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Stem Cell derived kidneys

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

HUMAN STEM CELL-DERIVED KIDNEYS CONNECT TO BLOOD VESSELS WHEN TRANSPLANTED INTO MICE

http://health-innovations.org/2015/11/20/human-stem-cell-derived-kidneys-connect-to-blood-vessels-when-transplanted-into-mice/

 

ft Stem cell-derived kidneys connect to blood vessels when transplanted into mice - healthinnovations

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The kidney tissues derived from human iPS cells
A.The kidney tissue generated in vitro, which shows green fluorescence in each glomerulus.
B.Vascularized glomerulus formed upon transplantation into the mouse. Many red blood cells (arrowhead) are observed, and the substance exists in the lumen (*), suggesting the possible filtration.
C.Mouse vascular endothelial cells (green) are incorporated into the glomerulus that consists of podocytes (magenta).
D.The slit diaphragm (arrow) formed between the cellular processes of the podocytes. Credit: The Institute of Molecular Embryology and Genetics (IMEG).

In the field of iPS cell-based regenerative medicine, advanced research with clinical applications for many organs and tissues, such as the retina, has steadily progressed. However, growing a kidney from scratch has been extremely difficult.  Although the number of renal failure patients on dialysis is increasing, opportunities for renal transplant have been limited with great attention given to the growth of kidneys to stem the shortage.

Now, a study from researchers at Kumamoto University shows mouse kidney capillaries successfully connecting to kidney tissue derived from human iPS cells. The team state that this achievement shows that human kidney glomeruli made in vitro can connect to blood vessels after transplantation and grow to maturity, a big step forward in gain-of-function for a urine-producing kidney.  The opensource study is published in the Journal of the American Society of Nephrology.

Earlier studies from the lab led to the development of an in vitro three-dimensional kidney structure from human iPS cells.  However, it was unclear how similar the kidney tissue made in vitro was to that formed in a living body. Additionally, the original kidney tissue was not connected to any blood vessels, even though the primary function of the organ is to filter waste products and excess fluid from the blood.  In many kidney diseases, the pathology is with the glomeruli that filter urine from the blood. Filtration in the glomerulus is performed by cells called podocytes that are in direct contact with the blood vessels. Through the special filtration membrane of the podocytes, proteins don’t leak into the urine and allows moisture to pass through.  Therefore, the group focused on analyzing the podocyte of the glomeruli in detail.  They achieved this by genetically modifying the iPS cells and growing human kidney tissue in vitro with green fluorescence then visualizing how human glomeruli became established.

The current study continued this analysis by taking out only the podocytes of the human glomeruli using the green fluorescence, and revealed that glomerular podocytes made in vitro express the same genes important for normal biological function.  Data findings show that after transplanting the human iPS cell-based kidney tissue into a mouse body, glomeruli connecting to mouse kidney capillaries formed. Results show that human glomerular podocytes further matured around adjacent blood vessels as in a living body and formed a characteristic filtration membrane structure.  The group state that to their knowledge the successful connection of capillaries with the podocytes of iPS cell-manufactured human glomeruli resulting in a distinct filtration membrane is the first of its kind in the world.

The team surmise that their findings should advance research into the manufactured kidney’s function to produce and excrete urine.  They go on to add that by using iPS cells from patients, development of new drugs and clarification of the causes of kidney disease are also expected.  For the future, the researchers state that they are now working to develop a discharge path for the kidney and combine it with findings on glomerular cells.

Source: The Institute of Molecular Embryology and Genetics (IMEG)

 

Human Induced Pluripotent Stem Cell–Derived Podocytes Mature into Vascularized Glomeruli upon Experimental Transplantation

Sazia Sharmin*Atsuhiro Taguchi*Yusuke Kaku*Yasuhiro Yoshimura*Tomoko Ohmori*Tetsushi Sakuma, et al.

JASN Nov 19; 2015 ASN.2015010096      http://dx.doi.org:/10.1681/ASN.2015010096    http://jasn.asnjournals.org/content/early/2015/11/18/ASN.2015010096.full

Glomerular podocytes express proteins, such as nephrin, that constitute the slit diaphragm, thereby contributing to the filtration process in the kidney. Glomerular development has been analyzed mainly in mice, whereas analysis of human kidney development has been minimal because of limited access to embryonic kidneys. We previously reported the induction of three-dimensional primordial glomeruli from human induced pluripotent stem (iPS) cells. Here, using transcription activator–like effector nuclease-mediated homologous recombination, we generated human iPS cell lines that express green fluorescent protein (GFP) in the NPHS1 locus, which encodes nephrin, and we show that GFP expression facilitated accurate visualization of nephrin-positive podocyte formation in vitro. These induced human podocytes exhibited apicobasal polarity, with nephrin proteins accumulated close to the basal domain, and possessed primary processes that were connected with slit diaphragm–like structures. Microarray analysis of sorted iPS cell–derived podocytes identified well conserved marker gene expression previously shown in mouse and human podocytes in vivo. Furthermore, we developed a novel transplantation method using spacers that release the tension of host kidney capsules, thereby allowing the effective formation of glomeruli from human iPS cell–derived nephron progenitors. The human glomeruli were vascularized with the host mouse endothelial cells, and iPS cell–derived podocytes with numerous cell processes accumulated around the fenestrated endothelial cells. Therefore, the podocytes generated from iPS cells retain the podocyte-specific molecular and structural features, which will be useful for dissecting human glomerular development and diseases.

 

The glomerulus is the filtering apparatus of the kidney and contains three types of cells: podocytes, vascular endothelial cells, and mesangial cells. Podocytes cover the basal domains of the endothelial cells via the basement membrane and play a major role in the filtration process.1,2 Podocytes possess multiple cytoplasmic protrusions. The primary processes are complicated by the further stemming of smaller protrusions (secondary processes or foot processes), which interdigitate with those from neighboring podocytes. The gaps between these foot processes are connected with the slit diaphragm, which is detectable only by electron microscopy. The molecular nature of the slit diaphragm was initially revealed by identification of NPHS1 as the gene responsible for Finnish-type congenital nephrotic syndrome.3 The nephrin protein encoded by NPHS1intercalates with those from neighboring cells, thus forming a molecular mesh that hinders serum proteins of high molecular weight from leaking into the urine.4,5 To date, many slit diaphragm–associated proteins have been identified, including NPHS2 (encoding podocin) and NEPH1, mutations that cause proteinuria in humans and/or mice.6,7

