The zebrafish model has been used in many different fields of research because of its high homology to the human genome, its easy genetic manipulation, its high fecundity, and its rapid development. For glomerular diseases, zebrafish larvae have proven to be a versatile tool to study the contribution of different genes, because the zebrafish pronephros is very comparable to the human kidney in function and ultrastructure. Here we describe the principle and use of a simple screening assay based on the measurement of the fluorescence in the retinal vessel plexus of the Tg(l-fabp:DBP:eGFP) zebrafish line (\"eye assay\") to indirectly determine proteinuria as a hallmark of podocyte dysfunction. Furthermore, we illustrate how to analyze the obtained data and outline methods to attribute the findings to podocyte impairment.

Biological and biomedical research using Drosophila melanogaster as a model organism has gained recognition through several Nobel prizes within the last 100 years. Drosophila exhibits several advantages when compared to other in vivo models such as mice and rats, as its life cycle is very short, animal maintenance is easy and inexpensive and a huge variety of transgenic strains and tools are publicly available. Moreover, more than 70% of human disease-causing genes are highly conserved in the fruit fly. Here, we explain the use of Drosophila in nephrology research and describe two kidney tissues, Malpighian tubules and the nephrocytes. The latter are the homologous cells to mammalian glomerular podocytes and helped to provide insights into a variety of signaling pathways due to the high morphological similarities and the conserved molecular make-up between nephrocytes and podocytes. In recent years, nephrocytes have also been used to study inter-organ communication as links between nephrocytes and the heart, the immune system and the muscles have been described. In addition, other tissues such as the eye and the reproductive system can be used to study the functional role of proteins being part of the kidney filtration barrier.

UNASSIGNED: Diabetic kidney diseases (DKDs) are the most common cause of dialysis-dependent kidney disease around the world. Previous studies have suggested that urinary level of podocyte-associated molecules may predict the prognosis of DKD.
UNASSIGNED: Observational cohort.
UNASSIGNED: 118 consecutive patients with biopsy-proven DKD; 13 nondiabetic patients with hypertensive nephrosclerosis as controls.
UNASSIGNED: Urinary podocalyxin and podocin levels were obtained by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) and the corresponding intrarenal levels by western blotting.
UNASSIGNED: Dialysis-free survival; kidney event-free survival; rate of kidney function decline in 12 months.
UNASSIGNED: Correlation and time to event analysis.
UNASSIGNED: Urinary podocalyxin level was closely correlated with its messenger RNA (mRNA) level (r = 0.562, P < 0.001), but this did not predict the progression of DKD. Intrarenal podocalyxin level had only modest correlation with its urinary mRNA and ELISA levels, was an independent predictor of dialysis-free survival (adjusted HR, 1.85; 95% CI, 1.21-2.82; P = 0.005), and showed an insignificant trend of predicting kidney event-free survival (adjusted HR, 1.36; 95% CI, 0.94-1.95; P = 0.10). Urinary podocin level by ELISA had a modest correlation with the rate of kidney function decline (r = 0.238, P = 0.01) but did not predict dialysis-free survival.
UNASSIGNED: Small sample size; lack of serial measurement.
UNASSIGNED: Intrarenal podocalyxin level, but not its urinary level, was an independent predictor of dialysis-free survival, whereas urinary podocin level by ELISA correlated with the rate of kidney function decline. Although intrarenal podocalyxin level has prognostic value, it may not be suitable for routine clinical use.

