Podocyte Development Research Articles

Amino Acid Transporter LAT3 Is Required for Podocyte Development and Function

LAT3 is a Na+-independent neutral l-amino acid transporter recently isolated from a human hepatocellular carcinoma cell line. Although liver, skeletal muscle, and pancreas are known to express LAT3, the tissue distribution and physiologic function of this transporter are not completely understood. Here, we observed that glomeruli express LAT3. Immunofluorescence, confocal microscopy, and immunoelectron microscopy revealed that LAT3 localizes to the apical plasma membrane of podocyte foot processes. In mice, starvation upregulated glomerular LAT3, phosphorylated AKT1, reconstituted the actin network, and elongated foot processes. In the fetal kidney, we observed intense LAT3 expression at the capillary loops stage of renal development. Finally, zebrafish morphants lacking lat3 function showed collapsed glomeruli with thickened glomerular basement membranes. Permeability studies of the glomerular filtration barrier in these zebrafish morphants demonstrated a disruption of selective glomerular permeability. Our data suggest that LAT3 may play a crucial role in the development and maintenance of podocyte structure and function by regulating protein synthesis and the actin cytoskeleton.

Wilms Tumor 1 (WT1) and Foxc2 are required for podocyte development in Xenopus

Single‐gene knockout studies have shown that Foxc2, Lmx1b, MafB, Tcf21 and WT1 are the necessary transcriptional regulators of podocyte development. It is still unclear, however, whether these five transcription factors (TFs) can explain all aspects of podocyte development. To this end, we have used the Xenopus pronephros as a tool to study podocyte development.To test the roles of these five TFs in podocyte development, each was individually knocked down with antisense morpholino oligomer technology. In situ hybridization for podocyte TFs (Foxc2, Lmx1b, MafB, Tcf21 and WT1) and markers of terminal differentiation (Nephrin, Neph1, Glepp1 and Podocin) was used to analyze the resultant phenotype. These data were compiled to construct a gene regulatory network for podocyte development (PGRN). The PGRN displayed classical characteristics of a gene regulatory network, including master regulators, multi‐gene loops and a high degree of redundancy. Based on the network, we hypothesized that WT1 and Foxc2 are the master regulators of podocyte development. Simultaneous depletion of WT1 and Foxc2 resulted in loss of expression of downstream podocyte TFs (Lmx1b, MafB and Tcf21) and markers of terminal differentiation (Nephrin, Neph1, Glepp1 and Podocin); moreover, these embryos were characterized by a complete loss of the glomus structure. This work was supported by F30DK082121.Grant Funding SourceNIDDK

MicroRNA: a new frontier in kidney and blood pressure research

MicroRNA (miRNA) has emerged rapidly as a major new direction in many fields of research including kidney and blood pressure research. A mammalian genome encodes several hundred miRNAs. These miRNAs potentially regulate the expression of thousands of proteins. miRNA expression profiles differ substantially between the kidney and other organs as well as between kidney regions. miRNAs may be functionally important in models of diabetic nephropathy, podocyte development, and polycystic disease. miRNAs may be involved in the regulation of arterial blood pressure, including possible involvement in genetic elements of hypertension. Studies of miRNAs could generate diagnostic biomarkers for kidney disease and new mechanistic insights into the complex regulatory networks underlying kidney disease and hypertension. Further progress in the understanding of miRNA biogenesis and action and technical improvements for target identification and miRNA manipulation will be important for studying miRNAs in renal function and blood pressure regulation.

Initial Insight on the Determinants of Podocyte Polarity

Glomerular disease manifest by proteinuria is associated with changes in the architecture of podocyte foot processes and the structure of podocyte intercellular junctions. For this reason, there is great interest in defining the cellular mechanisms that determine and maintain normal podocyte morphology in health, that results in changes in podocyte morphology in disease, and might affect recovery of normal podocyte structure after disease. Much research in this area has focused on protein complexes localized to the podocyte intercellular junction. This work has been propelled by identification of several genes that encode proteins targeted to the intercellular junction, which when mutated in human congenital disease result either in failure of podocyte development or in an inability of the podocyte to maintain its normal architecture. Among these disease genes is Nphs1 , which encodes Nephrin, a transmembrane protein of the cell adhesion family targeted to the intercellular junction of the mature podocyte foot process.1 With structurally similar Neph1 protein, Nephrin-Neph1 forms a complex in the plane of the cell membrane that recruits proteins of the actin polymerization complex regulating foot process actin cytoskeletal dynamics.2–4 In doing so, these proteins coordinate foot process intercellular junction formation and maintenance with development and homeostasis of podocyte morphology. Given the complexity of these cellular processes, our knowledge of the precise mechanisms by which nephrin operates remain rudimentary. Polarity, or the asymmetric distribution of cellular constituents, is a fundamental property of polarized cells that defines cellular architecture and tissue organization essential for cellular function.5 Renal tubular epithelial cells are polarized along the apical-basal axis that extends from the tubular basement membrane to the apical luminal surface. In this example, epithelial polarity is defined by the asymmetric distribution …

