Filtration Barrier Integrity Research Articles

Glomerular Endothelial Cell Receptor Adhesion G-Protein-Coupled Receptor F5 (ADGRF5) and the Integrity of the Glomerular Filtration Barrier.

Background: Glomerular endothelial cells are recognized to be important for maintaining the glomerular filtration barrier. ADGRF5, an adhesion G protein-coupled receptor, has been suggested to be involved in endothelial cell function. However, the role of ADGRF5 in the glomerular filtration barrier integrity remains elusive. Methods: Cellular expression of ADGRF5 in mouse glomerulus was determined by histological analyses. The impact of ADGRF5 deletion on the glomerular morphology, kidney function, and glomerular endothelial gene/protein expression was then analyzed using ADGRF5 knockout (Adgrf5 −/−) mice and human primary glomerular endothelial cells. Results: ADGRF5 was specifically expressed in the capillary endothelial cells within the glomerulus. Adgrf5 −/− mice developed albuminuria and impaired kidney function with morphological defects in the glomeruli, namely glomerular hypertrophy, glomerular basement membrane splitting and thickening, diaphragmed fenestration and detachment of the glomerular endothelial cells, and mesangial interposition. These defects were accompanied by the altered expression of genes responsible for glomerular basement membrane organization (type IV collagens and laminins) and Krüppel-like factor 2 (Klf2) in glomerular endothelial cells. Moreover, ADGRF5 knockdown decreased COL4A3 and COL4A4 expression and increased KLF2 expression in human primary glomerular endothelial cells. Conclusions: The loss of ADGRF5 resulted in altered gene expression in glomerular endothelial cells, and perturbed the structure and permselectivity of the glomerular filtration barrier.

Genome-wide association study of hospitalized patients and acute kidney injury

Acute kidney injury (AKI) is a common and devastating complication of hospitalization. Here, we identified genetic loci associated with AKI in patients hospitalized between 2002-2019 in the Million Veteran Program and data from Vanderbilt University Medical Center's BioVU. AKI was defined as meeting a modified KDIGO Stage 1 or more for two or more consecutive days or kidney replacement therapy. Control individuals were required to have one or more qualifying hospitalizations without AKI and no evidence of AKI during any other observed hospitalizations. Genome-wide association studies (GWAS), stratified by race, adjusting for sex, age, baseline estimated glomerular filtration rate (eGFR), and the top ten principal components of ancestry were conducted. Results were meta-analyzed using fixed effects models. In total, there were 54,488 patients with AKI and 138,051 non-AKI individuals included in the study. Two novel loci reached genome-wide significance in the meta-analysis: rs11642015 near the FTO locus on chromosome 16 (obesity traits) (odds ratio 1.07 (95% confidence interval, 1.05-1.09)) and rs4859682 near the SHROOM3 locus on chromosome 4 (glomerular filtration barrier integrity) (odds ratio 0.95 (95% confidence interval, 0.93-0.96)). These loci colocalized with previous studies of kidney function, and genetic correlation indicated significant shared genetic architecture between AKI and eGFR. Notably, the association at the FTO locus was attenuated after adjustment for BMI and diabetes, suggesting that this association may be partially driven by obesity. Both FTO and the SHROOM3 loci showed nominal evidence of replication from diagnostic-code-based summary statistics from UK Biobank, FinnGen, and Biobank Japan. Thus, our large GWA meta-analysis found two loci significantly associated with AKI suggesting genetics may explain some risk for AKI.

#2518 The decrease of the filtration slit density, not proteinuria, is the early indication of the filtration barrier dysfunction

Abstract Background and Aims Intact podocyte foot processes (FP) play a crucial role in ensuring proper selective filtration in the kidney. Changes in FP morphology are thought to be the initial trigger for proteinuria, but definitive proof is still missing. As FP structures are below the optical resolution of light microscopes, changes are investigated by electron microscopy (EM) on ultrathin sections. In this study, the correlation between morphology and proteinuria was investigated using the Podocyte Exact Morphology Measurement Procedure (PEMP), a 3D-SIM-based technique. Our investigation was focused on podocyte damage caused by treatment with nephrotoxic serum (NTS) as a model for focal segmental glomerulosclerosis. Method The mice were treated with nephrotoxic serum (NTS), and kidneys were taken at 24, 48, and 72 hours after injection. Kidney sections (3 µm thick) were prepared for 3D-SIM analysis after staining with podocin and integrin and then fixed in formalin and embedded in paraffin. Filtration slit density (FSD) was quantified using the Podocyte Exact Morphology Measurement Procedure (PEMP) as described earlier (Siegerist et al. 2017). In addition, EM was performed on glutaraldehyde-fixed tissue samples using standard protocols. The albumin to creatinine ratio was also determined. Results We observed that NTS-treated mice developed moderate proteinuria after 48 hours. Using the super-resolution microscopy-based 3D-SIM technique to quantify podocyte PF morphology by calculating the filtrations slit density (FSD) revealed a significant reduction of the FSD in NTS-treated mice already after 24 hours. This structural change was confirmed by transmission electron microscopy analysis, showing that the effacement of podocytes FPs is the first sign of a change in the filtration barrier function before proteinuria is measurable. Conclusion PEMP analysis of standard histological sections from NTS-injected and control mice demonstrated that the first event in the functional loss of the size selectivity of the filtration barrier is the effacement of FP morphology, followed by detectable proteinuria. Therefore, the assessment of the FSD is a promising strategy for early and accurate detection of the loss of filtration barrier integrity, providing opportunities for faster and more precise diagnostics.

