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Podocytes are critical components of the glomerular filtration barrier, sitting on the outside of the glomerular basement membrane. Primary and secondary foot processes are characteristic for podocytes, but cell processes that develop in culture were not studied much in the past. Moreover, protocols for diverse visualization methods mostly can only be used for one technique, due to differences in fixation, drying and handling. However, we detected by single-cell RNA sequencing (scRNAseq) analysis that cells reveal high variability in genes involved in cell type-specific morphology, even within one cell culture dish, highlighting the need for a compatible protocol that allows measuring the same cell with different methods. Here, we developed a new serial and correlative approach by using a combination of a wide variety of microscopic and spectroscopic techniques in the same cell for a better understanding of podocyte morphology. In detail, the protocol allowed for the sequential analysis of identical cells with light microscopy (LM), Raman spectroscopy, scanning electron microscopy (SEM) and atomic force microscopy (AFM). Skipping the fixation and drying process, the protocol was also compatible with scanning ion-conductance microscopy (SICM), allowing the determination of podocyte surface topography of nanometer-range in living cells. With the help of nanoGPS Oxyo®, tracking concordant regions of interest of untreated podocytes and podocytes stressed with TGF-β were analyzed with LM, SEM, Raman spectroscopy, AFM and SICM, and revealed significant morphological alterations, including retraction of podocyte process, changes in cell surface morphology and loss of cell-cell contacts, as well as variations in lipid and protein content in TGF-β treated cells. The combination of these consecutive techniques on the same cells provides a comprehensive understanding of podocyte morphology. Additionally, the results can also be used to train automated intelligence networks to predict various outcomes related to podocyte injury in the future. [ABSTRACT FROM AUTHOR]

The Chinese medicine Extractum trametes robiniophila murr (ETRM) is the extract of a type of fungus. Recent studies have suggested that ETRM efficiently improves the effacement of podocyte foot processes in adriamycin (ADR)-induced nephrotic rats. In the present study, we aimed to assess whether ETRM modulated the actin rearrangements of podocytes and involved signaling molecules, including α-actinin-4 and nephrin. Podocytes were treated with ADR (0.5 μmol/L), ADR (0.5 μmol/L) + dexamethasone (Dex) (1 μmol/L), ADR (0.5 μmol/L) + ETRM (10 mg/mL). The F-actin in the podocytes was stained by fluorescent phallotoxins and observed by confocal microscopy. The expression levels of α-actinin-4 and nephrin were tested by real-time PCR and Western blotting analysis. The administration of ETRM could significantly prevent ADR-treated podocytes from actin rearrangement. Both ETRM and Dex could stabilize podocyte actin cytoskeletons. Moreover, α-actinin-4 might act as a potential target for ETRM functionality in podocyte actin rearrangements. However, pretreatment with ETRM could not inhibit the up-regulation of nephrin as a result of ADR treatment. ETRM could improve the cytoskeleton stability in ADR-induced actin rearrangement of podocytes via regulating α-actinin-4 expression. [ABSTRACT FROM AUTHOR]

Background/Aim: Effacement of the epithelial cell foot processes of glomerular podocytes are thought to be diffuse in the primary form of focal and segmental glomerulosclerosis (FSGS). In contrast, effacements that occur in the secondary form of FSGS are thought to be focal. To evaluate this theory, the extent of epithelial cell foot process effacement was analyzed and compared in podocytes from cases of primary and secondary FSGS. Methods: Consecutive cases of adult primary and secondary FSGS that were diagnosed between January 1997 and December 2010 were retrospectively retrieved and analyzed. The electron microscopy materials of all specimens were reviewed. Glomerular podocytes from each sample were counted, recorded and analyzed for the percentage of diffuse versus focal epithelial foot process effacement. Results: Seventeen primary and 16 secondary FSGS specimens were reviewed. In the 17 primary FSGS cases, 35.1% of the podocytes were focally fused, and 64.9% were diffusely fused. In the 16 secondary FSGS cases, 72.1% of the podocytes were focally fused. There were significantly more focally fused podocytes in secondary FSGS than in primary FSGS. In contrast, significantly more diffusely fused podocytes were observed in primary FSGS than in secondary FSGS. Conclusion: This is the first study to perform a quantitative analysis of the extent of epithelial cell foot process effacement in podocytes in primary and secondary FSGS. This study also confirms that the electron microscopic characteristics of the podocyte effacement process can be used to facilitate the diagnosis of primary versus secondary FSGS. [ABSTRACT FROM AUTHOR]

