-
International Journal of Molecular... May 2024The mechanism underlying podocyte dysfunction in minimal change disease (MCD) remains unknown. This study aimed to shed light on the potential pathophysiology of MCD...
The mechanism underlying podocyte dysfunction in minimal change disease (MCD) remains unknown. This study aimed to shed light on the potential pathophysiology of MCD using glomerular proteomic analysis. Shotgun proteomics using label-free quantitative mass spectrometry was performed on formalin-fixed, paraffin-embedded (FFPE) renal biopsies from two groups of samples: control (CTR) and MCD. Glomeruli were excised from FFPE renal biopsies using laser capture microdissection (LCM), and a single-pot solid-phase-enhanced sample preparation (SP3) digestion method was used to improve yield and protein identifications. Principal component analysis (PCA) revealed a distinct separation between the CTR and MCD groups. Forty-eight proteins with different abundance between the two groups (-value ≤ 0.05 and |FC| ≥ 1.5) were identified. These may represent differences in podocyte structure, as well as changes in endothelial or mesangial cells and extracellular matrix, and some were indeed found in several of these structures. However, most differentially expressed proteins were linked to the podocyte cytoskeleton and its dynamics. Some of these proteins are known to be involved in focal adhesion (NID1 and ITGA3) or slit diaphragm signaling (ANXA2, TJP1 and MYO1C), while others are structural components of the actin and microtubule cytoskeleton of podocytes (ACTR3 and NES). This study suggests the potential of mass spectrometry-based shotgun proteomic analysis with LCM glomeruli to yield valuable insights into the pathogenesis of podocytopathies like MCD. The most significantly dysregulated proteins in MCD could be attributable to cytoskeleton dysfunction or may be a compensatory response to cytoskeleton malfunction caused by various triggers.
Topics: Humans; Nephrosis, Lipoid; Proteomics; Podocytes; Kidney Glomerulus; Male; Female; Adult; Proteome; Laser Capture Microdissection; Middle Aged
PubMed: 38891801
DOI: 10.3390/ijms25115613 -
Cells May 2024Podocyte health is vital for maintaining proper glomerular filtration in the kidney. Interdigitating foot processes from podocytes form slit diaphragms which regulate... (Review)
Review
Podocyte health is vital for maintaining proper glomerular filtration in the kidney. Interdigitating foot processes from podocytes form slit diaphragms which regulate the filtration of molecules through size and charge selectivity. The abundance of lipid rafts, which are ordered membrane domains rich in cholesterol and sphingolipids, near the slit diaphragm highlights the importance of lipid metabolism in podocyte health. Emerging research shows the importance of sphingolipid metabolism to podocyte health through structural and signaling roles. Dysregulation in sphingolipid metabolism has been shown to cause podocyte injury and drive glomerular disease progression. In this review, we discuss the structure and metabolism of sphingolipids, as well as their role in proper podocyte function and how alterations in sphingolipid metabolism contributes to podocyte injury and drives glomerular disease progression.
Topics: Podocytes; Sphingolipids; Humans; Animals; Lipid Metabolism; Kidney Diseases; Membrane Microdomains
PubMed: 38891023
DOI: 10.3390/cells13110890 -
Acta Physiologica (Oxford, England) Jun 2024Calcineurin inhibitors (CNIs) are the backbone for immunosuppression after solid organ transplantation. Although successful in preventing kidney transplant rejection,...
AIM
Calcineurin inhibitors (CNIs) are the backbone for immunosuppression after solid organ transplantation. Although successful in preventing kidney transplant rejection, their nephrotoxic side effects contribute to allograft injury. Renal parenchymal lesions occur for cyclosporine A (CsA) as well as for the currently favored tacrolimus (Tac). We aimed to study whether chronic CsA and Tac exposures, before reaching irreversible nephrotoxic damage, affect renal compartments differentially and whether related pathogenic mechanisms can be identified.
METHODS
CsA and Tac were administered chronically in wild type Wistar rats using osmotic minipumps over 4 weeks. Functional parameters were controlled. Electron microscopy, confocal, and 3D-structured illumination microscopy were used for histopathology. Clinical translatability was tested in human renal biopsies. Standard biochemical, RNA-seq, and proteomic technologies were applied to identify implicated molecular pathways.
RESULTS
Both drugs caused significant albeit differential damage in vasculature and nephron. The glomerular filtration barrier was more affected by Tac than by CsA, showing prominent deteriorations in endothelium and podocytes along with impaired VEGF/VEGFR2 signaling and podocyte-specific gene expression. By contrast, proximal tubule epithelia were more severely affected by CsA than by Tac, revealing lysosomal dysfunction, enhanced apoptosis, impaired proteostasis and oxidative stress. Lesion characteristics were confirmed in human renal biopsies.