Embryonic nervous system genes predominate in searches for dinucleotide simple sequence repeats flanked by conserved sequences

To study evolution of dinucleotide simple sequence repeats (diSSRs) we searched recently available mammalian genomes for UTR-localized diSSRs with conserved upstream flanking sequences (CFS). There were 252 reported Homo sapiens genes containing the repeats (AC) n, (GT) n, (AG) n or (CT) n in their UTRs including 22 (8.7%) with diSSR-upstream flanking sequences conserved comparing divergent mammalian lineages represented by Homo sapiens and the marsupial, Monodelphis domestica. Of these 22 genes, 19 had known functions including 18 (95%) that proved critical for mammalian nervous systems (Fishers exact test, P < 0.0001). The remaining gene, Cd2ap, proved critical for development of kidney podocytes, cells that have multiple similarities to neurons. Gene functions included voltage and chloride channels, synapse-associated proteins, neurotransmitter receptors, axon and dendrite pathfinders, a NeuroD potentiator and other neuronal activities. Repeat length polymorphism was confirmed for 68% of CFS diSSRs even though these repeats were nestled among highly conserved sequences. This finding supports a hypothesis that SSR polymorphism has functional implications. A parallel study was performed on the self-complementary diSSRs (AT) n and (GC) n. When flanked by conserved sequences, the self-complementary diSSR (AT) n was also associated with genes expressed in the developing nervous system. Our findings implicate functional roles for diSSRs in nervous system development.

Transcriptional control of podocyte development in Xenopus

Transcriptional control of podocyte development in Xenopus

Development of Glomerular Endothelial Cells, Podocytes and Mesangial Cells in the Human Fetus and Infant

The process of glomerular development consists of four developmental stages: vesicle (V) stage, S-shaped body (S) stage, capillary loop (C) stage and maturation (M) stage. However, the development of glomerular endothelial, mesangial and epithelial cells in fetal and infant kidneys remains unclear. In order to determine the characteristics of human glomerular development, we investigated the process of glomerular development by staining fetal and infant kidneys for CD31, CD34 and FB21, markers for endothelial cells, alpha-smooth muscle actin (alpha-SMA), a marker for mesangial cells, and nephrin, a marker for podocytes. These series of studies were carried out on kidneys obtained at autopsy from 27 fetuses and 5 infants. The fetuses were divided into the following 5 groups according to gestational age; 13-19, 20-24, 25-29, 30-34 and 35-39 weeks. In each group, glomerular development was classified according to the developmental stage and the staining patterns for CD31, CD34, FB21, alpha-SMA and nephrin. The proportion of V-stage development in 100 glomeruli examined was highest at 13-19 weeks. After 20 weeks, the V-stage proportion decreased gradually, and the proportion of S stage became highest at 20-24 weeks. The C-stage proportion was highest at 25-29 weeks, while the M-stage proportion was highest in infants aged 1-6 months. The staining patterns for CD31, CD34 and FB21 were similar in endothelial cells after 25 weeks of gestation. Staining of alpha-SMA and nephrin was first observed in the S stage. In conclusion, maturation of endothelial cells starts at 25 weeks and is completed by 35 weeks of gestation. Epithelial cells and mesangial cells first appear during the S stage.

Nck Links Nephrin to Actin in Kidney Podocytes

Two papers, one in Nature (Jones et al., 2006) and the other in the Journal of Clinical Investigation (Verma et al., 2006) show that Nck adaptor proteins connect phosphorylated nephrin with actin polymerization in podocyte foot processes, structures important for slit-diaphragm formation in the kidney. Their results further our understanding of podocyte development and repair in glomerular disease.

