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The signaling molecule stimulator of IFN genes (STING) was identified as a crucial regulator of the DNA-sensing cyclic GMP-AMP synthase (cGAS)-STING pathway, and this signaling pathway regulates inflammation and energy homeostasis under conditions of obesity, kidney fibrosis, and AKI. However, the role of STING in causing CKD, including diabetic kidney disease (DKD) and Alport syndrome, is unknown.To investigate whether STING activation contributes to the development and progression of glomerular diseases such as DKD and Alport syndrome, immortalized human and murine podocytes were differentiated for 14 days and treated with a STING-specific agonist. We used diabeticThe activation of the STING pathway acts as a mediator of disease progression in DKD and Alport syndrome. Targeting STING may offer a therapeutic option to treat glomerular diseases of metabolic and nonmetabolic origin or prevent their development, progression, or both.

Advances in the understanding of lipid droplet biology have revealed essential roles for these organelles in mediating proper cellular homeostasis and stress response. Lipid droplets were initially thought to play a passive role in energy storage. However, recent studies demonstrate that they have substantially broader functions, including protection from reactive oxygen species, endoplasmic reticulum stress, and lipotoxicity. Dysregulation of lipid droplet homeostasis is associated with various pathologies spanning neurological, metabolic, cardiovascular, oncological, and renal diseases. This review provides an overview of the current understanding of lipid droplet biology in both health and disease.

Introduction: Dysregulation of sphingolipid and cholesterol metabolism contributes to the pathogenesis of glomerular diseases (GDs). Apolipoprotein M (ApoM) promotes cholesterol efflux and modulates the bioactive sphingolipid sphingosine-1-phosphate (S1P). Glomerular ApoM expression is decreased in patients with focal segmental glomerulosclerosis (FSGS). We hypothesized that glomerular ApoM deficiency occurs in GD and that ApoM expression and plasma ApoM correlate with outcomes. Methods: Patients with GD from the Nephrotic Syndrome Study Network (NEPTUNE) were studied. We compared glomerular mRNA expression of ApoM (gApoM), sphingosine kinase 1 (SPHK1), and S1P receptors 1 to 5 (S1PR1–5) in patients (n = 84) and controls (n = 6). We used correlation analyses to determine associations between gApoM, baseline plasma ApoM (pApoM), and urine ApoM (uApoM/Cr). We used linear regression to determine whether gApoM, pApoM, and uApoM/Cr were associated with baseline estimated glomerular filtration rate (eGFR) and proteinuria. Using Cox models, we determined whether gApoM, pApoM, and uApoM/Cr were associated with complete remission (CR) and the composite of end-stage kidney disease (ESKD) or ≥40% eGFR decline. Results: gApoM was reduced (P < 0.01) and SPHK1 and S1PR1 to 5 expression was increased (P < 0.05) in patients versus controls, consistent with ApoM/S1P pathway modulation. gApoM positively correlated with pApoM in the overall cohort (r = 0.34, P < 0.01) and in the FSGS (r = 0.48, P < 0.05) and minimal change disease (MCD) (r = 0.75, P < 0.05) subgroups. Every unit decrease in gApoM and pApoM (log2) was associated with a 9.77 ml/min per 1.73 m2 (95% confidence interval [CI]: 3.96−15.57) and 13.26 ml/min per 1.73 m2 (95% CI: 3.57−22.96) lower baseline eGFR, respectively (P < 0.01). From Cox models adjusted for age, sex, or race, pApoM was a significant predictor of CR (hazard ratio [HR]: 1.85; 95% CI: 1.06–3.23). Conclusions: pApoM is a potential noninvasive biomarker of gApoM deficiency and strongly associates with clinical outcomes in GD.

Sodium-glucose cotransporter-2 inhibitors (SGLT2i) are anti-hyperglycemic agents that prevent glucose reabsorption in proximal tubular cells. SGLT2i improves renal outcomes in both diabetic and non-diabetic patients, indicating it may have beneficial effects beyond glycemic control. Here, we demonstrate that SGLT2i affects energy metabolism and renal lipotoxicity in experimental Alport syndrome (AS). In vitro, we found that SGLT2 protein expression levels in human and mouse podocytes were similar to tubular cells. Newly established immortalized podocytes from Col4a3 knockout mice (AS podocytes) accumulate lipid droplets along with increased apoptosis when compared to wildtype podocytes. Treatment with SGLT2i empagliflozin reduces lipid droplet accumulation and apoptosis in AS podocytes. Empagliflozin inhibits the utilization of glucose/pyruvate as a metabolic substrate in AS podocytes. In vivo, we demonstrate that empagliflozin reduces albuminuria and prolongs the survival of AS mice. Empagliflozin-treated AS mice show decreased serum blood urea nitrogen and creatinine levels in association with reduced triglyceride and cholesterol ester content in kidney cortices when compared to AS mice. Lipid accumulation in kidney cortices correlates with the decline in renal function. In summary, empagliflozin reduces renal lipotoxicity and improves kidney function in experimental AS in association with the energy substrates switch from glucose to fatty acids in podocytes.

