Urinary Podocyte Research Articles

Effect of SGLT2 Inhibitor Therapy on Urinary Podocyte Markers in Diabetic Kidney Disease

Effect of SGLT2 Inhibitor Therapy on Urinary Podocyte Markers in Diabetic Kidney Disease

Quantification of Urine Podocytes (Podo) Using Imagining Flow Cytometry (IFC)

Quantification of Urine Podocytes (Podo) Using Imagining Flow Cytometry (IFC)

Quantification of Urine Podocytes (Podo) and Their Globotriaosylceramide (GL3) Content Using Imaging Flow Cytometry

Quantification of Urine Podocytes (Podo) and Their Globotriaosylceramide (GL3) Content Using Imaging Flow Cytometry

Urine-derived podocytes from steroid resistant nephrotic syndrome patients as a model for renal-progenitor derived extracellular vesicles effect and drug screening

BackgroundPersonalized disease models are crucial for evaluating how diseased cells respond to treatments, especially in case of innovative biological therapeutics. Extracellular vesicles (EVs), nanosized vesicles released by cells for intercellular communication, have gained therapeutic interest due to their ability to reprogram target cells. We here utilized urinary podocytes obtained from children affected by steroid-resistant nephrotic syndrome with characterized genetic mutations as a model to test the therapeutic potential of EVs derived from kidney progenitor cells (nKPCs).MethodsEVs were isolated from nKPCs derived from the urine of a preterm neonate. Three lines of urinary podocytes obtained from nephrotic patients’ urine and a line of Alport syndrome patient podocytes were characterized and used to assess albumin permeability in response to nKPC-EVs or various drugs. RNA sequencing was conducted to identify commonly modulated pathways after nKPC-EV treatment. siRNA transfection was used to demonstrate the involvement of SUMO1 and SENP2 in the modulation of permeability.ResultsTreatment with the nKPC-EVs significantly reduced permeability across all the steroid-resistant patients-derived and Alport syndrome-derived podocytes. At variance, podocytes appeared unresponsive to standard pharmacological treatments, with the exception of one line, in alignment with the patient’s clinical response at 48 months. By RNA sequencing, only two genes were commonly upregulated in nKPC-EV-treated genetically altered podocytes: small ubiquitin-related modifier 1 (SUMO1) and Sentrin-specific protease 2 (SENP2). SUMO1 and SENP2 downregulation increased podocyte permeability confirming the role of the SUMOylation pathway.ConclusionsnKPCs emerge as a promising non-invasive source of EVs with potential therapeutic effects on podocytes with genetic dysfunction, through modulation of SUMOylation, an important pathway for the stability of podocyte slit diaphragm proteins. Our findings also suggest the feasibility of developing a non-invasive in vitro model for screening regenerative compounds on patient-derived podocytes.

#2493 Podocyturia: a novel biomarker of ageing

Abstract Background and Aims Urinary podocyte shedding (podocyturia) is a biomarker of glomerular injury in young and old individuals. Podocyturia is associated with diabetes and hypertension, and it is an early indicator of systemic cardiovascular disease. Importantly, podocyturia has also been noted in healthy adults and may indicate physiologic podocyte turnover. Whether age is a predictor of podocyte loss, independently of diabetes and hypertension, is still unclear. The aim of this study was to examine the association between age and urinary podocin levels. Method This was a secondary analysis of data from 5 studies, including 868 individuals, 18-90 years of age. Data on urinary podocin mRNA levels was present on all subjects, as measured by CFX96 Touch™ RT-PCR detection system (Biorad, USA). Descriptive statistics, Chi-square test, ANOVA, and post hoc analysis were performed. Linear regression was conducted with gender, diabetes status, and hypertension status as covariates. Significance was set at p < 0.05. Results The mean age of the total sample was 55.10 ± 14.84 years; 46% were female, 26.4% had diabetes, and 37.9% had hypertension. There were significant differences in mean podocin levels by age group (F = 25.27, p ≤ 0.001) as follows. Podocin levels were significantly lower in Group 1 (age 18-35 years; 0.79 ± 0.67 million copies), compared to Group 2 (36-55 years; 2.21 ± 2.15 million copies) and Group 3 (over 56 years; 2.86 ± 3.18 million copies). Moreover, mean podocin levels differed significantly between Group 2 and Group 3 (p = 0.003). Age was a predictor of podocyturia, independently of diabetes and hypertension (p < 0.001). Conclusion Podocyturia was present in younger adults. There was a significant rise in podocyturia in middle-aged adults (36-55 years), denoting a transitional phase. There were higher podocyturia levels in older adults compared to the youngest and middle-aged adults. These differences in podocin by age were observed while accounting for diabetes and hypertension.

