Nephrin and Podocin mRNA Detection in Urine Sediment of Dogs with Chronic Kidney Disease: Preliminary Observations.

Introduction: Dogs with chronic kidney disease (CKD) may have alterations in the glomerular filtration barrier, including podocyte loss. Detection of podocyte mRNA in urine could be useful for assessing podocyturia in dogs with kidney disease. The objective of this study was to evaluate the presence of nephrin mRNA (NPHS1) and podocin mRNA (NPHS2) in urine sediments of dogs with naturally occurring CKD and healthy dogs. Material and Methods: Twenty-four dogs, 14 with CKD and 10 as healthy controls, underwent clinical evaluation. The dogs with CKD were divided into two groups, according to the International Renal Interest Society criteria: stage 1 or 2 CKD (n = 5) and stage 3 or 4 CKD (n = 9). Urine was collected by catheterisation or free catch and RNA isolation from the urine sediments was optimised using glycogen as a co-precipitant. Detection of NPHS1 and NPHS2 in the sediment samples was performed using quantitative real-time PCR. Results: Both types of mRNA were detected in samples from all groups, but the percentages of detection were higher in the group of dogs with stage 1 or 2 CKD and lower in the group of dogs with stage 3 or 4 disease. Conclusion: Physiological podocyturia was observed in healthy dogs, and the results suggest differential podocyturia in dogs with CKD, according to the stage of the disease, i.e. an increase in podocyturia in dogs at stage 1 or 2 and a reduction in podocyturia in dogs at stage 3 or 4.

Lyso-Gb3 Increases αvß3 Integrin Gene Expression in Cultured Human Podocytes in Fabry Nephropathy.

BACKGROUND: Podocyturia in Fabry nephropathy leads to glomerulosclerosis and kidney disease progression. Integrins are involved in podocyte attachment to the glomerular basement membrane. We hypothesized that in Fabry nephropathy, lyso-Gb3 could modulate αvß3 expression in podocytes. Together with UPAR, the αvß3 integrin is a key mechanism involved in podocyte detachment and podocyturia. METHODS: In cultured human podocytes stimulated with lyso-Gb3, the mRNA expression of the ITGAV and ITGB3 genes encoding integrins αv and ß3, respectively, was analyzed by RT-qPCR. RESULTS: In cultured human podocytes, lyso-Gb3 at concentrations encountered in the serum of Fabry patients increased ITGAV and ITGB3 mRNA levels within 3 to 6 h. This pattern of gene expression is similar to that previously observed for PLAUR (UPAR) gene expression but is in contrast to the delayed (24 h) upregulation of other markers of podocyte stress and mediators of injury, such as CD80, TGFß1, CD74, Notch1, and HES. CONCLUSIONS: Human podocyte stress in response to glycolipid overload in Fabry nephropathy, exemplified by lyso-Gb3, is characterized by an early increase in the expression of components of the αvß3/UPAR system, which contrasts with the delayed rise in the expression of other mediators of podocyte injury. This suggests that the αvß3/UPAR system may be a therapeutic target in Fabry nephropathy.

Glomeruloesclerosis Focal y Segmentaria luego de Embarazo Complicado con Preeclampsia y Falla Renal Aguda. Reporte de Caso / Focal Segmental Glomerulosclerosis after Pregnancy Complicated with Preeclampsy and Acute Renal Injury. Case Report

Resumen Presentamos el caso de una paciente de 35 años, que cursó su última gestación con preeclampsia y falla renal aguda oligúrica, requiriendo ser intervenida mediante cesárea de emergencia y apoyo dialítico con un total de 16 sesiones de hemodiálisis, evolución favorable. Después de cuatro meses de alta desarrolló síndrome nefrótico, por lo que se la hospitalizó para un mejor manejo y una exhaustiva investigación de la etiología. Se tomaron medidas de soporte sin nuevo requerimiento de terapia de reemplazo renal, se descartó secundarismo y se le realizó biopsia renal. El estudio anatomopatológico concluyó que se trataba de una glomeruloesclerosis focal y segmentaria, en su variante perihiliar. La paciente evolucionó favorablemente durante su hospitalización y al darle el alta se la citó para continuar manejo por consulta externa. La aparición de síndrome nefrótico meses después del embarazo nos debe sugerir la presencia de una glomerulopatía subyacente o una glomerulopatía de novo.

