APOL1 risk variants affect podocyte lipid homeostasis and energy production in focal segmental glomerulosclerosis.

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.

Sphingosine-1-Phosphate Metabolism and Signaling in Kidney Diseases.

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.

Sterol-O-acyltransferase-1 has a role in kidney disease associated with diabetes and Alport syndrome.

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.

Hydroxypropyl-ß-cyclodextrin protects from kidney disease in experimental Alport syndrome and focal segmental glomerulosclerosis.

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-ß-cyclodextrin (HPßCD) 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. HPßCD 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 HPßCD improves renal function in experimental Alport Syndrome and FSGS.

Young capillary vessels rejuvenate aged pancreatic islets.

Pancreatic islets secrete hormones that play a key role in regulating blood glucose levels (glycemia). Age-dependent impairment of islet function and concomitant dysregulation of glycemia are major health threats in aged populations. However, the major causes of the age-dependent decline of islet function are still disputed. Here we demonstrate that aging of pancreatic islets in mice and humans is notably associated with inflammation and fibrosis of islet blood vessels but does not affect glucose sensing and the insulin secretory capacity of islet beta cells. Accordingly, when transplanted into the anterior chamber of the eye of young mice with diabetes, islets from old mice are revascularized with healthy blood vessels, show strong islet cell proliferation, and fully restore control of glycemia. Our results indicate that beta cell function does not decline with age and suggest that islet function is threatened by an age-dependent impairment of islet vascular function. Strategies to mitigate age-dependent dysregulation in glycemia should therefore target systemic and/or local inflammation and fibrosis of the aged islet vasculature.

Noninvasive in vivo model demonstrating the effects of autonomic innervation on pancreatic islet function.

The autonomic nervous system is thought to modulate blood glucose homeostasis by regulating endocrine cell activity in the pancreatic islets of Langerhans. The role of islet innervation, however, has remained elusive because the direct effects of autonomic nervous input on islet cell physiology cannot be studied in the pancreas. Here, we used an in vivo model to study the role of islet nervous input in glucose homeostasis. We transplanted islets into the anterior chamber of the eye and found that islet grafts became densely innervated by the rich parasympathetic and sympathetic nervous supply of the iris. Parasympathetic innervation was imaged intravitally by using transgenic mice expressing GFP in cholinergic axons. To manipulate selectively the islet nervous input, we increased the ambient illumination to increase the parasympathetic input to the islet grafts via the pupillary light reflex. This reduced fasting glycemia and improved glucose tolerance. These effects could be blocked by topical application of the muscarinic antagonist atropine to the eye, indicating that local cholinergic innervation had a direct effect on islet function in vivo. By using this approach, we found that parasympathetic innervation influences islet function in C57BL/6 mice but not in 129X1 mice, which reflected differences in innervation densities and may explain major strain differences in glucose homeostasis. This study directly demonstrates that autonomic axons innervating the islet modulate glucose homeostasis.

High-resolution, noninvasive longitudinal live imaging of immune responses.

Intravital imaging emerged as an indispensible tool in biological research, and a variety of imaging techniques have been developed to noninvasively monitor tissues in vivo. However, most of the current techniques lack the resolution to study events at the single-cell level. Although intravital multiphoton microscopy has addressed this limitation, the need for repeated noninvasive access to the same tissue in longitudinal in vivo studies remains largely unmet. We now report on a previously unexplored approach to study immune responses after transplantation of pancreatic islets into the anterior chamber of the mouse eye. This approach enabled (i) longitudinal, noninvasive imaging of transplanted tissues in vivo; (ii) in vivo cytolabeling to assess cellular phenotype and viability in situ; (iii) local intervention by topical application or intraocular injection; and (iv) real-time tracking of infiltrating immune cells in the target tissue.

Identification of Glomerular and Plasma Apolipoprotein M as Novel Biomarkers in Glomerular 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.

Empagliflozin reduces podocyte lipotoxicity in experimental Alport syndrome.

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 podocyte lipotoxicity in experimental Alport syndrome (AS). In vitro, we found that the SGLT2 protein was expressed in human and mouse podocytes to a similar extent in tubular cells. Newly established immortalized podocytes from Col4a3 knockout mice (AS podocytes) accumulate lipid droplets along with increased apoptosis when compared to wild-type 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 but not in AS tubular cells. 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 a decline in renal function. In summary, empagliflozin reduces podocyte lipotoxicity and improves kidney function in experimental AS in association with the energy substrates switch from glucose to fatty acids in podocytes.