CONCLUSION
We conclude that pathogenetic alterations in the renal compartments are specific for either treatment. Considering translation to the clinical setting, CNI choice should reflect individual risk factors for renal vasculature and tubular epithelia. As a step in this direction, we share protein signatures identified from multiomics with potential pathognomonic relevance.
PubMed: 38884453
DOI: 10.1111/apha.14190 -
Experimental Biology and Medicine... 2024Podocyte injury or dysfunction can lead to proteinuria and glomerulosclerosis. Zonula occludens 1 (ZO-1) is a tight junction protein which connects slit diaphragm (SD)...
Podocyte injury or dysfunction can lead to proteinuria and glomerulosclerosis. Zonula occludens 1 (ZO-1) is a tight junction protein which connects slit diaphragm (SD) proteins to the actin cytoskeleton. Previous studies have shown that the expression of ZO-1 is decreased in chronic kidney disease (CKD). Thus, elucidation of the regulation mechanism of ZO-1 has considerable clinical importance. Triptolide (TP) has been reported to exert a strong antiproteinuric effect by inhibiting podocyte epithelial mesenchymal transition (EMT) and inflammatory response. However, the underlying mechanisms are still unclear. We found that TP upregulates ZO-1 expression and increases the fluorescence intensity of ZO-1 in a puromycin aminonucleoside (PAN)-induced podocyte injury model. Permeablity assay showed TP decreases podocyte permeability in PAN-treated podocyte. TP also upregulates the DNA demethylase TET2. Our results showed that treatment with the DNA methyltransferase inhibitors 5-azacytidine (5-AzaC) and RG108 significantly increased ZO-1 expression in PAN-treated podocytes. Methylated DNA immunoprecipitation (MeDIP) and hydroxymethylated DNA immunoprecipitation (hMeDIP) results showed that TP regulates the methylation status of the ZO-1 promoter. Knockdown of TET2 decreased ZO-1 expression and increased methylation of its promoter, resulting in the increase of podocyte permeability. Altogether, these results indicate that TP upregulates the expression of ZO-1 and decreases podocyte permeability through TET2-mediated 5 mC demethylation. These findings suggest that TP may alleviate podocyte permeability through TET2-mediated hydroxymethylation of ZO-1.
Topics: Podocytes; Zonula Occludens-1 Protein; Phenanthrenes; Diterpenes; Epoxy Compounds; Dioxygenases; Animals; DNA-Binding Proteins; Mice; Proto-Oncogene Proteins; Permeability; Humans; DNA Methylation
PubMed: 38881848
DOI: 10.3389/ebm.2024.10051 -
Diabetologia Jun 2024Glomerular lipid accumulation is a defining feature of diabetic kidney disease (DKD); however, the precise underlying mechanism requires further elucidation. Recent...
AIMS/HYPOTHESIS
Glomerular lipid accumulation is a defining feature of diabetic kidney disease (DKD); however, the precise underlying mechanism requires further elucidation. Recent evidence suggests a role for proprotein convertase subtilisin/kexin type 9 (PCSK9) in intracellular lipid homeostasis. Although PCSK9 is present in kidneys, its role within kidney cells and relevance to renal diseases remain largely unexplored. Therefore, we investigated the role of intracellular PCSK9 in regulating lipid accumulation and homeostasis in the glomeruli and podocytes under diabetic conditions. Furthermore, we aimed to identify the pathophysiological mechanisms responsible for the podocyte injury that is associated with intracellular PCSK9-induced lipid accumulation in DKD.
METHODS
In this study, glomeruli were isolated from human kidney biopsy tissues, and glomerular gene-expression analysis was performed. Also, db/db and db/m mice were used to perform glomerular gene-expression profiling. We generated DKD models using a high-fat diet and low-dose intraperitoneal streptozocin injection in C57BL/6 and Pcsk9 knockout (KO) mice. We analysed cholesterol and triacylglycerol levels within the kidney cortex. Lipid droplets were evaluated using BODIPY staining. We induced upregulation and downregulation of PCSK9 expression in conditionally immortalised mouse podocytes using lentivirus and siRNA transfection techniques, respectively, under diabetic conditions.
RESULTS
A significant reduction in transcription level of PCSK9 was observed in glomeruli of individuals with DKD. PCSK9 expression was also reduced in podocytes of animals under diabetic conditions. We observed significantly higher lipid accumulation in kidney tissues of Pcsk9 KO DKD mice compared with wild-type (WT) DKD mice. Additionally, Pcsk9 KO mouse models of DKD exhibited a significant reduction in mitochondria number vs WT models, coupled with a significant increase in mitochondrial size. Moreover, albuminuria and podocyte foot process effacement were observed in WT and Pcsk9 KO DKD mice, with KO DKD mice displaying more pronounced manifestations. Immortalised mouse podocytes exposed to diabetic stimuli exhibited heightened intracellular lipid accumulation, mitochondrial injury and apoptosis, which were ameliorated by Pcsk9 overexpression and aggravated by Pcsk9 knockdown in mouse podocytes.
