Podocytes from hypertensive and obese mice acquire an inflammatory, senescent, and aged phenotype.

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.

Podocyte injury at young age causes premature senescence and worsens glomerular aging.

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 ß-galactosidase (SA-ß-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 black staining 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.NEW & NOTEWORTHY Glomerular function is decreased by aging. However, little is known about the molecular mechanisms involved in age-related glomerular changes and which factors could contribute to a worse glomerular aging process. Here, we reported that podocyte injury in young mice and culture podocytes induced senescence, a marker of aging, and accelerates glomerular aging when compared with healthy aging mice.

Podocyte Senescence and Aging.

As the population in many industrial countries is aging, the risk, incidence, and prevalence of CKD increases. In the kidney, advancing age results in a progressive decrease in nephron number and an increase in glomerulosclerosis. In this review, we focus on the effect of aging on glomerular podocytes, the post-mitotic epithelial cells critical for the normal integrity and function of the glomerular filtration barrier. The podocytes undergo senescence and transition to a senescence-associated secretory phenotype typified by the production and secretion of inflammatory cytokines that can influence neighboring glomerular cells by paracrine signaling. In addition to senescence, the aging podocyte phenotype is characterized by ultrastructural and functional changes; hypertrophy; cellular, oxidative, and endoplasmic reticulum stress; reduced autophagy; and increased expression of aging genes. This results in a reduced podocyte health span and a shortened life span. Importantly, these changes in the pathways/processes characteristic of healthy podocyte aging are also often similar to pathways in the disease-induced injured podocyte. Finally, the better understanding of podocyte aging and senescence opens therapeutic options to slow the rate of podocyte aging and promote kidney health.

Inhibiting NLRP3 signaling in aging podocytes improves their life- and health-span.

The decrease in the podocyte's lifespan and health-span that typify healthy kidney aging cause a decrease in their normal structure, physiology and function. The ability to halt and even reverse these changes becomes clinically relevant when disease is superimposed on an aged kidney. RNA-sequencing of podocytes from middle-aged mice showed an inflammatory phenotype with increases in the NLRP3 inflammasome, signaling for IL2/Stat5, IL6 and TNF, interferon gamma response, allograft rejection and complement, consistent with inflammaging. Furthermore, injury-induced NLRP3 signaling in podocytes was further augmented in aged mice compared to young ones. The NLRP3 inflammasome (NLRP3, Caspase-1, IL1ß IL-18) was also increased in podocytes of middle-aged humans. Higher transcript expression for NLRP3 in human glomeruli was accompanied by reduced podocyte density and increased global glomerulosclerosis and glomerular volume. Pharmacological inhibition of NLRP3 with MCC950, or gene deletion, reduced podocyte senescence and the genes typifying aging in middle-aged mice, which was accompanied by an improved podocyte lifespan and health-span. Moreover, modeling the injury-dependent increase in NLRP3 signaling in human kidney organoids confirmed the anti-senescence effect of MC9950. Finally, NLRP3 also impacted liver aging. Together, these results suggest a critical role for the NLRP3 inflammasome in podocyte and liver aging.

Loss of epigenetic information as a cause of mammalian aging.

All living things experience an increase in entropy, manifested as a loss of genetic and epigenetic information. In yeast, epigenetic information is lost over time due to the relocalization of chromatin-modifying proteins to DNA breaks, causing cells to lose their identity, a hallmark of yeast aging. Using a system called "ICE" (inducible changes to the epigenome), we find that the act of faithful DNA repair advances aging at physiological, cognitive, and molecular levels, including erosion of the epigenetic landscape, cellular exdifferentiation, senescence, and advancement of the DNA methylation clock, which can be reversed by OSK-mediated rejuvenation. These data are consistent with the information theory of aging, which states that a loss of epigenetic information is a reversible cause of aging.

Upregulated PD-1 signaling antagonizes glomerular health in aged kidneys and disease.

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.

Podocyte Aging: Why and How Getting Old Matters.

The effects of healthy aging on the kidney, and how these effects intersect with superimposed diseases, are highly relevant in the context of the population's increasing longevity. Age-associated changes to podocytes, which are terminally differentiated glomerular epithelial cells, adversely affect kidney health. This review discusses the molecular and cellular mechanisms underlying podocyte aging, how these mechanisms might be augmented by disease in the aged kidney, and approaches to mitigate progressive damage to podocytes. Furthermore, we address how biologic pathways such as those associated with cellular growth confound aging in humans and rodents.

