Emerging Roles of Xanthine Oxidoreductase in Chronic Kidney Disease.

Xanthine Oxidoreductase (XOR) is a ubiquitous, essential enzyme responsible for the terminal steps of purine catabolism, ultimately producing uric acid that is eliminated by the kidneys. XOR is also a physiological source of superoxide ion, hydrogen peroxide, and nitric oxide, which can function as second messengers in the activation of various physiological pathways, as well as contribute to the development and the progression of chronic conditions including kidney diseases, which are increasing in prevalence worldwide. XOR activity can promote oxidative distress, endothelial dysfunction, and inflammation through the biological effects of reactive oxygen species; nitric oxide and uric acid are the major products of XOR activity. However, the complex relationship of these reactions in disease settings has long been debated, and the environmental influences and genetics remain largely unknown. In this review, we give an overview of the biochemistry, biology, environmental, and current clinical impact of XOR in the kidney. Finally, we highlight recent genetic studies linking XOR and risk for kidney disease, igniting enthusiasm for future biomarker development and novel therapeutic approaches targeting XOR.

Open-Source System for Real-Time Functional Assessment of In Vitro Filtration Barriers.

The integrity of the barrier between blood and the selective filtrate of solutes is important for homeostasis and its disruption contributes to many diseases. Microphysiological systems that incorporate synthetic or natural membranes with human cells can mimic biological filtration barriers, such as the glomerular filtration barrier in the kidney, and they can readily be used to study cellular filtration processes as well as drug effects and interactions. We present an affordable, open-source platform for the real-time monitoring of functional filtration status in engineered microphysiological systems. Using readily available components, our assay can linearly detect real-time concentrations of two target molecules, FITC-labeled inulin and Texas Red-labeled human-serum albumin, within clinically relevant ranges, and it can be easily modified for different target molecules of varying sizes and tags. We demonstrate the platform's ability to determine the concentration of our target molecules automatically and consistently. We show through an acellular context that the platform enables real-time tracking of size-dependent diffusion with minimal fluid volume loss and without manual extraction of media, making it suitable for continuous operational monitoring of filtration status in microphysiological system applications. The platform's affordability and integrability with microphysiological systems make it ideal for many precision medicine applications, including evaluation of drug nephrotoxicity and other forms of drug discovery.

Redox regulation in diabetic kidney disease.

Diabetic kidney disease (DKD) is one of the most devastating complications of diabetes mellitus, where currently there is no cure available. Several important mechanisms contribute to the pathogenesis of this complication, with oxidative stress being one of the key factors. The past decades have seen a large number of publications with various aspects of this topic; however, the specific details of redox regulation in DKD are still unclear. This is partly because redox biology is very complex, coupled with a complex and heterogeneous organ with numerous cell types. Furthermore, often times terms such as "oxidative stress" or reactive oxygen species are used as a general term to cover a wide and rich variety of reactive species and their differing reactions. However, no reactive species are the same, and not all of them are capable of biologically relevant reactions or "redox signaling." The goal of this review is to provide a biochemical background for an array of specific reactive oxygen species types with varying reactivity and specificity in the kidney as well as highlight some of the advances in redox biology that are paving the way to a better understanding of DKD development and risk.

Effect of Aging and a Dual Orexin Receptor Antagonist on Sleep Architecture and Non-REM Oscillations Including an REM Behavior Disorder Phenotype in the PS19 Mouse Model of Tauopathy.