Impaired cellular cholesterol efflux is a key factor in the progression of renal, cardiovascular, and autoimmune diseases. Here we describe a class of 5-arylnicotinamide compounds, identified through phenotypic drug discovery, that upregulate ABCA1-dependent cholesterol efflux by targeting Oxysterol Binding Protein Like 7 (OSBPL7). OSBPL7 was identified as the molecular target of these compounds through a chemical biology approach, employing a photoactivatable 5-arylnicotinamide derivative in a cellular cross-linking/immunoprecipitation assay. Further evaluation of two compounds (Cpd A and Cpd G) showed that they induced ABCA1 and cholesterol efflux from podocytes in vitro and normalized proteinuria and prevented renal function decline in mouse models of proteinuric kidney disease: Adriamycin-induced nephropathy and Alport Syndrome. In conclusion, we show that small molecule drugs targeting OSBPL7 reveal an alternative mechanism to upregulate ABCA1, and may represent a promising new therapeutic strategy for the treatment of renal diseases and other disorders of cellular cholesterol homeostasis., This study describes a class of small molecule compounds that promote ABCA1-dependent cholesterol efflux via a non-transcriptional mechanism, the identification of the molecular target by a chemical biology approach, and the potential of these agents for the treatment of chronic kidney diseases and potentially other diseases where lipid accumulation drives disease progression.

Diabetic nephropathy (DN) is the leading cause of end-stage kidney disease. Besides glycemic and blood pressure control, environmental factors such as cigarette smoking (CS) adversely affect the progression of DN. The effects of CS on DN progression have been attributed to combustion-generated molecules without consideration to the role of nicotine (NIC), responsible for the addictive properties of both CS and electronic cigarettes (ECs). Podocytes are essential to preserve the structure and function of the glomerular filtration barrier, and strong evidence indicates that early podocyte loss promotes DN progression. We performed experiments in human podocytes and in a mouse model of diabetes that develops nephropathy resembling human DN. We determined that NIC binding to podocytes in concentrations achieved with CS and ECs activated NADPH oxidase, which sets in motion a dysfunctional molecular network integrated by cyclooxygenase 2, known to induce podocyte injury; downregulation of AMP-activated protein kinase, important for maintaining cellular energy stores and antioxidation; and upregulation of CD36, which increased lipid uptake and promoted apoptosis. In diabetic mice, NIC increased proteinuria, a recognized marker of chronic kidney disease progression, accompanied by reduced glomerular podocyte synaptopodin, a crucial stabilizer of the podocyte cytoskeleton, and increased fibronectin expression. This novel study critically implicates NIC itself as a contributor to DN progression in CS and EC users. NEW & NOTEWORTHY In this study, we demonstrate that nicotine increases the production of reactive oxygen species, increases cyclooxygenase-2 expression, and upregulates Cd36 while inducing downregulation of AMP-activated protein kinase. In vivo nicotine increases proteinuria and fibronectin expression in diabetic mice. This study demonstrates that effects of nicotine on podocytes are responsible, at least in part, for the deleterious effects of smoking in the progression of chronic kidney disease, including diabetic nephropathy.

In the past few decades, sphingolipids and sphingolipid metabolites have gained attention because of their essential role in the pathogenesis and progression of kidney diseases. Studies in models of experimental and clinical nephropathies have described accumulation of sphingolipids and sphingolipid metabolites, and it has become clear that the intracellular sphingolipid composition of renal cells is an important determinant of renal function. Proper function of the glomerular filtration barrier depends heavily on the integrity of lipid rafts, which include sphingolipids as key components. In addition to contributing to the structural integrity of membranes, sphingolipid metabolites, such as sphingosine-1-phosphate (S1P), play important roles as second messengers regulating biologic processes, such as cell growth, differentiation, migration, and apoptosis. This review will focus on the role of S1P in renal cells and how aberrant extracellular and intracellular S1P signaling contributes to the pathogenesis and progression of kidney diseases.

A critical link between metabolic disorders and a form of low-grade systemic and chronic inflammation has been recently established and named 'Metaflammation'. Metaflammation has been recognized as a key mediator of both microvascular and macrovascular complications of diabetes and as a significant contributor to the development of diabetic kidney disease (DKD). The goal of this review is to summarize the contribution of diabetes-induced inflammation and the related signaling pathways to diabetic complications, with a particular focus on how innate immunity and lipid metabolism influence each other.