Podocytes are derived from nephron progenitors that reside in the embryonic kidney and express transcription factor Six2.8 Upon Wnt stimulation, the nephron progenitors undergo mesenchymal-to-epithelial transition and form a tubule.9 This tubule changes its shape; one end forms the glomerulus with podocytes inside, which is surrounded by a Bowman’s capsule. Meanwhile, vascular endothelial cells and mesangial cells migrate into the developing glomeruli, thus connecting the glomeruli with circulation.2 In these processes, several transcription factors, including Wt1, regulate expression of nephrin in podocytes.10 Apical junctions are initially formed between the presumptive podocytes, but the apical domain loses its direct contact with that of the neighboring cells, thus forming the characteristic podocyte shape. Nephrin is eventually localized to the site close to the basal domain and contributes to the formation of the slit diaphragm.2 The molecular mechanisms underlying podocyte development have been extensively studied in mice. However, because of limited access to human embryos, relatively little is known regarding transcription profiles of podocytes and glomerulogenesis in humans.4,11,12

We have recently induced the nephron progenitors from mouse embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells by redefining the in vivo origin of the nephron progenitors.13 The induced progenitor aggregates readily form three-dimensional primordial glomeruli and renal tubules upon Wnt stimulation in vitro. To analyze the detailed structures and transcription profiles of the induced podocytes, we have here inserted the GFP gene into the NPHS1 locus of human iPS cells via homologous recombination using transcription activator–like effector nucleases (TALENs)14 and generated glomeruli with green fluorescent protein (GFP)-tagged podocytes.

 

Fluorescent Visualization of Human Glomerular Podocytes Generated fromNPHS1-GFP iPS Cells

To visualize developing human podocytes in vitro, we inserted a gene encoding GFP into the NPHS1 locus of human iPS cells by homologous recombination (Figure 1A). We first constructed a pair of plasmids expressing TALENs targeted in close proximity to the NPHS1 start codon. When tested in HEK 293 cells, these plasmids efficiently deleted the NPHS1 gene (Supplemental Figure 1A). We then introduced these TALEN plasmids, along with a targeting vector containing the GFP gene and the homology arms, into human iPS cells. This resulted in efficient homologous recombination and isolation of heterozygous GFP knock-in (NPHS1-GFP) clones (Figure 1B, Supplemental Figure 1, B and C).

Figure 1.

Successful generation ofNPHS1-GFP iPS cells by homologous recombination. (A) Strategy for targeting the human NPHS1 locus. TheGFP cassette was inserted upstream of the NPHS1 start codon. The puromycin-resistance cassette (PURO) is flanked by loxP sites. Positions for primers and probes for screening are indicated. E, EcoRV; N, NheI. (B) Southern blot of control (+/+) and NPHS1-GFP (GFP/+) clones. Genomic DNA was digested and hybridized with the indicated probes.

We differentiated these NPHS1-GFP iPS clones toward the nephron progenitors and subsequently combined them with murine embryonic spinal cord, which is a potent inducer of tubulogenesis, as we previously reported.13 Four days after recombination, spotty GFP signals could be observed, and the number and intensity of GFP signals increased thereafter until day 9 (Figure 2A,Supplemental Figure 2A). We observed GFP signals in all the examined samples from seven independent experiments (a total of 50 samples). Some of the signals started in a crescent shape and gradually changed into round structures (Figure 2A, lower panels), which suggests that human glomerular formation in vitro may be visualized. Therefore, we examined glomerulogenesis using sections of the explants. At day 3, only tubular structures were observed and GFP-positive cells were undetectable (Figure 2B). At day 4, structures that resembled S-shaped bodies were observed, in which proximo-distal specification occurred toward the presumptive distal (E-cadherin+) and proximal (cadherin-6+) renal tubules and glomerular podocytes (WT1+) (Figure 2C). At day 6, various forms of primordial glomeruli were observed, and most of the GFP signals overlapped with those of WT1 (Figure 2B). We ordered these glomeruli according to GFP intensity, which is likely to reflect the chronologic order of development. Weakly GFP-positive (and WT1-positive) limbs appeared at one end of the tubules, which elongated to surround the renal tubules. GFP intensity increased when the podocyte layers were convoluted. At day 9, strongly GFP-positive round glomeruli were formed. These histologic changes are consistent with the previous observations of human glomeruli in aborted fetuses.15 Thus, we succeeded in visualizing human podocyte development and glomerulogenesis in vitro. Interestingly, some, but not all, of the Bowman’s capsule cells were positive for GFP and nephrin (Supplemental Figure 2B), suggesting that these cells are not completely specified yet. Indeed, transient nephrin expression in some capsule cells was reported in vivo.16

Figure 2.

Fluorescent visualization of human glomerular podocytes generated fromNPHS1-GFP iPS cells. (A) Morphologic changes of GFP-positive glomeruli during differentiation in vitro. The nephron progenitors induced fromNPHS1-GFP iPS cells were combined with murine embryonic spinal cord and cultured for the indicated time. Lower panels: higher magnification of the areas marked by rectangles in the upper panels. Note the shape changes of the glomerulus (arrowheads). Scale bars: 500 μm. (B) Histologic sections of glomeruli developing in vitro. Tissues at day 3, 6, and 9 after recombination with the spinal cord were analyzed. Top panels: Hematoxylin-eosin (HE) staining. Middle panels: GFP (green) staining. Bottom panels: dual staining with GFP and WT1. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI: blue). Scale bars: 20 μm. (C) Presumptive S-shaped bodies observed at day 4 (left two panels) and day 6 (right two panels). Serial sections were stained with E-cadherin (Ecad: magenta)/cadherin-6 (cad6: green) and E-cadherin (magenta)/WT1 (green). Arrowheads: WT1-positive presumptive glomerular regions. Scale bars: 20 μm.

Induced Podocytes Exhibit Apicobasal Polarity and Basally Localized Nephrin

We analyzed day 9 sections at higher resolution to examine the apicobasal polarity of the induced podocytes. GFP was detected in the nuclei and cytoplasm of most cells in the round glomeruli (Figure 3A) because we did not attach any localization signal to GFP when generating NPHS1-GFP iPS cells. Nephrin proteins were distributed in a linear fashion in the iPS cell–derived glomeruli and at one end of the WT1-positive podocyte layer (Figure 3, A and B). These expression patterns significantly overlapped with those of type IV collagen, which was accumulated on the basal side of the podocytes (Figure 3C). In contrast, podocalyxin, an apical marker, was expressed in a manner mutually exclusive of nephrin (Figure 3D). Therefore, the induced podocytes exhibited a well established apicobasal polarity and nephrin proteins were properly localized at the basal side, where the presumptive slit diaphragm should be formed. We also observed nephrin-positive dots on the lateral side of the podocytes (Figure 3A, arrowheads), as reported in human developing podocytes in vivo.15 We found that these dots actually represent the filamentous structures encompassing the basal to the lateral side of the podocytes (Figure 3, B and C, arrowheads). Although further investigation is required, this may reflect the transit state of nephrin proteins shifting from the apical to the basal domain of the induced podocytes.

Figure 3.