Abstract We describe the case of a 35-year-old patient, who experienced preeclampsia and oliguric acute renal injury during her last pregnancy, requiring intervention by emergency cesarean section and dialysis support with a total of 16 hemodialysis sessions; she responded favorably. Four months after discharge, she developed nephrotic syndrome, so she was hospitalized for better management and a thorough etiologic research. Support measures were taken with no need for renal replacement therapy; secondary lesions were ruled out and a renal biopsy was performed. The pathological study concluded that it was a focal segmental glomerulosclerosis, in its perihilar variant. The patient responded favorably during her hospitalization and when discharged, she was summoned to continue management by external consultation. The appearance of nephrotic syndrome months after pregnancy may suggest the presence of an underlying glomerulopathy or de novo glomerulopathy.

Mechanisms of Podocyte Detachment, Podocyturia, and Risk of Progression of Glomerulopathies.

BACKGROUND: Glomerulopathies are the main cause of ESRD. Primary or secondary causes of glomerular diseases comprise more than 70% of cases that end up in renal replacement therapies. SUMMARY: The total glomerular mass that each individual contains is key to maintaining normal kidney function. Diabetes, hypertension, and any primary or secondary glomerulopathy may threaten the normal glomerular function. In fact, any glomerular insult may alter the glomerular filtration barrier, which in turn is composed by the podocyte, the glomerular basement membrane, and the capillary endothelial cell. Deposition of immune complexes, antibodies, or complement components at the subepithelial, intramembranous, or subendothelial space, and mutations in podocyte, slit diaphragm, or glomerular basement membrane proteins or enzymes are the main etiologies of glomerular alterations. Podocytes are glomerular cells that do not divide under normal circumstances. In this respect, maintenance of the absolute podocyte number per glomer-ulus is critical for normal glomerular function. As the insult progresses, podocytes start to detach from the glomerular basement membrane. When the podocyte loss is over 40% in a glomerulus, glomerulosclerosis develops, and obliteration of the glomerulus is the rule. In clinical grounds, this phenomenon is diagnosed mainly by proteinuria and a decline in glomerular filtration rate. KEY MESSAGES: In this review article, the impact of podocyturia in glomerular diseases and the main mechanisms of podocyte detachment are discussed. Finally, potential targets of therapeutic approach are suggested.

Early decrease in the podocalyxin to synaptopodin ratio in urinary Fabry podocytes.

BACKGROUND: In Fabry nephropathy, podocyturia is an early event that may lead to glomerulosclerosis and chronic kidney disease. The glycocalyx is a potential podocyte damaged compartment in glomerulopathies. We investigated glycocalyx podocalyxin in urinary detached podocytes compared with cytoplasmic synaptopodin. METHODS: This was a cross-sectional study including 68 individuals: Controls (n = 20) and Fabry patients (n = 48), 15 untreated and 33 treated. Variables included age, gender, urinary protein/creatinine ratio (UPCR), estimated glomerular filtration rate (eGFR), lyso-triasocylsphingosine (lyso-Gb3) levels and enzyme replacement therapy (ERT). Podocyturia was assessed by immunofluorescence and podocyte subpopulations were analyzed. RESULTS: Fabry patients displayed higher podocyturia than controls. Fabry treated subjects (n = 33) presented significantly higher UPCR compared with untreated ones (n = 15); podocyturia, eGFR and lyso-Gb3 levels were not different. All control podocytes colocalized synaptopodin and podocalyxin; 13 Fabry patients (27%) colocalized these proteins, while 35 (73%) were only synaptopodin positive. No podocalyxin-positive/synaptopodin-negative cells were encountered. In Fabry patients, podocyturia was significantly higher and proteinuria lower in those that colocalized. CONCLUSION: Fabry patients present higher podocyturia and a presumably more damaged glycocalyx assessed by podocalyxin. Treated patients had significant higher proteinuria suggesting ERT is initiated late, at advanced stages. The degree of podocalyxin-negative podocytes was similar in both groups, but colocalization was associated with lower proteinuria. Podocyturia assessed by podocalyxin alone may be underestimated. The implications of podocyte glycocalyx damage deserve further investigations.