Defective cholesterol metabolism primarily linked to reduced ATP-binding cassette transporter A1 (ABCA1) expression is closely associated with the pathogenesis and progression of kidney diseases, including diabetic kidney disease and Alport Syndrome. However, whether the accumulation of free or esterified cholesterol contributes to progression in kidney disease remains unclear. Here, we demonstrate that inhibition of sterol-O-acyltransferase-1 (SOAT1), the enzyme at the endoplasmic reticulum that converts free cholesterol to cholesterol esters, which are then stored in lipid droplets, effectively reduced cholesterol ester and lipid droplet formation in human podocytes. Furthermore, we found that inhibition of SOAT1 in podocytes reduced lipotoxicity-mediated podocyte injury in diabetic kidney disease and Alport Syndrome in association with increased ABCA1 expression and ABCA1-mediated cholesterol efflux. In vivo, Soat1 deficient mice did not develop albuminuria or mesangial expansion at 10-12 months of age. However, Soat1 deficiency/inhibition in experimental models of diabetic kidney disease and Alport Syndrome reduced cholesterol ester content in kidney cortices and protected from disease progression. Thus, targeting SOAT1-mediated cholesterol metabolism may represent a new therapeutic strategy to treat kidney disease in patients with diabetic kidney disease and Alport Syndrome, like that suggested for Alzheimer's disease and cancer treatments.

Kidney diseases affect more than 15% of adults in the US, yet drug development in the kidney field, when compared with that for other common diseases, has been lagging behind. Modifiers that increase the susceptibility to injury and contribute to the pathogenesis and progression of kidney disease include genetic and environmental factors and epigenetic mechanisms. In this issue of the JCI, Cao et al. and Doke et al. independently report the identification of a susceptibility factor called Dachshund homolog 1 (DACH1). Both groups identify an association of reduced DACH1 expression with kidney disease, using different screening approaches, studying different types of human kidney diseases, and using different experimental models, making the fact that both stumbled over the same protein very compelling. Combined, these studies highlight DACH1 as a key safeguard in the kidney, granting various cell types proper function by modulating several molecular pathways.

Lipid dysmetabolism is one of the main features of diabetes mellitus and manifests by dyslipidemia as well as the ectopic accumulation of lipids in various tissues and organs, including the kidney. Research suggests that impaired cholesterol metabolism, increased lipid uptake or synthesis, increased fatty acid oxidation, lipid droplet accumulation and an imbalance in biologically active sphingolipids (such as ceramide, ceramide-1-phosphate and sphingosine-1-phosphate) contribute to the development of diabetic kidney disease (DKD). Currently, the literature suggests that both quality and quantity of lipids are associated with DKD and contribute to increased reactive oxygen species production, oxidative stress, inflammation, or cell death. Therefore, control of renal lipid dysmetabolism is a very important therapeutic goal, which needs to be archived. This article will review some of the recent advances leading to a better understanding of the mechanisms of dyslipidemia and the role of particular lipids and sphingolipids in DKD.

The kidney is a radiosensitive late-responding normal tissue. Injury is characterized by radiation nephropathy and decline of glomerular filtration rate (GFR). The current study aimed to compare two rapid and cost-effective methodologies of assessing GFR against more conventional biomarker measurements.C57BL/6 mice were treated with bilateral focal X-irradiation (1x14Gy or 5x6Gy). Functional measurements of kidney injury were assessed 20 weeks post-treatment. GFR was estimated using a transcutaneous measurement of fluorescein-isothiocyanate conjugated (FITC)-sinistrin renal excretion and also dynamic contrast-enhanced CT imaging with a contrast agent (ISOVUE-300 Iopamidol).Hematoxylin and eosin (HE) and Periodic acid-Schiff staining identified comparable radiation-induced glomerular atrophy and mesangial matrix accumulation after both radiation schedules, respectively, although the fractionated regimen resulted in less diffuse tubulointerstitial fibrosis. Albumin-to-creatinine ratios (ACR) increased after irradiation (1x14Gy: 100.4 ± 12.2 µg/mg; 6x5Gy: 80.4 ± 3.02 µg/mg) and were double that of nontreated controls (44.9 ± 3.64 µg/mg). GFR defined by both techniques was negatively correlated with BUN, mesangial expansion score, and serum creatinine. The FITC-sinistrin transcutaneous method was more rapid and can be used to assess GFR in conscious animals, dynamic contrast-enhanced CT imaging technique was equally safe and effective.This study demonstrated that GFR measured by dynamic contrast-enhanced CT imaging is safe and effective compared to transcutaneous methodology to estimate kidney function.

Lipid accumulation in podocytes is a major determinant of diabetic kidney disease (DKD) and identification of potential therapeutic targets by mediating podocyte lipid metabolism has clinical importance. This study was to elucidate the role of JAML (junctional adhesion molecule-like protein) in the pathogenesis of DKD. We first confirmed the expression of JAML in podocytes and found that podocyte-specific deletion of Jaml ameliorated podocyte injury and proteinuria in two different models of diabetic mice. We further demonstrated a novel role of JAML in regulating podocyte lipid metabolism through SIRT1-mediated SREBP1 signaling. Similar results were also found in mice with adriamycin-induced nephropathy. Importantly, we observed a higher expression of JAML in glomeruli from subjects with DKD and other types of proteinuric kidney diseases, and the level of JAML was correlated with lipid accumulation and glomerular filtration rate, suggesting that JAML may be an attractive therapeutic target for proteinuric kidney disease.