The proteasome modulates endocytosis specifically in glomerular cells to promote kidney filtration

Kidney filtration is ensured by the interaction of podocytes, endothelial and mesangial cells. Immunoglobulin accumulation at the filtration barrier is pathognomonic for glomerular injury. The mechanisms that regulate filter permeability are unknown. Here, we identify a pivotal role for the proteasome in a specific cell type. Combining genetic and inhibitor-based human, pig, mouse, and Drosophila models we demonstrate that the proteasome maintains filtration barrier integrity, with podocytes requiring the constitutive and glomerular endothelial cells the immunoproteasomal activity. Endothelial immunoproteasome deficiency as well as proteasome inhibition disrupt the filtration barrier in mice, resulting in pathologic immunoglobulin deposition. Mechanistically, we observe reduced endocytic activity, which leads to altered membrane recycling and endocytic receptor turnover. This work expands the concept of the (immuno)proteasome as a control protease orchestrating protein degradation and antigen presentation and endocytosis, providing new therapeutic targets to treat disease-associated glomerular protein accumulations.

β-Arrestin pathway activation by selective ATR1 agonism promotes calcium influx in podocytes, leading to glomerular damage.

Angiotensin receptor blockers (ARBs) are the first-line treatment for hypertension; they act by inhibiting signaling through the angiotensin 1 receptor (AT1R). Recently, a novel biased AT1R agonist, TRV120027 (TRV), which selectively activates the β-arrestin cascade and blocks the G-protein-coupled receptor pathway has been proposed as a potential blood pressure medication. Here, we explored the effects of TRV and associated β-arrestin signaling in podocytes, essential cells of the kidney filter. We used human podocyte cell lines to determine β-arrestin's involvement in calcium signaling and cytoskeletal reorganization and Dahl SS rats to investigate the chronic effects of TRV administration on glomerular health. Our experiments indicate that the TRV-activated β-arrestin pathway promotes the rapid elevation of intracellular Ca2+ in a dose-dependent manner. Interestingly, the amplitude of β-arrestin-mediated Ca2+ influx was significantly higher than the response to similar Ang II concentrations. Single-channel analyses show rapid activation of transient receptor potential canonical (TRPC) channels following acute TRV application. Furthermore, the pharmacological blockade of TRPC6 significantly attenuated the β-arrestin-mediated Ca2+ influx. Additionally, prolonged activation of the β-arrestin pathway in podocytes resulted in pathological actin cytoskeleton rearrangements, higher apoptotic cell markers, and augmented glomerular damage. TRV-activated β-arrestin signaling in podocytes may promote TRPC6 channel-mediated Ca2+ influx, foot process effacement, and apoptosis, possibly leading to severe defects in glomerular filtration barrier integrity and kidney health. Under these circumstances, the potential therapeutic application of TRV for hypertension treatment requires further investigation to assess the balance of the benefits versus possible deleterious effects and off-target damage.

Transmembrane protein 14A protects glomerular filtration barrier integrity.

Transmembrane protein 14A (TMEM14A) is a relatively unknown protein that is now identified to be required for maintaining the integrity of the glomerular filtration barrier. It is an integral transmembrane protein of 99 amino acids with three transmembrane domains. TMEM14A has been implied to suppress Bax-mediated apoptosis in other studies. Other than that, little is currently known of its function. Here, we show that its expression is diminished before onset of proteinuria in a spontaneously proteinuric rat model. Knocking down tmem14a mRNA translation results in proteinuria in zebrafish embryos without affecting tubular reabsorption. Also, it is primarily expressed by podocytes. Lastly, an increase in glomerular TMEM14A expression is exhibited in various proteinuric renal diseases. Overall, these results suggest that TMEM14A is a novel factor in the protective mechanisms of the nephron to maintain glomerular filtration barrier integrity.