Integrin α3β1 is a cell adhesion receptor widely expressed in epithelial cells. Pathogenic variants in the gene encoding the integrin α3 subunit ITGA3 lead to a syndrome including interstitial lung disease, nephrotic syndrome, and epidermolysis bullosa (ILNEB). Renal involvement mainly consists of glomerular disease caused by loss of adhesion between podocytes and the glomerular basement membrane. The aim of this study was to characterize the impact of loss of integrin α3 on human podocytes. ITGA3 was stably knocked-out in the human podocyte cell line AB8/13, designated as PodoA3-, and in human proximal tubule epithelial cell line HK2 using the targeted genome editing technique CRISPR/Cas9. Cell clones were characterized by Sanger sequencing, quantitative PCR, Western Blot and immunofluorescence staining. RNASeq of integrin α3 negative cells and controls was performed to identify differential gene expression patterns. Differentiated PodoA3- did not substantially change morphology and adhesion under standard culture conditions, but displayed significantly reduced spreading and adhesion when seed on laminin 511 in serum free medium. Gene expression studies demonstrated a distinct dysregulation of the adhesion network with downregulation of most integrin α3 interaction partners. In agreement with this, biological processes such as \"extracellular matrix organization\" and \"cell differentiation\" as well as KEGG pathways such as \"ECM-receptor interaction\", \"focal adhesion\" and the \"PI3K-Akt signaling pathway\" were significantly downregulated in human podocytes lacking the integrin α3 subunit.

UNASSIGNED: Increasing evidence shows that Angptl4 affects proteinuria in podocytes injured kidney disease, however, whether there is a relationship between Angptl4 and IgA nephropathy (IgAN) has not been studied yet.
UNASSIGNED: Plasma and urine samples were obtained from 71 patients with IgAN and 61 healthy controls. Glomeruli from six renal biopsy specimens (three IgAN patients and three healthy controls) were separated by RNA-Seq. Differentially expressed genes (DEGs) related to podocytes and Angptl4 between IgAN patients and healthy controls were performed using the Limma package. Gene set enrichment analysis was used to determine whether there was a statistically significant difference between the two groups. STRING was used to create a protein-protein interaction network of DEGs. Association analysis between Angptl4 levels and clinical features of IgAN was performed.
UNASSIGNED: Thirty-three podocyte-related and twenty-three Angpt4-related DEGs were found between IgAN patients and healthy controls. By overlapping the genes, FOS and G6PC were found to be upregulated in IgAN patients, while MMP9 was downregulated in IgAN patients. Plasma and urine Angptl4 levels were closely related to the degree of podocyte injury and urine protein, but not to the protein-creatine ratio.
UNASSIGNED: Our findings show that Angptl4 levels in plasma and urine are related to podocyte damage and, therefore, may be a promising tool for assessing the severity of IgAN patients to identify and reverse the progression to ESRD.

The current study aimed to evaluate the associations between podocyte injury and clinicopathological features in renal thrombotic microangiopathy (TMA) based on a Chinese cohort, which might be underscored in this disease.
The clinical, laboratory, and renal histopathological data of patients with renal biopsy-proven TMA from 2000 to 2015 in our institute were collected. Foot process effacement (FPE) was quantified by foot process width (FPW) by electron microscopy. Podocytes in the renal specimens were also detected by stainings for podocyte-specific markers, including Wilms tumor 1 (WT-1), synaptopodin, and podocalyxin. The associations between FPW and clinico-histopathological data were further analyzed. A composite end-point was defined by all-cause death or end-stage renal disease to address the predictive value of FPW.
Sixty-three patients with renal biopsy-proven TMA were enrolled. The FPW of renal TMA patients was 1,090 ± 637 nm (range, 572-4,748 nm), which was significantly higher than the normal range in our center (p = 0.005). By immunohistochemistry and immunofluorescence assays, we found decreased expressions of synaptopodin, podocalyxin, and WT-1 and continued stainings of WT-1 in some podocytes without detectable synaptopodin stainings in the areas of sclerotic tufts and cellular crescents. The FPW value was correlated with the serum albumin concentration (rs = -0.281, p = 0.026), proteinuria amount (rs = 0.255, p = 0.047), serum creatinine levels (rs = 0.339, p = 0.007), and eGFR (rs = -0.335, p = 0.007). According to ROC curve analysis, the optimal cutoff level of FPW for predicting the composite end-point was 869 nm. In patients with FPW ≥ 869 nm, FPW levels were further correlated with the severity of mesangiolysis (rs = 0.351, p = 0.033) and glomerulosclerosis (rs = 0.369, p = 0.025) in pathological evaluations. Patients without clinical remission also had higher FPW than those with remission (1,240 ± 793 vs. 925 ± 344 nm, p = 0.013). The multivariate Cox hazard model showed that FPW ≥ 869 nm was an independent risk factor for the composite end-point (hazard ratio: 3.64, 95% CI: 1.37-9.66, p = 0.009).
The podocyte injury was prevalent and the FPW levels were closely associated with clinicopathological features, especially prognosis, in renal TMA patients.

