The effect of proteinase inhibitors on glomerular albumin permeability induced in vitro by serum from patients with idiopathic focal segmental glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS) is one of the primary glomerular disorders in both children and adults which can progress to end-stage renal failure. Although there are genetic and secondary causes, circulating factors have also been regarded as an important factor in the pathogenesis of FSGS, because about 40% of the patients with FSGS have recurrence after renal transplantation. Soluble urokinase-type plasminogen activator receptor (suPAR) is a soluble form of uPAR, which is a membrane-bound protein linked to GPI in various immunologically active cells, including podocytes. It has recently been suggested as a potential circulating factor in FSGS by in vitro podocyte experiments, in vivo mice models, and human studies. However, there have also been controversies on this issue, because subsequent studies showed conflicting results. suPAR levels were also increased in patients with other glomerular diseases and were inversely correlated with estimated glomerular filtration rate. Nevertheless, there has been no balanced review on this issue. In this review, we compare the conflicting data on the involvement of suPAR in the pathogenesis of FSGS and shed light on interpretation by taking into account many points and the potential variables and confounders influencing serum suPAR levels.

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Anti-apoptosis mechanism of triptolide based on network pharmacology in focal segmental glomerulosclerosis rats

Bioscience Reports ◽

10.1042/bsr20192920 ◽

2020 ◽

Vol 40 (4) ◽

Cited By ~ 2

Author(s):

Yayu Li ◽

Xue Jiang ◽

Litao Song ◽

Mengdie Yang ◽

Jing Pan

Keyword(s):

Focal Segmental Glomerulosclerosis ◽

Cell Apoptosis ◽

P53 Protein ◽

Network Pharmacology ◽

Tripterygium Wilfordii ◽

P53 Expression ◽

Chemical Structures ◽

Segmental Glomerulosclerosis

Abstract Triptolide (TPL), the active component of Tripterygium wilfordii, exhibits anti-cancer and antioxidant functions. We aimed to explore the anti-apoptosis mechanism of TPL based on network pharmacology and in vivo and in vitro research validation using a rat model of focal segmental glomerulosclerosis (FSGS). The chemical structures and pharmacological activities of the compounds reported in T. wilfordii were determined and used to perform the network pharmacology analysis. The Traditional Chinese Medicine Systems Pharmacology Database (TCMSP) was then used to identify the network targets for 16 compounds from Tripterygium wilfordii. Our results showed that 47 overlapping genes obtained from the GeneCards and OMIM databases were involved in the occurrence and development of FSGS and used to construct the protein–protein interaction (PPI) network using the STRING database. Hub genes were identified via the MCODE plug-in of the Cytoscape software. IL4 was the target gene of TPL in FSGS and was mainly enriched in the cell apoptosis term and p53 signaling pathway, according to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. TPL inhibited FSGS-induced cell apoptosis in rats and regulated IL4, nephrin, podocin, and p53 protein levels via using CCK8, TUNEL, and Western blot assays. The effects of IL4 overexpression, including inhibition of cell viability and promotion of apoptosis, were reversed by TPL. TPL treatment increased the expression of nephrin and podocin and decreased p53 expression in rat podocytes. In conclusion, TPL inhibited podocyte apoptosis by targeting IL4 to alleviate kidney injury in FSGS rats.

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First identification of PODXL nonsense mutations in autosomal dominant focal segmental glomerulosclerosis

Clinical Science ◽

10.1042/cs20180676 ◽

2019 ◽

Vol 133 (1) ◽

pp. 9-21 ◽

Cited By ~ 2

Author(s):

Fu-Jun Lin ◽

Lei Yao ◽

Xue-Qing Hu ◽

Fan Bian ◽

Gang Ji ◽

...

