Vincristine Treatment Protects Against Podocyte Damage in Focal Segmental Glomerulosclerosis

PAR-3 Activation by Activated Protein C: a Novel Podocyte Protective Signalling Pathway

Familial occurrence of systemic and cutaneous mastocytosis in an adult multicentre series.

A zebrafish model of conditional targeted podocyte ablation and regeneration

Podocytes, highly specified kidney epithelial cells, live under several pathological stimuli and stresses during which they adapt themselves to keep homeostasis. Nevertheless, under extreme stress, a complex scenario of podocyte damage and its consequences occur. Podocyte damage causes foot process effacement and their detachment from the glomerular basement membrane, leading to proteinuria. Podocyte-derived extracellular vesicles (pEVs), mainly microparticles and exosomes are considered as signaling mediators of intercellular communication. Recently, it has been shown that throughout the injury-related migration procedure, podocytes are capable of releasing the injury-related migrasomes. Evidence indicates that at the early stages of glomerular disorders, increased levels of pEVs are observed in urine. At the early stage of nephropathy, pEVs especially migrasomes seem to be more sensitive and reliable indicators of podocyte stress and/or damage than proteinuria. This review highlights the current knowledge of pEVs and their values for the diagnosis of different kidney diseases.

Background Focal segmental glomerulosclerosis (FSGS) is a relatively prevalent glomerular disorder that often progresses to end-stage renal disease. 30 to 80% of kidney transplant (KT) recipients with FSGS will experience recurrence characterized by proteinuria and podocyte damage. We hypothesized the degree of podocyte foot process (FP) effacement in post-reperfusion transplant biopsies can be used to predict the development of clinical recurrence of FSGS.

Podocytes have an intricate structure featured by numerous actin-based projections called foot processes. Rho-family of small GTPases, including Ras-related C3 botulinum toxin substrate 1 (Rac1), play important roles in actin cytoskeletal remodeling required for cell morphology and adhesion. We previously showed that Rac1 activation in podocytes causes foot process effacement and proteinuria, but the upstream and spatiotemporal regulatory mechanism directing Rac1 is largely unknown. Recently we identified the focal adhesion protein, GIT ArfGAP2 (GIT2) as one of the Rac1 interactors in human podocytes by proximity-dependent biotin identification (BioID) and proteomics. Systemic and podocyte-specific GIT2 knockout mice were generated and assessed for kidney phenotypes. Human podocytes with GIT2 knockdown and overexpression were established using lentiviral transduction and characterized. GIT2 was enriched in glomeruli including podocytes in the mouse kidney. Gene deletion of Git2 in podocytes caused exacerbated proteinuria and foot process effacement when subjected to the minimal change disease model and the salt-sensitive hypertension model, which were improved by pharmacological inhibition of Rac1. In cultured podocytes, GIT2 knockdown resulted in Rac1-dependent cell spreading with marked lamellipodial protrusions, accelerated focal adhesion disassembly, and a shorter focal adhesion lifetime. In GIT2 knockdown podocytes, tyrosine phosphorylation of the focal adhesion protein, p130 Crk-associated substrate (Cas), was significantly increased, accompanied by impaired localization of the tyrosine phosphatase, PTP1B, to focal adhesions. These phenotypes observed in GIT2 knockdown podocytes were reversed by GIT2 overexpression. The results indicate that GIT2 facilitates translocation of PTP1B to focal adhesions where it dephosphorylates p130Cas, thereby suppressing local Rac1 activity and protecting against podocyte injury and proteinuria.

Bradykinin receptor 1 activation exacerbates experimental focal and segmental glomerulosclerosis