Urinary podocyte markers in diabetic kidney disease.

Podocytes are involved in maintaining kidney function and are a major focus of research on diabetic kidney disease (DKD). Urinary biomarkers derived from podocyte fragments and molecules have been proposed for the diagnosis and monitoring of DKD. Various methods have been used to detect intact podocytes and podocyte-derived microvesicles in urine, including centrifugation, visualization, and molecular quantification. Quantification of podocyte-specific protein targets and messenger RNA levels can be performed by Western blotting or enzyme-linked immunosorbent assay and quantitative polymerase chain reaction, respectively. At present, many of these techniques are expensive and labor-intensive, all limiting their widespread use in routine clinical tests. While the potential of urinary podocyte markers for monitoring and risk stratification of DKD has been explored, systematic studies and external validation are lacking in the current literature. Standardization and automation of laboratory methods should be a priority for future research, and the added value of these methods to routine clinical tests should be defined.

Urinary podocyte stress marker as a prognostic indicator for diabetic kidney disease

BackgroundDiabetic kidney diseases (DKD) is a the most common cause of end-stage kidney disease (ESKD) around the world. Previous studies suggest that urinary podocyte stress biomarker, e.g. podocin:nephrin mRNA ratio, is a surrogate marker of podocyte injury in non-diabetic kidney diseases.MethodWe studied 118 patients with biopsy-proved DKD and 13 non-diabetic controls. Their urinary mRNA levels of nephrin, podocin, and aquaporin-2 (AQP2) were quantified. Renal events, defined as death, dialysis, or 40% reduction in glomerular filtration rate, were determined at 12 months.ResultsUrinary podocin:nephrin mRNA ratio of DKD was significantly higher than the control group (p = 0.0019), while urinary nephrin:AQP2 or podocin:AQP2 ratios were not different between groups. In DKD, urinary podocin:nephrin mRNA ratio correlated with the severity of tubulointerstitial fibrosis (r = 0.254, p = 0.006). and was associated with the renal event-free survival in 12 months (unadjusted hazard ratio [HR], 1.523; 95% confidence interval [CI] 1.157–2.006; p = 0.003). After adjusting for clinical and pathological factors, urinary podocin:nephrin mRNA ratio have a trend to predict renal event-free survival (adjusted HR, 1.327; 95%CI 0.980–1.797; p = 0.067), but the result did not reach statistical significance.ConclusionUrinary podocin:nephrin mRNA ratio has a marginal prognostic value in biopsy-proven DKD. Further validation is required for DKD patients without kidney biopsy.