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 reporter PEC-rtTA|LC1|tdTomato|Nphs1-FLPo|FRT-EGFP mice were generated. PECs were inducibly labeled with a tdTomato reporter, and podocytes were constitutively labeled with an EGFP reporter. With advancing age (14 and 24 months) glomeruli in the juxta-medullary cortex (JMC) were more severely injured than those in the outer cortex (OC). In aged mice (24m), injured glomeruli with lower podocyte number (41% decrease), showed more PEC migration and differentiation to a podocyte fate than mildly injured or healthy glomeruli. PECs differentiated to a podocyte fate had ultrastructural features of podocytes and co-expressed the podocyte markers podocin, nephrin, p57 and VEGF164, but not markers of mesangial (Perlecan) or endothelial (ERG) cells. PECs differentiated to a podocyte fate did not express CD44, a marker of PEC activation. Taken together, we demonstrate that a subpopulation of PECs differentiate to a podocyte fate predominantly in injured glomeruli in mice of advanced age.

Nanoparticles exhibit greater accumulation in kidney glomeruli during experimental glomerular kidney disease.

Loss and dysfunction of glomerular podocytes result in increased macromolecule permeability through the glomerular filtration barrier and nephrotic syndrome. Current therapies can induce and maintain disease remission, but cause serious and chronic complications. Nanoparticle drug carriers could mitigate these side effects by delivering drugs to the kidneys more efficiently than free drug through tailoring of carrier properties. An important extrinsic factor of nanoparticle biodistribution is local pathophysiology, which may drive greater nanoparticle deposition in certain tissues. Here, we hypothesized that a "leakier" filtration barrier during glomerular kidney disease would increase nanoparticle distribution into the kidneys. We examined the effect of nanoparticle size and disease state on kidney accumulation in male BALB/c mice. The effect of size was tested using a panel of fluorescent polystyrene nanoparticles of size 20-200 nm, due to the relevance of this size range for drug delivery applications.Experimental focal segmental glomerulosclerosis was induced using an anti-podocyte antibody that causes abrupt podocyte depletion. Nanoparticles were modified with carboxymethyl-terminated poly(ethylene glycol) for stability and biocompatibility. After intravenous injection, fluorescence from nanoparticles of size 20 and 100 nm, but not 200 nm, was observed in kidney glomeruli and peritubular capillaries. During conditions of experimental focal segmental glomerulosclerosis, the number of fluorescent nanoparticle punctae in kidney glomeruli increased by 1.9-fold for 20 and 100 nm nanoparticles compared to normal conditions. These findings underscore the importance of understanding and leveraging kidney pathophysiology in engineering new, targeted drug carriers that accumulate more in diseased glomeruli to treat glomerular kidney disease.

Feature-rich covalent stains for super-resolution and cleared tissue fluorescence microscopy.

Fluorescence microscopy is a workhorse tool in biomedical imaging but often poses substantial challenges to practitioners in achieving bright or uniform labeling. In addition, while antibodies are effective specific labels, their reproducibility is often inconsistent, and they are difficult to use when staining thick specimens. We report the use of conventional, commercially available fluorescent dyes for rapid and intense covalent labeling of proteins and carbohydrates in super-resolution (expansion) microscopy and cleared tissue microscopy. This approach, which we refer to as Fluorescent Labeling of Abundant Reactive Entities (FLARE), produces simple and robust stains that are modern equivalents of classic small-molecule histology stains. It efficiently reveals a wealth of key landmarks in cells and tissues under different fixation or sample processing conditions and is compatible with immunolabeling of proteins and in situ hybridization labeling of nucleic acids.

CD44 impacts glomerular parietal epithelial cell changes in the aged mouse kidney.

CD44 contributes to the activation of glomerular parietal epithelial cells (PECs). Although CD44 expression is higher in PECs of healthy aged mice, the biological role of CD44 in PECs in this context remains unclear. Accordingly, young (4 months) and aged (24 months) CD44-/- mice were compared to age-matched CD44+/+ mice, both aged in a nonstressed environment. Parietal epithelial cell densities were similar in both young and aged CD44+/+ and CD44-/- mice. Phosphorylated ERK 1/2 (pERK) was higher in aged CD44+/+ mice. Vimentin and α-SMA, markers of changes to the epithelial cell phenotype, were present in PECs in aged CD44+/+ mice, but absent in aged CD44-/- mice in both outer cortical (OC) and juxtamedullary (JM) glomeruli. Because age-related glomerular hypertrophy was lower in CD44-/- mice, mTOR activation was assessed by phospho-S6 ribosomal protein (pS6RP) staining. Parietal epithelial cells and glomerular tuft staining for pS6RP was lower in aged CD44-/- mice compared to aged CD44+/+ mice. Podocyte density was higher in aged CD44-/- mice in both OC and JM glomeruli. These changes were accompanied by segmental and global glomerulosclerosis in aged CD44+/+ mice, but absent in aged CD44-/- mice. These results show that the increase in CD44 in PECs in aged kidneys contributes to several changes to the glomerulus during healthy aging in mice, and may involve ERK and mTOR activation.

