important role on the internalization of albumin in the podocytes. In addition, albumin induces apoptosis by activation of Caspase‐12 in these cells, mainly via the endoplasmic reticulum stress/PKC δ/p38 MAPK pathway.Support or Funding InformationFinancial support: Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

Nitric oxide production by glomerular podocytes

Nitric Oxide (NO), a potent vasodilator and vital signaling molecule, has been shown to contribute to the regulation of glomerular ultrafiltration. However, whether changes in NO occur in podocytes during the pathogenesis of salt-sensitive hypertension has not yet been thoroughly examined. We showed here that podocytes produce NO, and further hypothesized that hypertensive animals would exhibit reduced NO production in these cells in response to various paracrine factors, which might contribute to the damage of glomeruli filtration barrier and development of proteinuria. To test this, we isolated glomeruli from the kidneys of Dahl salt-sensitive (SS) rats fed a low salt (LS; 0.4% NaCl) or high salt (HS; 4% NaCl, 3 weeks) diets and loaded podocytes with either a combination of NO and Ca2+ fluorophores (DAF-FM and Fura Red, respectively) or DAF-FM alone. Changes in fluorescence were observed with confocal microscopy in response to adenosine triphosphate (ATP), angiotensin II (Ang II), and hydrogen peroxide (H2O2). Application of Ang II resulted in activation of both NO and intracellular calcium ([Ca2+]i) transients. In contrast, ATP promoted [Ca2+]i transients, but did not have any effects on NO production. SS rats fed a HS diet for 3 weeks demonstrated impaired NO production: the response to Ang II or H2O2 in podocytes of glomeruli isolated from SS rats fed a HS diet was significantly reduced compared to rats fed a LS diet. Therefore, glomerular podocytes from hypertensive rats showed a diminished NO release in response to Ang II or oxidative stress, suggesting that podocytic NO signaling is dysfunctional in this condition and likely contributes to the development of kidney injury.

Abstract 337: Kidney Injury Affects Metabolism and Modification of Apolipoprotein A1 Promoting Renal Lymphangiogenesis

Kidney disease causes ApoAI/HDL to lose their vasculoprotective effects. A likely mechanism for the protective loss is modification of ApoAI/HDL reactive carbonyls which are increased. Kidney injury increases reactive carbonyls and permits more ApoAI to escape into the glomerular ultrafiltrate. We examined how apoAI was metabolized in the injured proteinuric kidney. Nphs1-hCD25 mice (NEP25) expressing human CD25 in podocytes were injured by immunotoxin, LMB2. Six weeks after LMB2, we assessed urinary excretion and intrarenal localization of ApoAI and examined the renal lymphatic network with podoplanin. In vitro, we studied proximal tubular cells (PTC) and lymphatic endothelial cells (LEC) exposed to ApoAI or modified ApoAI (IsoLG-ApoAI). NEP25 mice developed glomerulosclerosis, increased urinary excretion of albumin and ApoAI with high molecular weight forms of ApoAI appearing in urine of injured mice assessed by western blot. Proteinuric kidneys had more ApoAI and VEGFC staining that localized to tubular epithelial cells and showed a dramatically more dense and complex lymphatic network than wild type mice. ApoAI localized to lymphatic vessels. Cultured PTC and LEC responded differently to normal and isolevuglandin (IsoLG)-modified ApoAI. In PTC, IsoLG-ApoAI promoted more ApoAI uptake and VEGFC secretion, a powerful lymphaniogenic stimulus. In LEC, IsoLG-ApoAI led to greater cell viability, migration and ROS production than normal ApoAI. Notably, supernatant of PTC exposed to ApoAI secreted a high molecular weight form of ApoAI. In conclusion, injury that disrupts the glomerular filtration barrier causing proteinuria also leads to a dramatic increase in filtered ApoAI followed by greater uptake by the proximal tubules, interstitium and lymphatic vessels. Proximal tubules can modify ApoAI and activate tubular secretion of VEGFC, which in turn promotes renal lymphangiogenesis and encourages delivery of kidney-modified ApoAI into circulation.

Aging Selectively Modulates Vitamin C Transporter Expression Patterns in the Kidney

In the kidney, vitamin C is reabsorbed from the glomerular ultrafiltrate by sodium-vitamin C cotransporter isoform 1 (SVCT1) located in the brush border membrane of the proximal tubules. Although we know that vitamin C levels decrease with age, the adaptive physiological mechanisms used by the kidney for vitamin C reabsorption during aging remain unknown. In this study, we used an animal model of accelerated senescence (SAMP8 mice) to define the morphological alterations and aging-induced changes in the expression of vitamin C transporters in renal tissue. Aging induced significant morphological changes, such as periglomerular lymphocytic infiltrate and glomerular congestion, in the kidneys of SAMP8 mice, although no increase in collagen deposits was observed using 2-photon microscopy analysis and second harmonic generation. The most characteristic histological alteration was the dilation of intracellular spaces in the basolateral region of proximal tubule epithelial cells. Furthermore, a combination of laser microdissection, qRT-PCR, and immunohistochemical analyses allowed us to determine that SVCT1 expression specifically increased in the proximal tubules from the outer strip of the outer medulla (segment S3) and cortex (segment S2) during aging and that these tubules also express GLUT1. We conclude that aging modulates vitamin C transporter expression and that renal over-expression of SVCT1 enhances vitamin C reabsorption in aged animals that may synthesize less vitamin C. J. Cell. Physiol. 232: 2418-2426, 2017. © 2016 Wiley Periodicals, Inc.