#5978 SUBCAPSULAR INJECTION OF EXTRACELLULAR VESICLES FROM MESENCHYMAL STEM CELLS, PROMOTED ADDITIONAL RENOPROTECTION IN AN EXPERIMENTAL MODEL OF CKD

Abstract Background and Aims Chronic kidney disease (CKD) is considered a public health concern worldwide, both due to its insidious and highly debilitating feature and to its high global prevalence. Clinical management of CKD is often achieved by the blockade of the renin-angiotensin-aldosterone system (RAAS). However, this treatment alone is not efficient to prevent CKD progression completely, motivating the scientific community to search for alternative treatments to control CKD progression and to detain its evolution to the end-stage renal failure. In this context, we have recently shown that the association of a single renal subcapsular injection of 2 × 106 adipose-derived mesenchymal stem cells (ASC) to RAAS blockade with the AT1RB Losartan (LOS) promoted greater renoprotection when compared to LOS monotherapy, leading to the normalization of urinary protein excretion (UPE) and to the regression of established glomerulosclerosis (GS), in experimental CKD. Since there are methodological limits for the number of ASC that can be injected under the renal capsule, and based on the current literature, which suggests the main beneficial effects of ASC are not due to direct in situ cell differentiation, but to paracrine factors produced and released by the ASC; in the present study we aimed to investigate if the association of a renal subcapsular injection of extracellular vesicles (EV) derived from ASC, to the oral treatment with LOS would promote additional renoprotective effects in a model of experimental CKD. Method The present experimental protocol was approved by the Ethics Committee for the Use of Experimental Animals of the University of São Paulo Medical School (CEUA-FMUSP No 1761/2022). EV were obtained from ASC isolated from gonadal adipose tissue of 5 male Wistar rats. Cells were cultured until P4, characterized by flow cytometry and in vitro differentiation, and kept under serum deprivation for 24 h. The conditioned culture media, containing the EV released by these cells, was ultracentrifuged and the resulting EV pellets were diluted in 100 μL of sterile PBS and used for the renal subcapsular injections. Experimental CKD was induced in 25 male Wistar rats through the surgical ligation of 2 from the 3 branches of the left renal artery, followed by the total nephrectomy of the right kidney, resulting in a 5/6 renal ablation. Additional 10 Sham-operated rats were used as control. After 15 days from surgery, the animals submitted to renal ablation were stratified into 3 groups, with closely similar mean values of body weight (BW), systolic blood pressure (SBP), UPE and urinary albumin excretion (UAE), before the start of the different treatments. Animals from CKD group were kept untreated, LOS and LOS+EV animals received diary 50 mg/Kg/d of Losartan, diluted in drinking water and LOS+EV rats underwent a second surgery for the renal subcapsular application of EV. All animals were followed until 30 days after CKD induction, when BW, SBP, UPE and UAE were analyzed again. Animals where then euthanized for the assessment of serum creatinine (SCr) and blood urea nitrogen (BUN) concentration, as well as for histological studies to determine the percentage of GS and the renal interstitial infiltration by macrophages (CD68). Results The association of a single subcapsular injection of EV derived from ASC, to the oral treatment with the AT1RB LOS, significantly improved the effects of this antihypertensive drug. CKD+LOS+EV animals exhibited normal SBP, in spite of having only 1/6 of functioning renal mass. Moreover, the combined treatment significantly reduced UAE and numerically diminished de percentage of glomerulosclerosis, compared to LOS alone. Detailed obtained results are presented in Table 1. Data are presented as Mean ± SE. Differences among groups were analyzed by one-way ANOVA: p<0.05 vs.*CONT, #CKD, †LOS. as Mean ± SE. Conclusion Despite the small number of animals in the association group, our preliminary results suggest EV from ASC to exert additional renoprotective effects when associated to LOS, especially regarding the control of SBP and the protection of the glomerular filtration barrier integrity.