The impact of tau pathology on sleep microarchitecture features, including slow oscillations, spindles, and their coupling, has been understudied, despite the proposed importance of these electrophysiological features toward learning and memory. Dual orexin receptor antagonists (DORAs) are known to promote sleep, but whether and how they affect sleep microarchitecture in the setting of tauopathy is unknown. In the PS19 mouse model of tauopathy MAPT (microtubule-associated protein tau) P301S (both male and female), young PS19 mice 2-3 months old show a sleep electrophysiology signature with markedly reduced spindle duration and power and elevated slow oscillation (SO) density compared with littermate controls, although there is no significant tau hyperphosphorylation, tangle formation, or neurodegeneration at this age. With aging, there is evidence for sleep disruption in PS19 mice, characterized by reduced REM duration, increased non-REM and REM fragmentation, and more frequent brief arousals at the macrolevel and reduced spindle density, SO density, and spindle-SO coupling at the microlevel. In ∼33% of aged PS19 mice, we unexpectedly observed abnormal goal-directed behaviors in REM, including mastication, paw grasp, and forelimb/hindlimb extension, seemingly consistent with REM behavior disorder (RBD). Oral administration of DORA-12 in aged PS19 mice increased non-REM and REM duration, albeit with shorter bout lengths, and increased spindle density, spindle duration, and SO density without change to spindle-SO coupling, power in either the SO or spindle bands, or the arousal index. We observed a significant effect of DORA-12 on objective measures of RBD, thereby encouraging future exploration of DORA effects on sleep-mediated cognition and RBD treatment.SIGNIFICANCE STATEMENT The specific effect of tauopathy on sleep macroarchitecture and microarchitecture throughout aging remains unknown. Our key findings include the following: (1) the identification of a sleep EEG signature constituting an early biomarker of impending tauopathy; (2) sleep physiology deteriorates with aging that are also markers of off-line cognitive processing; (3) the novel observation that dream enactment behaviors reminiscent of RBD occur, likely the first such observation in a tauopathy model; and (4) a dual orexin receptor antagonist is capable of restoring several of the sleep macroarchitecture and microarchitecture abnormalities.

Genetic susceptibility to diabetic kidney disease is linked to promoter variants of XOR.

The lifetime risk of kidney disease in people with diabetes is 10-30%, implicating genetic predisposition in the cause of diabetic kidney disease (DKD). Here we identify an expression quantitative trait loci (QTLs) in the cis-acting regulatory region of the xanthine dehydrogenase, or xanthine oxidoreductase (Xor), a binding site for C/EBPß, to be associated with diabetes-induced podocyte loss in DKD in male mice. We examine mouse inbred strains that are susceptible (DBA/2J) and resistant (C57BL/6J) to DKD, as well as a panel of recombinant inbred BXD mice, to map QTLs. We also uncover promoter XOR orthologue variants in humans associated with high risk of DKD. We introduced the risk variant into the 5'-regulatory region of XOR in DKD-resistant mice, which resulted in increased Xor activity associated with podocyte depletion, albuminuria, oxidative stress and damage restricted to the glomerular endothelium, which increase further with type 1 diabetes, high-fat diet and ageing. Therefore, differential regulation of Xor contributes to phenotypic consequences with diabetes and ageing.

Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria.

Glomerular endothelial cell (GEC) dysfunction can initiate and contribute to glomerular filtration barrier breakdown. Increased mitochondrial oxidative stress has been suggested as a mechanism resulting in GEC dysfunction in the pathogenesis of some glomerular diseases. Historically the isolation of GECs from in vivo models has been notoriously challenging due to difficulties in isolating pure cultures from glomeruli. GECs have complex growth requirements in vitro and a very limited lifespan. Here, we describe the procedure for isolating and culturing conditionally immortalized GECs with fluorescent mitochondria, enabling the tracking of mitochondrial fission and fusion events. GECs were isolated from the kidneys of a double transgenic mouse expressing the thermolabile SV40 TAg (from the Immortomouse), conditionally promoting proliferation and suppressing cell differentiation, and a photo-convertible fluorescent protein (Dendra2) in all mitochondria (from the photo-activatable mitochondria [PhAMexcised] mouse). The stable cell line generated allows for cell differentiation after inactivation of the immortalizing SV40 TAg gene and photo-activation of a subset of mitochondria causing a switch in fluorescence from green to red. The use of mitoDendra2-GECs allows for live imaging of fluorescent mitochondria's distribution, fusion, and fission events without staining the cells.

Podocyte as the link between sterile inflammation and diabetic kidney disease.

Shahzad et al. examined the underlying mechanisms of sterile inflammation in diabetic kidney disease, specifically the role of NLRP3 inflammasome activation in podocytes. Using mouse models with gain-of-function and loss-of-function mutations in podocyte Nlrp3, or caspase-1 loss-of-function mutations in podocytes, they identified that Nlrp3 activation in these cells is central for development of diabetic kidney disease but not solely dependent on canonical mechanisms and caspase-1. These findings position podocyte-mediated immune cell-like functions as potential therapeutic targets for diabetic kidney disease.

Hypertrophy and glomerular cell adaptation through crosstalk leads to glomerular injury after kidney transplantation.