Keyword(s):

Focal Segmental Glomerulosclerosis ◽

Autosomal Dominant ◽

In Vitro Study ◽

Peripheral Blood Cell ◽

Fiber Formation ◽

Causative Role ◽

Gene Encoding ◽

Nonsense Mutations ◽

Segmental Glomerulosclerosis

Abstract Recently, a novel heterozygous missense mutation c.T1421G (p. L474R) in the PODXL gene encoding podocalyxin was identified in an autosomal dominant focal segmental glomerulosclerosis (AD-FSGS) pedigree. However, this PODXL mutation appeared not to impair podocalyxin function, and it is necessary to identify new PODXL mutations and determine their causative role for FSGS. In the present study, we report the identification of a heterozygous nonsense PODXL mutation (c.C976T; p. Arg326X) in a Chinese pedigree featured by proteinuria and renal insufficiency with AD inheritance by whole exome sequencing (WES). Total mRNA and PODXL protein abundance were decreased in available peripheral blood cell samples of two affected patients undergoing hemodialysis, compared with those in healthy controls and hemodialysis controls without PODXL mutation. We identified another novel PODXL heterozygous nonsense mutation (c.C1133G; p.Ser378X) in a British–Indian pedigree of AD-FSGS by WES. In vitro study showed that, human embryonic kidney 293T cells transfected with the pEGFP-PODXL-Arg326X or pEGFP-PODXL-Ser378X plasmid expressed significantly lower mRNA and PODXL protein compared with cells transfected with the wild-type plasmid. Blocking nonsense-mediated mRNA decay (NMD) significantly restored the amount of mutant mRNA and PODXL proteins, which indicated that the pathogenic effect of PODXL nonsense mutations is likely due to NMD, resulting in podocalyxin deficiency. Functional consequences caused by the PODXL nonsense mutations were inferred by siRNA knockdown in cultured podocytes and podocalyxin down-regulation by siRNA resulted in decreased RhoA and ezrin activities, cell migration and stress fiber formation. Our results provided new data implicating heterozygous PODXL nonsense mutations in the development of FSGS.

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Urinary myo-inositol is associated with the clinical outcome in focal segmental glomerulosclerosis

Scientific Reports ◽

10.1038/s41598-019-51276-9 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Jung Nam An ◽

Jin Seong Hyeon ◽

Youngae Jung ◽

Young Wook Choi ◽

Jin Hyuk Kim ◽

...

Keyword(s):

Focal Segmental Glomerulosclerosis ◽

Urinary Metabolites ◽

Urine Samples ◽

Resonance Spectroscopy ◽

Important Indicator ◽

Clinical Prognosis ◽

Segmental Glomerulosclerosis ◽

Healthy Control ◽

Validation Set

Abstract Focal segmental glomerulosclerosis (FSGS) and minimal change disease (MCD) have similar initial histological findings; however, their prognoses are distinct. Therefore, it is of great importance to discriminate FSGS from MCD in the early phase of disease and predict clinical prognosis. A discovery set of 184 urine samples (61 healthy control, 80 MCD, and 43 FSGS) and a validation set of 61 urine samples (12 healthy control, 26 MCD, and 23 FSGS) were collected at the time of kidney biopsy. Metabolic profiles were examined using nuclear magnetic resonance spectroscopy. Of 70 urinary metabolites, myo-inositol was significantly higher in FSGS patients than in control patients (discovery set, 2.34-fold, P < 0.001; validation set, 2.35-fold, P = 0.008) and MCD patients (discovery set, 2.48-fold, P = 0.002; validation set, 1.69-fold, P = 0.042). Myo-inositol showed an inverse relationship with the initial estimated glomerular filtration rate (eGFR) and was associated with the plasma level of soluble urokinase-type plasminogen activator receptor in FSGS patients. Myo-inositol treatment ameliorated the decreased expression of ZO-1 and synaptopodin in an in vitro FSGS model, and as myo-inositol increased, myo-inositol oxygenase tissue expression decreased proportionally to eGFR. Furthermore, urinary myo-inositol exhibited an increase in the power to discriminate FSGS patients, and its addition could better predict the response to initial treatment. In conclusion, urinary myo-inositol may be an important indicator in the diagnosis and treatment of FSGS patients.