Clinical parameters, LysoGb3, podocyturia, and kidney biopsy in children with Fabry disease: is a correlation possible?

BACKGROUND: Fabry disease is an X-linked lysosomal storage disorder caused by α-galactosidase enzyme deficiency. We present clinical, biochemical, and histologic findings in children with classical phenotypic presentation of Fabry disease. METHODS: A retrospective analysis was performed using charts from 14 children with confirmed diagnosis. Clinical parameters were evaluated. Globotriaosylsphingosine -lysoGb3- detection in plasma, podocyturia, and kidney biopsy were carried out in all cases. RESULTS: All patients except one demonstrated at least one symptom of Fabry disease. LysoGb3 levels were above the normal range in all patients. Podocyturia was documented in all patients. Kidney biopsy revealed glomerular, interstitial, vascular, and tubular changes on light microscopy in nearly all patients. Electron microscopy showed podocyte inclusions in all patients. CONCLUSIONS: No difference in symptomatology was discernible between boys and girls. Podocyturia was detectable in children serving as a possible early marker of kidney injury. LysoGb3 was elevated in all cases, emphasizing the importance for diagnosis especially in female patients with normal αGal A activity. A possible association between lysoGb3 and symptom severity and histological involvement in kidney biopsy should be assessed in prospective studies with enough statistical power to determine if lysoGb3 can be used to predict nephropathy in children with Fabry disease.

Podocyturia: Potential applications and current limitations.

Chronic kidney disease is a prevalent condition that affects millions of people worldwide and is a major risk factor of cardiovascular morbidity and mortality. The main diseases that lead to chronic kidney disease are frequent entities as diabetes mellitus, hypertension and glomerulopathies. One of the clinical markers of kidney disease progression is proteinuria. Moreover, the histological hallmark of kidney disease is sclerosis, located both in the glomerular and in the interstitial compartments. Glomerulosclerosis underscores an irreversible lesion that is clinically accompanied by proteinuria. In this regard, proteinuria and glomerular sclerosis are linked by the cell that has been conserved phylogenetically not only to prevent the loss of proteins in the urine, but also to maintain the health of the glomerular filtration barrier: The podocyte. It can then be concluded that the link between proteinuria, kidney disease progression and chronic kidney disease is mainly related to the podocyte. What is this situation due to? The podocyte is unable to proliferate under normal conditions, and a complex molecular machinery exists to avoid its detachment and eventual loss. When the loss of podocytes in the urine, or podocyturia, is taking place and its glomerular absolute number decreased, glomerulosclerosis is the predominant histological feature in a kidney biopsy. Therefore, tissular podocyte shortage is the cause of proteinuria and chronic kidney disease. In this regard, podocyturia has been demonstrated to precede proteinuria, showing that the clinical management of proteinuria cannot be considered an early intervention. The identification of urinary podocytes could be an additional tool to be considered by nephrologists to assess the activity of glomerulopathies, for follow-up purposes and also to unravel the pathophysiology of podocyte detachment in order to tailor the therapy of glomerular diseases more appropriately.

In IgA Nephropathy, Glomerulosclerosis Is Associated with Increased Urinary CD80 Excretion and Urokinase-Type Plasminogen Activator Receptor-Positive Podocyturia.

