SPTLC3 Is Essential for Complex I Activity and Contributes to Ischemic Cardiomyopathy.

BACKGROUND: Dysregulated metabolism of bioactive sphingolipids, including ceramides and sphingosine-1-phosphate, has been implicated in cardiovascular disease, although the specific species, disease contexts, and cellular roles are not completely understood. Sphingolipids are produced by the serine palmitoyltransferase enzyme, canonically composed of 2 subunits, SPTLC1 (serine palmitoyltransferase long chain base subunit 1) and SPTLC2 (serine palmitoyltransferase long chain base subunit 2). Noncanonical sphingolipids are produced by a more recently described subunit, SPTLC3 (serine palmitoyltransferase long chain base subunit 3). METHODS: The noncanonical (d16) and canonical (d18) sphingolipidome profiles in cardiac tissues of patients with end-stage ischemic cardiomyopathy and in mice with ischemic cardiomyopathy were analyzed by targeted lipidomics. Regulation of SPTLC3 by HIF1α under ischemic conditions was determined with chromatin immunoprecipitation. Transcriptomics, lipidomics, metabolomics, echocardiography, mitochondrial electron transport chain, mitochondrial membrane fluidity, and mitochondrial membrane potential were assessed in the cSPTLC3KO transgenic mice we generated. Furthermore, morphological and functional studies were performed on cSPTLC3KO mice subjected to permanent nonreperfused myocardial infarction. RESULTS: Herein, we report that SPTLC3 is induced in both human and mouse models of ischemic cardiomyopathy and leads to production of atypical sphingolipids bearing 16-carbon sphingoid bases, resulting in broad changes in cell sphingolipid composition. This induction is in part attributable to transcriptional regulation by HIF1α under ischemic conditions. Furthermore, cardiomyocyte-specific depletion of SPTLC3 in mice attenuates oxidative stress, fibrosis, and hypertrophy in chronic ischemia, and mice demonstrate improved cardiac function and increased survival along with increased ketone and glucose substrate metabolism utilization. Depletion of SPTLC3 mechanistically alters the membrane environment and subunit composition of mitochondrial complex I of the electron transport chain, decreasing its activity. CONCLUSIONS: Our findings suggest a novel essential role for SPTLC3 in electron transport chain function and a contribution to ischemic injury by regulating complex I activity.

Nicotinamide riboside activates renal metabolism and protects the kidney in a model of Alport syndrome.

Chronic kidney disease (CKD) is associated with renal metabolic disturbances, including impaired fatty acid oxidation (FAO). Nicotinamide adenine dinucleotide (NAD + ) is a small molecule that participates in hundreds of metabolism-related reactions. NAD + levels are decreased in CKD, and NAD + supplementation is protective. However, both the mechanism of how NAD + supplementation protects from CKD, as well as the cell types most responsible, are poorly understood. Using a mouse model of Alport syndrome, we show that nicotinamide riboside (NR), an NAD + precursor, stimulates renal peroxisome proliferator-activated receptor α signaling and restores FAO in the proximal tubules, thereby protecting from CKD in both sexes. Bulk RNA-sequencing shows that renal metabolic pathways are impaired in Alport mice and dramatically activated by NR in both sexes. These transcriptional changes are confirmed by orthogonal imaging techniques and biochemical assays. Single nuclei RNA-sequencing and spatial transcriptomics, both the first of their kind from Alport mice, show that NAD + supplementation restores FAO in the proximal tubules with minimal effects on the podocytes. Finally, we also report, for the first time, sex differences at the transcriptional level in this Alport model. Male Alport mice had more severe inflammation and fibrosis than female mice at the transcriptional level. In summary, the data herein identify both the protective mechanism and location of NAD + supplementation in this model of CKD.

Fibrosis quantification using multiphoton excitation imaging of picrosirius red stained cardiac tissue.