Background/Aims: Previously we have shown that activation of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-antioxidant response element (ARE) attenuated hyperglycemia-induced damage in podocytes, but the molecular mechanism remains unknown. Methods: Tert-butylhydroquinone (t-BHQ) and small interfering RNAs (siRNAs) were used to regulate Nrf2 expression, while nicotinamide and siRNAs were used to regulate sirtuin 1 (Sirt1) activity and expression, respectively. Mitochondrial superoxide, membrane potential and ATP levels were measured to assess changes in mitochondrial function. Nephrin and synaptopodin expression were measured by western blot analysis. Human podocytes and db/db diabetic mice were used in this study. Results: t-BHQ pretreatment of human podocytes exposed to high glucose (HG) alleviated mitochondrial dysfunction, enhanced the expression of Sirt1, nephrin and synaptopodin and lowered BSA permeability compared with podocytes exposed to HG without t-BHQ pretreatment (p< 0.05). Human podocytes exposed to HG had more severe mitochondrial dysfunction, lower expression of Sirt1, synaptopodin and nephrin and higher BSA permeability than podocytes exposed to HG when Nrf2 expression was downregulated by siRNAs (p< 0.05). The protection provided by activation of the Nrf-ARE pathway in podocytes exposed to HG was partially diminished when Sirt1 expression or activity was decreased by siRNAs or inhibitor compared with podocytes exposed to HG and pretreated with t-BHQ (p< 0.05). When nicotinamide and t-BHQ were both administered to db/db mice, we observed higher levels of urinary albumin/creatinine, lower nephrin and synaptopodin expression, more severe mesangial matrix deposition, collagen deposition on pathological slides and mitochondrial structural damage in podocytes compared to db/db mice treated only with t-BHQ. Conclusions: Our findings suggest that crosstalk between Sirt1 and the Nrf2-ARE anti-oxidative pathway forms a positive feedback loop and that protection provided by t-BHQ activation of the Nrf2-ARE pathway in db/db mice is partly dependent on Sirt1.

Introduction: Early human immunodeficiency virus (HIV) testing of partners or families of the index case is an innovative type of testing which is performed within 14 days after contact elicitation using a contractual referral approach. Testing within this time is very important because it helps to identify contacts early and enroll them for intervention timely. In most health facilities, the contractual approach was not practiced; by implementing this early index case testing, most contacts could be tested early and it could prevent them from HIV-related death.Objective: This study aimed to assess the magnitude and associated factors of early index case testing among adult HIV index cases in Debre Markos town high case load health facilities.Methods: Institution-based cross-sectional study was conducted on adult index cases who started antiretroviral therapy from December 1/2018 to August 30/2022. A total of 384 index case charts were selected by the systematic random sampling technique using their medical record number from January 23/2023 up to February 28/2023. After the data were collected using a structured checklist, they were entered into EpiData Version 4.6 and then exported to SPSS software Version 25 for data cleaning, coding, categorizing, and further analysis. The Hosmer–Lemeshow test goodness of fit was checked for model fitness. Both bivariable and multivariable logistic regression analyses were used to identify significant factors with early testing of index cases' families. Finally, variables having pvalue < 0.05 with 95% CI in the multivariable logistic regression are considered as significant factors.Result: In this study, the magnitude of early index case testing among adult index patients was 28.6% (95% CI: 27.17%–30.0%). Being female (AOR = 1.89, 95% CI (1.17–3.06)), urban resident index cases (AOR = 1.88, 95% CI (1.16–3.03)), and having disclosure status (AOR = 2.34, 95% CI (1.40–3.92)) were significantly associated with early index case testing.Conclusion: This study examines the prevalence and key factors influencing early index case HIV testing among adult patients in Debre Markos town. By identifying critical determinants such as gender, residence, and disclosure status, it provides valuable insights into how early testing can be enhanced to reduce transmission and improve health outcomes.

Background and hypothesis. Steroid-resistant nephrotic syndrome (SRNS) is the most severe form of nephrotic syndrome, with genetic or unidentified immunological origins and rapidly progressing to the need for kidney replacement therapy. Lipotoxicity can affect podocytes inducing kidney damage. In this study, we evaluate the effects of SRNS serum on podocyte functionality and lipid metabolism. Methods. A three-dimensional (3D) dynamic in vitro glomerulus was incubated with serum from multi-drug resistant (MDR) and genetic SRNS or healthy controls. The glomerular filtration barrier (GFB) integrity, podocyte viability, and fatty acids (FAs) composition were evaluated by serum albumin permeability estimation, cytofluorimetric analysis and gas chromatography, respectively. Expression of slit diaphragm molecules and FA-related enzymes was analyzed by immunofluorescence and PCR. Results. Serum from SRNS patients induced cell granularity, increased GFB permeability, and disrupted slit diaphragm protein structure. The podocyte damage was most severe when MDR serum was administered compared to the serum of genetic-SRNS. This was associated with a significant upregulation of the transcripts coding for nephrin, synaptopodin, and CD2AP. An alteration of fatty acid profile in MDR-treated podocytes was observed, with increased monounsaturated FAs following the decrease of saturated FAs. The exposure of cultured podocytes to MDR- and genetic-SRNS serum induced disruption of arachidonic acid (AA) synthesis pathway, with different intermediate players involved. Conclusion. This study highlights the detrimental effects of serum from SRNS patients on podocyte function and the association of AA synthesis pathway with the podocyte damage.