Urinary Podocyte Stress Marker as a Prognostic Indicator for Diabetic Kidney Disease

Urinary Podocyte Stress Marker as a Prognostic Indicator for Diabetic Kidney Disease

Quantitative Analysis of Mitochondrial DNA Heteroplasmy in Urinary Podocytes of Myoclonus Epilepsy With Ragged-Red Fibers Syndrome

Quantitative Analysis of Mitochondrial DNA Heteroplasmy in Urinary Podocytes of Myoclonus Epilepsy With Ragged-Red Fibers Syndrome

PS-C13-7: THE ROLE OF WATER HAMMER PRESSURE IN THE GLOMERULAR PODOCYTE SHEDDING AND RENAL PROGNOSIS

Objective: Urinary podocyte excretion is related to a reduction in glomerular podocyte numbers, glomerulosclerosis, and urinary protein selectivity. To elucidate the factors of urinary podocyte shedding and renal prognosis, we examined urinary podocytes in 120 renal biopsy patients. Design and method: Podocytes were identified in urinary sediments stained with fluorescent-labelled anti-podocalyxin antibodies in ten high power fields. The amounts of protein bands, separated by SDS-polyacrylamide gel electrophoresis, were calculated using an image software program. The renal prognosis, including induction of hemodialysis or 30% reduction in eGFR, was investigated. Results: Urinary podocyte excretion showed a higher positive correlation with albumin excretion compared to IgG, prealbumin, and transferrin, but not with low molecular weight proteins, including beta2-microglobulin and alpha1-microglobulin. The hemodynamic pressure on the glomerular capillary wall, including products of pulse pressure and pulse rate (water hammer pressure), was positively correlated with urinary podocyte excretion. Urinary podocyte excretion and Tamm-Horsfall protein were independent risk factors for renal prognosis. Conclusions: Urinary podocytes shed from glomerular capillaries by water hammer and were involved in albuminuria and renal prognosis of renal biopsy patients.

Cell Cycle and Senescence Regulation by Podocyte Histone Deacetylase 1 and 2.

The loss of integrity of the glomerular filtration barrier results in proteinuria that is often attributed to podocyte loss. Yet how damaged podocytes are lost remains unknown. Germline loss of murine podocyte-associated Hdac1 and Hdac2 ( Hdac1/2 ) results in proteinuria and collapsing glomerulopathy due to sustained double-stranded DNA damage. Hdac1/2 deletion induces loss of podocyte quiescence, cell cycle entry, arrest in G1, and podocyte senescence, observed both in vivo and in vitro . Through the senescence secretory associated phenotype, podocytes secrete proteins that contribute to their detachment. These results solidify the role of HDACs in cell cycle regulation and senescence, providing important clues in our understanding of how podocytes are lost following injury. Intact expression of podocyte histone deacetylases (HDAC) during development is essential for maintaining a normal glomerular filtration barrier because of its role in modulating DNA damage and preventing premature senescence. Germline podocyte-specific Hdac1 and 2 ( Hdac1 / 2 ) double-knockout mice were generated to examine the importance of these enzymes during development. Podocyte-specific loss of Hdac1 / 2 in mice resulted in severe proteinuria, kidney failure, and collapsing glomerulopathy. Hdac1 / 2 -deprived podocytes exhibited classic characteristics of senescence, such as senescence-associated β-galactosidase activity and lipofuscin aggregates. In addition, DNA damage, likely caused by epigenetic alterations such as open chromatin conformation, not only resulted in podocyte cell-cycle entry as shown in vivo by Ki67 expression and by FUCCI-2aR mice, but also in p21-mediated cell-cycle arrest. Through the senescence secretory associated phenotype, the damaged podocytes secreted proinflammatory cytokines, growth factors, and matrix metalloproteinases, resulting in subsequent podocyte detachment and loss, evidenced by senescent podocytes in urine. Hdac1 / 2 plays an essential role during development. Loss of these genes in double knockout mice leads to sustained DNA damage and podocyte senescence and loss.