OBJECTIVE: Catalpol (Cat) can ameliorate oxide stress and inflammation caused by diabetic nephropathy (DN), but the molecular mechanisms are unclear. This study was designed to investigate the anti-diabetic effects of Cat and its potential mechanism.
METHODS: We constructed high-fat diet/streptozotocin (HFD/STZ)-induced DN mice and high glucose (HG)-induced podocyte model. The hypoglycemic effect of Cat was analyzed by general features of DN mice. Kidney function was detected via ELISA assay and Western blotting. Renal histopathology analysis was conducted via hematoxylin and eosin (H&E), Masson and periodic acid-silver metheramine (PASM) staining. Cellular viability was measured by TUNEL assay. In order to further study the potential mechanisms of Cat, various proteins in AMPK/SIRT1/NF-κB pathway were detected in DN mice and podocytes with siRNA-AMPK intervention using Western blotting, respectively.
RESULTS: We found hyperglycemia, renal structural and function abnormalities, and increased renal inflammation in DN mice. However, Cat effectively attenuated kidney damage caused by inflammation and increased AMPK, p-AMPK and SIRT1 levels. After AMPK-siRNA transfected into HG-induced podocyte model, AMPK, p-AMPK and SIRT1 levels were obviously decreased, while Cat reversed these chandes. The levels of p-NF-κB, ASC, Cleaved IL-1β, NLRP3, Cleaved caspase1 and GSDMD-N significantly decreased by Cat treatment both in DN mice and podocyte model, which indicated that Cat could activate AMPK/SIRT1/NF-κB pathway.
CONCLUSIONS: Cat could effectively inhibit oxide stress and inflammation accompanied with pyroptosis and its mechanism might be related to AMPK/SIRT1/NF-κB pathway, indicating that Cat possessed potential value in the treatment of DN.

In this chapter we describe conventional methods used for preparing renal tissue for transmission electron microscopy. We also describe a relatively new technique, serial block face scanning electron microscopy. Protocols are given for processing, sectioning, and imaging of tissue along with methods for obtaining quantitative data from the results.

Diabetic nephropathy is associated with injury and loss of podocytes, specialized epithelial cells that are critical for glomerular filtration. This chapter describes a method of isolating and culturing podocyte cells from mouse adult kidneys. In this way, podocytes with genetic modifications can be obtained from transgenic animals and they can be used to study the effects of the diabetic environment in vitro.

