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With an aging population, kidney health becomes an important medical and socioeconomic factor. Kidney aging mechanisms are not well understood. We previously showed that podocytes isolated from aged mice exhibit increased expression of programmed cell death protein 1 (PD-1) surface receptor and its 2 ligands (PD-L1 and PD-L2). PDCD1 transcript increased with age in microdissected human glomeruli, which correlated with lower estimated glomerular filtration rate and higher segmental glomerulosclerosis and vascular arterial intima-to-lumen ratio. In vitro studies in podocytes demonstrated a critical role for PD-1 signaling in cell survival and in the induction of a senescence- associated secretory phenotype. To prove PD-1 signaling was critical to podocyte aging, aged mice were injected with anti-PD- 1 antibody. Treatment significantly improved the aging phenotype in both kidney and liver. In the glomerulus, it increased the life span of podocytes, but not that of parietal epithelial, mesangial, or endothelial cells. Transcriptomic and immunohistochemistry studies demonstrated that anti-PD-1 antibody treatment improved the health span of podocytes. Administering the same anti-PD-1 antibody to young mice with experimental focal segmental glomerulosclerosis (FSGS) lowered proteinuria and improved podocyte number. These results suggest a critical contribution of increased PD-1 signaling toward both kidney and liver aging and in FSGS., Introduction The US population is aging, and the number of Americans aged 65 and older will more than double over the next 40 years (US Census Bureau); Eurostat forecasts that [...]

Patients with hypertension or obesity can develop glomerular dysfunction characterized by injury and depletion of podocytes. To better understand the molecular processes involved, young mice were treated with either deoxycorticosterone acetate (DOCA) or fed a high-fat diet (HFD) to induce hypertension or obesity, respectively. The transcriptional changes associated with these phenotypes were measured by unbiased bulk mRNA sequencing of isolated podocytes from experimental models and their respective controls. Key findings were validated by immunostaining. In addition to a decrease in canonical proteins and reduced podocyte number, podocytes from both hypertensive and obese mice exhibited a sterile inflammatory phenotype characterized by increases in NLR family pyrin domain containing 3 (NLRP3) inflammasome, protein cell death-1, and Toll-like receptor pathways. Finally, although the mice were young, podocytes in both models exhibited increased expression of senescence and aging genes, including genes consistent with a senescence-associated secretory phenotype. However, there were differences between the hypertension- and obesity-associated senescence phenotypes. Both show stress-induced podocyte senescence characterized by increased p21 and p53. Moreover, in hypertensive mice, this is superimposed upon age-associated podocyte senescence characterized by increased p16 and p19. These results suggest that senescence, aging, and inflammation are critical aspects of the podocyte phenotype in experimental hypertension and obesity in mice. NEW & NOTEWORTHY: Hypertension and obesity can lead to glomerular dysfunction in patients, causing podocyte injury and depletion. Here, young mice given deoxycorticosterone acetate or a high-fat diet to induce hypertension or obesity, respectively. mRNA sequencing of isolated podocytes showed transcriptional changes consistent with senescence, a senescent-associated secretory phenotype, and aging, which was confirmed by immunostaining. Ongoing studies are determining the mechanistic roles of the accelerated aging podocyte phenotype in experimental hypertension and obesity. [ABSTRACT FROM AUTHOR]

As life expectancy continues to rise, age-related diseases are becoming more prevalent. For example, proteinuric glomerular diseases typified by podocyte injury have worse outcomes in the elderly compared with young patients. However, the reasons are not well understood. We hypothesized that injury to nonaged podocytes induces senescence, which in turn augments their aging processes. In primary cultured human podocytes, injury induced by a cytopathic antipodocyte antibody, adriamycin, or puromycin aminonucleoside increased the senescence-related genes CDKN2A (p16INK4a/p14ARF), CDKN2D (p19INK4d), and CDKN1A (p21). Podocyte injury in human kidney organoids was accompanied by increased expression of CDKN2A, CDKN2D, and CDKN1A. In young mice, experimental focal segmental glomerulosclerosis (FSGS) induced by adriamycin and antipodocyte antibody increased the glomerular expression of p16, p21, and senescence-associated β-galactosidase (SA-β-gal). To assess the long-term effects of early podocyte injury-induced senescence, we temporally followed young mice with experimental FSGS through adulthood (12 m of age) and middle age (18 m of age). p16 and Sudan blackstaining were higher at middle age in mice with earlier FSGS compared with age-matched mice that did not get FSGS when young. This was accompanied by lower podocyte density, reduced canonical podocyte protein expression, and increased glomerular scarring. These results are consistent with injury-induced senescence in young podocytes, leading to increased senescence of podocytes by middle age accompanied by lower podocyte lifespan and health span. [ABSTRACT FROM AUTHOR]