Global transcriptomic changes occur in aged mouse podocytes.

Glomerular podocytes undergo structural and functional changes with advanced age, that increase susceptibility of aging kidneys to worse outcomes following superimposed glomerular diseases. To delineate transcriptional changes in podocytes in aged mice, RNA-seq was performed on isolated populations of reporter-labeled (tdTomato) podocytes from multiple young (two to three months) and advanced aged mice (22 to 24 months, equivalent to 70 plus year old humans). Of the 2,494 differentially expressed genes, 1,219 were higher and 1,275 were lower in aged podocytes. Pathway enrichment showed that major biological processes increased in aged podocytes included immune responses, non-coding RNA metabolism, gene silencing and MAP kinase signaling. Conversely, aged podocytes showed downregulation of developmental, morphogenesis and metabolic processes. Canonical podocyte marker gene expression decreased in aged podocytes, with increases in apoptotic and senescence genes providing a mechanism for the progressive loss of podocytes seen with aging. In addition, we revealed aberrations in the podocyte autocrine signaling network, identified the top transcription factors perturbed in aged podocytes, and uncovered candidate gene modulations that might promote healthy aging in podocytes. The transcriptional signature of aging is distinct from other kidney diseases. Thus, our study provides insights into biomarker discovery and molecular targeting of the aging process itself within podocytes.

Differential expression of parietal epithelial cell and podocyte extracellular matrix proteins in focal segmental glomerulosclerosis and diabetic nephropathy.

In healthy glomeruli, parietal epithelial cell (PEC)-derived extracellular matrix (ECM) proteins include laminin-ß1, perlecan, and collagen type IV-α2 and podocyte-specific ECM proteins include laminin-ß2, agrin, and collagen type IV-α4. This study aimed to define individual ECM protein isoform expression by PECs in both experimental and human focal segmental glomerulosclerosis (FSGS) and diabetic nephropathy (DN) and to determine if changes were CD44 dependent. In experimental FSGS induced with a cytotoxic podocyte antibody and in the BTBR ob/ob mouse model of DN, PEC-derived protein staining was significantly increased in PECs. Dual staining also showed de novo expression of the podocyte-specific ECM proteins laminin-ß2 and agrin in PECs. Similar findings were observed in biopsies from patients with FSGS and DN. Increases in individual ECM proteins colocalized with CD44 in PECs in disease. To determine the role of CD44, FSGS was induced in CD44-/- and CD44+/+ mice. PEC staining for perlecan, collagen type IV-α2, laminin-ß2, and agrin were significantly lower in diseased CD44-/- mice compared with diseased CD44+/+ mice. These results show that in experimental and human FSGS and DN, PECs typically in an activated state, produce both PEC-derived and podocyte-specific ECM protein isoforms, and that the majority of these changes were dependent on CD44.

Dual lineage tracing shows that glomerular parietal epithelial cells can transdifferentiate toward the adult podocyte fate.

Podocytes are differentiated post-mitotic cells that cannot replace themselves after injury. Glomerular parietal epithelial cells are proposed to be podocyte progenitors. To test whether a subset of parietal epithelial cells transdifferentiate to a podocyte fate, dual reporter PEC-rtTA|LC1|tdTomato|Nphs1-FLPo|FRT-EGFP mice, named PEC-PODO, were generated. Doxycycline administration permanently labeled parietal epithelial cells with tdTomato reporter (red), and upon doxycycline removal, the parietal epithelial cells (PECs) cannot label further. Despite the presence or absence of doxycycline, podocytes cannot label with tdTomato, but are constitutively labeled with an enhanced green fluorescent protein (EGFP) reporter (green). Only activation of the Nphs1-FLPo transgene by labeled parietal epithelial cells can generate a yellow color. At day 28 of experimental focal segmental glomerulosclerosis, podocyte density was 20% lower in 20% of glomeruli. At day 56 of experimental focal segmental glomerulosclerosis, podocyte density was 18% lower in 17% of glomeruli. TdTomato+ parietal epithelial cells were restricted to Bowman's capsule in healthy mice. However, by days 28 and 56 of experimental disease, two-thirds of tdTomato+ parietal epithelial cells within glomerular tufts were yellow in color. These cells co-expressed the podocyte markers podocin, nephrin, p57 and VEGF164, but not markers of endothelial (ERG) or mesangial (Perlecan) cells. Expansion microscopy showed primary, secondary and minor processes in tdTomato+EGFP+ cells in glomerular tufts. Thus, our studies provide strong evidence that parietal epithelial cells serve as a source of new podocytes in adult mice.

