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International Journal of Biochemistry... 2024Glomerular podocytes are specialized epithelial cells localized to the blood-urine interface of the kidney. Podocyte slit-diaphragm (SD), a size-and-charge-selective...
INTRODUCTION
Glomerular podocytes are specialized epithelial cells localized to the blood-urine interface of the kidney. Podocyte slit-diaphragm (SD), a size-and-charge-selective junction, is instrumental in blood ultrafiltration and the formation of protein-free urine. The SD consists of macromolecular complexes of several proteins, such as nephrin, podocin, and CD2-associated protein (CD2AP). CD2AP is an adapter protein and is considered to be crucial for the integrity of SD. Mutations in the SD proteins cause nephrotic syndrome (NS), characterized by proteinuria. SD proteins' structural features must be elucidated to understand the mechanism of proteinuria in NS. In this study, we expressed, purified, and biophysically characterized heterologously expressed human CD2AP.
METHODS
Codon-optimized human CD2AP was expressed in Rosetta cells. The recombinant protein was induced with 1 mM IPTG and purified by Ni-NTA affinity chromatography. Analytical size-exclusion chromatography, blue native-PAGE, circular dichroism, and fluorescence spectroscopy were performed to decipher the oligomeric nature, secondary structural content, and tertiary packing of CD2AP.
RESULTS
Our analysis revealed that CD2AP adopts a predominantly disordered secondary structure despite exhibiting moderate tertiary packing, characterized by low helical and β-sheet content. CD2AP readily assembles into homo-oligomers, with octamers and tetramers constituting the primary population. Interestingly, the inherent flexibility of CD2AP's secondary structural elements appears resistant to thermal denaturation. Frameshift mutation (p.K579Efs*7) that leads to loss of the coiled-coil domain promotes aberrant oligomerization of CD2AP through SH3 domains.
CONCLUSION
We successfully expressed full-length human CD2AP in a heterologous system, wherein the secondary structure of CD2AP is predominantly disordered. CD2AP can form higher-order oligomers, and the significance of these oligomers and the impact of mutations in the context of size-selective permeability of SD needs further investigation.
PubMed: 38765876
DOI: 10.62347/UVSH8436 -
Kidney Medicine Jun 2024Focal segmental glomerulosclerosis (FSGS) defines a distinct histologic pattern observed in kidney tissue that is linked to several distinct underlying causes, all...
Focal segmental glomerulosclerosis (FSGS) defines a distinct histologic pattern observed in kidney tissue that is linked to several distinct underlying causes, all converging on the common factor of podocyte injury. It presents a considerable challenge in terms of classification because of its varied underlying causes and the limited correlation between histopathology and clinical outcomes. Critically, precise nomenclature is key to describe and delineate the pathogenesis, subsequently guiding the selection of suitable and precision therapies. A proposed pathomechanism-based approach has been suggested for FSGS classification. This approach differentiates among primary, secondary, genetic, and undetermined causes, aiming to provide clarity. Genetic FSGS from monogenic mutations can emerge during childhood or adulthood, and it is advisable to conduct genetic testing in cases in which there is a family history of chronic kidney disease, nephrotic syndrome, or resistance to treatment. Genome-wide association studies have identified several genetic risk variants, such as those in apolipoprotein L1 (), that play a role in the development of FSGS. Currently, no specific treatments have been approved to treat genetic FSGS; however, interventions targeting underlying cofactor deficiencies have shown potential in some cases. Furthermore, encouraging results have emerged from a phase 2 trial investigating inaxaplin, a novel small molecule APOL1 channel inhibitor, in -associated FSGS.
PubMed: 38765809
DOI: 10.1016/j.xkme.2024.100826 -
Kidney International Reports Apr 2024The phase 3 DUPLEX trial is evaluating sparsentan, a novel, nonimmunosuppressive, single-molecule dual endothelin angiotensin receptor antagonist, in patients with focal...