Traditional methodologies for fibrosis quantification involve histological measurements, staining with Masson's trichrome and picrosirius red (PSR), and label-free imaging using second harmonic generation (SHG). The difficulty of label-free cardiac SHG imaging is that both collagen (i.e., collagen 1 fibrils) and myosin are harmonophores that generate SHG signals, and specific identification of either collagen or myosin is difficult to achieve. Here we present an alternate method of quantifying cardiac fibrosis by using PSR staining followed by multiphoton excitation fluorescence imaging. Our data from the deoxycorticosterone model of cardiac fibrosis shows that this imaging method and downstream analyses, including background correction, are robust and easy to perform. These advantages are due to the high signal-to-noise ratio provided by PSR in areas of collagen fibers. Furthermore, the hyperspectral and fluorescence lifetime information of PSR-stained area of fibrosis shows better quantification can eventually be obtained using more complex instrumentation.

Farnesoid X receptor prevents neutrophil extracellular traps via reduced sphingosine-1-phosphate in chronic kidney disease.

Farnesoid X receptor (FXR) activation reduces renal inflammation, but the underlying mechanisms remain elusive. Neutrophil extracellular traps (NETs) are webs of DNA formed when neutrophils undergo specialized programmed cell death (NETosis). The signaling lipid sphingosine-1-phosphate (S1P) stimulates NETosis via its receptor on neutrophils. Here, we identify FXR as a negative regulator of NETosis via repressing S1P signaling. We determined the effects of the FXR agonist obeticholic acid (OCA) in mouse models of adenosine phosphoribosyltransferase (APRT) deficiency and Alport syndrome, both genetic disorders that cause chronic kidney disease. Renal FXR activity is greatly reduced in both models, and FXR agonism reduces disease severity. Renal NETosis and sphingosine kinase 1 (Sphk1) expression are increased in diseased mice, and they are reduced by OCA in both models. Genetic deletion of FXR increases Sphk1 expression, and Sphk1 expression correlates with NETosis. Importantly, kidney S1P levels in Alport mice are two-fold higher than controls, and FXR agonism restores them back to baseline. Short-term inhibition of sphingosine synthesis in Alport mice with severe kidney disease reverses NETosis, establishing a causal relationship between S1P signaling and renal NETosis. Finally, extensive NETosis is present in human Alport kidney biopsies (six male, nine female), and NETosis severity correlates with clinical markers of kidney disease. This suggests the potential clinical relevance of the newly identified FXR-S1P-NETosis pathway. In summary, FXR agonism represses kidney Sphk1 expression. This inhibits renal S1P signaling, thereby reducing neutrophilic inflammation and NETosis.NEW & NOTEWORTHY Many preclinical studies have shown that the farnesoid X receptor (FXR) reduces renal inflammation, but the mechanism is poorly understood. This report identifies FXR as a novel regulator of neutrophilic inflammation and NETosis via the inhibition of sphingosine-1-phosphate signaling. Additionally, NETosis severity in human Alport kidney biopsies correlates with clinical markers of kidney disease. A better understanding of this signaling axis may lead to novel treatments that prevent renal inflammation and chronic kidney disease.

Estrogen-Related Receptor Agonism Reverses Mitochondrial Dysfunction and Inflammation in the Aging Kidney.

A gradual decline in renal function occurs even in healthy aging individuals. In addition to aging, per se, concurrent metabolic syndrome and hypertension, which are common in the aging population, can induce mitochondrial dysfunction and inflammation, which collectively contribute to age-related kidney dysfunction and disease. This study examined the role of the nuclear hormone receptors, the estrogen-related receptors (ERRs), in regulation of age-related mitochondrial dysfunction and inflammation. The ERRs were decreased in both aging human and mouse kidneys and were preserved in aging mice with lifelong caloric restriction (CR). A pan-ERR agonist, SLU-PP-332, was used to treat 21-month-old mice for 8 weeks. In addition, 21-month-old mice were treated with a stimulator of interferon genes (STING) inhibitor, C-176, for 3 weeks. Remarkably, similar to CR, an 8-week treatment with a pan-ERR agonist reversed the age-related increases in albuminuria, podocyte loss, mitochondrial dysfunction, and inflammatory cytokines, via the cyclic GMP-AMP synthase-STING and STAT3 signaling pathways. A 3-week treatment of 21-month-old mice with a STING inhibitor reversed the increases in inflammatory cytokines and the senescence marker, p21/cyclin dependent kinase inhibitor 1A (Cdkn1a), but also unexpectedly reversed the age-related decreases in PPARG coactivator (PGC)-1α, ERRα, mitochondrial complexes, and medium chain acyl coenzyme A dehydrogenase (MCAD) expression. These studies identified ERRs as CR mimetics and as important modulators of age-related mitochondrial dysfunction and inflammation. These findings highlight novel druggable pathways that can be further evaluated to prevent progression of age-related kidney disease.