Role of urinary podocytes excretion versus renal biopsy in assessment of lupus nephritis patients

Background: Systemic lupus erythematosus often affects the kidneys, and its treatment continues to provide new challenges to clinicians every day. Proteinuria caused by either direct or indirect podocyte damage is diagnostic of lupus nephritis. Aim: The purpose of this study was to evaluate the prognostic usefulness of urinary podocytes in predicting renal complication in individuals with SLE. Subjects and methods: Twenty individuals with SLE participated in this cross-sectional observational research. Complete history collection, clinical examination, and laboratory investigation were performed on all individuals. Podocytes detection in the urine and a kidney biopsy were performed. Results: The relationship between urinary podocytes and histological subtype is quite significant. Also, urinary podocytes significantly positively correlated with all parameters except with albumin significantly negatively correlated. Conclusion: Lupus patients who have renal damage may exhibit urinary podocytes, however this is not a diagnostic indication for lupus nephritis per se. Flares of lupus nephritis may be defined with the use of renal function tests and kidney biopsies.

Enhanced SHP-1 Expression in Podocyturia Is Associated with Kidney Dysfunction in Patients with Diabetes

Diabetic kidney disease (DKD) remains the leading cause of end stage kidney disease worldwide. Despite significant advances in kidney care, there is a need to improve noninvasive techniques to predict the progression of kidney disease better for patients with diabetes. After injury, podocytes are shed in urine and may be used as a biologic tool. We previously reported that SHP-1 is upregulated in the kidney of diabetic mice, leading to podocyte dysfunction and loss. Our objective was to evaluate the expression levels of SHP-1 in urinary podocytes and kidney tissues of patients with diabetes. In this prospective study, patients with and without diabetes were recruited for the quantification of SHP-1 in kidney tissues, urinary podocytes, and peripheral blood monocytes. Immunochemistry and mass spectrometry techniques were applied for kidney tissues. Urinary podocytes were counted, and expression of SHP-1 and podocyte markers were measured by quantitative PCR. A total of 66 participants (diabetic n=48, nondiabetic n=18) were included in the analyses. Diabetes was associated with increased SHP-1 expression in kidney tissues (P=0.03). Nephrin and podocin mRNA was not significantly increased in urinary podocytes from patients with diabetes compared with those without diabetes, whereas levels of SHP-1 mRNA expression significantly correlated with HbA1c and estimated glomerular filtration rate (eGFR). Additionally, follow-up (up to 2 years post recruitment) evaluation indicated that SHP-1 mRNA expression continued to increase with eGFR decline. Levels of SHP-1 in urinary podocytes may serve as an additional marker of glomerular disease progression in this population.

Transcriptomic Analysis of Human Podocytes In Vitro: Effects of Differentiation and APOL1 Genotype

The mechanisms in podocytes that mediate the pathologic effects of the APOL1 high-risk (HR) variants remain incompletely understood, although various molecular and cellular mechanisms have been proposed. We previously established conditionally immortalized human urine-derived podocyte-like epithelial cell (HUPEC) lines to investigate APOL1 HR variant-induced podocytopathy. We conducted comprehensive transcriptomic analysis, including mRNA, microRNA (miRNA), and transfer RNA fragments (tRFs), to characterize the transcriptional profiles in undifferentiated and differentiated HUPEC with APOL1 HR (G1/G2, 2 cell lines) and APOL1 low-risk (LR) (G0/G0, 2 cell lines) genotypes. We reanalyzed single-cell RNA-seq data from urinary podocytes from focal segmental glomerulosclerosis (FSGS) subjects to characterize the effect of APOL1 genotypes on podocyte transcriptomes. Differential expression analysis showed that the ribosomal pathway was one of the most enriched pathways, suggesting that altered function of the translation initiation machinery may contribute to APOL1 variant-induced podocyte injury. Expression of genes related to the elongation initiation factor 2 pathway was also enriched in the APOL1 HR urinary podocytes from single-cell RNA-seq, supporting a prior report on the role of this pathway in APOL1-associated cell injury. Expression of microRNA and tRFs were analyzed, and the profile of small RNAs differed by both differentiation status and APOL1 genotype. We have profiled the transcriptomic landscape of human podocytes, including mRNA, miRNA, and tRF, to characterize the effects of differentiation and of different APOL1 genotypes. The candidate pathways, miRNAs, and tRFs described here expand understanding of APOL1-associated podocytopathies.