ABCA1 deficiency contributes to podocyte pyroptosis priming via the APE1/IRF1 axis in diabetic kidney disease.

Decreased ATP Binding Cassette Transporter A1 (ABCA1) expression and caspase-4-mediated noncanonical inflammasome contribution have been described in podocytes in diabetic kidney disease (DKD). To investigate a link between these pathways, we evaluated pyroptosis-related mediators in human podocytes with stable knockdown of ABCA1 (siABCA1) and found that mRNA levels of IRF1, caspase-4, GSDMD, caspase-1 and IL1ß were significantly increased in siABCA1 compared to control podocytes and that protein levels of caspase-4, GSDMD and IL1ß were equally increased. IRF1 knockdown in siABCA1 podocytes prevented increases in caspase-4, GSDMD and IL1ß. Whereas TLR4 inhibition did not decrease mRNA levels of IRF1 and caspase-4, APE1 protein expression increased in siABCA1 podocytes and an APE1 redox inhibitor abrogated siABCA1-induced expression of IRF1 and caspase-4. RELA knockdown also offset the pyroptosis priming, but ChIP did not demonstrate increased binding of NFκB to IRF1 promoter in siABCA1 podocytes. Finally, the APE1/IRF1/Casp1 axis was investigated in vivo. APE1 IF staining and mRNA levels of IRF1 and caspase 11 were increased in glomeruli of BTBR ob/ob compared to wildtype. In conclusion, ABCA1 deficiency in podocytes caused APE1 accumulation, which reduces transcription factors to increase the expression of IRF1 and IRF1 target inflammasome-related genes, leading to pyroptosispriming.

Noninvasive assessment of radiation-induced renal injury in mice.

PURPOSE: 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. METHODS: 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). RESULTS: Hematoxylin and eosin (H&E) 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. CONCLUSION: This study demonstrated that GFR measured by dynamic contrast-enhanced CT imaging is safe and effective compared to transcutaneous methodology to estimate kidney function.

Compounds targeting OSBPL7 increase ABCA1-dependent cholesterol efflux preserving kidney function in two models of kidney disease.

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.

Mechanism and effects of pulsatile GABA secretion from cytosolic pools in the human beta cell.

Pancreatic beta cells synthesize and secrete the neurotransmitter γ-aminobutyric acid (GABA) as a paracrine and autocrine signal to help regulate hormone secretion and islet homeostasis. Islet GABA release has classically been described as a secretory vesicle-mediated event. Yet, a limitation of the hypothesized vesicular GABA release from islets is the lack of expression of a vesicular GABA transporter in beta cells. Consequentially, GABA accumulates in the cytosol. Here we provide evidence that the human beta cell effluxes GABA from a cytosolic pool in a pulsatile manner, imposing a synchronizing rhythm on pulsatile insulin secretion. The volume regulatory anion channel (VRAC), functionally encoded by LRRC8A or Swell1, is critical for pulsatile GABA secretion. GABA content in beta cells is depleted and secretion is disrupted in islets from type 1 and type 2 diabetic patients, suggesting that loss of GABA as a synchronizing signal for hormone output may correlate with diabetes pathogenesis.

ATP-binding cassette A1 deficiency causes cardiolipin-driven mitochondrial dysfunction in podocytes.

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 (Abca1fl/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 Abca1fl/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.

Human Beta Cells Produce and Release Serotonin to Inhibit Glucagon Secretion from Alpha Cells.

In the pancreatic islet, serotonin is an autocrine signal increasing beta cell mass during metabolic challenges such as those associated with pregnancy or high-fat diet. It is still unclear whether serotonin is relevant for regular islet physiology and hormone secretion. Here, we show that human beta cells produce and secrete serotonin when stimulated with increases in glucose concentration. Serotonin secretion from beta cells decreases cyclic AMP (cAMP) levels in neighboring alpha cells via 5-HT1F receptors and inhibits glucagon secretion. Without serotonergic input, alpha cells lose their ability to regulate glucagon secretion in response to changes in glucose concentration, suggesting that diminished serotonergic control of alpha cells can cause glucose blindness and the uncontrolled glucagon secretion associated with diabetes. Supporting this model, pharmacological activation of 5-HT1F receptors reduces glucagon secretion and has hypoglycemic effects in diabetic mice. Thus, modulation of serotonin signaling in the islet represents a drug intervention opportunity.

Parasitological cure of acute and chronic experimental Chagas disease using the long-acting experimental triazole TAK-187. Activity against drug-resistant Trypanosoma cruzi strains.