Lipotoxicity was recently reported in several forms of kidney disease, including focal segmental glomerulosclerosis (FSGS). Susceptibility to FSGS in African Americans is associated with the presence of genetic variants of the Apolipoprotein L1 gene (APOL1) named G1 and G2. If and how endogenous APOL1 may alter mitochondrial function by the modifying cellular lipid metabolism is unknown. Using transgenic mice expressing the APOL1 variants (G0, G1 or G2) under endogenous promoter, we show that APOL1 risk variant expression in transgenic mice does not impair kidney function at baseline. However, APOL1 G1 expression worsens proteinuria and kidney function in mice characterized by the podocyte inducible expression of nuclear factor of activated T-cells (NFAT), which we have found to cause FSGS. APOL1 G1 expression in this FSGS-model also results in increased triglyceride and cholesterol ester contents in kidney cortices, where lipid accumulation correlated with loss of renal function. In vitro, we show that the expression of endogenous APOL1 G1/G2 in human urinary podocytes is associated with increased cellular triglyceride content and is accompanied by mitochondrial dysfunction in the presence of compensatory oxidative phosphorylation (OXPHOS) complexes elevation. Our findings indicate that APOL1 risk variant expression increases the susceptibility to lipid-dependent podocyte injury, ultimately leading to mitochondrial dysfunction.

Sphingolipids are well-recognized as major players in the pathogenesis of many human diseases, including chronic kidney disease. The kidney is a very sensitive organ to alterations in sphingolipid metabolism. The critical issues to be addressed in this review relate to the role of sphingolipids and enzymes involved in sphingolipid metabolism in the pathogenesis of glomerular diseases with a special focus on podocytes, a key cellular component of the glomerular filtration barrier. Among several sphingolipids, we will highlight the role of ceramide, sphingosine, sphingosine-1-phosphate and ceramide-1-phosphate. Additionally, we will summarize the current knowledge with regard to the use of sphingolipids as therapeutic agents for the treatment of podocyte injury in kidney disease.

Although dyslipidemia is associated with chronic kidney disease (CKD), it is more common in nephrotic syndrome (NS), and guidelines for the management of hyperlipidemia in NS are largely opinion-based. In addition to the role of circulating lipids, an increasing number of studies suggest that intrarenal lipids contribute to the progression of glomerular diseases, indicating that proteinuric kidney diseases may be a form of “fatty kidney disease” and that reducing intracellular lipids could represent a new therapeutic approach to slow the progression of CKD. In this review, we summarize recent progress made in the utilization of lipid-modifying agents to lower renal parenchymal lipid accumulation and to prevent or reduce kidney injury. The agents mentioned in this review are categorized according to their specific targets, but they may also regulate other lipid-relevant pathways.

Focal segmental glomerulosclerosis (FSGS) accounts for about 40% of all nephrotic syndrome cases in adults. The presence of several potential circulating factors has been suggested in patients with primary FSGS and particularly in patients with recurrent disease after transplant. Irrespectively of the nature of the circulating factors, this study was aimed at identifying early glomerular/podocyte-specific pathways that are activated by the sera of patients affected by FSGS. Kidney biopsies were obtained from patients undergoing kidney transplantation due to primary FSGS. Donor kidneys were biopsied pre-reperfusion (PreR) and a subset 1-2 hours after reperfusion of the kidney (PostR). Thirty-one post reperfusion (PostR) and 36 PreR biopsy samples were analyzed by microarray and gene enrichment KEGG pathway analysis. Data were compared to those obtained from patients with incident primary FSGS enrolled in other cohorts as well as with another cohort to correct for pathways activated by ischemia reperfusion. Using an ex-vivo cell-based assay in which human podocytes were cultured in the presence of sera from patients with recurrent and non recurrent FSGS, the molecular signature of podocytes exposed to sera from patients with REC was compared to the one established from patients with NON REC. We demonstrate that inflammatory pathways, including the TNF pathway, are primarily activated immediately after exposure to the sera of patients with primary FSGS, while phagocytotic pathways are activated when proteinuria becomes clinically evident. The TNF pathway activation by one or more circulating factors present in the sera of patients with FSGS supports prior experimental findings from our group demonstrating a causative role of local TNF in podocyte injury in FSGS. Correlation analysis with clinical and histological parameters of disease was performed and further supported a possible role for TNF pathway activation in FSGS. Additionally, we identified a unique set of genes that is specifically activated in podocytes when cultured in the presence of serum of patients with REC FSGS. This clinical translational study supports our prior experimental findings describing a potential role of the TNF pathway in the pathogenesis of FSGS. Validation of these findings in larger cohorts may lay the ground for the implementation of integrated system biology approaches to risk stratify patients affected by FSGS and to identify novel pathways relevant to podocyte injury.