Steroid-resistant nephrotic syndrome (SRNS) is the most severe form of nephrotic syndrome, with genetic or unidentified immunological origins and rapidly progressing to the need for kidney replacement therapy. Lipotoxicity can affect podocytes inducing kidney damage. In this study, we evaluate the effects of SRNS serum on podocyte functionality and lipid metabolism. A three-dimensional (3D) dynamic in vitro glomerulus was incubated with serum from multi-drug resistant (MDR) and genetic SRNS or healthy controls. The glomerular filtration barrier (GFB) integrity, podocyte viability, and fatty acids (FAs) composition were evaluated by serum albumin permeability estimation, cytofluorimetric analysis and gas chromatography, respectively. Expression of slit diaphragm molecules and FA-related enzymes was analyzed by immunofluorescence and PCR. Serum from SRNS patients induced cell granularity, increased GFB permeability, and disrupted slit diaphragm protein structure. The podocyte damage was most severe when MDR serum was administered compared to the serum of genetic-SRNS. This was associated with a significant upregulation of the transcripts coding for nephrin, synaptopodin, and CD2AP. An alteration of fatty acid profile in MDR-treated podocytes was observed, with increased monounsaturated FAs following the decrease of saturated FAs. The exposure of cultured podocytes to MDR- and genetic-SRNS serum induced disruption of arachidonic acid (AA) synthesis pathway, with different intermediate players involved. This study highlights the detrimental effects of serum from SRNS patients on podocyte function and the association of AA synthesis pathway with the podocyte damage.

Pathophysiologic Implications of Reduced Podocyte Number in a Rat Model of Progressive Glomerular Injury

Introduction: Patients with hypertension or renal disease are more likely to exhibit glomerular pathology associated with smoking. Smoking has been linked to lower nitric oxide (NO) bioavailability, oxidative and nitrosative stress. Nicotine triggers the formation of peroxynitrite (ONOO - ), a highly damaging redox molecule, and was also recently implicated as a contributor to hypertensive renal disease. Here we hypothesize that nicotine induces podocyte damage through peroxynitrite formation, thus causing glomerular function decline and aggravation of kidney damage in the Dahl SS rat. Methods: We performed live confocal imaging on human podocytes to detect the production of ONOO - (HPF), Ca 2+ (Fluo-8), and nitric oxide NO (DAF-FM) in response to nicotine. Seahorse assay was used to detect changes in mitochondrial health. Then, nicotine was chronically infused in the Dahl SS rats using Alzet pumps (0.2 mg/kg/day s.c., 0.4% NaCl, 21 days). Electron microscopy (EM) and Masson's trichrome staining were used to blindly evaluate glomerular and mitochondrial damage. ANOVA with post-hoc comparisons was used for statistical analysis. Results: IHC indicated that human and rat glomerular podocytes express nAChRs. Acute application of nicotine promoted intracellular Ca 2+ and ONOO - transients. The ONOO - response was blocked in the presence of SOD, indicating that ONOO - production requires superoxide. The application of specific nAChR agonists elicited Ca 2+ transients but did not produce ONOO - , suggesting that nitrosative processes may occur independently from Ca 2+ influx or nAChR activation. Incubation with nicotine (12-hrs) resulted in a significant decrease in podocytes’ NO bioavailability (p&lt;0.05) and mitochondrial respiration (p&lt;0.05). In vivo, in the Dahl SS rat nicotine infusion promoted mitochondrial damage in podocytes, which exhibited swelling, loss of cristae, and signs of mitophagy. Masson's trichrome staining and the assessment of glomerular damage showed deterioration of glomerular health. Conclusion: The pathological shift in redox balance in the podocyte is directly associated with nicotine. It may be the primary mechanism responsible for podocyte damage, renal function decline, and blood pressure development in smokers.

“Role of NADPH oxidases Nox1 and Nox4 in diabetic nephropathy: genetic deletion and pharmacological inhibition studies”

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