In response to injury, the highly specialized and terminally differentiated glomerular visceral epithelial cell, or podocyte, may undergo several cell fates, including dedifferentiation and proliferation, persistent cell cycle arrest, hypertrophy, apoptosis, or necrosis. Common to these potential outcomes of injury is their ultimate regulation at the level of the cell cycle. There is now a large body of literature confirming the importance of cell cycle regulatory proteins in the cellular response to injury. Although CDK inhibitor p21 levels increase in podocytes following injury, the role of p21 is unclear in focal segmental glomerulosclerosis (FSGS), in part because its function depends heavily on the cytotoxic stimulus and the cellular context. Adriamycin (ADR) is a podocyte toxin used to induce experimental FSGS. The purpose of this study was to define the role of p21 in ADR-induced podocyte injury. BALB/c mice, a strain carrying the recessive ADR susceptibility gene, were backcrossed against c57B6 p21-/- mice to yield a 12th generation BALB/c p21-/- strain. Experimental FSGS was induced by injection of ADR 12 mg/kg x 2 doses (n = 8/group), with mice killed at 1, 2, 8, and 11 wk. Diseased p21-/- mice demonstrated worse albuminuria, more widespread glomerulosclerosis, and higher blood urea nitrogen compared with diseased p21 +/+ mice. In diseased p21-/- mice vs. p21+/+ mice, apoptosis [measured by TdT-mediated dUTP nick end labeling (TUNEL) assay] was increased, and podocyte number (measured by WT-1 immunostaining) was decreased. To validate these findings in vitro, we utilized differentiated mouse podocytes, p21-/- and p21+/+, exposed to 0.125 [micro]g/ml ADR. Apoptosis, measured by Hoechst 33342 staining and TUNEL assay, was greater in cultured p21-/- podocytes compared with p21 +/+ podocytes. Reconstitution of p21 via retroviral transfection rescued the p21-/- podocytes from apoptosis. We conclude that p21 is prosurvival in the podocyte's response to ADR-induced injury. Ongoing studies are defining the mechanisms of this protective effect as it relates to DNA damage and apoptosis. apoptosis; adriamycin nephropathy; focal glomerulosclerosis doi: 10.1152/ajprenal.00216.2009.

Studies have shown that certain cells of the glomerular tuft begin to express proteins considered unique to other cell types upon injury. Little is known about the response of parietal epithelial cells (PEC) to injury. To determine whether PECs change their phenotype upon injury to also express proteins traditionally considered podocyte specific, the following four models of glomerular disease were studied: the transforming growth factor (TGF)-[beta]1 transgenic mouse model of global glomerulosclerosis, the adriamycin model of focal segmental glomerulosclerosis (FSGS), the anti-glomerular basement membrane (GBM) model of crescentic glomerulonephritis, and the passive Heymann nephritis model of membranous nephropathy. Double immunostaining was performed with antibodies to podocyte-specific proteins (synaptopodin and Wilms' tumor 1) and antibodies to PEC specific proteins (paired box gene 8 and claudin-1). No double staining was detected in normal mice. In contrast, the results showed a statistical increase in the number of cells attached to Bowman basement membrane that were double-positive for both podocyte/PEC proteins in TGF-[beta];1 transgenic, anti-GBM, and membranous animals. Double-positive cells for both podocyte and PEC proteins were also statistically increased in the glomerular tuft in TGF-[beta]1 transgenic, anti-GBM, and FSGS mice. These results are consistent with glomerular cells coexpressing podocyte and PEC proteins in experimental glomerular disease, but not under normal circumstances. glomerulonephritis; differentiation; phenotype; FSGS; membranous nephropathy doi:10.1152/ajprenal.00428.2009