Induced podocytes exhibit apicobasal polarity and basally localized nephrin. (A) Nephrin (magenta) and GFP (green) staining of the induced glomerulus at day 9. (B) Nephrin (magenta) and WT1 (green) staining. (C) Nephrin (magenta) and type IV collagen (COL: green) staining. (D) Nephrin (magenta) and podocalyxin (PODXL: green) staining. The left columns are at lower magnification to show a whole glomerulus. The right two columns are singly stained, while the left two columns represent merged images. Arrows: nephrin proteins localized to the basal domain; arrowheads: nephrin-positive dot-like or filamentous structures. Scale bars: 10 μm.

Induced Podocytes Possess Primary Processes with the Slit Diaphragm–Like Structures

We further analyzed the morphology of the induced glomeruli by electron microscopy. Both scanning and transmission electron microscopy showed well organized glomeruli surrounded by Bowman’s capsules (Figure 4, A and B). Interestingly, numerous microvilli were detected in the apical domain of the induced podocytes (Figure 4, C and D). Similar microvilli were reported in developing in vivo podocytes in humans.17,18 The podocytes were attached to each other at sites close to the basal region (Figure 4D). Inspection of the basal side of the induced podocytes by scanning microscopy identified multiple protrusions (Figure 4E), which were confirmed by transmission microscopy (Figure 4F). Higher magnification clearly showed bridging structures between the protrusions, which may represent an immature form of the slit diaphragm (Figure 4, G and H, Supplemental Figure 3, A–C). Thus, this is the first in vitrogeneration of mammalian podocytes with slit diaphragm–like structures from pluripotent stem cells. However, because typical interdigitation of the protrusions is lacking, they are likely to represent primary processes but not secondary processes (foot processes).

Figure 4.

Induced podocytes possess primary processes with the immature slit diaphragm–like structures. (A and B) Induced glomerulus covered with a Bowman’s capsule shown by (A) scanning and (B) transmission electron microscopy. (C) Induced podocytes observed by scanning electron microscopy. Multiple microvilli are observed on the apical surface (arrowheads). (D) Aligned podocytes, which attach to each other at sites close to the basal region, shown by transmission electron microscopy. Multiple microvilli are observed on the apical surface (arrowheads). (E) Primary processes shown by scanning electron microscopy (asterisks). Podocytes from the basal side are shown. (F) Primary processes shown by transmission electron microscopy (asterisks). (G) Slit diaphragm–like structures between the primary processes (arrows), shown by scanning electron microscopy. (H) Primary processes with slit diaphragm–like structures (arrows), shown by transmission electron microscopy. Scale bars: A and B: 10μm; C–F: 2 μm; G and H: 0.2 μm.

Induction of Podocytes from Human NPHS1-GFP iPS Cells Enables Their Efficient Isolation

We next tried to purify the GFP-positive podocytes at day 9 by FACS. Of the induced cells, 7.45%±0.72% (mean±SEM from five independent induction experiments) were positive for GFP (Figure 5A, left panel). We also found that the monoclonal antibody against the extracellular domain of nephrin (48E11),19in combination with the anti-podocalyxin antibody, was useful for sorting developing podocytes. Of the GFP-positive cells, 94.0% were positive for both nephrin and podocalyxin (Figure 5A, middle panel), while most of the GFP-negative cells (97.5%) were negative for both markers (Figure 5A, right panel). Thus, GFP faithfully mimics nephrin expression and podocytes were enriched in the GFP-positive population. Quantitative RT-PCR analysis of sorted cells confirmed the differential expression of several podocyte markers, such asNPHS2 (encoding podocin) and synaptopodin (Figure 5B). When the sorted GFP-positive cells were cultured for 3 days, the cells expressed WT1 in nuclei and podocalyxin on the cell surface (Figure 5C). Nephrin and GFP were detected on the cell surface membrane and in the cytoplasm, respectively, at day 7 of culture, although expression levels were lower than before the start of the culture. These results indicate that induction from NPHS1-GFP iPS cells enables efficient isolation of developing human podocytes.

Figure 5.

Induction of podocytes from human NPHS1-GFP iPS cells enables their efficient isolation. (A) FACS analysis of induced tissues at day 9. Almost 8% of cells are positive for GFP in this representative experiment (left panel). Nephrin and podocalyxin (PODXL) expression in the GFP-positive or -negative fraction (middle and right panel, respectively). (B) Quantitative RT-PCR analysis of GFP-positive and -negative fractions. Average and SEM from three independent experiments are shown. β-ACT, β-actin; SYNPO, synaptopodin. (C) Immunostaining of podocytes cultured for the indicated times after sorting GFP-positive cells. Scale bars: 5 μm. (D) Venn diagram of the transcription profiles of podocytes. Microarray data of GFP-positive podocytes are compared with those of human adult glomeruli and murine podocytes.

GFP-Positive–Induced Podocytes Show Transcriptional Profiles That Overlap with Those of Mouse and Human Podocytes In Vivo

To obtain comprehensive transcription profiles of the iPS cell–derived podocytes, we performed microarray analysis at day 9. We detected 2985 probes that were enriched in GFP-positive podocytes compared with GFP-negative cells. Of these, the top 300 genes were used for unbiased cluster analysis against microarray data from a wide variety of human tissues (Supplemental Figure 4, A and C).20 Genes enriched in the GFP-positive podocytes had variable tissue specificity. For example, NPHS2 was selectively expressed in the kidney or fetal kidney tissues. However, synaptopodin andFOXC2 were sorted into the ubiquitously expressing cluster. Dendrin was assigned to a cluster enriched in the neuronal tissues. These results suggest a single molecule is not sufficient to confirm the identity of podocytes. Therefore, we compared our gene list of GFP-positive human podocytes with the published microarray analyses of adult human glomeruli and adult podocytes from Mafb-GFP transgenic mice.11,21 Overall, 190 probes were overlapping among the three gene sets (Figure 5D, Supplemental Table 1, Table 1). These included typical slit diaphragm–related genes, such as NPHS1, NPHS2,CD2AP,22 chloride intracellular channel protein 5 (CLIC5),23 and dendrin,24,25and basolateral adhesion molecules such as claudin 5 and integrinα3.26,27Phospholipase ε1 and nonmuscle myosin heavy chain 9 (Myh9), causative genes for hereditary kidney diseases,2830 were also included. Transcription factors that have important roles in podocyte development, including WT1, MAFB, FOXD1, and TCF21, as well as vascular attractants such as VEGFA and semaphorin, were also expressed.1,2,31 Interestingly, when these selected overlapping genes were used for the cluster analysis against the microarray data from various organs described above, kidney and fetal kidney were segregated as separate clusters, suggesting the kidney-biased features of the overlapping gene set (Supplemental Figure 4B).

Table 1.

Genes common to iPS cell–derived podocytes in vitro, human glomeruli, and mouse podocytes in vivo

We also identified genes common to GFP-positive podocytes and adult human glomeruli (Figure 5D, Supplemental Table 2), and genes common to GFP-positive podocytes and mouse adult podocytes (Figure 5D, Supplemental Table 3). The former includes BMP7,32 while the latter includes NEPH1 (KIRREL),FOXC2, ROBO2, and EPHRIN-B1.7,3336 These results indicated that the typical transcriptional profiles are well conserved among our podocytes generated in vitro as well as mouse and human podocytes in vivo. In addition, extracellular matrix components characteristic of glomeruli at the capillary loop stage,lamininα5/β2/γ1 isoforms (corresponding to laminin 521) and type IV collagenα4/α5,37 were detected, the latter of which is the causative gene for Alport syndrome. These data indicate that the transition to these mature forms from immature laminin 111 and collagen α1/α2 has already occurred in vitro.