Clinical Significance of Urine Podocyte Messenger Ribonucleic Acids in Idiopathic Nephrotic Syndrome and Lupus Nephritis Patients

Clinical Significance of Urine Podocyte Messenger Ribonucleic Acids in Idiopathic Nephrotic Syndrome and Lupus Nephritis Patients

Activation of necroptosis pathway in podocyte contributes to the pathogenesis of focal segmental glomerular sclerosis.

Focal segmental glomerulosclerosis (FSGS) is characterized by podocyte damage and severe proteinuria. The exact mechanism of podocyte damage and loss remains unclear. Necroptosis, a lytic form of programmed cell death mediated by RIP3 and MLKL, has emerged as an important cell death pattern in multiple tissues and cell types. Necroptosis in FSGS has not been investigated. Public GEO data regarding podocyte treated with vehicle or adriamycin (ADR) was identified and analyzed. Cultured human podocytes were used to explore the activation of necroptosis upon ADR stimulation. The expression of necroptosis pathway molecules, p-RIP3 and p-MLKL, was examined in the glomeruli and defoliated urinary podocytes of patients with FSGS. The effect of necroptosis inhibition was assessed in ADR-induced glomerulopathy mice using GSK872. Publicly available RNA-sequencing data analysis showed that both necroptosis and NLRP3 inflammasome pathway were up-regulated in ADR-injured podocyte. Immunofluorescent staining showed increased expression of p-RIP3 and p-MLKL, the active forms of RIP3 and MLKL, in podocytes of FSGS patients and ADR-induced glomerulopathy mice but not in the normal control. GSK872, an RIP3 kinase inhibitor, significantly inhibited the expression of p-RIP3, p-MLKL and activation of NLRP3 in cultured podocytes treated with ADR. GSK872 treatment of mice with ADR-induced nephropathy resulted in the reduced expression of p-RIP3, p-MLKL, NLRP3 and caspase-1 p20. GSK872 also significantly inhibited the expression of p-MLKL in the podocytes of ADR-induced nephropathy, resulting in the attenuation of proteinuria and renal histological lesions. Necroptosis pathway might be a valuable target for the treatment of FSGS.

Urinary Podocyte Excretion Predicts Urinary Protein Selectivity and Renal Prognosis.

Background Urinary podocyte excretion is related to a reduction in glomerular podocyte numbers, glomerulosclerosis, and urinary protein selectivity. To elucidate the role of urinary podocytes in proteinuria and renal prognosis and to identify the factors that cause podocyte detachment, we examined urinary podocytes in 120 renal biopsy patients. Methods Podocytes were identified in urinary sediments stained with fluorescent-labeled anti-podocalyxin antibodies in ten high power fields. The amounts of protein bands, separated by SDS-polyacrylamide gel electrophoresis, were calculated using an image software program and the correlation with urinary podocytes was analyzed. Podocyte surface pores were observed using a low-vacuum scanning electron microscope. The renal prognosis, including induction of hemodialysis or 30% reduction in eGFR, was investigated. Results Urinary podocyte excretion showed a higher positive correlation with albumin excretion compared to IgG, prealbumin, and transferrin. There were no significant correlations between urinary podocyte count and low molecular weight proteins, including β2-microglobulin and α1-microglobulin. The number of podocyte surface pores was positively correlated with proteinuria, suggesting enhanced albumin transcytosis. The hemodynamic pressure on the glomerular capillary wall, including products of pulse pressure and pulse rate (water hammer pressure), was positively correlated with urinary podocyte excretion. Urinary podocyte excretion and Tamm–Horsfall protein (THP) were independent risk factors for renal prognosis but were not related to response to treatment. Conclusion Urinary podocyte excretion was correlated with urinary albumin excretion, indicating specific albumin transport by podocytes. Podocytes were detached from the glomerular capillaries by water hammer pressure and THP was involved in the renal prognosis.