Cells of NG2 lineage increase in glomeruli of mice following podocyte depletion.

Under certain circumstances, podocytes can be partially replaced following their loss in disease. The inability of podocytes to proliferate suggests that replacement derives from other cell types. Because neural/glial antigen 2 (NG2)-expressing cells can serve as progenitors in other organs and because herein we showed increased NG2 staining in podocytes following their loss in experimental focal segmental glomerulosclerosis, we used lineage tracing in NG2-CreER tdTomato mice to test the hypothesis that partial podocyte replacement might derive from this cell population. The percentage of glomeruli with red fluorescence protein (RFP)-labeled NG2 cells increased following podocyte depletion, which was augmented by enalapril. However, BrdU was not detected in RFP-labeled cells, consistent with the migration of these cells to the glomerulus. Within glomeruli, RFP-labeled cells did not coexpress podocyte proteins (p57, synaptopodin, nephrin, or podocin) but did coexpress markers for mesangial (α8 integrin, PDGFß receptor) and parietal epithelial cells (PAX8, src-suppressed C-kinase substrate). These results suggest that following podocyte depletion, cells of NG2 lineage do not serve as adult podocyte progenitors but have the ability to transdifferentiate to mesangial and parietal epithelial cell fates.

Volumetric, Nanoscale Optical Imaging of Mouse and Human Kidney via Expansion Microscopy.

Although light microscopy is a powerful tool for the assessment of kidney physiology and pathology, it has traditionally been unable to resolve structures separated by less than the ~250 nm diffraction limit of visible light. Here, we report on the optimization, validation, and application of a recently developed super-resolution fluorescence microscopy method, called expansion microscopy (ExM), for volumetric interrogation of mouse and human kidney tissue with 70-75 nm lateral and ~250 nm axial spatial resolution. Using ExM with a standard confocal microscope, we resolve fine details of structures that have traditionally required visualization by electron microscopy, including podocyte foot processes, the glomerular basement membrane, and the cytoskeleton. This inexpensive and accessible approach to volumetric, nanoscale imaging enables visualization of fine structural details of kidney tissues that were previously difficult or impossible to measure by conventional methodologies.

Glomerular disease augments kidney accumulation of synthetic anionic polymers.

Polymeric drug carriers can alter the pharmacokinetics of their drug cargoes, thereby improving drug therapeutic index and reducing side effects. Understanding and controlling polymer properties that drive tissue-specific accumulation is critical in engineering targeted drug delivery systems. For kidney disease applications, targeted drug delivery to renal cells that reside beyond the charge- and size-selective glomerular filtration barrier could have clinical potential. However, there are limited reports on polymer properties that might enhance kidney accumulation. Here, we studied the effects of molecular weight and charge on the in vivo kidney accumulation of polymers in health and disease. We synthesized a panel of well-defined polymers by atom transfer radical polymerization to answer several questions. First, the biodistribution of low molecular weight (23-27 kDa) polymers composed of various ratios of neutral:anionic monomers (1:0, 1:1, 1:4) in normal mice was determined. Then, highly anionic (1:4 monomer ratio) low molecular and high molecular weight (47 kDa) polymers were tested in both normal and experimental focal segmental glomerulosclerosis (FSGS) mice, a model that results in loss of glomerular filtration selectivity. Through these studies, we observed that kidney-specific polymer accumulation increases with anionic monomer content, but not molecular weight; experimental FSGS increases kidney accumulation of anionic polymers; and anionic polymers accumulate predominantly in proximal tubule cells, with some distribution in kidney glomeruli. These findings can be applied to the design of polymeric drug carriers to enhance or mitigate kidney accumulation.

High-Throughput Screening Enhances Kidney Organoid Differentiation from Human Pluripotent Stem Cells and Enables Automated Multidimensional Phenotyping.

Organoids derived from human pluripotent stem cells are a potentially powerful tool for high-throughput screening (HTS), but the complexity of organoid cultures poses a significant challenge for miniaturization and automation. Here, we present a fully automated, HTS-compatible platform for enhanced differentiation and phenotyping of human kidney organoids. The entire 21-day protocol, from plating to differentiation to analysis, can be performed automatically by liquid-handling robots, or alternatively by manual pipetting. High-content imaging analysis reveals both dose-dependent and threshold effects during organoid differentiation. Immunofluorescence and single-cell RNA sequencing identify previously undetected parietal, interstitial, and partially differentiated compartments within organoids and define conditions that greatly expand the vascular endothelium. Chemical modulation of toxicity and disease phenotypes can be quantified for safety and efficacy prediction. Screening in gene-edited organoids in this system reveals an unexpected role for myosin in polycystic kidney disease. Organoids in HTS formats thus establish an attractive platform for multidimensional phenotypic screening.