Beta-arrestin-biased agonism promotes TRPC6-mediated calcium influx in human podocytes

Background: Angiotensin Receptor blockers (ARBs) are the first-line treatment for hypertension and other chronic kidney diseases and act by inhibiting signaling through the angiotensin 1 receptor (AT1R). AT1R activation initiates signaling through G protein-coupled receptor (GPCR) effector signaling and β-arrestins. Recently, a novel AT1R agonist TRV120027 (TRV) that selectively activates the β-arrestin cascade without impacting the GCPR pathway has become available. Therefore, we hypothesized that β-arrestin and G-protein signaling pathways via AT1R have distinct downstream effects in podocytes. Furthermore, we propose that β-arrestin signaling initiates TRPC6-mediated Ca 2+ entry. Methods: We used a conditionally immortalized human podocyte cell line to determine β-arrestin’s involvement in podocyte calcium signaling and cytoskeletal reorganization. Intracellular Ca 2+ influx (Fluo-4 AM) and NO response (DAF-FM) to Ang II or TRV applications were analyzed with confocal microscopy. The role of TRPC channels was tested by single-channel electrophysiology and confocal imaging using a variety of commercially available inhibitors (AC1903 for TRPC5 and GSK283350A, SAR7334 for TRPC6). Western blotting for apoptosis-associated proteins, TUNEL staining to detect DNA cleavage, and actin cytoskeleton rearrangements were performed in podocytes exposed to TRV or Ang II treatment. Results: Our experiments determined that TRV-mediated β-arrestin pathway activation in podocytes promotes rapid elevation of intracellular Ca 2+ in a dose-dependent manner (IC 50 =15μM, ligand-receptor binding fit modeled with Levenberg–Marquardt algorithm, adj. R 2 =0.93). Interestingly, the amplitude of β-arrestin-mediated Ca 2+ influx was four times higher than the response to similar Ang II concentrations (t-test, n≥30, p<0.05). Moreover, the TRV response was significantly increased under hyperglycemic stress conditions, and promoted rapid apoptosis in podocytes (one-way ANOVA, n≥21, p=0.003). The pharmacological blockade of TRPC6, but not TRPC5, significantly attenuated Ca 2+ influx in response to β-arrestin. In contrast to Ang II, TRV does not induce AT1R-mediated NO production, suggesting that β-arrestin activation only targets AT1R, and not AT2R. Single-channel analyses show rapid activation of TRPC activity in response to acute TRV application in podocytes (one-way ANOVA, n=8, p=0.004). Overall prolonged activation of the β-arrestin pathway in podocytes results in enhanced actin bundle thickness, abnormal actin cytoskeleton distribution, and increased apoptotic cell markers. Conclusions: TRV-mediated β-arrestin signaling in podocytes promotes high ionotropic TRPC6 channel-mediated Ca 2+ influx, cytoskeleton rearrangement, and apoptosis, possibly leading to severe defects in glomerular filtration barrier integrity and kidney health. Under these circumstances, the potential therapeutic application of TRV for hypertension treatment is controversial and requires further investigation. DK126720 (to OP), DK129227 (to AS and OP), and NIH/NCATS/SCTR UL1TR001450/SCTR 2214 (to OP), endowed funds from the SC SmartState Centers of Excellence (to OP), Veterans Affairs Support Veterans Affairs (Merit Award I01 BX000820 to JL), research grant from Dialysis Clinic, Inc (to JL). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Huangkui capsule alleviates doxorubicin-induced proteinuria via protecting against podocyte damage and inhibiting JAK/STAT signaling.

Huangkui capsule (HKC), a Chinese patent medicine, has been widely used in China as adjuvant therapy for chronic kidney disease (CKD). It displays superior anti-proteinuria efficacy than losartan in patients with CKD at stages 1-2, however, the mechanism of HKC alleviating proteinuria has not been well elucidated. This study aims to confirm the therapeutic effect and investigate associated underlying mechanism of HKC against proteinuria by in vivo and in vitro experiments. We established a doxorubicin (DOX) induced proteinuria mouse model to evaluate kidney function by biochemical markers measurement and to observe histopathological alterations by hematoxylin and eosin (H&E), Masson's trichrome and Periodic Acid-Schiff (PAS)-stained sections of renal, respectively. Moreover, the expressions of Nephrin and Podocin were measured by immunohistochemistry (IHC) and western blotting analysis to investigate podocyte damage. Furthermore, we established Mouse Podocyte Clone-5 (MPC-5) injury model to identify the active components of HKC against podocyte damage by detecting the expressions of Nephrin, Podocin, and ZO-1 proteins. At last, the key protein levels of Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway were assessed by western blotting analysis to explore the underlying mechanism of HKC against proteinuria. Our results showed that HKC administration for three consecutive weeks dose-dependently ameliorated both renal function and histopathological damages, elevated the expressions of Nephrin and Podocin, the pivotal molecules maintaining filtration function of the podocyte, indicating the promising protective effect against podocyte injury under DOX exposure. Consistently, in vitro experiments showed HKC administration effectively reversed the abnormal expressions of Nephrin and Podocin in MPC-5 cells treated with DOX, suggesting its protective effect against podocyte injury to maintain filtration barrier integrity. In addition, Hibifolin was identified as the most active ingredients in HKC, which suppressed upstream JAK2/STAT3 and PI3K/Akt pathway phosphorylation to maintain the structural and functional integrity of podocyte filtration barrier. Of note, AG490, a selective JAK2 inhibitor, was used to further affirm the role of Hibifolin involving in regulation JAK2/STAT3. Our study suggested that HKC may protect podocytes via JAK2/STAT3 and PI3K/Akt pathway to display its effects of ameliorating proteinuria.

