Parietal epithelial cell differentiation to a podocyte fate in the aged mouse kidney

Healthy aging is typified by a progressive and absolute loss of podocytes over the lifespan of animals and humans. To test the hypothesis that a subset of glomerular parietal epithelial cell (PEC) progenitors transition to a podocyte fate with aging, dual reporte

Astrocytes promote progression of breast cancer metastases to the brain via a KISS1-mediated autophagy.

Formation of metastases, also known as cancer dissemination, is an important stage of breast cancer (BrCa) development. KISS1 expression is associated with inhibition of metastases development. Recently we have demonstrated that BrCa metastases to the brain exhibit low levels of KISS1 expression at both mRNA and protein levels. By using multicolor immunofluorescence and coculture techniques here we show that normal adult astrocytes in the brain are capable of promoting metastatic transformation of circulating breast cancer cells localized to the brain through secretion of chemokine CXCL12. The latter was found in this study to downregulate KISS1 expression at the post-transcriptional level via induction of microRNA-345 (MIR345). Furthermore, we demonstrated that ectopic expression of KISS1 downregulates ATG5 and ATG7, 2 key modulators of autophagy, and works concurrently with autophagy inhibitors, thereby implicating autophagy in the mechanism of KISS1-mediated BrCa metastatic transformation. We also found that expression of KISS1 in human breast tumor specimens inversely correlates with that of MMP9 and IL8, implicated in the mechanism of metastatic invasion, thereby supporting the role of KISS1 as a potential regulator of BrCa metastatic invasion in the brain. This conclusion is further supported by the ability of KISS1, ectopically overexpressed from an adenoviral vector in MDA-MB-231Br cells with silenced expression of the endogenous gene, to revert invasive phenotype of those cells. Taken together, our results strongly suggest that human adult astrocytes can promote brain invasion of the brain-localized circulating breast cancer cells by upregulating autophagy signaling pathways via the CXCL12-MIR345- KISS1 axis

Tamoxifen overrides autophagy inhibition in Beclin-1-deficient glioma cells and their resistance to adenovirus-mediated oncolysis via upregulation of PUMA and BAX.

Autophagy is an evolutionarily conserved process regulating cellular homeostasis via digestion of dysfunctional proteins and whole cellular organelles by mechanisms, involving their enclosure into double-membrane vacuoles that are subsequently fused to lysosomes. Glioma stem cells utilize autophagy as a main mechanism of cell survival and stress response. Most recently, we and others demonstrated induction of autophagy in gliomas in response to treatment with chemical drugs, such as temozolomide (TMZ) or oncolytic adenoviruses (Ads). As autophagy has been implicated in the mechanism of Ad-mediated cell killing, autophagy deficiency in some glioma tumors could be the reason for their resistance to oncolysis. Despite the observed connection, the exact relationship between autophagy-activating cell signaling and adenoviral infection remains unclear. Here, we report that inhibition of autophagy in target glioma cells induces their resistance to killing by oncolytic agent CRAd-S-5/3. Furthermore, we found that downregulation of autophagy inducer Beclin-1 inhibits replication-competent Ad-induced oncolysis of human glioma by suppressing cell proliferation and inducing premature senescence. To overcome the autophagy-deficient state of such glioma cells and restore their susceptibility to oncolytic Ad infection, we propose treating glioma tumors with an anticancer drug tamoxifen (TAM) as a means to induce apoptosis in Ad-targeted cancer cells via upregulation of BAX/PUMA genes. In agreement with the above hypothesis, our data suggest that TAM improves susceptibility of Beclin-1-deficient glioma cells to CRAd-S-5/3 oncolysis by means of activating autophagy and pro-apoptotic signaling pathways in the target cancer cells

Tracking the stochastic fate of cells of the renin lineage after podocyte depletion using multicolor reporters and intravital imaging