Taken together, our podocytes induced in vitro possessed the typical features of those in vivo, not only in morphology but also in transcription profiles, further supporting the authenticity of our human iPS cell induction protocol. In addition, genes exclusively expressed in the GFP-positive podocytes are worthy of further investigation because they may include genes specific to developing human podocytes, a possibility that has not been addressed to date (Figure 5D,Supplemental Table 4).

 

Transplanted iPS Cell–Derived Nephron Progenitors Form Vascularized Glomeruli

We next examined whether glomeruli generated from iPS cells integrated with the vascular endothelial cells. The iPS cell–derived nephron progenitor spheres were induced by spinal cord for 1 day in vitro to initiate tubulogenesis and were then transplanted beneath the kidney capsule of immunodeficient mice. We also cotransplanted mixed aggregates of human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cells (MSCs) because these cells are useful for the generation of vascularized organ buds in vitro.38,39 When these aggregates were transplanted using a conventional method that we used for the transplantation of mouse ES cell–derived nephron progenitors,13 minimal nephron differentiation was observed at 10 days after transplantation (n=4) (Figure 6A). Because human iPS cell–derived aggregates were larger (approximately 1000 µm in diameter) than those from mouse ES cells (approximately 600 µm) and were instantly flattened upon transplantation (Supplemental Figure 5A), we hypothesized that mechanical tension of the capsule may have hampered nephron differentiation. Therefore, we inserted two agarose rods of 1100 µm diameter in a V-shaped position to release tension and secure a space for the transplanted aggregates (Figure 6B). We also soaked the rods with VEGF to enhance vasculogenesis.31 As a result, we observed immature glomerular formation at day 10 in the transplants, accompanied by blood vessels integrating into these glomeruli (n=5) (Figure 6, C and D). The vessels were preferentially clustered in the glomeruli among the grafted tissue (Figure 6D), suggesting that the iPS cell–derived podocytes possess the potential to attract vasculature. This is also consistent with microarray data showing VEGFA expression in our induced podocytes.

Figure 6.

Transplanted iPS cell–derived nephron progenitors form vascularized glomeruli. (A) Hematoxylin-eosin sections of tissues at 10 days after transplantation using a conventional method. Right panel: magnified image of the square in the right panel. kid, kidney of the host mouse. (B) Method for transplantation using solid agarose rods. Right panel: macroscopic view of transplanted tissue under the kidney capsule. Ag, agarose rods. (C) Hematoxylin-eosin sections of the transplanted tissue at day 10 in the presence of the rods. Right panel: magnified images of the square. (D) Vascularized glomeruli at day 10. Staining of WT1 and CD31. Right panel: magnified image of the square in the left panel. (E) Hematoxylin-eosin section of the transplanted tissue at day 20. Middle and right panel: magnified images of the squares in the panels on their left, respectively. *Stromal cells. kid, kidney of the host mouse. (F) Vascularized glomeruli formed upon transplantation at day 20. Left panel: magnified images of panel E. Right panel: magnified image of the square in the left panel. Note the enlarged Bowman’s space. (G) The endothelial cells are of mouse origin. Staining of WT1 (magenta) and MECA-32, a marker for mouse-specific endothelial cells (green). (H) Hematoxylin-eosin staining showing red blood cells in the induced glomeruli. (I) Hematoxylin-eosin staining showing the eosin-positive precipitates in the Bowman’s space. (J) Staining of nephrin (magenta) and CD31 (green). Right panel shows the basal localization of nephrin. Scale bars: A, C–F, I: 100 μm; B: 1 mm; G, H, J: 10 μm.

At day 20 after transplantation, we observed enlarged transplanted tissues beneath the capsule (Supplemental Figure 5B). Histologic examination revealed excessive growth of stromal cells of human origin, which were presumably derived from nonrenal tissues that were coinduced with nephron progenitors from iPS cells (n=4) (Figure 6E, Supplemental Figure 5C). Nonetheless, glomeruli were formed and the blood vessels were well integrated into the glomeruli (Figure 6, F and G). Moreover, 90% (135 of 150) of the glomeruli contained red blood cells (Figure 6H). Indeed, some of the glomeruli showed an enlarged Bowman’s space and contained eosin-positive precipitation (Figure 6I), which might imply a small amount of urine production. Interestingly, endothelial cells in the induced glomeruli were of mouse origin (Figure 6G,Supplemental Figure 5D). HUVEC-derived endothelial cells were not integrated into the iPS cell–derived glomeruli but were located separately from the sites of nephron formation (Supplemental Figure 5E). Therefore, HUVEC may not be competent to interact with human podocytes.

The anti-human specific podocalyxin antibody stained the apical domains of the iPS cell–derived podocytes, but not those of the host mouse podocytes (Supplemental Figure 5F). Nephrin protein in induced podocytes was localized at the basal side that faced the vascular endothelial cells (Figure 6J), suggesting the emergence of filtering apparatus. Electron microscopic analyses of two additional samples at day 20 showed that iPS cell–derived podocytes accumulated around, and were closely associated with, endothelial cells (Figure 7A). The induced podocytes developed numerous complex cell processes, as well as a linear basement membrane, at interfaces with endothelial cells (Figure 7B). The distances between the cell processes of some podocytes were enlarged, and slit diaphragm–like structures were formed between the processes located above the basement membrane (Figure 7C). Each of these diaphragms appeared as an electron-dense line (approximately 35 nm wide, 10 nm thick) bridging adjacent cell processes of the iPS cell–derived podocytes (Figure 7D). This feature was also observed in vivo and differed from the immature ladder-like structure that was seen between adjacent podocytes cultured exclusively in vitro without transplantation (Figure 4). Endothelial cells also produced basement membrane, but it was not fused to that of the podocytes in most cases, thus forming double-layered structures (Figure 7E). Interestingly, endothelial cells were fenestrated with residual diaphragm, a characteristic feature of embryonic glomerular endothelial cells (Figure 7F).40Furthermore, an electron-dense substance was detected in the Bowman’s space (Figure 7C), as in Figure 6I, implying the possible presence of filtration. Taken together, glomeruli generated from human iPS cells were vascularized and had many morphologic features present in glomeruli in vivo.