INTRODUCTION
The phase 3 DUPLEX trial is evaluating sparsentan, a novel, nonimmunosuppressive, single-molecule dual endothelin angiotensin receptor antagonist, in patients with focal segmental glomerulosclerosis (FSGS).
METHODS
DUPLEX (NCT03493685) is a global, multicenter, randomized, double-blind, parallel-group, active-controlled study evaluating the efficacy and safety of sparsentan 800 mg once daily versus irbesartan 300 mg once daily in patients aged 8 to 75 years (USA/UK) and 18 to 75 years (ex-USA/UK) weighing ≥20 kg with biopsy-proven FSGS or documented genetic mutation in a podocyte protein associated with FSGS, and urine protein-to-creatinine ratio (UP/C) ≥1.5 g/g. Baseline characteristics blinded to treatment allocation are reported descriptively.
RESULTS
The primary analysis population includes 371 patients (336 adult, 35 pediatric [<18 years]) who were randomized and received study drug (median age, 42 years). Patients were White (73.0%), Asian (13.2%), Black/African American (6.7%), or Other race (7.0%); and from North America (38.8%), Europe (36.1%), South America (12.7%), or Asia Pacific (12.4%). Baseline median UP/C was 3.0 g/g; 42.6% in nephrotic-range (UP/C >3.5 g/g [adults]; >2.0 g/g [pediatrics]). Patients were evenly distributed across estimated glomerular filtration rate (eGFR) categories corresponding to chronic kidney disease (CKD) stages 1 to 3b. Thirty-three patients (9.4% of 352 evaluable samples) had pathogenic or likely pathogenic (P/LP) variants of genes essential to podocyte structural integrity and function, 27 (7.7%) had P/LP collagen gene () variants, and 14 (4.0%) had high-risk genotypes.
CONCLUSIONS
Patient enrollment in DUPLEX, the largest interventional study in FSGS to date, will enable important characterization of the treatment effect of sparsentan in a geographically broad and clinically diverse FSGS population.
PubMed: 38765567
DOI: 10.1016/j.ekir.2024.01.032 -
Kidney International Reports Apr 2024During glomerular diseases, podocyte-specific pathways can modulate the intensity of histological disease and prognosis. The therapeutic targeting of these pathways...
INTRODUCTION
During glomerular diseases, podocyte-specific pathways can modulate the intensity of histological disease and prognosis. The therapeutic targeting of these pathways could thus improve the management and prognosis of kidney diseases. The Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway, classically described in immune cells, has been recently described in detail in intrinsic kidney cells.
METHODS
We describe STAT5 expression in human kidney biopsies from patients with focal segmental glomerulosclerosis (FSGS) and studied mice with a podocyte-specific deletion in experimental glomerular diseases.
RESULTS
Here, we show, for the first time, that STAT5 is activated in human podocytes in FSGS. In addition, podocyte-specific inactivation aggravates the structural and functional alterations in a mouse model of FSGS. This could be due, at least in part, to an inhibition of autophagic flux. Finally, interleukin 15 (IL-15), a classical activator of STAT5 in immune cells, increases STAT5 phosphorylation in human podocytes, and its administration alleviates glomerular injury by maintaining autophagic flux in podocytes.
CONCLUSION
Activating podocyte STAT5 with commercially available IL-15 represents a potential new therapeutic avenue for FSGS.
PubMed: 38765560
DOI: 10.1016/j.ekir.2024.01.010 -
Clinical Proteomics May 2024The early identification of patients at high-risk for end-stage renal disease (ESRD) is essential for providing optimal care and implementing targeted prevention...
BACKGROUND
The early identification of patients at high-risk for end-stage renal disease (ESRD) is essential for providing optimal care and implementing targeted prevention strategies. While the Kidney Failure Risk Equation (KFRE) offers a more accurate prediction of ESRD risk compared to static eGFR-based thresholds, it does not provide insights into the patient-specific biological mechanisms that drive ESRD. This study focused on evaluating the effectiveness of KFRE in a UK-based advanced chronic kidney disease (CKD) cohort and investigating whether the integration of a proteomic signature could enhance 5-year ESRD prediction.