Podocytes are postmitotic renal glomerular cells with multiple ramifications that extend from the cell body. Processes departing from a podocyte interdigitate with corresponding projections from neighboring cells and form an intricate web that enwraps the glomerular capillary completely. Podocyte processes are interconnected by the slit diaphragm, an adhesion junction mostly formed by Ig-like molecules, cadherins/protocadherins, ephrin/eph, and neurexin molecules organized in an assembly that resembles synaptic junctions. Podocyte failure is primarily or secondarily implicated in all forms of proteinuric glomerular diseases, as confirmed by the morphological changes of their elaborate cell architecture detectable by electron microscopy. Importantly, mutations of podocyte proteins are responsible for the most severe forms of congenital nephrotic syndrome. In the last 15 years, progressive technological advances have aided the study of podocyte biology and pathology, confirming the relevance of podocyte molecules and signaling pathways for the function of the glomerular filter. This review will examine the most important and newest discoveries in the field, which is rapidly evolving, hopefully leading to a detailed knowledge of this fascinating cell and to the development of specific therapeutic options for proteinuric diseases. [ABSTRACT FROM AUTHOR]

Abstract: In the renal glomerulus, podocytes envelop the external side of the capillary basement membrane with their intertwining ramifications, and ensure elimination of metabolic waste within the urine, while proteins and important blood components are retained into the circulation. To preserve the integrity of the glomerular filter, which is constantly exposed to a high variety of stimuli, podocytes need to communicate by rapid and precise signaling, likely similar to that used by neuronal cells. In the last years, we and others have shown that podocytes are indeed molecularly equipped for communicating in a synaptic-like way, where glutamate and its receptors seem to have a pivotal role, because altering glutamatergic communication leads to podocyte damage and increased filter permeability. Major components of glutamatergic signaling are organized at foot process junctions by adhesion molecules, chiefly by nephrin, and are connected to the actin cytoskeleton, that governs the health of podocytes. Further advances in understanding podocyte physiological behavior and signaling properties have the potential to improve the knowledge of podocyte diseases, first among them idiopathic focal segmental glomerulosclerosis that still needs more precise molecular-based diagnosis and targeted treatment. [Copyright &y& Elsevier]

Palladin, a cytoskeletal protein with essential functions for stress fiber formation, is found in developing and mature tissues, including the kidney. To define its role in the kidney, we measured its expression in mouse kidney and found it co-localized with F-actin in smooth muscle cells of renal arterial vessels, mesangial cells, and podocytes but not in tubular epithelium. Using immunoelectron microscopy, we confirmed that palladin was present in podocytes. In cultured mouse podocytes, palladin co-localized with F-actin in dense regions of stress fibers, focal adhesions, cell–cell contacts and motile cell margins. Transfection with the N-terminal half of palladin targeted it to F-actin-containing structures in podocytes while the C-terminal half accumulated in the nucleus, a result also found for endogenous palladin in cultured cells after leptomycin B was used to block nuclear export. Green fluorescent protein (GFP)-tagged palladin was found in dynamic ring-like F-actin structures and ruffles in cultured podocytes after stimulation with epidermal growth factor. Inhibition of palladin expression by transfection of an antisense construct reduced the formation of ring-like structures. Photo-bleaching analysis showed that GFP-palladin turned over with a half-time of 10 s in focal adhesions and dense regions of stress fibers, suggesting that palladin is a dynamic scaffolding protein. Our study shows that palladin is expressed in podocytes and plays an important role in actin dynamics.Kidney International (2009) 75, 214–226; doi:10.1038/ki.2008.486; published online 1 October 2008 [ABSTRACT FROM AUTHOR]

Abstract: Patients with nail-patella syndrome, which among other symptoms also includes podocyte-associated renal failure, suffer from mutations in the LMX1B gene. The disease severity among patients is quite variable and has given rise to speculations on the presence of modifier genes. Promising candidates for modifier proteins are the proteins interacting with LMX1B, such as LDB1 and E47. Since human kidney samples from patients are difficult to obtain, conventional Lmx1b knock-out mice have been extremely valuable to study the role of Lmx1b in podocyte differentiation. In contrast to findings in these mice, however, in which a downregulation of the Col4a3, Col4a4 and Nphs2 genes has been described, no such changes have been detected in kidney biopsies from patients. We now report on our results on the characterization of constitutive podocyte-specific Lmx1b, Ldb1 and E2a knock-out mice. Constitutive podocyte-specific Lmx1b knock-out mice survive for approximately 2 weeks after birth and do not present with a downregulation of the Col4a3, Col4a4 and Nphs2 genes, therefore they mimic the human disease more closely. The podocyte-specific Ldb1 knock-out mice survive longer, but then also succumb to renal failure, whereas the E2a knock-out mice show no renal symptoms for at least 6 months after birth. We conclude that LDB1, but not E2A is a promising candidate as a modifier gene in patients with nail-patella syndrome. [Copyright &y& Elsevier]