We investigated the activity of TAK-187, an experimental antifungal triazole with a long terminal half-life in several experimental animals, against Trypanosoma cruzi. In vitro studies showed that the minimal inhibitory concentration (MIC) against the (extracellular) epimastigote form was 0.3-1 microM, while the corresponding concentration against clinically relevant intracellular amastigotes was 1 nM. At the MIC the endogenous epimastigote C4,14-desmethyl sterols were replaced by di- and tri-methylated sterols, supporting the notion that the primary target of TAK-187 is the parasite's sterol C14alpha demethylase. We investigated the in vivo activity of the compound in a murine model of acute Chagas disease, using T. cruzi strains with different susceptibilities to the drugs currently used clinically (nitrofurans and nitroimidazoles). It was found that TAK-187 given orally at 20 mg/kg induced complete protection against death and high levels (60-100%) of parasitological cures, independently of the infecting strain and even when administered every other day (e.o.d.), consistent with its long terminal half-life in mice. Other experiments, using longer treatment periods were carried out in both acute and chronic models of the disease and showed that TAK-187 given at 10-20 mg/kg e.o.d. induced 80-100% survival with 80-100% of parasitological cures of survivors in both models. No toxic side effects were observed in any of the experimental protocols. TAK-187 is a potent anti-T. cruzi compound with trypanocidal activity in vivo and should be considered for further studies as a potential specific treatment of human Chagas disease.

Control of insulin secretion by cholinergic signaling in the human pancreatic islet.

Acetylcholine regulates hormone secretion from the pancreatic islet and is thus crucial for glucose homeostasis. Little is known, however, about acetylcholine (cholinergic) signaling in the human islet. We recently reported that in the human islet, acetylcholine is primarily a paracrine signal released from α-cells rather than primarily a neural signal as in rodent islets. In this study, we demonstrate that the effects acetylcholine produces in the human islet are different and more complex than expected from studies conducted on cell lines and rodent islets. We found that endogenous acetylcholine not only stimulates the insulin-secreting ß-cell via the muscarinic acetylcholine receptors M3 and M5, but also the somatostatin-secreting δ-cell via M1 receptors. Because somatostatin is a strong inhibitor of insulin secretion, we hypothesized that cholinergic input to the δ-cell indirectly regulates ß-cell function. Indeed, when all muscarinic signaling was blocked, somatostatin secretion decreased and insulin secretion unexpectedly increased, suggesting a reduced inhibitory input to ß-cells. Endogenous cholinergic signaling therefore provides direct stimulatory and indirect inhibitory input to ß-cells to regulate insulin secretion from the human islet.

Maternal inheritance of an inactive type III deiodinase gene allele affects mouse pancreatic ß-cells and disrupts glucose homeostasis.

Dio3 is the most distal gene of the imprinted Dlk1-Dio3 gene locus and is expressed according to parental origin. Dio3 encodes the type 3 deiodinase (D3), a thioredoxin-fold like containing selenoenzyme that inactivates thyroid hormone and dampens thyroid hormone signaling. Here we used heterozygous animals with disruption of the Dio3 gene to study the allelic expression pattern of Dio3 in pancreatic ß-cells and the metabolic phenotype resulting from its inactivation. Adult heterozygous mice with disruption of the Dio3 gene with maternal inheritance of the inactive Dio3 allele exhibited a total loss of D3 activity in isolated pancreatic islets, approximately 30% reduction in total pancreatic islet area, a marked decrease in insulin2 mRNA and in vivo glucose intolerance. In contrast, inheritance of the inactive Dio3 allele from the father did not affect D3 activity in isolated pancreatic islets and did not result in a pancreatic phenotype. Furthermore, exposure of pancreatic explants, D3-expressing MIN6-C3 cells or isolated pancreatic islets to 100 nM T3 for 24 hours reduced insulin2 mRNA by approximately 50% and the peak of glucose-induced insulin secretion. An unbiased analysis of T3-treated pancreatic islets revealed the down-regulation of 21 gene sets (false discovery rate q value < 25%) involved in nucleolar function and transcription of rRNA, ribonucleotide binding, mRNA translation, and membrane organization. We conclude that the Dio3 gene is preferentially expressed from the maternal allele in pancreatic islets and that the inactivation of this allele is sufficient to disrupt glucose homeostasis by reducing the pancreatic islet area, insulin2 gene expression, and glucose-stimulated insulin secretion.