Co-Culture of Glomerular Endothelial Cells and Podocytes in a Custom-Designed Glomerulus-on-a-Chip Model Improves the Filtration Barrier Integrity and Affects the Glomerular Cell Phenotype.

Crosstalk between glomerular endothelial cells and glomerular epithelial cells (podocytes) is increasingly becoming apparent as a crucial mechanism to maintain the integrity of the glomerular filtration barrier. However, in vitro studies directly investigating the effect of this crosstalk on the glomerular filtration barrier are scarce because of the lack of suitable experimental models. Therefore, we developed a custom-made glomerulus-on-a-chip model recapitulating the glomerular filtration barrier, in which we investigated the effects of co-culture of glomerular endothelial cells and podocytes on filtration barrier function and the phenotype of these respective cell types. The custom-made glomerulus-on-a-chip model was designed using soft lithography. The chip consisted of two parallel microfluidic channels separated by a semi-permeable polycarbonate membrane. The glycocalyx was visualized by wheat germ agglutinin staining and the barrier integrity of the glomerulus-on-a-chip model was determined by measuring the transport rate of fluorescently labelled dextran from the top to the bottom channel. The effect of crosstalk on the transcriptome of glomerular endothelial cells and podocytes was investigated via RNA-sequencing. Glomerular endothelial cells and podocytes were successfully cultured on opposite sides of the membrane in our glomerulus-on-a-chip model using a polydopamine and collagen A double coating. Barrier integrity of the chip model was significantly improved when glomerular endothelial cells were co-cultured with podocytes compared to monocultures of either glomerular endothelial cells or podocytes. Co-culture enlarged the surface area of podocyte foot processes and increased the thickness of the glycocalyx. RNA-sequencing analysis revealed the regulation of cellular pathways involved in cellular differentiation and cellular adhesion as a result of the interaction between glomerular endothelial cells and podocytes. We present a novel custom-made glomerulus-on-a-chip co-culture model and demonstrated for the first time using a glomerulus-on-a-chip model that co-culture affects the morphology and transcriptional phenotype of glomerular endothelial cells and podocytes. Moreover, we showed that co-culture improves barrier function as a relevant functional readout for clinical translation. This model can be used in future studies to investigate specific glomerular paracrine pathways and unravel the role of glomerular crosstalk in glomerular (patho) physiology.

A high-impact FN1 variant correlates with fibronectin-mediated glomerulopathy via decreased binding to collagen type IV

The glomerular basement membrane (GBM) consists of laminins, collagen IV, nidogens, and fibronectin and is essential for filtration barrier integrity in the kidney. Critically, structural and functional abnormalities in the GBM are involved in chronic kidney disease (CKD) occurrence and development. Fibronectin is encoded by FN1 and is essential for podocyte-podocyte and podocyte-matrix interactions. However, disrupted or disordered fibronectin occurs in many kidney diseases. In this study, we identified a novel mutation (c.3415G>A) in FN1 that causes glomerular fibronectin-specific deposition in a gain-of-function manner, that may be associated with thin basement membrane nephropathy (TBMN) and expand the spectrum of phenotypes seen in glomerulopathy with fibronectin deposits (GFND). Our studies confirmed this variant increased fibronectin's ability to bind to integrin, thereby maintaining podocyte adhesion. Also, we hypothesised that TBMN arose as the fibronectin variant exhibited a decreased capacity to bind COL4A3/4. Our study is the first to identify and link this novel pathogenic mutation (c.3415G>A) in FN1 to GFND as well as TBMN, which may broaden the phenotype and mutation spectrums of the FN1 gene. We believe our data will positively impact genetic counselling and prenatal diagnostics for GFND with TBMN and other associated conditions that may be commonly benign conditions in humans, and may not require proteinuria-lowering treatments or renal biopsy.