Mutations in the ACTN4 gene, encoding the actin crosslinking protein α-actinin-4, are associated with a familial form of focal segmental glomerulosclerosis (FSGS). Mice with podocyte-specific expression of K256E α-actinin-4 develop foot process effacement and glomerulosclerosis, highlighting the importance of the cytoskeleton in podocyte structure and function. K256E α-actinin-4 exhibits increased affinity for F-actin. However, the downstream effects of this aberrant binding on podocyte dynamics remain unclear. Wild-type and K256E α-actinin-4 were expressed in cultured podocytes via adenoviral infection to determine the effect of the mutation on α-actinin-4 subcellular localization and on cytoskeletal-dependent processes such as adhesion, spreading, migration, and formation of foot process-like peripheral projections. Wild-type α-actinin-4 was detected primarily in the Triton-soluble fraction of podocyte lysates and localized to membrane-associated cortical actin and focal adhesions, with some expression along stress fibers. Conversely, K256E α-actinin-4 was detected predominantly in the Triton-insoluble fraction, was excluded from cortical actin, and localized almost exclusively along stress fibers. Both wild-type and K256E α-actinin-4-expressing podocytes adhered equally to an extracellular matrix (collagen-I). However, podocytes expressing K256E α-actinin-4 showed a reduced ability to spread and migrate on collagen-I. Lastly, K256E α-actinin-4 expression reduced the mean number of actin-rich peripheral projections. Our data suggest that aberrant sequestering of K256E α-actinin-4 impairs podocyte spreading, motility, and reduces the number of peripheral projections. Such intrinsic cytoskeletal derangements may underlie initial podocyte damage and foot process effacement encountered in ACTN4-associated FSGS.Kidney International (2006) 70, 1054–1061. doi:10.1038/sj.ki.5001665; published online 12 July 2006 [ABSTRACT FROM AUTHOR]

We compared renal morphology in normoalbuminuric adolescents with type 1 diabetes with age-and sex-matched controls. Renal morphological measurements in 46 unselected adolescents with diabetes for approx. 10 years were compared with those in 20 healthy kidney donors. Renal volume estimated by ultra-sound, renal function determined by clearances of inulin and para-aminohippurate, and long-term mean HbA1c were measured in the patient group. Basement membrane thickness (BMT), mesangial matrix and capillary volume fractions per glomerulus [VV(matrix/glom), VV(cap/glom)] were significantly greater in patients than in controls (505 nm, 10.5%, 46.7% vs. 320 nm, 7.9% and 39.3%). The foot processes were wider in patients than in living donors (414 nm vs. 372 nm). There was no difference in mean kidney volume of patients with diabetes and healthy subjects. Glomerular filtration rate, body weight, and slit pore length density explained 60% of the variance in renal volume. After about 10 years’ duration of type 1 diabetes, BMT, VV(matrix/glom), VV(cap/glom), and foot process width were greater in normoalbuminuric adolescents than in healthy controls. This increase in foot processes of normoalbuminuric patients has not been reported before. [ABSTRACT FROM AUTHOR]

Podocyte depletion and glomerulosclerosis have a direct relationship in the PAN-treated rat. Background. Podocytes are highly differentiated glomerular epithelial cells with limited potential to divide. They are responsible for maintaining and supporting the glomerular basement membrane so as to facilitate efficient filtration. The hypothesis tested was whether the development of glomerulosclerosis in the puromycin aminonucleoside (PAN)-treated rat could be attributed to podocyte depletion. Methods. PAN was injected in Sprague-Dawley rats once, twice, or three times at 30-day intervals. Podocytes were counted in glomeruli using immunoperoxidase histochemistry and antibodies to both GLEPP1 (PTPRO) and WT-1. Podocytes were assayed in urine using reverse transcription-quantitative polymerase chain reaction (RT-QPCR). Glomerular areas were measured by computerized morphometry. Results. In a preliminary experiment, a single injection of PAN caused a reduction in the glomerular podocyte count by 25%. Additional independent confirmation that podocytes were lost from glomeruli after PAN injection was obtained identifying detached podocytes in Bowman's space, measurement of nephrin and GLEPP1 mRNAs in urine, ultrastructural analysis of glomeruli, and identification of TUNEL-positive apoptotic podocytes in glomeruli. In a second experiment, sequential podocyte depletion by 15, 31, and 53% was achieved by the administration of one, two, or three injections of PAN at 30-day intervals. The region of the glomerulus devoid of podocytes developed glomerulosclerosis, and this area progressively increased as podocytes were progressively depleted. The correlation coefficient (r2) value for the relationship between percent podocyte depletion and glomerulosclerotic area was 0.99. The Y intercept of this plot showed that glomerulosclerosis was initiated when only 10 to 20% of podocytes were lost. Conclusion. This report supports the growing body of data linking... [ABSTRACT FROM AUTHOR]

Despite a wealth of information on structural proteins, comparatively little is known on the transcriptional regulation of podocyte structure and function. In this review we will highlight those transcription factors which, by gene inactivation or classical transgenic experiments, have been shown to be essential for podocytes or probably will turn out to be so. The tumor suppressor protein WT1 is not only indispensable for the initial stages of kidney development, but also very likely maintains the integrity of the fully differentiated podocyte. In the kidney, the LIM homeodomain transcription factor LMX1B is specifically synthesized in podocytes, and mutations in LMX1B lead to nail-patella syndrome and the associated nephropathy. Other transcription factors such as hypoxia-inducible factors and PAX2 are likely to play a role in podocytes, whereas the significance of others, e.g. of POD1 and CITED2, is more speculative at this point. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]