CONCLUSIONS/INTERPRETATION
The downregulation of PCSK9 in podocytes is associated with lipid accumulation, which leads to mitochondrial dysfunction, cell apoptosis and renal injury. This study sheds new light on the potential involvement of PCSK9 in the pathophysiology of glomerular lipid accumulation and podocyte injury in DKD.
PubMed: 38879617
DOI: 10.1007/s00125-024-06191-8 -
European Journal of Medicinal Chemistry Jun 2024Transient receptor potential canonical 5 (TRPC5) is a calcium-permeable non-selective cation channel involved in various pathophysiological processes, including renal...
Discovery of pyridazinone derivatives bearing tetrahydroimidazo[1,2-a]pyrazine scaffold as potent inhibitors of transient receptor potential canonical 5 to ameliorate hypertension-induced renal injury in rats.
Transient receptor potential canonical 5 (TRPC5) is a calcium-permeable non-selective cation channel involved in various pathophysiological processes, including renal injury. Recently, GFB-887, an investigational pyridazinone TRPC5 inhibitor, demonstrated significant therapeutic potential in a Phase II clinical trial for focal segmental glomerulosclerosis (FSGS), a rare and severe form of chronic kidney disease (CKD). In the current study, based on the structure of GFB-887, we conducted extensive structural modification to explore novel TRPC5 inhibitors with desirable drug-like properties and robust nephroprotective efficacy. A series of pyridazinone derivatives featuring a novel tetrahydroimidazo[1,2-a]pyrazine scaffold were synthesized and their activities were evaluated in HEK-293 cells stably expressing TRPC5 using a fluorescence-based Ca mobilization assay. Among these compounds, compound 12 is turned out to be a potent TRPC5 inhibitor with apparent affinity comparable to the parent compound GBF-887. Compound 12 is highly selective on TRPC4/5 over TRPC3/6/7 and hERG channels, along with acceptable pharmacokinetic properties and a favorable safety profile. More importantly, in a rat model of hypertension-induced renal injury, oral administration of compound 12 (10 mg/kg, BID) efficaciously reduced mean blood pressure, inhibited proteinuria, and protected podocyte damage. These findings further confirmed the potential of TRPC5 inhibitors on the CKD treatment and provided compound 12 to be a valuable tool for exploring TRPC4/5 pathophysiology.
PubMed: 38878518
DOI: 10.1016/j.ejmech.2024.116565 -
Cellular and Molecular Life Sciences :... Jun 2024Blood ultrafiltration in nephrons critically depends on specialized intercellular junctions between podocytes, named slit diaphragms (SDs). Here, by studying a...
Blood ultrafiltration in nephrons critically depends on specialized intercellular junctions between podocytes, named slit diaphragms (SDs). Here, by studying a homologous structure found in Drosophila nephrocytes, we identify the phospholipid scramblase Scramb1 as an essential component of the SD, uncovering a novel link between membrane dynamics and SD formation. In scramb1 mutants, SDs fail to form. Instead, the SD components Sticks and stones/nephrin, Polychaetoid/ZO-1, and the Src-kinase Src64B/Fyn associate in cortical foci lacking the key SD protein Dumbfounded/NEPH1. Scramb1 interaction with Polychaetoid/ZO-1 and Flotillin2, the presence of essential putative palmitoylation sites and its capacity to oligomerize, suggest a function in promoting SD assembly within lipid raft microdomains. Furthermore, Scramb1 interactors as well as its functional sensitivity to temperature, suggest an active involvement in membrane remodeling processes during SD assembly. Remarkably, putative Ca-binding sites in Scramb1 are essential for its activity raising the possibility that Ca signaling may control the assembly of SDs by impacting on Scramb1 activity.
Topics: Animals; Podocytes; Drosophila Proteins; Phospholipid Transfer Proteins; Membrane Proteins; Drosophila melanogaster; Membrane Microdomains; Intercellular Junctions
PubMed: 38878170
DOI: 10.1007/s00018-024-05287-z -
Renal Failure Dec 2024Podocyte loss in glomeruli is a fundamental event in the pathogenesis of chronic kidney diseases. Currently, mitotic catastrophe (MC) has emerged as the main cause of...