BACKGROUND: Podocyturia may determine the evolution to podocytopenia, glomerulosclerosis, and renal failure. According to the Oxford classification of IgA nephropathy (IgAN), the S1 lesion describes glomerulosclerosis. Urokinase-type plasminogen activator receptor (uPAR) participates in podocyte attachment, while CD80 increases in glomerulosclerosis. We measured uPAR-positive urinary podocytes and urinary CD80 (uCD80) in controls and in IgAN subjects with M1E0S0T0 and M1E0S1T0 Oxford scores to assess a potential association between podocyturia, inflammation, and glomerulosclerosis. METHODS: The groups were as follows: controls (G1), n = 20 and IgAN group (G2), n = 39, subdivided into M1E0S0T0 (G2A), n = 21 and M1E0S1T0 (G2B), n = 18. Among the included variables, we determined uPAR-positive podocytes/gram of urinary creatinine (gUrCr) and uCD80 ng/gUrCr. Biopsies with interstitial fibrosis and tubular atrophy <10% were included. RESULTS: Groups were not different in age and gender; urinary protein-creatinine (uP/C) ratio, Chronic Kidney Disease-Epidemiology Collaboration (CKD-EPI) equation, uPAR-positive podocytes/gUrCr, and uCD80 were significantly increased in G2 versus G1. G2A and G2B were not different in age, gender, hypertension, and follow-up. G2B displayed significantly higher uP/C, uPAR-positive podocytes, uCD80, and lower CKD-EPI versus G2A. Strong significant correlations were encountered between uCD80 and podocyturia in G2A and G2B. However, when G1 was compared to G2A and G2B separately, the differences with respect to uP/C, uPAR-positive podocytes, and podocyturia were significantly stronger versus G2B than versus G2A. CONCLUSIONS: IgAN presents elevated uCD80 excretion and uPAR-positive podocyturia, while CD80 correlates with podocyturia. Glomerulosclerosis (S1) at the time of biopsy is associated with higher uP/C, lower renal function, increased uPAR-positive podocyturia, and CD80 excretion, and is independent of M1. In IgAN, uPAR may participate in podocyte detachment.

Increased urinary CD80 excretion and podocyturia in Fabry disease.

BACKGROUND: Certain glomerulopathies are associated with increased levels of CD80 (B7-1). We measured the urinary excretion of CD80, podocyturia and proteinuria in controls and in subjects with Fabry disease either untreated or on enzyme replacement therapy (ERT). METHODS: Cross-sectional study including 65 individuals: controls (n = 20) and Fabry patients (n = 45, 23 of them not on ERT and 22 on ERT). Variables included age, gender, urinary protein/creatinine ratio (UPCR), estimated glomerular filtration rate (eGFR), urinary uCD80/creatinine ratio (uCD80) and podocyturia. CD80 mRNA expression in response to lyso-Gb3, a bioactive glycolipid accumulated in Fabry disease, was studied in cultured human podocytes. RESULTS: Controls and Fabry patients did not differ in age, eGFR and gender. However, UPCR, uCD80 and podocyturia were significantly higher in Fabry patients than in controls. As expected, Fabry patients not on ERT were younger and a higher percentage were females. Non-ERT Fabry patients had less advanced kidney disease than ERT Fabry patients: UPCR was lower and eGFR higher, but uCD80 and podocyturia did not differ between non-ERT or ERT Fabry patients. There was a significant correlation between uCD80 and UPCR in the whole population (r 0.44, p 0.0005) and in Fabry patients (r 0.42, p 0.0046). Lyso-Gb3 at concentrations found in the circulation of Fabry patients increased uCD80 expression in cultured podocytes. CONCLUSIONS: Fabry disease is characterized by early occurrence of increased uCD80 excretion that appears to be a consequence of glycolipid accumulation. The potential for uCD80 excretion to reflect early, subclinical renal Fabry involvement should be further studied.

Podocyturia is significantly elevated in untreated vs treated Fabry adult patients.