Menon et al. report cell-specific transcriptional changes in podocytes and glomerular endothelial cells that indicate cell stress and increased bidirectional crosstalk among these cells in apparently healthy human allografts. They identified common and independent podocytes and glomerular endothelial cell-specific responses in nondiabetic and diabetic transplant recipients, as well as parallels in genes related to podocyte and glomerular endothelial cell stress in experimental focal segmental glomerular sclerosis. These findings could explain hypertrophy-associated glomerular disease progression associated with podocyte detachment after transplantation.

Loss of Functional SCO2 Attenuates Oxidative Stress in Diabetic Kidney Disease.

Increased oxidative stress in glomerular endothelial cells (GEnCs) contributes to early diabetic kidney disease (DKD). While mitochondrial respiratory complex IV activity is reduced in DKD, it remains unclear whether this is a driver or a consequence of oxidative stress in GEnCs. Synthesis of cytochrome C oxidase 2 (SCO2), a key metallochaperone in the electron transport chain, is critical to the biogenesis and assembly of subunits required for functional respiratory complex IV activity. Here, we investigated the effects of Sco2 hypomorphs (Sco2KO/KI, Sco2KI/KI), with a functional loss of SCO2, in the progression of DKD using a murine model of Type II Diabetes Mellitus, db/db mice. Diabetic Sco2KO/KI and Sco2KI/KI hypomorphs exhibited a reduction in complex IV activity, but an improvement in albuminuria, serum creatinine, and histomorphometric evidence of early DKD as compared to db/db mice. Single-nucleus RNA sequencing with gene set enrichment analysis of differentially expressed genes in the endothelial cluster of Sco2KO/KI;db/db mice demonstrated an increase in genes involved in VEGF-VEGFR2 signaling and reduced oxidative stress as compared to db/db mice. These data suggest that reduced complex IV activity due to a loss of functional SCO2 might be protective in GEnCs in early DKD.

Loss of Functional SCO2 Attenuates Oxidative Stress in Diabetic Kidney Disease.

Increased oxidative stress in glomerular endothelial cells (GEnCs) contributes to early diabetic kidney disease (DKD). While mitochondrial respiratory complex IV activity is reduced in DKD, it remains unclear whether this is a driver or a consequence of oxidative stress in GEnCs. Synthesis of cytochrome C oxidase 2 (SCO2), a key metallochaperone in the electron transport chain, is critical to the biogenesis and assembly of subunits required for functional respiratory complex IV activity. Here, we investigated the effects of Sco2 hypomorphs (Sco2 KO/KI , Sco2 KI/KI ), with a functional loss of SCO2, in the progression of DKD using a murine model of Type II Diabetes Mellitus, db/db mice. Diabetic Sco2 KO/KI and Sco2 KI/KI hypomorphs exhibited a reduction in complex IV activity, but an improvement in albuminuria, serum creatinine, and histomorphometric evidence of early DKD as compared to db/db mice. Single-nucleus RNA sequencing with gene set enrichment analysis of differentially expressed genes in the endothelial cluster of Sco2 KO/KI ;db/db mice demonstrated an increase in genes involved in VEGF-VEGFR2 signaling and reduced oxidative stress as compared to db/db mice. These data suggest that reduced complex IV activity due to a loss of functional SCO2 might be protective in GEnCs in early DKD.

The glomerular filtration barrier: a structural target for novel kidney therapies.

Loss of normal kidney function affects more than 10% of the population and contributes to morbidity and mortality. Kidney diseases are currently treated with immunosuppressive agents, antihypertensives and diuretics with partial but limited success. Most kidney disease is characterized by breakdown of the glomerular filtration barrier (GFB). Specialized podocyte cells maintain the GFB, and structure-function experiments and studies of intercellular communication between the podocytes and other GFB cells, combined with advances from genetics and genomics, have laid the groundwork for a new generation of therapies that directly intervene at the GFB. These include inhibitors of apolipoprotein L1 (APOL1), short transient receptor potential channels (TRPCs), soluble fms-like tyrosine kinase 1 (sFLT1; also known as soluble vascular endothelial growth factor receptor 1), roundabout homologue 2 (ROBO2), endothelin receptor A, soluble urokinase plasminogen activator surface receptor (suPAR) and substrate intermediates for coenzyme Q10 (CoQ10). These molecular targets converge on two key components of GFB biology: mitochondrial function and the actin-myosin contractile machinery. This Review discusses therapies and developments focused on maintaining GFB integrity, and the emerging questions in this evolving field.

