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.

Repurposing drugs for diseases associated with podocyte dysfunction.

The majority of podocyte disorders are progressive in nature leading to chronic kidney disease and often kidney failure. The scope of current therapies is typically nonspecific immunosuppressant medications, which are accompanied by unwanted and serious side effects. However, many exciting clinical trials are underway to reduce the burden of podocyte diseases in our patients. Major advances and discoveries have recently been made experimentally in our understanding of the molecular and cellular mechanisms underlying podocyte injury in disease. This begs the question of how best to take advantage of these impressive strides. One approach to consider is the repurposing of therapeutics that have already been approved by the Food and Drug Administration, European Medicines Agency, and other regulatory agencies for indications beyond the kidney. The advantages of therapy repurposing include known safety profiles, drug development that has already been completed, and overall reduced costs for studying alternative indications for selected therapies. The purpose of this mini review is to examine the experimental literature of podocyte damage and determine if there are mechanistic targets in which prior approved therapies can be considered for repurposing to podocyte disorders.

An open microfluidic coculture model of fibroblasts and eosinophils to investigate mechanisms of airway inflammation.

Interactions between fibroblasts and immune cells play an important role in tissue inflammation. Previous studies have found that eosinophils activated with interleukin-3 (IL-3) degranulate on aggregated immunoglobulin G (IgG) and release mediators that activate fibroblasts in the lung. However, these studies were done with eosinophil-conditioned media that have the capacity to investigate only one-way signaling from eosinophils to fibroblasts. Here, we demonstrate a coculture model of primary normal human lung fibroblasts (HLFs) and human blood eosinophils from patients with allergy and asthma using an open microfluidic coculture device. In our device, the two types of cells can communicate via two-way soluble factor signaling in the shared media while being physically separated by a half wall. Initially, we assessed the level of eosinophil degranulation by their release of eosinophil-derived neurotoxin (EDN). Next, we analyzed the inflammation-associated genes and soluble factors using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and multiplex immunoassays, respectively. Our results suggest an induction of a proinflammatory fibroblast phenotype of HLFs following the coculture with degranulating eosinophils, validating our previous findings. Additionally, we present a new result that indicate potential impacts of activated HLFs back on eosinophils. This open microfluidic coculture platform provides unique opportunities to investigate the intercellular signaling between the two cell types and their roles in airway inflammation and remodeling.

Zoning in on podocytes.

Podocytes undergo defined morphologic changes during development, homeostasis, and aging, and on injury. Quantitative podometric assessments of podocyte endowment provide a powerful tool to interrogate glomerular health. Expanding this approach to a regional assessment demonstrates that the podocytes from cortical, subcortical, and juxtamedullary glomeruli are not only morphologically heterogeneous per se, but respond differently to stressors, such as age and hypertension. This suggests that zonal glomerular changes harbor critical information to understand glomerulopathies.

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.

The proliferative and the antifibrotic side of PAX2 in tubular repair.

Regenerative repair following injury to proximal tubular epithelial cells (PTECs) is essential to restore the kidney to normal function in acute kidney injury. Failure to accomplish this leads to chronic kidney disease. Expression of the paired-box transcription factor Pax2 in PTECs is required for their regenerative proliferation and repair. However, a loss-of-function study now shows that the absence of Pax2 not only impacts PTEC proliferation but also causes myofibroblast recruitment leading to excessive tubulointerstitial fibrosis.

Kidney repair and regeneration: perspectives of the NIDDK (Re)Building a Kidney consortium.

Acute kidney injury impacts âˆ¼13.3 million individuals and causes âˆ¼1.7 million deaths per year globally. Numerous injury pathways contribute to acute kidney injury, including cell cycle arrest, senescence, inflammation, mitochondrial dysfunction, and endothelial injury and dysfunction, and can lead to chronic inflammation and fibrosis. However, factors enabling productive repair versus nonproductive, persistent injury states remain less understood. The (Re)Building a Kidney (RBK) consortium is a National Institute of Diabetes and Digestive and Kidney Diseases consortium focused on both endogenous kidney repair mechanisms and the generation of new kidney tissue. This short review provides an update on RBK studies of endogenous nephron repair, addressing the following questions: (i) What is productive nephron repair? (ii) What are the cellular sources and drivers of repair? and (iii) How do RBK studies promote development of therapeutics? Also, we provide a guide to RBK's open access data hub for accessing, downloading, and further analyzing data sets.

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.

The Atypical Cyclin-Dependent Kinase 5 (Cdk5) Guards Podocytes from Apoptosis in Glomerular Disease While Being Dispensable for Podocyte Development.

Cyclin-dependent kinase 5 (Cdk5) is expressed in terminally differentiated cells, where it drives development, morphogenesis, and survival. Temporal and spatial kinase activity is regulated by specific activators of Cdk5, dependent on the cell type and environmental factors. In the kidney, Cdk5 is exclusively expressed in terminally differentiated glomerular epithelial cells called podocytes. In glomerular disease, signaling mechanisms via Cdk5 have been addressed by single or combined conventional knockout of known specific activators of Cdk5. A protective, anti-apoptotic role has been ascribed to Cdk5 but not a developmental phenotype, as in terminally differentiated neurons. The effector kinase itself has never been addressed in animal models of glomerular disease. In the present study, conditional and inducible knockout models of Cdk5 were analyzed to investigate the role of Cdk5 in podocyte development and glomerular disease. While mice with podocyte-specific knockout of Cdk5 had no developmental defects and regular lifespan, loss of Cdk5 in podocytes increased susceptibility to glomerular damage in the nephrotoxic nephritis model. Glomerular damage was associated with reduced anti-apoptotic signals in Cdk5-deficient mice. In summary, Cdk5 acts primarily as master regulator of podocyte survival during glomerular disease and-in contrast to neurons-does not impact on glomerular development or maintenance.

Advancing Nephrology: Division Leaders Advise ASN.

New treatments, new understanding, and new approaches to translational research are transforming the outlook for patients with kidney diseases. A number of new initiatives dedicated to advancing the field of nephrology-from value-based care to prize competitions-will further improve outcomes of patients with kidney disease. Because of individual nephrologists and kidney organizations in the United States, such as the American Society of Nephrology, the National Kidney Foundation, and the Renal Physicians Association, and international nephrologists and organizations, such as the International Society of Nephrology and the European Renal Association-European Dialysis and Transplant Association, we are beginning to gain traction to invigorate nephrology to meet the pandemic of global kidney diseases. Recognizing the timeliness of this opportunity, the American Society of Nephrology convened a Division Chief Retreat in Dallas, Texas, in June 2019 to address five key issues: (1) asserting the value of nephrology to the health system; (2) productivity and compensation; (3) financial support of faculty's and divisions' educational efforts; (4) faculty recruitment, retention, diversity, and inclusion; and (5) ensuring that fellowship programs prepare trainees to provide high-value nephrology care and enhance attraction of trainees to nephrology. Herein, we highlight the outcomes of these discussions and recommendations to the American Society of Nephrology.

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.