Figure 7.

iPS cell–derived glomeruli in the transplants exhibited many morphologic features of those in vivo. (A) Induced podocytes surrounding the vascular endothelial cells and extending many cell processes, shown by transmission electron microcopy. (B) Complex cell processes of podocytes formed between the cells and above the basement membrane. (C and D) Formation of slit diaphragm–like structures (arrows) between the cell processes of induced podocytes. Note the electron-dense substance in the Bowman’s capsule (asterisk). (E) Formation of double-layered basement membranes, each derived from endothelial cells (white arrowheads) and induced podocytes. (F) Fenestrated endothelial cells with diaphragms (black arrowheads). bm, basement membrane derived from induced podocytes; en, endothelial cells. Scale bars: A: 1 μm; B, E: 0.5 μm; C, D, F: 0.2 μm.

Discussion

We have inserted GFP into the NPHS1 locus of human iPS cells and successfully differentiated them toward three-dimensional glomeruli. The GFP-positive–induced podocytes possessed apicobasal polarity and were equipped with primary processes and slit diaphragm–like structures. Furthermore, sorted podocytes exhibited typical transcription profiles that overlap with those in vivo. These findings underscore the authenticity of our induction protocol.NPHS1 promoter–driven GFP expression is a good indicator of glomerulus formation. Several groups have reported the induction of kidney tissues in vitro,13,4143 and our iPS cell lines will be useful for assessing the induction efficiency of glomeruli by each protocol. In addition, we successfully sorted human podocytes using a combination of anti-nephrin and anti-podocalyxin antibodies. These reagents will make genetic GFP integration unnecessary for the purification of podocytes from patient-derived iPS cells, and possibly from more complex in vivo tissues.

It is surprising that well organized glomeruli are formed without the other two components of glomeruli: mesangial and vascular endothelial cells. These two cell types are not derived from nephron progenitors, as shown by cell lineage analysis in mice,8,44,45 and indeed we did not detect these lineages in the induced glomeruli (Supplemental Figure 3D). Thus, glomeruli can self-organize their structures solely from the podocytes derived from nephron progenitors, without any inductive signals from mesangial cells or the vasculature. However, further maturation will be required to reproduce hereditary glomerular diseases. We developed a new transplantation technique using agarose rods to secure a space against the tension evoked by kidney capsules. This technical improvement led to the successful generation, for the first time, of vascularized glomeruli derived from human iPS cells. The induced podocytes exhibited complex cell processes with slit diaphragm–like structures, and linear basement membrane that ran along that of the endothelial cells was formed. Furthermore, endothelial cells were fenestrated, which is a characteristic feature of glomerular endothelial cells. Most experiments used agarose rods soaked with VEGF to potentially accelerate vasculogenesis; however, the absence of VEGF in the rods also caused the formation of vascularized glomeruli (Supplemental Figure 5G). Thus, we can at least conclude that the human iPS cell–derived podocytes expressed sufficient attractants, including VEGF, to recruit endothelial cells.

It is noteworthy that the integrated endothelial cells were of mouse origin from the host animals but were not derived from HUVECs, although both vascular sources were initially located in proximity to the iPS cell–derived transplants. Therefore, human podocytes recruited mouse endothelial cells despite species differences, while HUVECs may not be competent to interact with human podocytes. Even when we performed transplantation without HUVECs or MSCs, we observed vascularized glomeruli, suggesting that paracrine effects of these cells may also be minimal (Supplemental Figure 5H). The presence of double-layered basement membrane might be caused by the incomplete fusion between those derived from human podocytes and mouse endothelial cells, as observed when mouse embryonic kidney was transplanted onto a quail chorioallantoic membrane.46 Therefore, the identification of optimal sources for human endothelial cells is necessary.

While it is difficult to estimate the gestational age on the basis of the morphology of the individual glomeruli,47,48 waiting for a longer period after transplantation may help further maturation of induced podocytes. However, we observed an excessive growth of stromal, presumably nonrenal, cells in the transplants. Thus, it will be essential to develop methods to purify nephron progenitors for transplantation. At the same time, it is necessary to induce genuine stromal cells because both interstitial cells and mesangial cells are derived from renal stromal progenitors.45 At present, we have no evidence that proper mesangial cells exist in our vascularized glomeruli. Ideally, human endothelial and mesangial cells that correspond to those in the developing kidney should be combined. Although further induction studies, as well as imaging techniques to visualize the slit diaphragm with a higher resolution,49are needed to achieve this goal, our results will accelerate the understanding of human podocyte biology both in developmental and diseased states.

 

 

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Targeting Kidney Glomerulus

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Targeting the podocyte to treat glomerular kidney disease

Lal MA, Young KW, and Andag Uwe    1 CVMD iMED, AstraZeneca, Molndal, Sweden 2 Evotec AG, Germany
Drug Discovery Today  http://www.e-ditionsbyfry.com/olive/ODE/DDT/Default.aspx?href=DDT/2015/10/01

The number of randomized clinical trials addressing renal disease continues to be outpaced by most other specialties and the failure of a number of recent kidney clinical trials suggests a need for new thinking and new strategies to address CKD [5,58]. Delineation of the crucial molecular species and signaling pathways underlying podocyte function will undoubtedly lead to the identification of cell-specific targets that can be exploited for treating various glomerulopathies and CKD.

 

The majority of chronic kidney disease (CKD) cases have their origin in the glomerulus, the microvascular unit of the nephron that serves as a filter tasked with forming primary urine. This selective filtration process is determined to a large extent by the functional capacity of the podocyte, a highly differentiated cell type that enwraps the outer aspect of the glomerular capillary wall. In this short review, we describe the biology of the podocyte, its central role in the etiology of various glomerulopathies and highlight current and future opportunities to exploit the unique properties of this cell type for developing kidney-specific therapeutics.  

The kidneys   The kidneys are a remarkable set of paired organs that receive › 20% of cardiac output and that filter some 180 l blood daily, which is subsequently modified to give a final urine volume of approximately 1.5 l. This phenomenal physiological capacity is carried out by the concerted actions of the glomerular filter and renal tubular systems that together represent the single functional unit of the kidney known as the nephron. In performing this outstanding feat, the kidneys participate in establishing wholebody ion homeostasis, acid-base balance and blood pressure regulation. It should therefore come as no surprise that loss of kidney function and a compromised ability to carry out such fundamental processes is associated with considerable risks to overall health [1].  

Chronic kidney disease   Chronic kidney disease (CKD) is a progressive malady defined by reduced glomerular filtration rate, increased urinary albumin excretion or both, and is a major global public health concern with an extremely high unmet medical need. CKD is estimated to occur in 8–16% of the worldwide population and results in a substantially reduced life expectancy [1]. Diabetes itself is recognized as the primary cause of kidney failure in almost half of all new CKD cases and, given the current global diabetes pandemic, the prevalence of renal complications will continue to grow.  