<div><p>Podocyte depletion plays a major role in focal segmental glomerular sclerosis (FSGS). Because cells of the renin lineage (CoRL) serve as adult podocyte and parietal epithelial cell (PEC) progenitor candidates, we generated <i>Ren1cCre/R26R-ConfettiTG/WT</i> and <i>Ren1dCre/R26R-ConfettiTG/WT</i> mice to determine CoRL clonality during podocyte replacement. Four CoRL reporters (GFP, YFP, RFP, CFP) were restricted to cells in the juxtaglomerular compartment (JGC) at baseline. Following abrupt podocyte depletion in experimental FSGS, all four CoRL reporters were detected in a subset of glomeruli at day 28, where they co-expressed de novo four podocyte proteins (podocin, nephrin, WT-1 and p57) and two glomerular parietal epithelial cell (PEC) proteins (claudin-1, PAX8). To monitor the precise migration of a subset of CoRL over a 2w period following podocyte depletion, intravital multiphoton microscopy was used. Our findings demonstrate direct visual support for the migration of single CoRL from the JGC to the parietal Bowman’s capsule, early proximal tubule, mesangium and glomerular tuft. In summary, these results suggest that following podocyte depletion, multi-clonal CoRL migrate to the glomerulus and replace podocyte and PECs in experimental FSGS.</p></div

Partial podocyte replacement in <i>Ren1cCre /R26R-ConfettiTG/WT</i> mice with experimental FSGS.

<p>(A-C) Double staining was performed for the podocyte marker p57 (brown, nuclear) and counterstain with Periodic acid Schiff's (pink stains matrix, blue stains nuclei) in reporter mice at baseline (A), day 14 (D14) FSGS (B) and D28 FSGS (C). (D) Podocyte number was lower at D14 compared to baseline, and partial recovered by D28. (E) Glomerulosclerosis was highest at D14 FSGS, with a significant reduction by D28. (F) The urinary albumin to creatinine ratio (ACR) was significantly higher at D14 FSGS, with a significant decrease by D28 of FSGS.</p

Labeled cells of renin lineage (CoRL) co-express nephrin in glomeruli of <i>Ren1cCre /R26R-ConfettiTG/WT</i> mice with experimental FSGS.

<p>The confocal images in the left column (A-C) represent nephrin staining (magenta) detected by antibody, and 4 CoRL reporters (green, red, blue, yellow) detected without antibody. The confocal images in the right column represents the same image on the left, but for ease of viewing, all 4 confetti reporter channels have been converted to green, and nephrin has been converted to red, so that co-localization can be visualized as yellow. (A, B) At baseline: All four CoRL reporter colors are restricted to the JGC (dashed white box), and nephrin staining is restricted to the glomerular tuft. (B) all four Confetti CoRL reporters (green) are seen in the JGC, with no overlap with nephrin (red). (C, D) At D14 FSGS: (C) There is a segmental decrease in nephrin staining in the right upper quadrant of the glomerular tuft. Multi-clonal CoRL are detected in glomerular tuft, but do not co-localize with nephrin. (D) The CoRL reporters in the tuft do not co-localize with nephrin staining. (E, F) At D28 FSGS: (E) Multi-clonal CoRL are detected in the glomerular tuft. (F) CoRL reporters co-localize with nephrin, creating a yellow color (arrows indicate examples).</p

Multi-colored reporters of CoRL are detected in glomerular tufts of <i>Ren1cCre /R26R-ConfettiTG/WT</i> mice with FSGS.

<p>Confocal images showing four CoRL reporter colors detected without the use of antibodies–nGFP (green), cRFP (red), mCFP (blue) and cYFP (yellow). (A) All four reporters are restricted to the JGC at baseline, and are not detected in the glomerular tuft (dashed white circles). At D14 FSGS (B) and at D28 FSGS (C), all four CoRL reporter colors were detected in a subpopulation of cells in the glomerular tufts. (D) Graph showing that the percentage of glomeruli with reporter positive CoRL within the tuft was higher at D28 of FSGS and that these glomeruli contained 2–4 clones. (E) Represenative image showing all four reporters (converted to green color) and BRDU(red) do not co-localize at baseline. BRDU is present in some tubular epithelial cells as expected, but is not readily detected in JGC or the glomerular tuft. (F) BRDU staining increased at D14 of FSGS, but BRDU positive cells are not present in the JGC and glomerular tuft. (G) At D28 of FSGS there is an increase in the number of reporter labeled cells present on the glomerular tuft, however there is no overlap of reporters with BRDU.</p