Fibroblasts from patients with Tangier disease carrying ATP-binding cassette A1 (ABCA1) loss-of-function mutations are characterized by cardiolipin accumulation, a mitochondrial-specific phospholipid. Suppression of ABCA1 expression occurs in glomeruli from patients with diabetic kidney disease (DKD) and in human podocytes exposed to DKD sera collected prior to the development of DKD. We demonstrated that siRNA ABCA1 knockdown in podocytes led to reduced oxygen consumption capabilities associated with alterations in the oxidative phosphorylation (OXPHOS) complexes and with cardiolipin accumulation. Podocyte-specific deletion of Abca1 (Abca1(fl/fl)) rendered mice susceptible to DKD, and pharmacological induction of ABCA1 improved established DKD. This was not mediated by free cholesterol, as genetic deletion of sterol-o-acyltransferase-1 (SOAT1) in Abca1(fl/fl) mice was sufficient to cause free cholesterol accumulation but did not cause glomerular injury. Instead, cardiolipin mediates ABCA1-dependent susceptibility to podocyte injury, as inhibition of cardiolipin peroxidation with elamipretide improved DKD in vivo and prevented ABCA1-dependent podocyte injury in vitro and in vivo. Collectively, we describe a pathway definitively linking ABCA1 deficiency to cardiolipin-driven mitochondrial dysfunction. We demonstrated that this pathway is relevant to DKD and that ABCA1 inducers or inhibitors of cardiolipin peroxidation may each represent therapeutic strategies for the treatment of established DKD.

Sphingomyelin phosphodiesterase acid-like 3b (SMPDL3b) is a lipid raft enzyme that regulates plasma membrane (PM) fluidity. Here we report that SMPDL3b excess, as observed in podocytes in diabetic kidney disease (DKD), impairs insulin receptor isoform B-dependent pro-survival insulin signaling by interfering with insulin receptor isoforms binding to caveolin-1 in the PM. SMPDL3b excess affects the production of active sphingolipids resulting in decreased ceramide-1-phosphate (C1P) content as observed in human podocytes in vitro and in kidney cortexes of diabetic db/db mice in vivo. Podocyte-specific Smpdl3b deficiency in db/db mice is sufficient to restore kidney cortex C1P content and to protect from DKD. Exogenous administration of C1P restores IR signaling in vitro and prevents established DKD progression in vivo. Taken together, we identify SMPDL3b as a modulator of insulin signaling and demonstrate that supplementation with exogenous C1P may represent a lipid therapeutic strategy to treat diabetic complications such as DKD., Sphingomyelin phosphodiesterase acid-like 3b (SMPDL3b) is a lipid raft enzyme known to affect membrane lipid composition. Here, Mitrofanova et al. show that increased expression of SMPDL3b in diabetes impairs insulin signaling and ceramide-1-phosphate (C1P) availability in podocytes, and that C1P supplementation protects mice from diabetic kidney disease.

Lipid droplets (LDs) are dynamic organelles that regulate the storage and homeostasis of intracellular triglycerides and other neutral lipids. Studies show that the number, morphology, and subcellular localization of LDs are altered in a growing number of diseases. As such, methodologies for imaging and quantifying LDs have become essential tools for detecting changes in cellular lipid metabolism, which could be an important indicator of disease onset or progression. We previously reported on the accumulation of LDs in podocytes of the kidney glomerulus in nephrological diseases of metabolic and non-metabolic origin. Here, we describe a high-content analysis (HCA) method for automated detection and quantification of LDs in differentiated human podocytes.

Studies suggest that altered renal lipid metabolism plays a role in the pathogenesis of diabetic kidney disease and that genetic or pharmacological induction of cholesterol efflux protects from the development of diabetic kidney disease and focal segmental glomerulosclerosis (FSGS). Here we tested whether altered lipid metabolism contributes to renal failure in the Col4a3 knockout mouse model for Alport Syndrome. There was an eight-fold increase in the cholesterol content in renal cortexes of mice with Alport Syndrome. This was associated with increased glomerular lipid droplets and cholesterol crystals. Treatment of mice with Alport Syndrome with hydroxypropyl-β-cyclodextrin (HPβCD) reduced cholesterol content in the kidneys of mice with Alport Syndrome and protected from the development of albuminuria, renal failure, inflammation and tubulointerstitial fibrosis. Cholesterol efflux and trafficking-related genes were primarily affected in mice with Alport Syndrome and were differentially regulated in the kidney cortex and isolated glomeruli. HPβCD also protected from proteinuria and mesangial expansion in a second model of non-metabolic kidney disease, adriamycin-induced nephropathy. Consistent with our experimental findings, microarray analysis confirmed dysregulation of several lipid-related genes in glomeruli isolated from kidney biopsies of patients with primary FSGS enrolled in the NEPTUNE study. Thus, lipid dysmetabolism occurs in non-metabolic glomerular disorders such as Alport Syndrome and FSGS, and HPβCD improves renal function in experimental Alport Syndrome and FSGS.