BACKGROUND: Proteinuria suggests kidney involvement in Fabry disease. We assessed podocyturia, an early biomarker, in controls and patients with and without enzyme therapy, correlating podocyturia with proteinuria and renal function. METHODS: Cross-sectional study (n = 67): controls (Group 1, n = 30) vs. Fabry disease (Group 2, n = 37) subdivided into untreated (2A, n = 19) and treated (2B, n = 18). Variables evaluated: age, gender, creatinine, CKD-EPI, proteinuria, podocyte count/10 20× microscopy power fields, podocytes/100 ml urine, podocytes/g creatininuria (results expressed as median and range). RESULTS: Group 1 vs. 2 did not differ concerning age, gender and CKD-EPI, but differed regarding proteinuria and podocyturia. Group 2A vs. 2B: age: 29 (18-74) vs. 43 (18-65) years (p = ns); gender: males n = 3 (16 %) vs. n = 9 (50 %). Proteinuria was significantly higher in Fabry treated patients, while CKD-EPI and podocyturia were significantly elevated in untreated individuals. Significant correlations: group 2A: age-proteinuria, ρ = 0.62 (p = 0.0044); age-CKD-EPI, ρ = -0.84 (p < 0.0001); podocyturia-podocytes/100 ml urine, ρ = 0.99 (p = 0.0001); podocyturia-podocytes/g creatininuria ρ = 0.86 (p = 0.0003), podocytes/100 ml urine-podocytes/g urinary creatinine, ρ = 0.84 (p = 0.0004); proteinuria-CKD-EPI, ρ = -0.68 (p = 0.0013). Group 2B: podocyturia-podocytes/100 ml urine, ρ = 0.88 (p < 0.0001); podocyturia-podocytes/g creatininuria, ρ = 0.84 (p < 0.0001); podocytes/100 ml urine-podocytes/g creatininuria, ρ = 0.94 (p < 0.0001); CKD-EPI-proteinuria, ρ = -0.66 (p = 0.0028). CONCLUSIONS: Patients with Fabry disease display heavy podocyturia; those untreated present significantly higher podocyturia, lower proteinuria and better renal function than those who are treated, suggesting that therapy may be started at advanced stages. Podocyturia may antedate proteinuria, and enzyme therapy may protect against podocyte loss.

Is there a role for urinary podocyte excretion assessment in lupus nephritis?

OBJECTIVE: To establish the occurrence and intensity of podocyturia and its relation to grade of disease activity, as defined by clinical and laboratory criteria. METHODS: Prospective, cross-sectional study involving 50 patients with lupus nephritis and 29 controls, which had podocyturia levels determined from random urine samples using an immunofluorescence technique. Disease activity was graded by BILAG (renal criteria) and an additional system used in the service (S2). RESULTS: Fifty patients with lupus nephritis (WHO classes III, IV and V), with a median age of 37 years, were evaluated. Of these, 86.5% were female, and 52% were BILAG A. Podocyturia quantification in the lupus nephritis and control groups differed significantly (p = 0.009). This score was higher in relation to classes III, IV and V. The correlation with C3 consumption was stronger (p = 0.011) than with C4. The highest levels were found in the most active groups (A and B of BILAG and S2). Lower podocyturia correlated with a lower dose of prednisone. There was no association with the intensity of proteinuria, hematuria or pyuria, serum creatinine levels, among others. CONCLUSIONS: Podocyturia assessment, which was performed by immunofluorescence in this study, can be used as an indicator of disease activity with the advantage of being a urinary biomarker. The levels proved to be higher in patients with lupus nephritis than in the controls and were particularly higher in class IV.

Podocyturia: What is in a name?

The podocyte is a highly differentiated cell located in the outer space of the glomerular basement membrane that deals with many different functions. This phylogenetically preserved cell that is responsible for the virtually absence of proteins in the urine lacks of the capacity to divide under normal conditions. When podocytes receive molecular insults, which normally occur during stress conditions as glomerulonephritis, hyperfiltration or metabolic disturbances, they adapt to the new situation by contracting their actin fibers. This adaptive behavior puts at risk the quality of the plasmatic filtration due to the denudation of the glomerular basement membrane, the potential mesangial inflammation and the appearance of proteinuria; podocytes run the risk of detachment from the basement membrane due to a decrease in the adherence to the surrounding matrix after contraction, a process called foot processes effacement. Podocytes change their shape and under constant mechanical stress they finally detach, rendering the glomerular basement membrane unprotected unless other contiguous podocytes are capable of covering the surface. However, these still anchored podocytes are generally also under the same stress situation and follow the same pathway. Podocyturia refers to the presence of these differentiated cells in the urinary sediment. Noteworthy, the podocytes that are encountered in the urine are viable despite the glomerular hostile environment and the urinary acidity. Podocyturia can precede proteinuria and can aggravate it. Therefore, in diseases that can threaten the glomerular normal environment, the presence and the quantification of urinary podocytes can be of remarkable relevance, as it can herald or accompany the appearance of proteinuria, and could offer another view to the interpretation and clinical approach and outcome of proteinuria. However, its identification needs a wide-spread training among biochemists and technicians, as well as commercially available kits.