Modeling the Glomerular Filtration Barrier and Intercellular Crosstalk.

The glomerulus is a compact cluster of capillaries responsible for blood filtration and initiating urine production in the renal nephrons. A trilaminar structure in the capillary wall forms the glomerular filtration barrier (GFB), composed of glycocalyx-enriched and fenestrated endothelial cells adhering to the glomerular basement membrane and specialized visceral epithelial cells, podocytes, forming the outermost layer with a molecular slit diaphragm between their interdigitating foot processes. The unique dynamic and selective nature of blood filtration to produce urine requires the functionality of each of the GFB components, and hence, mimicking the glomerular filter in vitro has been challenging, though critical for various research applications and drug screening. Research efforts in the past few years have transformed our understanding of the structure and multifaceted roles of the cells and their intricate crosstalk in development and disease pathogenesis. In this review, we present a new wave of technologies that include glomerulus-on-a-chip, three-dimensional microfluidic models, and organoids all promising to improve our understanding of glomerular biology and to enable the development of GFB-targeted therapies. Here, we also outline the challenges and the opportunities of these emerging biomimetic systems that aim to recapitulate the complex glomerular filter, and the evolving perspectives on the sophisticated repertoire of cellular signaling that comprise the glomerular milieu.

Molecular Mechanisms in Early Diabetic Kidney Disease: Glomerular Endothelial Cell Dysfunction.

Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease (ESRD), with prevalence increasing at an alarming rate worldwide and today, there are no known cures. The pathogenesis of DKD is complex, influenced by genetics and the environment. However, the underlying molecular mechanisms that contribute to DKD risk in about one-third of diabetics are still poorly understood. The early stage of DKD is characterized by glomerular hyperfiltration, hypertrophy, podocyte injury and depletion. Recent evidence of glomerular endothelial cell injury at the early stage of DKD has been suggested to be critical in the pathological process and has highlighted the importance of glomerular intercellular crosstalk. A potential mechanism may include reactive oxygen species (ROS), which play a direct role in diabetes and its complications. In this review, we discuss different cellular sources of ROS in diabetes and a new emerging paradigm of endothelial cell dysfunction as a key event in the pathogenesis of DKD.

The diabetic microenvironment causes mitochondrial oxidative stress in glomerular endothelial cells and pathological crosstalk with podocytes.

BACKGROUND: In the setting of diabetes mellitus, mitochondrial dysfunction and oxidative stress are important pathogenic mechanisms causing end organ damage, including diabetic kidney disease (DKD), but mechanistic understanding at a cellular level remains obscure. In mouse models of DKD, glomerular endothelial cell (GEC) dysfunction precedes albuminuria and contributes to neighboring podocyte dysfunction, implicating GECs in breakdown of the glomerular filtration barrier. In the following studies we wished to explore the cellular mechanisms by which GECs become dysfunctional in the diabetic milieu, and the impact to neighboring podocytes. METHODS: Mouse GECs were exposed to high glucose media (HG) or 2.5% v/v serum from diabetic mice or serum from non-diabetic controls, and evaluated for mitochondrial function (oxygen consumption), structure (electron microscopy), morphology (mitotracker), mitochondrial superoxide (mitoSOX), as well as accumulation of oxidized products (DNA lesion frequency (8-oxoG, endo-G), double strand breaks (γ-H2AX), endothelial function (NOS activity), autophagy (LC3) and apoptotic cell death (Annexin/PI; caspase 3). Supernatant transfer experiments from GECs to podocytes were performed to establish the effects on podocyte survival and transwell experiments were performed to determine the effects in co-culture. RESULTS: Diabetic serum specifically causes mitochondrial dysfunction and mitochondrial superoxide release in GECs. There is a rapid oxidation of mitochondrial DNA and loss of mitochondrial biogenesis without cell death. Many of these effects are blocked by mitoTEMPO a selective mitochondrial anti-oxidant. Secreted factors from dysfunctional GECs were sufficient to cause podocyte apoptosis in supernatant transfer experiments, or in co-culture but this did not occur when GECs had been previously treated with mitoTEMPO. CONCLUSION: Dissecting the impact of the diabetic environment on individual cell-types from the kidney glomerulus indicates that GECs become dysfunctional and pathological to neighboring podocytes by increased levels of mitochondrial superoxide in GEC. These studies indicate that GEC-signaling to podocytes contributes to the loss of the glomerular filtration barrier in DKD. Video abstract.