Sphingolipids have important functions as structural components of cells but they also function as signaling molecules regulating different cellular processes such as apoptosis, cell proliferation, cell migration, cell division and inflammation. Hence, a tight regulation of the sphingolipid homeostasis is essential to maintain proper cellular functions. Mammalian ORMDL proteins are orthologues of the yeast ORM1/2 proteins, which regulate ceramide synthesis in yeast. ORMDL proteins inhibit serine palmitoyltransferase (SPT), the enzyme regulating a rate-limiting step of the sphingolipid pathway to control the levels of ceramides and other sphingolipids. Sphingomyelinase phosphodiesterase like 3b (SMPDL3b) is a glycosylphosphatidylinositol (GPI) anchored protein in the plasma membrane (PM) and determines membrane fluidity in macrophages. We previously showed that differential expression of SMPDL3b alters the availability of Ceramide-1-phosphate (C1P) in human podocytes, which are terminally differentiated cells of the kidney filtration barrier. This observation lead us to investigate if SMPDL3b controls C1P availability in human podocytes by interfering with ceramide kinase (CERK) expression and function. We found that SMPDL3b interacts with CERK and can bind to C1P in vitro. Furthermore, CERK expression is reduced when SMPDL3b expression is silenced. These observations led us to propose that one of the mechanisms by which SMPDL3b influences the amount of C1P available in the podocytes is by interfering with the function of CERK thereby maintaining a balance in the levels of the C1P in podocytes.

Dysregulation of the actin cytoskeleton in podocytes represents a common pathway in the pathogenesis of proteinuria across a spectrum of chronic kidney diseases (CKD). The GTPase dynamin has been implicated in the maintenance of cellular architecture in podocytes through its direct interaction with actin. Furthermore, the propensity of dynamin to oligomerize into higher-order structures in an actin-dependent manner and to cross-link actin microfilaments into higher-order structures has been correlated with increased actin polymerization and global organization of the actin cytoskeleton in the cell. We found that use of the small molecule Bis-T-23, which promotes actin-dependent dynamin oligomerization and thus increased actin polymerization in injured podocytes, was sufficient to improve renal health in diverse models of both transient kidney disease and CKD. In particular, administration of Bis-T-23 in these renal disease models restored the normal ultrastructure of podocyte foot processes, lowered proteinuria, lowered collagen IV deposits in the mesangial matrix, diminished mesangial matrix expansion and extended lifespan. These results further establish that alterations in the actin cytoskeleton of kidney podocytes is a common hallmark of CKD, while also underscoring the substantial regenerative potential of injured glomeruli and identifying the oligomerization cycle of dynamin as an attractive potential therapeutic target to treat CKD.

Esterified cholesterol content is often lower than free cholesterol content in biological systems and thus the determination of the esterified cholesterol content of cells is often challenging. Traditional methods use enzymatic assays in which an indirect measurement of the esterified cholesterol content is obtained by subtracting the measurements of the free from the total cholesterol content. However, this approach fails in the case where the total cholesterol content of cells is unchanged while the ratio of free to esterified cholesterol changes such that total and free cholesterol content are very similar and thus the difference may fall within the background noise of the enzymatic assay. To overcome this challenge, we here describe a method that utilizes a TLC-based technique to isolate esterified cholesterol. Isolated esterified cholesterol can then be measured using traditional enzymatic methods. Therefore, this method provides a practical and more sensitive assay to measure esterified cholesterol content in cellular extracts.

Fornoni et al. discuss the role of lipid biology in podocytes in the pathogenesis of glomerulopathies focusing on diabetic kidney disease and focal segmental glomerulosclerosis. They outline recent findings from genetic disorders that highlight the function of apolipoproteins in the podocyte and discuss the therapeutic implications. In the past 15 years, major advances have been made in understanding the role of lipids in podocyte biology. First, susceptibility to focal segmental glomerulosclerosis (FSGS) and glomerular disease is associated with an APOL1 sequence variant, is expressed in podocytes and encodes apolipoprotein L1, an important component of HDL. Second, acid sphingomyelinase-like phosphodiesterase 3b encoded by SMPDL3b has a role in the conversion of sphingomyelin to ceramide and its levels are reduced in renal biopsy samples from patients with recurrent FSGS. Furthermore, decreased SMPDL3b expression is associated with increased susceptibility of podocytes to injury after exposure to sera from these patients. Third, in many individuals with membranous nephropathy, autoantibodies against the phospholipase A2 (PLA2) receptor, which is expressed in podocytes, have been identified. Whether these autoantibodies affect the activity of PLA2, which liberates arachidonic acid from glycerophospholipids and modulates podocyte function, is unknown. Fourth, clinical and experimental evidence support a role for ATP-binding cassette sub-family A member 1-dependent cholesterol efflux, free fatty acids and glycerophospolipids in the pathogenesis of diabetic kidney disease. An improved understanding of lipid biology in podocytes might provide insights to develop therapeutic targets for primary and secondary glomerulopathies.