Urine podocyte mRNA loss in preterm infants and related perinatal risk factors

BackgroundPreterm birth has been identified as a risk factor for development of long-term chronic kidney disease. Podocyte loss has been reported to contribute to this process in preterm animal models. However, details about podocyte loss in preterm infants and related perinatal risk factors have not been well clarified.MethodsForty full-term infants and 106 preterm infants were enrolled. Urine samples were collected from full-term infants within 4–7 days of birth and preterm infants at 37–40 weeks of corrected age. Levels of urine podocin mRNA, urine protein (UP), and urine microalbumin (UMA) were measured, and the relationship between these markers was evaluated. Clinical information in these infants was collected, and potential correlates that may lead to increased podocyte loss during the perinatal period were identified using linear regression analysis.ResultsUrine podocyte loss indicated by the urine podocin mRNA to creatinine ratio (UpodCR) was higher in preterm infants than in full-term infants. UpodCR was correlated with the levels of UP and UMA. Multiple linear regression analysis also showed that lower gestational age (GA) at birth and small for gestational age (SGA) were high risk factors for urine podocyte loss.ConclusionsIncreasing urine podocyte loss was identified in preterm infants. Moreover, perinatal factors were associated with podocyte loss and may be a potential direction for comprehensive research and intervention in this field.Graphical A higher resolution version of the Graphical abstract is available as Supplementary information

MO168: Urinary Podocin Cell Count in Relation to Glomerular Damage Markers in Patients with Primary Nephrotic Syndrome

Abstract BACKGROUND AND AIMS Progressive renal failure in patients with glomerular disease is driven by podocyte depletion. Several methods have been proposed to monitor urinary podocyte loss. Here, we investigated the process of podocyte depletion in patients with primary nephrotic syndrome (PNS) by flow cytometric detection of podocin-positive cells in urine. METHOD We included nine patients with PNS. Urinary cell pellets were spun down, washed and stained for podocin and subsequently counted using FACS. Urinary protein and creatinine levels were determined from the same portion of urine. Normal values of urinary podocin-positive cells were obtained in urine samples of four healthy controls. RESULTS Mean urinary podocyte count (podocin-positive cells) was on average 100.0 (3.1–228.1) times higher in patients compared to healthy controls. Podocyte excretion showed significant correlations with urinary protein/creatinine ratios (R2 = 0.86, P < .01). CONCLUSION Urinary excretion of podocin-positive podocytes was significantly increased in patients with PNS, and correlated significantly with protein/creatinine ratios in patients with PNS. We conclude that this method can be used to monitor the process of podocyte depletion, and impact of treatment. We are planning prospective studies to evaluate the prognostic value of urinary podocin+ podocyte excretion in patients with PNS.

Urinary Podocyte Count as a Potential Routine Laboratory Test for Glomerular Disease: A Novel Method Using Liquid-Based Cytology and Immunoenzyme Staining

Introduction: This study investigated whether our urinary podocyte detection method using podocalyxin (PDX) and Wilms tumor 1 (WT1) immunoenzyme staining combined with liquid-based cytology can serve as a noninvasive routine laboratory test for glomerular disease. Methods: The presence of PDX- and WT1-positive cells was investigated in 79 patients with glomerular disease and 51 patients with nonglomerular disease. Results: The frequencies and numbers of PDX- and WT1-positive cells were significantly higher in the glomerular disease group than in the nonglomerular disease group. The best cutoffs for PDX- and WT1-positive cell counts for identifying patients with glomerular disease were 3.5 (sensitivity = 67.1% and specificity = 100%) and 1.2 cells/10 mL (sensitivity = 43.0% and specificity = 100%), respectively. Conclusion: Because our urinary podocyte detection method using PDX immunoenzyme staining can be standardized and it detected glomerular disease with high accuracy, it can likely serve as a noninvasive routine laboratory test for various glomerular diseases.