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Erratum to: Novel in vitro assays to detect circulating permeability factor(s) in idiopathic focal segmental glomerulosclerosis

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfab021 ◽

2021 ◽

Author(s):

Dirk J W den Braanker ◽

Rutger J Maas ◽

Jeroen K Deegens ◽

Cansu Yanginlar ◽

Jack F M Wetzels ◽

...

Keyword(s):

Focal Segmental Glomerulosclerosis ◽

In Vitro Assays ◽

Permeability Factor ◽

Segmental Glomerulosclerosis

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Melanocortin therapy ameliorates podocytopathy and proteinuria in experimental focal segmental glomerulosclerosis involving a podocyte specific non-MC1R-mediated melanocortinergic signaling

Clinical Science ◽

10.1042/cs20200016 ◽

2020 ◽

Vol 134 (7) ◽

pp. 695-710

Author(s):

Yingjin Qiao ◽

Pei Wang ◽

Mingyang Chang ◽

Bohan Chen ◽

Yan Ge ◽

...

Keyword(s):

Beneficial Effect ◽

Focal Segmental Glomerulosclerosis ◽

De Novo ◽

Protective Action ◽

Clinical Effectiveness ◽

Podocyte Injury ◽

Segmental Glomerulosclerosis ◽

Foot Process

Abstract The clinical effectiveness of adrenocorticotropin in inducing remission of steroid-resistant nephrotic syndrome points to a steroidogenic-independent anti-proteinuric activity of melanocortins. However, which melanocortin receptors (MCR) convey this beneficial effect and if systemic or podocyte-specific mechanisms are involved remain uncertain. In vivo, wild-type (WT) mice developed heavy proteinuria and kidney dysfunction following Adriamycin insult, concomitant with focal segmental glomerulosclerosis (FSGS) and podocytopathy, marked by loss of podocin and synaptopodin, podocytopenia and extensive foot process effacement on electron microscopy. All these pathologic findings were prominently attenuated by NDP-MSH, a potent non-steroidogenic pan-MCR agonist. Surprisingly, MC1R deficiency in MC1R-null mice barely affected the severity of Adriamycin-elicited injury. Moreover, the beneficial effect of NDP-MSH was completely preserved in MC1R-null mice, suggesting that MC1R is likely non-essential for the protective action. A direct podocyte effect seems to contribute to the beneficial effect of NDP-MSH, because Adriamycin-inflicted cytopathic signs in primary podocytes prepared from WT mice were all mitigated by NDP-MSH, including apoptosis, loss of podocyte markers, de novo expression of the podocyte injury marker desmin, actin cytoskeleton derangement and podocyte hypermotility. Consistent with in vivo findings, the podoprotective activity of NDP-MSH was fully preserved in MC1R-null podocytes. Mechanistically, MC1R expression was predominantly distributed to glomerular endothelial cells in glomeruli but negligibly noted in podocytes in vivo and in vitro, suggesting that MC1R signaling is unlikely involved in direct podocyte protection. Ergo, melanocortin therapy protects against podocyte injury and ameliorates proteinuria and glomerulopathy in experimental FSGS, at least in part, via a podocyte-specific non-MC1R-mediated melanocortinergic signaling.

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Novel in vitro assays to detect circulating permeability factor(s) in idiopathic focal segmental glomerulosclerosis

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfaa211 ◽

2020 ◽

Author(s):

Dirk J W den Braanker ◽

Rutger J Maas ◽

Jeroen K Deegens ◽

Cansu Yanginlar ◽

Jack F M Wetzels ◽

...