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

Changes in podocyte number or density have been suggested to play an important role in renal disease progression. Here, we investigated the temporal relationship between glomerular podocyte number and development of proteinuria and glomerulosclerosis in the male Munich Wistar Fromter (MWF) rat. We also assessed whether changes in podocyte number affect podocyte function and focused specifically on the slit diaphragm-associated protein nephrin. Age-matched Wistar rats were used as controls. Estimation of podocyte number per glomerulus was determined by digital morphometry of WT1-positive cells. MWF rats developed moderate hypertension, massive proteinuria, and glomerulosclerosis with age. Glomerular hypertrophy was already observed at 10 weeks of age and progressively increased thereafter. By contrast, mean podocyte number per glomerulus was lower than normal in young animals and further decreased with time. As a consequence, the capillary tuft volume per podocyte was more than threefold increased in older rats. Electron microscopy showed important changes in podocyte structure of MWF rats, with expansion of podocyte bodies surrounding glomerular filtration membrane. Glomerular nephrin expression was markedly altered in MWF rats and inversely correlated with both podocyte loss and proteinuria. Our findings suggest that reduction in podocyte number is an important determinant of podocyte dysfunction and progressive impairment of the glomerular permselectivity that lead to the development of massive proteinuria and ultimately to renal scarring.

Insulin-like growth factors inhibit podocyte apoptosis through the PI3 kinase pathway

Abnormal podocyte development and progressive podocyte injury have been implicated in a number of human kidney diseases. Factors necessary for regulating development and maintenance of this cell type are only beginning to emerge. To study the role of the insulin-like growth factor (IGF) system in regulating podocyte survival, we induced human fetal podocytes to undergo apoptosis. We demonstrated a significant increase in apoptosis when these cells were incubated in the presence of etoposide, as measured by DNA fragmentation and nuclear membrane condensation and blebbing. Podocyte apoptosis was reduced to control levels when the cells were coincubated in the presence of IGF-1. We showed that the protective effect of IGFs in this cell type was mediated through the activation of the phosphatidylinositol 3'-kinase (PI3K) pathway. IGF-1 stimulation resulted in the formation of the insulin receptor substrate (IRS)-1-p85 complex, an increase in PI3 kinase activity, and activation of protein kinase B (AKT/PKB) and the bcl-2 family member bad. Incubation of the podocytes with inhibitors of the PI3 kinase pathway resulted in a loss of this IGF-1 protective effect. These data demonstrate an important role for the IGF system in fetal podocyte survival in vitro, and suggest potential mediators to slow or alleviate the loss or damage of the podocyte in progressive renal disease.

Podocyte expression of the CDK-inhibitor p57 during development and disease

The mature podocyte is a terminally differentiated cell with a limited proliferative capacity. The precise cell cycle proteins necessary for establishing podocyte quiescence during development or permitting podocyte cell cycle re-entry in disease states have not been fully defined. Accordingly, we studied the role of the cyclin dependent kinase (CDK)-inhibitor p57Kip2 (p57) in modulating these processes. The expression of p57 protein in relation to markers of DNA synthesis was examined in developing mouse kidneys, and in the passive Heymann nephritis (PHN) and anti-glomerular antibody models of glomerular disease by immunohistochemistry. The role of p57 in glomerulogenesis was explored by examining renal tissue from embryonic p57-/- mice, and the expression of p21, p27 and p57 protein and mRNA was examined in podocytes in vitro. The de novo expression of p57 during glomerulogenesis coincides with the cessation of podocyte proliferation, and the establishment of a mature phenotype, and p57 is expressed exclusively in podocytes in mature glomeruli. However, p57 knockout mice have normal glomerular podocyte development. In addition, mRNA but not protein levels of p57 increased upon differentiation of podocytes in vitro. There was a marked decrease in p57 expression in both animal models of podocyte injury. This was diffuse in PHN, whereas in the murine model, loss of expression of p57 occurred predominantly in proliferating podocytes, expressing proliferating cell nuclear antigen (PCNA). Despite the de novo expression of p57 protein coinciding with the cessation of primitive podocyte proliferation during glomerulogenesis, embryonic p57-/- mice glomeruli were histologically normal. Cultured podocytes did not require changes in p57 protein levels to undergo differentiation. These data suggest that p57 alone is not required for podocyte differentiation, and that other cell cycle regulators may play a role. Furthermore, although injury to mature podocytes in experimental glomerular disease is associated with a decrease in p57, the levels of all three members of the Cip/Kip family of CDK inhibitors appear to determine the capability of podocytes to proliferate.