In vitro study of emodin-induced nephrotoxicity in human renal glomerular endothelial cells on a microfluidic chip

Emodin is an effective component of rhubarb with positive pharmacological effects on human health. However, it is also toxic to different cells or tissues to varying degrees. The effects of emodin on glomerular endothelial cells (GECs) remain to be tested, and the documented works were always performed in vitro and hardly reflect the real physiological situation. To study the effects of emodin on GECs in a biomimetic environment, we utilized a microfluidic chip to assess the physiological reaction of human renal glomerular endothelial cells to various concentrations of emodin in this work. The results showed that emodin caused cytotoxicity, impaired glomerular filtration barrier integrity to macromolecules, and increased barrier permeability in a dose-dependent manner. With the increase in emodin concentration, the concentration of the pro-inflammatory cytokine tumor necrosis factor-α, interleukin (IL)-6, transforming growth factor-β1, and monocyte chemoattractant protein (MCP-1) increased while the production of inflammatory cytokine IL-6 first increased and then decreased with the increase in emodin concentration. Our findings shed new light on emodin-induced nephrotoxicity and provide insights for the application of microfluidic chip devices to reveal drug-cell interactions.

Geranylgeranylation of Small GTPase Is Critical to Preserving the Glomerular Filtration Barrier Integrity

Geranylgeranylation of Small GTPase Is Critical to Preserving the Glomerular Filtration Barrier Integrity

Role of protease‐activated receptor‐1 (PAR‐1) in the glomerular filtration barrier integrity

Protease‐activated receptors (PAR) play an important role in the regulation of cellular function by the coagulation system, and they are activated by thrombin. PAR‐1 is expressed in both endothelial cells and podocytes in the kidney. The role of PAR1 in the maintenance of the glomerular filtration barrier is not clear. Anticoagulant‐related nephropathy (ARN) is a kidney disease with glomerular hematuria and red blood cell tubular casts. We validated 5/6 nephrectomy (5/6NE) in rats as a model of ARN and had demonstrated that direct thrombin inhibitor (dabigatran) induces ARN. The aim of this study was to investigate the role of PAR‐1 in the ARN pathogenesis. 5/6NE rats were treated with dabigatran (150 mg/kg/day), PAR‐1 inhibitor SCH79797 (1 and 3 mg/kg/day) and PAR‐1 agonist TFLLR‐NH2 (0.25 and 0.50 µmol/kg/day) for 7 days. Serum creatinine and hematuria were assessed daily. Kidney morphology was evaluated at the end of the study. In 5/6NE rats treated with either dabigatran or combination with a PAR‐1 modulator, there was an elevation in serum creatinine, glomerular hematuria, red blood casts in the tubules, and acute tubular epithelial cell injury. Interestingly, both PAR‐1 modulators in a dose‐depended manner had similar effects on the serum creatinine levels and hematuria as those of dabigatran. Dabigatran‐induced increase in the systolic blood pressure was not affected by PAR‐1 modulators. In conclusion, the normal function of PAR‐1 is crucial to maintain the glomerular filtration barrier integrity. Either activation or blockage of PAR‐1 leads to glomerular hematuria and subsequent acute tubular epithelial cell injury.

Complementary Nck1/2 Signaling in Podocytes Controls α Actinin-4-Mediated Actin Organization, Adhesion, and Basement Membrane Composition.

Maintenance of the kidney filtration barrier requires coordinated interactions between podocytes and the underlying glomerular basement membrane (GBM). GBM ligands bind podocyte integrins, which triggers actin-based signaling events critical for adhesion. Nck1/2 adaptors have emerged as essential regulators of podocyte cytoskeletal dynamics. However, the precise signaling mechanisms mediated by Nck1/2 adaptors in podocytes remain to be fully elucidated. We generated podocytes deficient in Nck1 and Nck2 and used transcriptomic approaches to profile expression differences. Proteomic techniques identified specific binding partners for Nck1 and Nck2 in podocytes. We used cultured podocytes and mice deficient in Nck1 and/or Nck2, along with podocyte injury models, to comprehensively verify our findings. Compound loss of Nck1/2 altered expression of genes involved in actin binding, cell adhesion, and extracellular matrix composition. Accordingly, Nck1/2-deficient podocytes showed defects in actin organization and cell adhesion in vitro, with podocyte detachment and altered GBM morphology present in vivo. We identified distinct interactomes for Nck1 and Nck2 and uncovered a mechanism by which Nck1 and Nck2 cooperate to regulate actin bundling at focal adhesions via α actinin-4. Furthermore, loss of Nck1 or Nck2 resulted in increased matrix deposition in vivo, with more prominent defects in Nck2-deficient mice, consistent with enhanced susceptibility to podocyte injury. These findings reveal distinct, yet complementary, roles for Nck proteins in regulating podocyte adhesion, controlling GBM composition, and sustaining filtration barrier integrity.