METHODS
Using the Salford Kidney Study biobank, a UK-based prospective cohort of over 3000 non-dialysis CKD patients, 433 patients met our inclusion criteria: a minimum of four eGFR measurements over a two-year period and a linear eGFR trajectory. Plasma samples were obtained and analysed for novel proteomic signals using SWATH-Mass-Spectrometry. The 4-variable UK-calibrated KFRE was calculated for each patient based on their baseline clinical characteristics. Boruta machine learning algorithm was used for the selection of proteins most contributing to differentiation between patient groups. Logistic regression was employed for estimation of ESRD prediction by (1) proteomic features; (2) KFRE; and (3) proteomic features alongside KFRE.
RESULTS
SWATH maps with 943 quantified proteins were generated and investigated in tandem with available clinical data to identify potential progression biomarkers. We identified a set of proteins (SPTA1, MYL6 and C6) that, when used alongside the 4-variable UK-KFRE, improved the prediction of 5-year risk of ESRD (AUC = 0.75 vs AUC = 0.70). Functional enrichment analysis revealed Rho GTPases and regulation of the actin cytoskeleton pathways to be statistically significant, inferring their role in kidney function and the pathogenesis of renal disease.
CONCLUSIONS
Proteins SPTA1, MYL6 and C6, when used alongside the 4-variable UK-KFRE achieve an improved performance when predicting a 5-year risk of ESRD. Specific pathways implicated in the pathogenesis of podocyte dysfunction were also identified, which could serve as potential therapeutic targets. The findings of our study carry implications for comprehending the involvement of the Rho family GTPases in the pathophysiology of kidney disease, advancing our understanding of the proteomic factors influencing susceptibility to renal damage.
PubMed: 38762513
DOI: 10.1186/s12014-024-09486-5 -
Cell Reports May 2024Signal-regulatory protein alpha (SIRPα) has recently been found to be highly expressed in podocytes and is essential for maintaining podocyte function. However, its...
Signal-regulatory protein alpha (SIRPα) has recently been found to be highly expressed in podocytes and is essential for maintaining podocyte function. However, its immunoregulatory function in podocytes remains elusive. Here, we report that SIRPα controls podocyte antigen presentation in specific T cell activation via inhibiting spleen tyrosine kinase (Syk) phosphorylation. First, podocyte SIRPα under lupus nephritis (LN) conditions is strongly downregulated. Second, podocyte-specific deletion of SIRPα exacerbates renal disease progression in lupus-prone mice, as evidenced by an increase in T cell infiltration. Third, SIRPα deletion or knockdown enhances podocyte antigen presentation, which activates specific T cells, via enhancing Syk phosphorylation. Supporting this, Syk inhibitor GS-9973 prevents podocyte antigen presentation, resulting in a decrease of T cell activation and mitigation of renal disease caused by SIRPα knockdown or deletion. Our findings reveal an immunoregulatory role of SIRPα loss in promoting podocyte antigen presentation to activate specific T cell immune responses in LN.
Topics: Podocytes; Lupus Nephritis; Animals; Receptors, Immunologic; T-Lymphocytes; Mice; Syk Kinase; Mice, Inbred C57BL; Inflammation; Phosphorylation; Lymphocyte Activation; Humans; Antigen Presentation; Female
PubMed: 38758648
DOI: 10.1016/j.celrep.2024.114249 -
Kidney Diseases (Basel, Switzerland) Apr 2024Unsaturated fatty acids play an essential role in the progression of diabetic nephropathy (DN). However, previous studies were mainly focused on the role of individual...
INTRODUCTION
Unsaturated fatty acids play an essential role in the progression of diabetic nephropathy (DN). However, previous studies were mainly focused on the role of individual unsaturated fatty acid. The serum unsaturated fatty acid patterns (FAPs) in patients with DN remain to be determined.