Glomerular laminin isoform transitions: errors in metanephric culture are corrected by grafting.

Glomerular basement membrane (GBM) assembly and maturation are marked by the replacement of laminin-1 (containing alpha 1-, beta 1-, and gamma 1-chains) with laminin-11 (consisting of alpha 5-, beta 2-, and gamma 1-chains). Similarly, the alpha 1- and alpha 2-chains of type IV collagen are replaced by collagen alpha 3-, alpha 4-, and alpha 5(IV)-chains. The cellular origins of these molecules and mechanisms for isoform removal and substitution are unknown. To explore glomerular laminin isoform transitions in vitro, we assessed metanephric organ cultures. Standard culture conditions do not support endothelial cell differentiation, and glomerular structures that form in vitro are avascular. Nevertheless, extensive podocyte development occurs in these cultures, including the formation of foot processes and assembly of a GBM-like matrix. Here, we show that the podocyte-specific markers, glomerular epithelial protein 1 and nephrin, which are normally expressed in capillary loop stage glomeruli in vivo, are also expressed by glomerular figures that form in organ culture. However, the GBM-like segments that form in vitro do not undergo normal laminin isoform switching. Instead, both laminin alpha 1- and alpha 5-chains are present, as is the beta 1-chain, but not beta 2. When avascular organ-cultured kidneys are grafted into anterior eye chambers, however, kidney-derived angioblasts establish extensive vasculature by 6 days, and glomeruli are lined by endothelial cells. We evaluated embryonic day 12 (E12) vascular endothelial growth factor receptor (Flk1)-lacZ kidneys that had first been grown in organ culture for 6--7 days and then grafted into wild-type mice. Correct laminin isoform substitution occurred and correlated with the appearance of endothelial cells expressing Flk1. Our findings indicate that endothelial cells, and/or factors present in the circulation, mediate normal GBM laminin isoform transitions in vivo.

Structure and function of podocytes: an update.

Glomerular visceral epithelial cells, also termed podocytes, are highly specialized epithelial cells that cover the outer aspect of the glomerular basement membrane. Recent studies point to an important role of podocytes in the physiology and pathophysiology of the glomerulus. This review summarizes the structure-function relationships of podocytes. Following a description of the general morphology of podocytes, the technical problems associated with studying these cells are discussed. A survey of podocyte function forms the center of this review. Finally, selected aspects of podocyte development and cell division are discussed.

Origin of chemical variations with ignimbrite cooling units

<h3>Background</h3> Podocytes are critical to maintaining the glomerular filtration barrier, and mutations in nephrotic syndrome genes are known to affect podocyte calcium signaling. However, the role of calcium signaling during podocyte development remains unknown. <h3>Methods</h3> We undertook live imaging of calcium signaling in developing podocytes, using zebrafish larvae and human kidney organoids. To evaluate calcium signaling during development and in response to channel blockers and genetic defects, the calcium biosensor GCaMP6s was expressed in zebrafish podocytes. We used electron microscopy to evaluate filtration barrier formation in zebrafish, and Fluo-4 to detect calcium signals in differentiating podocytes in human kidney organoids. <h3>Results</h3> Immature zebrafish podocytes (2.5 days postfertilization) generated calcium transients that correlated with interactions with forming glomerular capillaries. Calcium transients persisted until 4 days postfertilization, and were absent after glomerular barrier formation was complete. We detected similar calcium transients in maturing human organoid glomeruli, suggesting a conserved mechanism. In both models, inhibitors of SERCA or IP3 receptor calcium-release channels blocked calcium transients in podocytes, whereas lanthanum was ineffective, indicating the calcium source is from intracellular podocyte endoplasmic-reticulum stores. Calcium transients were not affected by blocking heartbeat or by blocking development of endothelium or endoderm, and they persisted in isolated glomeruli, suggesting podocyte-autonomous calcium release. Inhibition of expression of phospholipase C-<i>γ</i>1, but not nephrin or phospholipase C-<i>ε</i>1, led to significantly decreased calcium activity. Finally, blocking calcium release affected glomerular shape and podocyte foot process formation, supporting the critical role of calcium signaling in glomerular morphogenesis. <h3>Conclusions</h3> These findings establish podocyte cell–autonomous calcium signaling as a prominent and evolutionarily conserved feature of podocyte differentiation and demonstrate its requirement for podocyte foot process formation.