Podocyte loss in glomeruli is a fundamental event in the pathogenesis of chronic kidney diseases. Currently, mitotic catastrophe (MC) has emerged as the main cause of podocyte loss. However, the regulation of MC in podocytes has yet to be elucidated. The current work aimed to study the role and mechanism of p53 in regulating the MC of podocytes using adriamycin (ADR)-induced nephropathy. podocyte stimulation with ADR triggered the occurrence of MC, which was accompanied by hyperactivation of p53 and cyclin-dependent kinase (CDK1)/cyclin B1. The inhibition of p53 reversed ADR-evoked MC in podocytes and protected against podocyte injury and loss. Further investigation showed that p53 mediated the activation of CDK1/cyclin B1 by regulating the expression of Wee1. Restraining Wee1 abolished the regulatory effect of p53 inhibition on CDK1/cyclin B1 and rebooted MC in ADR-stimulated podocytes p53 inhibition. In a mouse model of ADR nephropathy, the inhibition of p53 ameliorated proteinuria and podocyte injury. Moreover, the inhibition of p53 blocked the progression of MC in podocytes in ADR nephropathy mice through the regulation of the Wee1/CDK1/cyclin B1 axis. Our findings confirm that p53 contributes to MC in podocytes through regulation of the Wee1/CDK1/Cyclin B1 axis, which may represent a novel mechanism underlying podocyte injury and loss during the progression of chronic kidney disorder.
Topics: Podocytes; Animals; CDC2 Protein Kinase; Tumor Suppressor Protein p53; Mice; Protein-Tyrosine Kinases; Doxorubicin; Cyclin B1; Cell Cycle Proteins; Mitosis; Disease Models, Animal; Humans; Male
PubMed: 38874119
DOI: 10.1080/0886022X.2024.2365408 -
Liver International : Official Journal... Jun 2024Accumulating epidemiological evidence shows that the patatin-like phospholipase domain-containing protein-3 (PNPLA3) rs738409 G allele, which is the most robust genetic... (Review)
Review
Accumulating epidemiological evidence shows that the patatin-like phospholipase domain-containing protein-3 (PNPLA3) rs738409 G allele, which is the most robust genetic variant associated with greater susceptibility to metabolic dysfunction-associated steatotic liver disease (MASLD), is significantly associated with impaired kidney function in both adults and children, regardless of the presence of common renal risk factors, MASLD severity, and other potential confounders. Although some prospective studies have reported a significant association between the PNPLA3 rs738409 G allele and the increased risk of developing chronic kidney disease (CKD), the epidemiological evidence about a possible direct effect of the PNPLA3 rs738409 G allele on the risk of developing CKD is still limited. Experimentally, PNPLA3 is expressed in renal podocytes, pericytes, and proximal tubule cells, thus supporting the notion that the mutant PNPLA3 protein may play a role in developing renal steatosis and fibrosis. However, it cannot be ruled out that a part of the adverse effect of the PNPLA3 rs738409 G allele on kidney function may be driven by a direct impact of this genetic variant on the development and progression of MASLD. It is possible to hypothesize that identifying the PNPLA3 genotype might help identify individuals at higher risk of CKD and those at greater risk of advanced MASLD. In this narrative minireview, we summarize the current epidemiological data about the association between the PNPLA3 rs738409 G allele and the risk of CKD and abnormal albuminuria. We also briefly discuss the putative biological mechanisms underpinning this association and its potential and future clinical implications.
PubMed: 38873992
DOI: 10.1111/liv.16010 -
IScience Jun 2024Podocytopathies, such as focal segmental glomerulosclerosis (FSGS), are characterized by podocyte injury and can easily progress to end-stage kidney disease. However,...
Podocytopathies, such as focal segmental glomerulosclerosis (FSGS), are characterized by podocyte injury and can easily progress to end-stage kidney disease. However, the mechanisms underlying podocyte injury remain unclear. We observed podocyte injury along with pyroptosis in patients with FSGS. Bioinformatic analysis of public datasets revealed that transmembrane protein 30a (Tmem30a) might be associated with FSGS. The expression of Temem30a and the podocyte-related protein, nephrin, were significantly downregulated in patients with FSGS, adriamycin (ADR)-induced mice, and podocyte-specific mice, whereas the expression of NLR family pyrin domain containing 3 (NLRP3) and ASC, two pyroptosis-related proteins, were significantly upregulated. Meanwhile, the pyroptosis inhibitor MCC950 and disulfiram (DSF) increased Tmem30a and podocyte-related proteins expression, and inhibited pyroptosis-related proteins expression in ADR-induced mouse podocytes and knockdown (KD) mouse podocytes. Therefore, Tmem30a might protect against podocyte injury by inhibiting pyroptosis, suggesting a potential therapeutic target for podocytopathies.
PubMed: 38868200
DOI: 10.1016/j.isci.2024.109976