Keyword(s):

Focal Segmental Glomerulosclerosis ◽

Calf Serum ◽

In Vitro Assays ◽

Future Research ◽

Foetal Calf Serum ◽

Dependent Manner ◽

Segmental Glomerulosclerosis ◽

Human And Mouse ◽

Active Disease

Abstract Background Many patients with idiopathic focal segmental glomerulosclerosis (FSGS) develop recurrence of proteinuria after kidney transplantation (TX). Although several circulating permeability factors (CPFs) responsible for recurrence have been suggested, there is no consensus. To facilitate CPF identification and predict recurrence after TX, there is a need for robust methods that demonstrate the presence of CPFs. Methods Cultured human podocytes (hPods) and human and mouse glomerular endothelial cells (ciGEnC, mGEnC) were exposed to plasmas of FSGS patients with presumed CPFs, and of (disease) controls. A visual scoring assay and flow cytometry analysis of side scatter were used to measured changes in cellular granularity after exposure to plasma. Results Nine out of 13 active disease plasmas of 10 FSGS patients with presumed CPFs induced granularity in hPod in a dose- and time-dependent manner. Corresponding remission plasmas induced no or less granularity in hPod. Similar results were obtained with ciGEnC and mGEnC, although induced granularity was less compared with hPod. Notably, foetal calf serum, healthy plasma and a remission plasma partially blocked FSGS plasma-induced hPod granularity. Conclusions We developed a novel assay in which active disease, presumably CPF-containing, FSGS plasmas induced granularity in cultured hPod. Our results may indicate the presence of CPF inhibitor(s) in healthy and remission plasma. We suggest the presence of a delicate balance between CPF and a CPF inhibitory factor, which is disturbed in patients with active disease. Our novel assays can be applied in future research to identify CPF and CPF inhibitors, and possibly to predict recurrence after TX.

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Apolipoproteins Prevent Glomerular Albumin Permeability Induced In Vitro by Serum from Patients with Focal Segmental Glomerulosclerosis

Journal of the American Society of Nephrology ◽

10.1681/asn.v121143 ◽

2001 ◽

Vol 12 (1) ◽

pp. 143-150

Author(s):

GIOVANNI CANDIANO ◽

LUCA MUSANTE ◽

MICHELE CARRARO ◽

LUIGI FACCINI ◽

LUCIANO CAMPANACCI ◽

...

Keyword(s):

High Density Lipoprotein ◽

Focal Segmental Glomerulosclerosis ◽

Inhibitory Activity ◽

Density Lipoprotein ◽

Normal Serum ◽

High Density ◽

Segmental Glomerulosclerosis ◽

Apo E ◽

Permeability Alterations

Abstract. Glomerular albumin permeability alterations can be induced in vitro by serum from patients with end-stage renal disease caused by primary focal segmental glomerulosclerosis (FSGS). It was hypothesized that inhibitory substances may be present in normal serum, which may prevent the permeability alterations in isolated glomeruli, and the present study sought to isolate and characterize these factors. Albumin permeability was determined from the change in glomerular volume induced by applying oncotic gradients across the basement membrane of healthy isolated rat glomeruli preincubated with FSGS serum and normal serum fractionated using standard techniques. Fractions of normal serum with inhibitory activity obtained by a multistep chromatographic procedure underwent two-dimensional electrophoresis and staining. Approximately 50 protein spots were recovered, renatured, and tested for antipermeability activity. Five of these proteins demonstrated consistent inhibitory activity, and desorption ionization and mass spectrometry proved them to be components of high-density lipoprotein: apolipoproteins (apo) E2 and E4, high-molecular-weight J and L, and a 28-kD fragment of A-IV. Polyclonal antibodies to apo E or apo J added to the whole normal serum restored the permeability activity of the FSGS serum in the bioassay. Commercially available apo E and apo J also demonstrated antipermeability activity when added to FSGS serum. Cyanogen bromide digestion of apo A-IV produced fragments that inhibited the permeability activity of the FSGS serum, whereas the intact protein did not. Thus, components of high-density lipoprotein are capable of preventing glomerular albumin permeability induced by serum from patients with FSGS in an in vitro system. The specificity and mechanism of the inhibition remain to be determined; the alteration of normal inhibitory activity in vivo may be a component in the pathophysiology of FSGS.