There is a strong relationship between CKD and cardiovascular disease which itself is twice as common in CKD patients as compared with the general population [2]. Accordingly, the current clinical paradigm for treating CKD patients is primarily focused on reducing cardiovascular risk and is, not surprisingly, inadequate to prevent patients from continuing their inexorable loss of renal function because such strategies do not directly address the principal mechanisms accounting for renal disease itself. Despite overall advances in renal replacement therapy and dialysis for CKD patients ultimately succumbing to end-stage renal failure, these last resort options are insufficient in terms of limited organ availability and high mortality and/or cost. The potential identification and implementation of novel, kidney-specific treatment strategies therefore represents a significant untapped opportunity to improve the prognosis of these patients. The future development of such kidney-centric therapeutics will require the identification of suitable renal targets and can only be achieved through a comprehensive understanding of disease pathophysiology, the underlying molecular mechanisms of disease initiation and progression, and the implementation of translatable models and technologies [3].  

The glomerular filtration barrier   The vast majority of all cases of CKD have their origin in the glomerulus [4]. Clinically, although kidney disease progression shows the strongest correlation with the degree of fibrosis in the tubules and interstitium, it is lesions within the highly specialized microvascular unit that initiate disease. The primary function of the glomerulus is to filter blood selectively across the capillary wall and to elaborate an ultrafiltrate that is secondarily modified by the renal tubule system. The filtration capacity of the glomerulus is defined by the functional properties of the glomerular filtration barrier (GFB), a trilaminar molecular sieve comprising endothelial cells and visceral podocytes that medially elaborate the constituents of the glomerular basement membrane (GBM) that lies between them (Fig. 1). Although each individual component of the GFB is necessary for its concerted function, the podocyte is largely recognized as the final determinant of size-selective filtration and the ultimate barrier to albumin [4]. This is of particular clinical relevance because even small increases in albuminuria confer clinical risk and, at increasing levels of proteinuria, life expectancy is substantially reduced [5].  

FIGURE 1   Ultrastructural images of the glomerular capillary and filtration barrier. (a,b) Scanning electron micrographs of the exterior surface of the glomerular capillary depicting the intricate pattern in which podocyte foot processes interdigitate, surround and enwrap the outer capillary surface. (c) Transmission electron micrograph of the glomerular filtration barrier in cross section. The * indicates the slit diaphragm. Size marker 1 mm (a) and 200 nm (b,c). Abbreviations: FP, foot process; GBM, glomerular basement membrane; EC, endothelial cell; RBC, red blood cell; CL, capillary lumen; US, urinary space.

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The podocyte   The podocyte is a terminally differentiated, highly specialized cell type with a remarkable morphology exquisitely designed to match its function. The podocyte establishes the integrity and function of the GFB and slit diaphragm, maintains capillary structure and resists intraglomerular blood pressure, contributes to the formation and modulation of the glomerular basement membrane and also determines endothelial cell homeostasis [6]. Ultrastructurally, the podocyte is like no other cell. From its voluminous cell body exposed in the urinary space extend primary and secondary processes that arborize into slender foot processes that interdigitate with those of neighboring podocytes to enwrap the outer surface of the glomerular capillary firmly (Fig. 1). One of the most unique features of the podocytes and their foot processes is the slit diaphragm, a modified cell–cell adherens junction that spans the length of their interaction. The slit diaphragm, visible upon electron microscopy as a bridge connecting juxtaposed foot processes, comprises a host of unique structural proteins, the founding member of which is nephrin [4]. It is the initial identification of nephrin and genetic mutations therein as the cause of the massive proteinuria that characterizes patients with congenital nephrotic syndrome of the Finnish type (CNSF) that has placed the podocyte at the center of current activities aimed at understanding the molecular and cellular determinants of proteinuria. Since the discovery of nephrin, the identification of a host of additional human genetic mutations in various proteins implicit in podocyte function has highlighted the central involvement of this cell type in the etiology of various inherited diseases of the glomerulus [7] (Table 1). Collectively, glomerular diseases can be classified as a related spectrum of podocytopathies where abnormalities in podocyte biology (i.e. dysfunction, injury and loss) are shared among them. They encompass purely genetic forms of podocyte disease [such as CNSF, Alport syndrome and some forms of focal and segmental glomerular sclerosis (FSGS) and membranous nephropathy] and also include nonhereditary glomerular diseases that occur secondary to conditions such as hypertension and diabetic nephropathy [8]. Taken together, glomerular diseases account for the vast majority of all end-stage kidney disease cases with diabetic nephropathy representing the single largest cause.  

 

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One of the earliest cellular lesions observed in the various podocytopathies that lead to glomerulosclerosis is a loss of podocytes or, more specifically, a reduction in the number of podocytes per glomerulus. Because podocytes are terminally differentiated and have a limited capacity for repair or regeneration, glomerular function is particularly sensitive to situations in which there is a mismatch between podocyte number and the glomerular filtration surface area. Experimental animal models show that a loss of <20% of podocytes causes transient proteinuria and that further podocyte loss results in progressive proteinuria, glomerulosclerosis and eventually loss of renal function [9]. In a similar manner, if glomerular volume exceeds the threshold for podocytes to maintain a functional GFB, glomerular disease will ensue. Data obtained from humans with diabetic nephropathy corroborate these preclinical observations [10]. Based on this understanding of disease etiology, therapeutic strategies aimed specifically at improving the health and function of the podocyte represent a promising avenue to address CKD [11].  

Current therapeutic options with unpredicted effects on podocyte biology  

The current state of the art for the treatment of CKD is focused on optimizing renal and cardiovascular risk factors and includes controlling blood pressure, albuminuria, blood glucose and blood lipids [5]. These current treatment options are based largely on repurposing of existing therapeutics and are not kidney-targetbased nor designed to address the underlying mechanisms of CKD progression. Nevertheless, there are now considerable amounts of data supporting the intriguing possibility that the beneficial effects on kidney function of a number of currently available drugs (including glucocorticoids, calcineurin inhibitors, rituximab, inhibitors of the renin–angiotensin–aldosterone system, among others) could be at least partially accounted for by their direct effects on the podocyte. Table 2 lists additional pathways and targets central to podocyte function for which therapeutic interventions have been described using preclinical models. Such examples will not be further described here so we invite the reader to consult some recent reviews on the subject [11–13].  

 

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The most intriguing and contentious clinical evidence available thus far supporting the possibility to improve renal function and prevent CKD progression by specifically targeting the podocyte comes from research examining the role of B7-1 (CD80) in this cell type [14–16]. Targeting of the podocyte at this molecular moiety with abatacept, an antibody-based inhibitor of B7-1, induced remission in five patients with FSGS [16]. Mechanistically, B7-1 overexpression in injured podocytes appears to interfere with b1-integrin-mediated cellular attachment to the glomerular basement membrane and abatacept exerts its protective effects by facilitating the stabilization of b1-integrin activation and podocyte adhesion. Although these results might be compelling, they are not without significant debate because the detection of B7-1 expression in podocytes as well as the efficacy of B7-1 blockers in treating FSGS patients are both highly controversial [17]. Future studies with sufficient sample size will hopefully clarify the situation.  