Podocytes are a major component of the glomerular filtration barrier, and their ability to sense insulin is essential to prevent proteinuria. Here we identify the insulin downstream effector GLUT4 as a key modulator of podocyte function in diabetic nephropathy (DN). Mice with a podocyte-specific deletion of GLUT4 (G4 KO) did not develop albuminuria despite having larger and fewer podocytes than wild-type (WT) mice. Glomeruli from G4 KO mice were protected from diabetes-induced hypertrophy, mesangial expansion, and albuminuria and failed to activate the mammalian target of rapamycin (mTOR) pathway. In order to investigate whether the protection observed in G4 KO mice was due to the failure to activate mTOR, we used three independent in vivo experiments. G4 KO mice did not develop lipopolysaccharide-induced albuminuria, which requires mTOR activation. On the contrary, G4 KO mice as well as WT mice treated with the mTOR inhibitor rapamycin developed worse adriamycin-induced nephropathy than WT mice, consistent with the fact that adriamycin toxicity is augmented by mTOR inhibition. In summary, GLUT4 deficiency in podocytes affects podocyte nutrient sensing, results in fewer and larger cells, and protects mice from the development of DN. This is the first evidence that podocyte hypertrophy concomitant with podocytopenia may be associated with protection from proteinuria.

Apolipoprotein L1 (APOL1) genetic variants G1 and G2, compared to the common allele G0, are major risk factors for non-diabetic kidney disease in African descent populations. APOL1 is a minor protein component of HDL, as well as being expressed in podocytes and vascular cells. Reverse cholesterol transport involves the transport of cholesterol to HDL by cellular ATP-binding cassette; ABCA1 and ABCG1 with subsequent delivery from peripheral tissues to the liver. With impaired reverse cholesterol transport, lipid accumulation occurs and macrophages morphologically transform into foam cells, releasing inflammatory factors. We asked whether the APOL1 risk variants alter peripheral cholesterol metabolism and specifically affect macrophage cholesterol efflux. Tissues and bone marrow (BM)-derived monocytes were isolated from wild-type mice (WT) and from BAC/APOL1 transgenic (APOL1-G0, APOL1-G1, and APOL1-G2) mice, which carry a bacterial artificial chromosome that contains the human APOL1 genomic region. Monocytes were differentiated into macrophages using M-CSF, and then polarized into M1 and M2 macrophages. Cholesterol content, cholesterol efflux, and ABCA1 and ABCG1 mRNA expression were measured. Kidney, spleen, and bone marrow-derived macrophages from APOL1-G1 and -G2 mice showed increased cholesterol accumulation and decreased ABCA1 and ABCG1 mRNA levels. BM-derived macrophages from APOL1-G1 and -G2 mice showed significantly reduced cholesterol efflux compared to WT or APOL1-G0 macrophages. Taken together, the evidence suggests that APOL1-G1 and -G2 risk variants impaired reverse cholesterol transport through decreased expression of cholesterol efflux transporters suggesting a possible mechanism to promote macrophage foam cell formation, driving inflammation in the glomerulus and renal interstitium.

Diabetic kidney disease (DKD) remains the most common cause of end-stage kidney disease despite multifactorial intervention. We demonstrated that increased cholesterol in association with downregulation of ATP-binding cassette transporter ABCA1 occurs in normal human podocytes exposed to the sera of patients with type 1 diabetes and albuminuria (DKD+) when compared with diabetic patients with normoalbuminuria (DKD−) and similar duration of diabetes and lipid profile. Glomerular downregulation of ABCA1 was confirmed in biopsies from patients with early DKD (n = 70) when compared with normal living donors (n = 32). Induction of cholesterol efflux with cyclodextrin (CD) but not inhibition of cholesterol synthesis with simvastatin prevented podocyte injury observed in vitro after exposure to patient sera. Subcutaneous administration of CD to diabetic BTBR (black and tan, brachiuric) ob/ob mice was safe and reduced albuminuria, mesangial expansion, kidney weight, and cortical cholesterol content. This was followed by an improvement of fasting insulin, blood glucose, body weight, and glucose tolerance in vivo and improved glucose-stimulated insulin release in human islets in vitro. Our data suggest that impaired reverse cholesterol transport characterizes clinical and experimental DKD and negatively influences podocyte function. Treatment with CD is safe and effective in preserving podocyte function in vitro and in vivo and may improve the metabolic control of diabetes.