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Unravelling the Role of PAX2 Mutation in Human Focal Segmental Glomerulosclerosis

Biomedicines ◽

10.3390/biomedicines9121808 ◽

2021 ◽

Vol 9 (12) ◽

pp. 1808

Author(s):

Lorena Longaretti ◽

Piera Trionfini ◽

Valerio Brizi ◽

Christodoulos Xinaris ◽

Caterina Mele ◽

...

Keyword(s):

Focal Segmental Glomerulosclerosis ◽

Cell Injury ◽

Normal Structure ◽

Branching Morphogenesis ◽

Patient Specific ◽

Developmental Defects ◽

Segmental Glomerulosclerosis

No effective treatments are available for familial steroid-resistant Focal Segmental Glomerulosclerosis (FSGS), characterized by proteinuria due to ultrastructural abnormalities in glomerular podocytes. Here, we studied a private PAX2 mutation identified in a patient who developed FSGS in adulthood. By generating adult podocytes using patient-specific induced pluripotent stem cells (iPSC), we developed an in vitro model to dissect the role of this mutation in the onset of FSGS. Despite the PAX2 mutation, patient iPSC properly differentiated into podocytes that exhibited a normal structure and function when compared to control podocytes. However, when exposed to an environmental trigger, patient podocytes were less viable and more susceptible to cell injury. Fixing the mutation improved their phenotype and functionality. Using a branching morphogenesis assay, we documented developmental defects in patient-derived ureteric bud-like tubules that were totally rescued by fixing the mutation. These data strongly support the hypothesis that the PAX2 mutation has a dual effect, first in renal organogenesis, which could account for a suboptimal nephron number at birth, and second in adult podocytes, which are more susceptible to cell death caused by environmental triggers. These abnormalities might translate into the development of proteinuria in vivo, with a progressive decline in renal function, leading to FSGS.

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APOL1 Risk Variants Affect Podocyte Lipid Homeostasis and Energy Production in Focal Segmental Glomerulosclerosis

Human Molecular Genetics ◽

10.1093/hmg/ddab022 ◽

2021 ◽

Author(s):

Mengyuan Ge ◽

Judith Molina ◽

G Michelle Ducasa ◽

Shamroop K Mallela ◽

Javier Varona Santos ◽

...

Keyword(s):

Transgenic Mice ◽

Mitochondrial Dysfunction ◽

Kidney Function ◽

Focal Segmental Glomerulosclerosis ◽

Activated T Cells ◽

Risk Variant ◽

Segmental Glomerulosclerosis ◽

Risk Variants ◽

Triglyceride Content

Abstract Lipotoxicity was recently reported in several forms of kidney disease, including focal segmental glomerulosclerosis (FSGS). Susceptibility to FSGS in African Americans is associated with the presence of genetic variants of the Apolipoprotein L1 gene (APOL1) named G1 and G2. If and how endogenous APOL1 may alter mitochondrial function by modifying cellular lipid metabolism is unknown. Using transgenic mice expressing the APOL1 variants (G0, G1 or G2) under endogenous promoter, we show that APOL1 risk variant expression in transgenic mice does not impair kidney function at baseline. However, APOL1 G1 expression worsens proteinuria and kidney function in mice characterized by the podocyte inducible expression of nuclear factor of activated T-cells (NFAT), which we have found to cause FSGS. APOL1 G1 expression in this FSGS-model also results in increased triglyceride and cholesterol ester contents in kidney cortices, where lipid accumulation correlated with loss of renal function. In vitro, we show that the expression of endogenous APOL1 G1/G2 in human urinary podocytes is associated with increased cellular triglyceride content and is accompanied by mitochondrial dysfunction in the presence of compensatory oxidative phosphorylation (OXPHOS) complexes elevation. Our findings indicate that APOL1 risk variant expression increases the susceptibility to lipid-dependent podocyte injury, ultimately leading to mitochondrial dysfunction.

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