Ultrastructural changes in podocytes are an important diagnostic and prognostic marker for nephropathies. However, the biomedical understanding of detected submicroscopic changes in podocytes remains controversial.
OBJECTIVE: To investigate the relationship between the ultrastructural changes of podocytes (fusion of cytopodia and denudation of the basement membrane as a result of their desquamation) with a number of clinical and laboratory indicators of kidney dysfunction in case of non-proliferative glomerulopathies (NPGP). Thirty-seven patients (23 men, 14 women) with NPGP, including 8 with focal segmental glomerulosclerosis (FSGS), 17 with membranous nephropathy (MN), and 12 with minimal change disease (MCD), were examined.
METHODS: All the patients underwent standard laboratory and instrumental studies: determinations of the levels of total serum cholesterol (mmol/l), total serum protein (g/l); serum albumin (g/l); CKD-EPI glomerular filtration rate (GFR) (ml/min/1.73 m2), and daily protein loss (g/day). Light optical changes were measured; completely sclerotic and/or focally segmentally sclerotic glomeruli were taken into account. Quantitative ultrastructural stereological analysis was carried out estimating the cytopodium width (CPW) and the degree of glomerular basement membrane denudation (GBMD) (%).
RESULTS: NPGP cases showed the largest number of sclerotic glomeruli in FSGS, which was accompanied by the lowest level of daily proteinuria and GFR. Quantitative values of CPW were associated with the level of daily protein loss (r=0.47; p < 0.05) and serum albumin (r=-0.57; p <0.05) in patients with nephrotic syndrome. In MN, the absolute value of CPW was larger than that in the other two patient groups. A correlation analysis of CPW and GBMD values among patients with NPGP revealed a statistically insignificant negative relation between these morphometric parameters. However, when a subgroup of patients with podocytopathies (only MCD and FSGS) was identified in the study group, this relationship was found to be significant (r=-0.54; p=0.012).
CONCLUSIONS: The patients with NPGP exhibited a relationship between the severity of nephrotic syndrome and proteinuria/hypoalbuminemia, on the one hand, and CPW, on the other. The established negative relationship between CPW and the percentage of GBMD in the subgroup of patients with podocytopathies may be due to the early stages of podocyte injury, which are accompanied by transient GBMD.
Ультраструктурные изменения подоцитов являются важным диагностическим и прогностическим маркером нефропатий. Однако медико-биологическое понимание выявляемых субмикроскопических изменений подоцитов остается противоречивым. Цель исследования - исследование взаимосвязи ультраструктурных изменений подоцитов (слияние цитоподий и оголение базальной мембраны в результате их слущивания) с рядом клинико-лабораторных показателей дисфункции почек на примере непролиферативных гломерулопатий. Обследованы 37 пациентов (23 мужчины, 14 женщин) с непролиферативными гломерулопатиями (НПГП): у 8 был фокально-сегментарный гломерулосклероз (ФСГС), у 17 - мембранозная нефропатия (МН), у 12 - болезнь минимальных изменений (БМИ). Материал и методы. Всем пациентам выполнены стандартные лабораторные и инструментальные исследования: определены уровень общего холестерина сыворотки крови (в ммоль/л); содержание общего белка сыворотки крови (в г/л); альбумина сыворотки крови (в г/л); скорость клубочковой фильтрации (СКФ) по EPI (в мл/мин/1,73 м2); суточная потеря белка (г/сут). Светооптические изменения оценивали количественно; учитывали клубочки полностью склерозированные и/или с фокально-сегментарным склерозом. Количественный ультраструктурный стереологический анализ выполнен посредством оценки ширины цитоподий (ШЦП), а также степени оголения (%) гломерулярной базальной мембраны (ОГБМ) клубочков. Результаты. При НПГП наибольшее количество склерозированных клубочков наблюдалось при ФСГС, что сопровождалось наиболее низким уровнем суточной протеинурии и СКФ. Количественные значения ШЦП были связаны с уровнем суточной потери белка (r=0,47; p<0,05) и уровнем альбумина сыворотки крови (r =–0,57; p<0,05) у больных с нефротическим синдромом. При МН абсолютное значение ширины цитоподий было больше, чем в двух других группах больных. Корреляционный анализ значений ШЦП и ОГБМ среди больных НПГП показал статистически недостоверную отрицательную зависимость этих морфометрических показателей. Однако при выделении из основной группы подгруппы больных с подоцитопатиями (только БМИ и ФСГС) данная зависимость была достоверна (r=–0,54; p=0,012). Выводы. У больных НПГП выявлена зависимость между выраженностью нефротического синдрома и протеинурии/гипоальбуминемии, с одной стороны, и ШЦП - с другой. Установленная отрицательная связь между ШЦП и процентом ОГБМ в подгруппе больных с подоцитопатиями может быть следствием ранних этапов повреждения подоцитов, сопровождающихся транзиторным оголением гломерулярной базальной мембраны.