A critical role of the podocyte cytoskeleton in the pathogenesis of glomerular proteinuria and autoimmune podocytopathies.

Selective glomerular filtration relies on the membrane separating the glomerular arterioles from the Bowman space. As a major component of the glomerular filtration barrier, podocytes form foot processes by the actin cytoskeleton, which dynamically adjusts in response to environmental changes to maintain filtration barrier integrity. The slit diaphragms bridge the filtration slits between neighboring foot processes and act as signaling hubs interacting with the actin cytoskeleton. Focal adhesions relay signals to regulate actin dynamics while allowing podocyte adherence to the basement membrane. Mutations in actin regulatory and signaling proteins may disrupt the actin cytoskeleton, resulting in foot process retraction, effacement, and proteinuria. Large-scale gene expression profiling platforms, transgenic animal models, and other in vivo gene delivery methods now enhance our understanding of the interactions among podocyte focal adhesions, slit diaphragms, and actin dynamics. In addition, our team found that at least 66% of idiopathic nephrotic syndrome (INS) children have podocyte autoantibodies, which was defined as a new disease subgroup-, autoimmune podocytopathies. This review outlines the pathophysiological mechanisms of podocyte cytoskeleton protein interactions in proteinuria and glomerular podocytopathy.

COMBINED EXOSOMAL AND PLASMA NON-CODING RNA SIGNATURE ASSOCIATED WITH URINARY ALBUMIN EXCRETION IN HYPERTENSION

Objective: Non-coding RNA (ncRNA), released into circulation or packaged into exosomes, play important roles in many biological processes in the kidney. The purpose of the present study is to identify a common ncRNA signature associated with early renal damage and its related molecular pathways by constructing a RNA-based transcriptional network. Design and method: This is an observational case-control study which included 43 hypertensives, twenty-one patients with essential hypertension and twenty-two without persistent elevated urinary albuminuria (UAE) (higher or equal to 30 mg/g urinary creatinine). Three individual libraries (plasma and urinary exosomes and total plasma) were prepared from each hypertensive patient for ncRNA sequencing analysis. Next, a RNA-based transcriptional regulatory network was constructed. Results: The three RNA biotypes with the greatest number of differentially expressed transcripts were long-ncRNA (lncRNA), microRNA (miRNA) and piwi-interacting RNA (piRNA). We identified a common 24 ncRNA molecular signature related to hypertension-associated albuminuria, of which lncRNA was the most representative. In addition, the transcriptional regulatory network analysis showed five lncRNA (LINC02614, BAALC-AS1, FAM230B, LOC100505824 and LINC01484), and the miR-301a-3p to play a significant role in network organization and to target critical pathways regulating filtration barrier integrity, tubule reabsorption and systemic endothelial dysfunction. Conclusions: Our study found a combined ncRNA signature associated with albuminuria, independently of biofluid origin (urine or plasma, circulating or in exosomes) that identifies a handful of potential targets involved in filtration barrier, tubule reabsorption and endothelial function that may be utilized to treating hypertension-associated albuminuria and cardiovascular damage progression.

Application of Scanning Ion Conductance Microscopy in the Studies of Podocytes Morphological Changes

High‐resolution imaging of glomerular podocytes morphology is becoming an essential and helpful tool for studying their role in renal disease progression. Electron microscopy (EM) is commonly used to analyze the effacement of the podocytes’ cell foot processes and the glomerular filtration barrier integrity loss. However, EM does not allow studying living samples and requires multiple complex procedures for sample preparation. Here, we propose and demonstrate the successful application of Scanning Ion Conductance Microscopy (SICM) for studying morphological changes of cultured podocytes and freshly isolated glomeruli.SICM provides a high‐resolution (comparable to EM) non‐optical imaging of cell topography using hopping probe microscopy principles and new dynamic reconstruction software. This technique can be used for analysis of cell membrane morphology and different functional characteristics, such as cell volume, membrane potentials, single ion‐channel currents, and other parameters. The major advantage of the current method is that it can be applied to live samples with complex morphology, such as intact glomeruli, under physiologically relevant liquid conditions.We have established and demonstrated a successful application of the SICM method in the live human glomerulus and provided a proof of principle for future dynamic analysis of membrane morphology and varied functional parameters in the living glomerulus. As a proof of principle, we applied this method for estimation the pathological structural changes in podocyte foot processes in type 2 diabetic nephropathy (T2DN) rats. Using topographical quantification of the length and irregular shape of the podocyte foot processes, we showed an absence of interdigitated foot processes on the selected region of the glomerular slit diaphragm in isolated T2DN glomeruli.In addition to the application of SICM with freshly isolated glomeruli, we used this approach to visualize and quantify foot process dynamics in cultured human podocytes during pharmacological applications. Thus, we showed that destroying the F‐actin with cytochalasin D completely stopped the migration of the podocyte filopodium through the remodeling of the actin cytoskeleton. Here, we further applied SICM to detect morphological changes in human podocytes after activation of Protease‐activated receptor‐1 (PAR1) by acute application of agonist TFLLR‐NH2. A series of experiments revealed that PAR1 activation led to retraction of the lamellipodium and a significant decrease in cell surface area (+51±46 vs. ‐15±8 µm2, 30 min after application of vehicle or TFLLR, respectively; n=5, p<0.05).In summary, SICM has numerous advantages and has excellent potential for precise quantitative analyses of the morphological changes in the glomerulus and cultured podocytes. Moreover, it opens the possibility of performing patch‐clamp recordings on the specific parts of the podocyte cell body and advances our understanding of the function and mechanism of the glomerulus filtration barrier.