Ohse T, Chang AM, Pippin JW, Jarad G, Hudkins KL, Alpers CE, Miner JH, Shankland SJ. A new function for parietal epithelial cells: a second glomerular barrier. Am J Physiol Renal Physiol 297: FI566-F1574, 2009. First published September 20, 2009; doi: 10.1152/ajprenal.00214.2009.--The functional role of glomerular parietal epithelial cells (PECs) remains poorly understood. To test the hypothesis that PECs form an impermeable barrier to filtered protein through the formation of tight junctions (TJ), studies were performed in normal animals and in the anti-glomerular basement membrane (GBM) model of crescentic nephritis. Electron microscopy showed well-defined TJ between PECs in normal mice, which no longer could be identified when these cells became extensively damaged or detached from their underlying Bowman's basement membrane. The TJ proteins claudin-1, zonula occludens-1, and occludin stained positive in PECs; however, staining decreased in anti-GBM disease. To show that these events were associated with increased permeability across the PEC-Bowman's basement membrane barrier, control and diseased animals were injected intravenously with either Texas red-conjugated dextran (3 kDa) or ovalbumin (45 kDa) tracers. As expected, both tracers were readily filtered across the glomerular filtration barrier and taken up by proximal tubular cells. However, when the glomerular filtration barrier was injured in anti-GBM disease, tracers were taken up by podocytes and PECs. Moreover, tracers were also detected between PECs and the underlying Bowman's basement membrane, and in many instances were detected in the extraglomerular space. We propose that together with its underlying Bowman's basement membrane, the TJ of PECs serve as a second barrier to protein. When disturbed following PEC injury, the increase in permeability of this layer to filtered protein is a mechanism underlying periglomerular inflammation characteristic of anti-GBM disease. tight junction; tracer study doi: 10.1152/ajprenal.00214.2009

Cyclin I is an atypical cyclin because it is most abundant in postmitotic cells. We previously showed that cyclin I does not regulate proliferation, but rather controls survival of podocytes, terminally differentiated epithelial cells that are essential for the structural and functional integrity of kidney glomeruli. Here, we investigated the mechanism by which cyclin I safeguards against apoptosis and found that cyclin I bound and activated cyclin-dependent kinase 5 (Cdk5) in isolated mouse podocytes and neurons. Cdk5 activity was reduced in glomeruli and brain lysates from cyclin I-deficient mice, and inhibition of Cdk5 increased in vitro the susceptibility to apoptosis in response to cellular damage. In addition, levels of the prosurvival proteins Bcl-2 and Bcl-XL were reduced in podocytes and neurons from cyclin I-deficient mice, and restoration of Bcl-2 or Bcl-XL expression prevented injury-induced apoptosis. Furthermore, we found that levels of phosphorylated MEK1/2 and ERK1/2 were decreased in cyclin I-deficient podocytes and that inhibition of MEK1/2 restored Bcl2 and Bcl-XL protein levels. Of interest, this pathway was also defective in mice with experimental glomerulonephritis. Taken together, these data suggest that a cyclin I-Cdk5 complex forms a critical antiapoptotic factor in terminally differentiated cells that functions via MAPK signaling to modulate levels of the prosurvival proteins Bcl-2 and Bcl-XL., Introduction Neurons and kidney podocytes share many characteristics, including being postmitotic and terminally differentiated cells (1). As such, they typically do not reengage the cell cycle, leading to a limited [...]

Glomerular diseases remain the leading cause of chronic and end-stage kidney disease. Significant advances in our understanding of human glomerular diseases have been enabled by the development and better characterization of animal models. Diseases of the glomerular epithelial cells (podocytes) account for the majority of proteinuric diseases. Rodents have been extensively used experimentally to better define mechanisms of disease induction and progression, as well as to identify potential targets and therapies. The development of podocyte-specific genetically modified mice has energized the research field to better understand which animal models are appropriate to study acquired podocyte diseases. In this review we discuss inducible experimental models of acquired nondiabetic podocyte diseases in rodents, namely, passive Heymann nephritis, puromycin aminonucleoside nephrosis, adriamycin nephrosis, liopolysaccharide, crescentic glomerulonephritis, and protein overload nephropathy models. Details are given on the model backgrounds, how to induce each model, the interpretations of the data, and the benefits and shortcomings of each. Genetic rodent models of podocyte injury are excluded. glomerulus; animal models