High levels of circulating TNF and its receptors, TNFR1 and TNFR2, predict the progression of diabetic kidney disease (DKD), but their contribution to organ damage in DKD remains largely unknown. Here, we investigated the function of local and systemic TNF in podocyte injury. We cultured human podocytes with sera collected from DKD patients, who displayed elevated TNF levels, and focal segmental glomerulosclerosis (FSGS) patients, whose TNF levels resembled those of healthy patients. Exogenous TNF administration or local TNF expression was equally sufficient to cause free cholesterol-dependent apoptosis in podocytes by acting through a dual mechanism that required a reduction in ATP-binding cassette transporter A1-mediated (ABCA1-mediated) cholesterol efflux and reduced cholesterol esterification by sterol-O-acyltransferase 1 (SOAT1). TNF-induced albuminuria was aggravated in mice with podocyte-specific ABCA1 deficiency and was partially prevented by cholesterol depletion with cyclodextrin. TNF-stimulated free cholesterol-dependent apoptosis in podocytes was mediated by nuclear factor of activated T cells 1 (NFATc1). ABCA1 overexpression or cholesterol depletion was sufficient to reduce albuminuria in mice with podocyte-specific NFATc1 activation. Our data implicate an NFATc1/ABCA1-dependent mechanism in which local TNF is sufficient to cause free cholesterol-dependent podocyte injury irrespective of TNF, TNFR1, or TNFR2 serum levels.

Background: Cisplatin is a widely used radiosensitizer. Dose-related and cumulative renal insufficiency, including acute renal failure, are the primary dose-limiting toxicities of cisplatin. The lipid-modulating enzyme sphingomyelin phosphodiesterase acid-like 3B (SMPDL3b) and Sphingosine 1-Phosphate Receptor-1 (S1P1) are critical determinants of renal podocyte injury. In the current study, we investigated the functions of SMPDL3b and S1P in cisplatin and radiation-induced renal injury, and the use of rituximab (RTX) to attenuate nephrotoxicity. Material/Methods: Podocytes were incubated for 2 h with S1P (100 nM) or vehicle (2% dodecane/98% ethanol) and irradiated with 0-8 Gy, or podocytes were irradiated (8 Gy) in the presence of RTX (100 mg/ml) or IgG (100 mg/ml). In other experiments, 10-14-week old C57BL/6 mice were given bilateral kidney X-irradiation (4 Gy) using an image-guided small animal arc radiation treatment system (iSMAART), 6mg/kg cisplatin or combined cisplatin plus radiation. Some animals received a single intravenous (IV) injection of rituximab mAb or IgG (50 mg/kg) prior to treatment. Functional kidney parameters and immuno-histopathological assessments of renal damage (e.g. oil red staining, H & E, Periodic Acid-Schiff (PAS) and SMPDL3B expression) were measured post-RT. iSMAART dynamic contrast-enhanced (DCE) imaging was used for estimating glomerular filtration rate 20 weeks post radiation. Results: Exogenous S1P rescued podocytes from concurrent chemoradiotherapy-induced cellular cytoskeletal remodeling, and RTX pretreatment mitigated podocytopathy in vitro. iSMAART dynamic contrast-enhanced (DCE) imaging analysis showed that rituximab pretreatment improved the GFR and kidney function parameters post concurrent chemoradiotherapy. SMPDL3b expression at the protein level and podocyte counts dropped significantly in irradiated kidney post-RT in cisplatin-treated mice. Rituximab pretreatment in cisplatin-treated mice, improved GFR, kidney functional parameters, vascular structure, normalization of pericyte coverage, suppress the development of fibrosis and tubular damage post-irradiation. Conclusion: This study shows that rituximab pretreatment protects mice against concurrent chemoradiation induced kidney injury. SMPDL3b and Sphingosine 1-Phosphate Receptor-1 may be important therapeutic targets for radiation-induced kidney injuries in cancer patients. Citation Format: Anis Ahmad, Alla Mitrofanova, Saba Ansari, Junwei Shi, Yidong Yang, Sandra Merscher, Alessia Fornoni, Youssef Zeidan, Brian Marples. Protecting Sphingomyelin Phosphodiesterase Acid Like 3B (SMPDL3b) enhances kidney function and reduces concurrent chemoradiotherapy-induced nephrotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4161.

The immunosuppressive action of the calcineurin inhibitor cyclosporine A (CsA) stems from the inhibition of nuclear factor of activated T cells (NFAT) signaling in T cells. CsA is also used for the treatment of proteinuric kidney diseases. As it stands, the antiproteinuric effect of CsA is attributed to its immunosuppressive action. Here we show that the beneficial effect of CsA on proteinuria is not dependent on NFAT inhibition in T cells, but rather results from the stabilization of the actin cytoskeleton in kidney podocytes. CsA blocks the calcineurin-mediated dephosphorylation of synaptopodin, a regulator of Rho GTPases in podocytes, thereby preserving the phosphorylation-dependent synaptopodin-14-3-3 beta interaction. Preservation of this interaction, in turn, protects synaptopodin from cathepsin L-mediated degradation. These results represent a new view of calcineurin signaling and shed further light on the treatment of proteinuric kidney diseases. Novel calcineurin substrates such as synaptopodin may provide promising starting points for antiproteinuric drugs that avoid the serious side effects of long-term CsA treatment.