METHODS
A total of 135 patients with DN (DN group) and 322 patients with type II diabetes without nephropathy (non-DN group) were included in this study. Clinical data, serum levels of unsaturated fatty acids, and other laboratory indicators were collected. Multivariate logistic regression was applied to identify risk factors for serum unsaturated fatty acid level in both groups. Serum unsaturated fatty acids were subjected to factor analysis to identify distinct FAPs. Multivariable logistic regression was employed to assess the risk of DN associated with different serum FAPs.
RESULTS
After adjusting for confounders, three types of unsaturated fatty acid including C20:5 (eicosapentaenoic acid [EPA]), C22:6 (docosahexaenoic acid [DHA]), and C22:5 -3 (docosapentaenoic acid -3) were significantly associated with DN in the population. The odds ratios (ORs) (95% confidence interval [CI]) of DN were 0.583 (0.374, 0.908), 0.826 (0.716, 0.954), and 0.513 (0.298, 0.883), respectively. Factor analysis revealed five major FAPs, among which FAP2 (enriched with EPA and DHA) exhibited a significant inverse association with DN. In the multivariate-adjusted model, the OR (95% CI) was 0.678 (0.493, 0.933). Additionally, a combination of DHA and EPA enriched in FAP2 further decreased extracellular matrix production induced by transforming growth factor beta 1 in podocytes and tubular cells.
CONCLUSIONS
Our findings suggest that FAP2 which is enriched with DHA and EPA is associated with a reduced risk of DN. This highlights the potential of targeting FAP2 for the patients with DN.
PubMed: 38751794
DOI: 10.1159/000536532 -
Free Radical Biology & Medicine Aug 2024Podocytes are crucial for regulating glomerular permeability. They have foot processes that are integral to the renal filtration barrier. Understanding their energy...
Podocytes are crucial for regulating glomerular permeability. They have foot processes that are integral to the renal filtration barrier. Understanding their energy metabolism could shed light on the pathogenesis of filtration barrier injury. Lactate has been increasingly recognized as more than a waste product and has emerged as a significant metabolic fuel and reserve. The recent identification of lactate transporters in podocytes, the expression of which is modulated by glucose levels and lactate, highlights lactate's relevance. The present study investigated the impact of lactate on podocyte respiratory efficiency and mitochondrial dynamics. We confirmed lactate oxidation in podocytes, suggesting its role in cellular energy production. Under conditions of glucose deprivation or lactate supplementation, a significant shift was seen toward oxidative phosphorylation, reflected by an increase in the oxygen consumption rate/extracellular acidification rate ratio. Notably, lactate dehydrogenase A (LDHA) and lactate dehydrogenase B (LDHB) isoforms, which are involved in lactate conversion to pyruvate, were detected in podocytes for the first time. The presence of lactate led to higher intracellular pyruvate levels, greater LDH activity, and higher LDHB expression. Furthermore, lactate exposure increased mitochondrial DNA-to-nuclear DNA ratios and resulted in upregulation of the mitochondrial biogenesis markers peroxisome proliferator-activated receptor coactivator-1α and transcription factor A mitochondrial, regardless of glucose availability. Changes in mitochondrial size and shape were observed in lactate-exposed podocytes. These findings suggest that lactate is a pivotal energy source for podocytes, especially during energy fluctuations. Understanding lactate's role in podocyte metabolism could offer insights into renal function and pathologies that involve podocyte injury.
Topics: Podocytes; Animals; Rats; Lactic Acid; L-Lactate Dehydrogenase; Mitochondrial Dynamics; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Mitochondria; Glucose; Energy Metabolism; Lactate Dehydrogenase 5; Oxidative Phosphorylation; DNA, Mitochondrial; Oxygen Consumption; Cells, Cultured; Pyruvic Acid; Isoenzymes
PubMed: 38740101
DOI: 10.1016/j.freeradbiomed.2024.05.022 -
Archives of Endocrinology and Metabolism May 2024Lipodystrophies are characterized by complete or selective loss of adipose tissue and can be acquired or inherited. Familial partial lipodystrophy (FPLD) is a hereditary...