Focusing on the podocyte as a specific therapeutic  target   Based on the above genetic evidence and efficacy of the described pharmacological approaches to targeting the podocyte clinically, combined with the plethora of preclinical animal studies conclusively demonstrating that podocyte dysfunction per se results in foot process effacement, albuminuria and glomerular disease, there is tantalizing hope of a future where the pursuit of podocentric therapeutic strategies will be realized. Although the ability to alter the podocyte genetically in mice has led to an explosion in the number of gene products identified as requisite for podocyte biology and potentially amenable to pharmacological manipulation, the ubiquitous nature of many of these targets and their pathways in other cell types makes them difficult to exploit. However, by identifying the molecular components that uniquely define and contribute to podocyte signaling and function, it should be possible to identify and develop cell-specific targeting opportunities.  

One of the best-studied aspects of podocyte biology concerns that of the mechanisms and factors regulating its cytoskeleton. Indeed, podocyte effacement and loss of the slit diaphragm as seen in glomerular diseases are phenomena intricately tied to disrupted actin cytoskeletal homeostasis in this cell type [12,18–20]. Not surprisingly, podocyte-specific manipulation of the mouse genome clearly indicates that the RhoGTPase family of actin master regulators is intricately involved in determining podocyte function [21,22]. The ultimate question that remains to be addressed, however, is how to exploit such a ubiquitous cell biological process driven by canonical signaling pathways therapeutically. Perhaps, the best opportunity for such a scenario could come from further studies aimed at defining the unique, principal molecular components that impact podocyte actin cytoskeletal architecture. A number of actin cytoskeleton effectors with podocyte-enriched expression have been identified and they could provide novel entry points to facilitate the fine-tuning and manipulation of the actin cytoskeleton specifically in this cell type to promote the reestablishment of normal foot process architecture in disease [23–25].  

Defining the molecular footprint of the podocyte   The ability to isolate pure populations of glomeruli from the renal parenchyma and further selectively separate the podocyte from its neighboring endothelial and mesangial cells by lineage-tracking and fluorescently activated cell sorting combined with various omics technologies has allowed researchers to catalogue the podocyte transcriptome and proteome [26–28]. Further definition of the miRNA transcriptional profile, alternative gene splicing events, phosphoproteome and epigenome represent additional levels of molecular complexity that further enhance our understanding of the underpinnings of podocyte biology [26,29–31]. A computational, machine-learning-based approach has also been developed that is able to infer podocyte-specific expression from whole-tissue samples through iterative statistical analysis of shared gene expression patterns and will be particularly useful to elucidate the roles of individual cell-specific transcripts in human glomerular disease [32]. The daunting task remains to unravel the cellular role of these individual proteins in podocyte health and disease and to consider whether they represent viable therapeutic targets. Significant inroads have been made over the past two decades and it is not unreasonable to envision a not too distant future when the involvement of all individual genes in podocyte biology is achieved. Acquired glomerular diseases, such as diabetic nephropathy, are not due to genetic defects but rather can be seen as a consequence of crucial signaling pathways of the podocyte that have gone awry. Determining which of the proteins expressed in podocytes and their pathways that are most amenable to therapeutic intervention will require considerable efforts from the research community.  

Use of podocytes in phenotypic screens   The ability to propagate conditionally immortalized podocytes in culture combined with rapid advances in molecular biology has greatly complemented our knowledge of the molecular machinery that defines this cell type [33,34]. Although there are important limitations to consider when utilizing such podocyte cell lines, podocyte cell culture has been an instrumental tool for simulating various aspects of glomerular disease biology. From the drug discovery perspective, there remain significant opportunities to take advantage of this cellular system for target evaluation and discovery [13,35].  

Target-agnostic high-content or phenotypic screening approaches are a recent addition to the drug discovery process [36]. Here, hit compounds are identified as having a positive phenotypic readout in a cell-based assay. Key to this process is the disease relevance of the phenotype being investigated and ultimately the translatability of the assay to human disease states. In this regard, we have used human conditionally immortalized podocytes [34] treated with a combination of palmate and high glucose to mimic conditions observed in diabetic nephropathy. The podocyte response to this ‘disease-relevant’ stimulus can be measured using high-content imaging technology, such as the OperaTM confocal imaging platform, and manifests as changes in the actin cytoskeleton and overall cell morphology, followed by onset of cell apoptosis (Fig. 2).

 

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FIGURE 2   Podocyte high-content screening. Conditionally immortalized human podocytes were screened using a combination of palmitate and high glucose to mimic conditions observed in diabetic nephropathy. Small-molecule inhibitors of phenotypic changes were identified using the OperaTM high-content imaging platform. The primary screen was run using activation of caspase 3/7 as a marker of podocyte apoptosis. Hit compounds were also tested in an orthogonal assay which used Alexa-labeled phalloidin to detect changes in the actin cytoskeleton.

 

These events provide suitably robust outputs for compound screening. Evotec, in combination with AstraZeneca, has screened >120 000 compounds using protection from palmitate and highglucose-induced podocyte apoptosis as a primary readout. Protection against effects on the actin cytoskeleton was used as a secondary orthogonal assay. To this end, we have identified a number of targets, some previously recognized as important regulators of podocyte biology [e.g. mammalian target of rapamycin (mTOR), glucocorticoid receptor, all-trans retinoic acid, among others]. Interestingly , a number of compounds that had the capability to attenuate palmitate and high-glucose-driven podocyte apoptosis in the screen were identified as glycogen synthase kinase (GSK)3b inhibitors. 1-Azakenpaullone (1-AZK), a selective GSK3b inhibitor identified by the podocyte screening approach, was further analyzed for its potency to protect ex vivo isolated porcine glomeruli against palmitate and high-glucose injury. 1-AZK significantly increased cell viability of glomeruli exposed to palmitate and high glucose, highlighting again the protective effect of GSK3b inhibitors in this injury setting (Fig. 3). Recently, GSK3b inhibitors have been demonstrated to exert protective effects on injured podocytes in culture as well as in mouse models of kidney disease including diabetic nephropathy [37,38]. These findings suggest that pharmacologically targeting GSK3b could represent a therapeutic strategy to protect podocytes against injury.  

 

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FIGURE 3   Glycogen synthase kinase (GSK)3b inhibition protects against podocyte injury. Representative images of the degree of podocyte apoptosis measured as caspase 3/7 activation (green) under control (a) and 200 mM palmitate with 25 mM glucose (b) conditions. Podocyte nuclei are counterstained red. (c) 1-Azakenpaullone (1-AZK) dose-dependently reduces caspase 3/7 activation in human podocytes challenged with 200 mM palmitate and 25 mM glucose for 48 h. Podocyte apoptosis was quantified and normalized to confluence level by measuring caspase 3/7 activity and confluence using IncuCyte live cell imaging (n = 4). (d) Overall cell viability, quantified by AlamarBlueWassay (n = 3), of glomeruli freshly isolated from Gottingen minipig via graded sieving technique and cultured in 11 mM glucose ( › ) or 400 mM palmitate with 25 mM glucose (+) with indicated 1-AZK concentrations for three days.