Astragaloside IV Improves the Barrier Damage in Diabetic Glomerular Endothelial Cells Stimulated by High Glucose and High Insulin.

Objective To investigate the protective effect and mechanism of astragaloside IV (AS-IV) on damage in human glomerular endothelial cells (GEnCs) stimulated by high glucose and high insulin. Methods The transwell method was used to detect the integrity of the cell barrier after AS-IV intervention in a high glucose and high insulin environment for 24 h; immunofluorescence and Western blot methods were used to detect the tight junction protein ZO-1 and claudin-5 expression; intracellular and extracellular 1β (IL-1β) and tumor necrosis factor α (TNFα) were determined by ELISA; expression and activation of AKT, p-AKT, GSK3α/β, and p-GSK3α/β were evaluated by Western blot. Results The results showed that AS-IV had a significant protective effect on the cell barrier of GEnCs. High glucose or insulin inhibited cell viability in a concentration-dependent manner. High glucose or insulin significantly inhibited glucose uptake and promoted release of reactive oxygen species in GEnCs. Administration with AS-IV dramatically preserved viability of the cells; moreover, the expression of intracellular tight junction proteins was upregulated, inflammatory cytokines including IL-1β and TNFα were decreased, and the AKT-GSK3 pathway participated in modulation of AS-IV in GEnCs cells. Conclusion We found in the present study that AS-IV can preserve filtration barrier integrity in glomerular endothelial cells under diabetic settings, its effects on increasing the cell energy metabolism and cell viability, inhibiting inflammation and oxidative stress damage, and enhancing tight junction between cells play a role in it; and the intracellular signaling pathway AKT-GSK modulated the above function. Our present finding supplied a new understanding towards development of DN and provided an alternative method on ameliorating DN.

Vasohibin-1 has α-tubulin detyrosinating activity in glomerular podocytes

Podocytes are highly specialized epithelial cells in glomeruli, with a complex morphology composed of a cell body, primary processes, and foot processes, which maintain barrier function in glomerular filtration. The microtubule-based cytoskeleton is necessary for podocyte morphology. Microtubule structure and function can be affected by post-translational modification of tubulin, including detyrosination. Recent studies have shown that vasohibin-1 (VASH1), an antiangiogenic factor, has tubulin carboxypeptidase activity that causes detyrosination of α-tubulin. We aimed to examine the role of VASH1 in regulating α-tubulin detyrosination in podocytes and the potential involvement of VASH1 deficiency in renal morphology. In normal mouse kidneys, detyrosinated α-tubulin was mainly identified in glomeruli, especially in podocytes; meanwhile, in cultured immortalized podocytes, α-tubulin detyrosination was promoted with cell differentiation. Notably, α-tubulin detyrosination in glomeruli was diminished in Vash1 homozygous knockout (Vash1−/−) mice, and knockdown of VASH1 in cultured podocytes prevented α-tubulin detyrosination. Although VASH1 deficiency-induced downregulation of detyrosination caused no remarkable glomerular lesions, urinary albuminuria excretion and glomerular volume were significantly higher in Vash1−/− mice than in wild-type mice. Furthermore, decreased glomerular nephrin expression and narrower slit diaphragms width were observed in Vash1−/− mice. Taken together, we demonstrated that α-tubulin detyrosination in podocytes was mainly regulated by VASH1 and that VASH1 deficiency-mediated decreases in α-tubulin detyrosination led to minor alterations in podocyte morphology and predisposition to albuminuria. VASH1 expression and α-tubulin detyrosination may be novel targets for maintaining glomerular filtration barrier integrity.