Lipodystrophies are characterized by complete or selective loss of adipose tissue and can be acquired or inherited. Familial partial lipodystrophy (FPLD) is a hereditary lipodystrophy commonly caused by mutations in the LMNA gene. Herein, we report two cases of FPLD associated with podocytopathies. Patient 1 was diagnosed with FPLD associated with the heterozygous p.Arg482Trp variant in LMNA and had normal glucose tolerance and hyperinsulinemia. During follow-up, she developed nephroticrange proteinuria. Renal biopsy was consistent with minimal change disease. Patient 2 was diagnosed with FPLD associated with a de novo heterozygous p.Arg349Trp variant in LMNA. Microalbuminuria progressed to macroalbuminuria within 6 years and tonephrotic range proteinuria in the last year. He remained without diabetes and with hyperinsulinemia. Renal biopsy revealed focal segmental glomerulosclerosis not otherwise specified. This report provides further evidence of variable features of lipodystrophy associated with LMNA variants and the importance of long-term follow-up with evaluation of kidney dysfunction.
Topics: Humans; Lamin Type A; Lipodystrophy, Familial Partial; Female; Male; Adult; Podocytes; Mutation
PubMed: 38739524
DOI: 10.20945/2359-4292-2023-0204 -
Kidney & Blood Pressure Research 2024Hyperglycaemia induces the production of a large quantity of reactive oxygen species (ROS) and activates the transforming growth factor β1 (TGF-β1)/Smad signalling...
INTRODUCTION
Hyperglycaemia induces the production of a large quantity of reactive oxygen species (ROS) and activates the transforming growth factor β1 (TGF-β1)/Smad signalling pathway, which is the main initiating factor in the formation of diabetic nephropathy. Indoxyl sulphate (IS) is a protein-binding gut-derived uraemic toxin that localizes to podocytes, induces oxidative stress, and inflames podocytes. The involvement of podocyte damage in diabetic nephropathy through the TGF-β1 signalling pathway is still unclear.
METHODS
In this study, we cultured differentiated rat podocytes in vitro and measured the expression levels of nephrin, synaptopodin, CD2AP, SRGAP2a, and α-SMA by quantitative real-time PCR (qRT-PCR) and Western blotting after siRNA-mediated TGF-β1 silencing, TGF-β1 overexpression, and the presence of the ROS inhibitor acetylcysteine. We detected the expression levels of nephrin, synaptopodin, CD2AP, SRGAP2a, small mother against decapentaplegic (Smad)2/3, phosphorylated-Smad2/3 (p-Smad2/3), Smad7, NADPH oxidase 4 (NOX4), and ROS levels under high glucose (HG) and IS conditions.
RESULTS
The results indicated that nephrin, synaptopodin, CD2AP, and SRGAP2a expressions were significantly upregulated, and α-SMA expression was significantly downregulated in the presence of HG under siRNA-mediated TGF-β1 silencing or after the addition of acetylcysteine. However, in the presence of HG, the expressions of nephrin, synaptopodin, CD2AP, and SRGAP2a were significantly downregulated, and the expression of α-SMA was significantly upregulated with the overexpression of TGF-β1. IS supplementation under HG conditions further significantly reduced the expressions of nephrin, synaptopodin, CD2AP, and SRGAP2a; altered the expressions of Smad2/3, p-Smad2/3, Smad7, and NOX4; and increased ROS production in podocytes.
CONCLUSION
This study suggests that IS may modulate the expression of nephrin, synaptopodin, CD2AP, and SRGAP2a by regulating the ROS and TGF-β1/Smad signalling pathways, providing new theoretical support for the treatment of diabetic nephropathy.
Topics: Indican; Podocytes; Animals; Rats; Reactive Oxygen Species; Transforming Growth Factor beta1; Signal Transduction; Diabetic Nephropathies; Smad Proteins; Membrane Proteins; Microfilament Proteins; Cells, Cultured; Adaptor Proteins, Signal Transducing; Cytoskeletal Proteins
PubMed: 38735279
DOI: 10.1159/000538858