 

The second, often more complex phase, of phenotypic screening is the need to identify the target or targets of hits found in the screening phase. Here, the process involves expansion of the chemical matter around the initial hit chemotype to understand the basic structure-related properties for the molecule. Next, chemical proteomics is employed to identify cellular binding partners for the hits and ultimately these targets need to be confirmed using the screening assay set up [39,40]. Together, Evotec and AstraZeneca have successfully progressed podocyte hit compounds to target identification and are currently developing compounds around these novel targets.  

Tools to evaluate podocyte function   Although the ability to culture podocytes has tremendously enhanced our understanding of the function of individual proteins in this cell type at a biochemical level, such methods and the conclusions drawn from their results need to be integrated within the complex framework of podocyte function in vivo as a requisite component of the GFB. This is important to consider because the podocyte in vitro is an imperfect representation of its in vivo counterpart at a molecular and functional level [25–27,41]. The isolated podocyte certainly mimics certain facets of podocyte biology observed in vivo, but there is a need for additional unbiased and integrated strategies that can facilitate our understanding of the mechanistic determinants of podocyte function in its native microenvironment. A number of novel and exciting strategies have been developed to fill the void between cell culture and whole-animal, mammalian studies of podocyte biology.  

The concept of the podocyte as a dynamic, motile cell type in vitro has been around for some time, but the corollary of this phenomenon in vivo and its putative physiological significance in human health and disease is a subject of active debate. Through the use of transgenic animals (i.e. mice and zebrafish) expressing fluorescently labeled podocytes and advanced microscopic imaging techniques it is possible to study and interrogate the occurrence of podocyte motility in vivo and its relevance to glomerular disease [42,43]. The conclusions drawn from such studies are not in complete harmony and continue to fuel the debate over podocyte plasticity and the cellular nature of their origin. Regardless, such strategies are valuable tools that will be important to evaluating the efficacy of novel therapeutic approaches designed to exploit podocyte dynamics and the potential to control podocyte regeneration from progenitor cell niches.  

Another novel and very interesting imaging-based approach for the study of glomerular function is a recently published technique based on repetitive noninvasive in vivo imaging of GFB integrity in isolated glomeruli transplanted into the anterior chamber of the mouse eye [44]. Podocytes from such glomeruli maintained their differentiation and displayed interdigitating podocyte foot processes for up to six months after transplantation and are amenable to repeated imaging. Transplanting healthy or diseased human glomeruli into mouse eyes might provide a means to image functioning human glomeruli and acutely examine the effect of therapeutics on glomerular function.  

The fruit fly has also recently seen its debut as a model organism to study podocyte biology [45]. Despite gross anatomical differences in the excretory system of humans and fruit flies, the Drosophila pericardial nephrocyte bears a remarkable evolutionary conservation of molecular components and functional properties with the mammalian podocyte. To exploit this observation, and to harness the power of Drosophila genetics, a reporter system of nephrocyte filtration function has been combined with a large RNA interference genetic screen that enables the scanning of the entire genome for genes specifically required for pericardial nephrocyte function [46]. An initial genetic screen of about 1000 genes resulted in the identification of about 7% that were essential for nephrocyte function, notable among them the mammalian homologs of nephrin and podocin [46]. In the near future, the complete repertoire of genes required for nephrocyte-specific function will be defined and this will be an invaluable source of unbiased information ready to be translated to higher animal species.  

The zebrafish, as mentioned above, has taken its place as an exceptional model for studying podocyte biology and glomerular function. The ability to knockdown individual genes and subsequently to follow functional parameters of filtration capacity has been an important tool in the armamentarium of the podocyte biologist to accelerate the study of podocyte gene function [47]. Reverse genetic screens can be readily used to investigate the potential role of novel podocyte proteins. Additionally, novel transgenic zebrafish models that incorporate inducible podocyte injury with a fluorescent tracer for proteinuria make it possible to use this functional model for the screening of therapeutic candidates that could improve overall podocyte health and GFB integrity [48].  

Another strategy not yet fully realized for the kidney, but demonstrated in principal for other organ systems including that of the lung and liver, is the microfluidic organ-on-a-chip which aims to facilitate the in vitro organomimetic modeling of complex human physiology [49,50]. Similar to recapitulating the alveolarcapillary interface of the lung in a mechanical environment that mirrors physiological function, the GFB of the kidney would appear to be a particularly well-suited functional unit for such a strategy. One can envisage the culturing of human podocytes and glomerular endothelial cells on either side of a structural support in a self-contained unit where separated perfusion chambers bath cells in appropriate media and biomechanical properties such as hydrostatic pressure, fluid flow and shear stress can be modulated to simulate in vivo properties. Such a biomimetic device could be what is required to promote the differentiation of cultured podocytes to develop foot processes and slit diaphragms as is functionally required for GFB function. Interestingly, it has also been demonstrated through the cellular repopulation of decellularized kidneys that the native kidney extracellular matrix, including that of the GBM, provides cellular cues that might promote the potential differentiation of podocytes in the glomerulus [51]. The future development of a human GFB biomimetic represents a significant hurdle but holds great promise as a means to study podocyte function and to evaluate drug pharmacology and toxicity.  

Podocyte delivery and targeting   Briefly, cell-specific targeting of therapeutic agents to podocytes of the glomerulus might potentially be an attractive method to increase their efficacy and/or minimize their side-effects. Size and charge characteristics could be exploited to facilitate the capability of drug–carrier conjugates to reach and interact specifically with the correct target cells. Within the kidney, the podocyte is an attractive target cell because of its unique properties. Large molecules that accumulate in the GBM might not need to be filtered to reach the podocyte because of the robust endocytic capacity of this cell type [52]. This raises the possibility of using tissue-specific homing strategies to deliver large proteins, antibodies and siRNAs into cells in a biologically active form [53,54]. Ultrasound-microbubble-mediated delivery of gene therapies to the kidney is an interesting approach that has been used to achieve drug targeting to the kidney [55]. Efficacy of ultrasound-microbubble-mediated gene delivery has been demonstrated in rat models of renal fibrosis but upregulation of the transgene of interest was noted in all kidney tissues [56]. The ability to combine such ultrasound microbubble technology with cell-specific targeting should be possible for the podocyte as has been demonstrated for achieving endothelium-specific transgene expression of a shRNA [57].  

Concluding remarks   The number of randomized clinical trials addressing renal disease continues to be outpaced by most other specialties and the failure of a number of recent kidney clinical trials suggests a need for new thinking and new strategies to address CKD [5,58]. Delineation of the crucial molecular species and signaling pathways underlying podocyte function will undoubtedly lead to the identification of cell-specific targets that can be exploited for treating various glomerulopathies and CKD. Realizing this goal will require a holistic approach where the short-comings of any single strategy in isolation will be overcome only by integrating their individual strengths. With this in mind, the future for developing podocyte specific therapeutics looks bright.

 

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