NAD metabolism modulates inflammation and mitochondria function in diabetic kidney disease.

Diabetes mellitus is the leading cause of cardiovascular and renal disease in the United -States. Despite the beneficial interventions available for patients with diabetes, there remains a need for additional therapeutic targets and therapies in diabetic kidney disease (DKD). Inflammation and oxidative stress are increasingly recognized as important causes of renal diseases. Inflammation is closely associated with mitochondrial damage. The molecular connection between inflammation and mitochondrial metabolism remains to be elucidated. Recently, nicotinamide adenine nucleotide (NAD+) metabolism has been found to regulate immune function and inflammation. In the present studies, we tested the hypothesis that enhancing NAD metabolism could prevent inflammation in and progression of DKD. We found that treatment of db/db mice with type 2 diabetes with nicotinamide riboside (NR) prevented several manifestations of kidney dysfunction (i.e., albuminuria, increased urinary kidney injury marker-1 (KIM1) excretion, and pathologic changes). These effects were associated with decreased inflammation, at least in part via inhibiting the activation of the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) signaling pathway. An antagonist of the serum stimulator of interferon genes (STING) and whole-body STING deletion in diabetic mice showed similar renoprotection. Further analysis found that NR increased SIRT3 activity and improved mitochondrial function, which led to decreased mitochondrial DNA damage, a trigger for mitochondrial DNA leakage which activates the cGAS-STING pathway. Overall, these data show that NR supplementation boosted NAD metabolism to augment mitochondrial function, reducing inflammation and thereby preventing the progression of diabetic kidney disease.

The role of FXR and TGR5 in reversing and preventing progression of Western diet-induced hepatic steatosis, inflammation, and fibrosis in mice.

Nonalcoholic steatohepatitis (NASH) is the most common chronic liver disease in the US, partly due to the increasing incidence of metabolic syndrome, obesity, and type 2 diabetes. The roles of bile acids and their receptors, such as the nuclear receptor farnesoid X receptor (FXR) and the G protein-coupled receptor TGR5, on the development of NASH are not fully clear. C57BL/6J male mice fed a Western diet (WD) develop characteristics of NASH, allowing determination of the effects of FXR and TGR5 agonists on this disease. Here we show that the FXR-TGR5 dual agonist INT-767 prevents progression of WD-induced hepatic steatosis, inflammation, and fibrosis, as determined by histological and biochemical assays and novel label-free microscopy imaging techniques, including third harmonic generation, second harmonic generation, and fluorescence lifetime imaging microscopy. Furthermore, we show INT-767 decreases liver fatty acid synthesis and fatty acid and cholesterol uptake, as well as liver inflammation. INT-767 markedly changed bile acid composition in the liver and intestine, leading to notable decreases in the hydrophobicity index of bile acids, known to limit cholesterol and lipid absorption. In addition, INT-767 upregulated expression of liver p-AMPK, SIRT1, PGC-1α, and SIRT3, which are master regulators of mitochondrial function. Finally, we found INT-767 treatment reduced WD-induced dysbiosis of gut microbiota. Interestingly, the effects of INT-767 in attenuating NASH were absent in FXR-null mice, but still present in TGR5-null mice. Our findings support treatment and prevention protocols with the dual FXR-TGR5 agonist INT-767 arrest progression of WD-induced NASH in mice mediated by FXR-dependent, TGR5-independent mechanisms.

A user-friendly tool for cloud-based whole slide image segmentation with examples from renal histopathology.

Background: Image-based machine learning tools hold great promise for clinical applications in pathology research. However, the ideal end-users of these computational tools (e.g., pathologists and biological scientists) often lack the programming experience required for the setup and use of these tools which often rely on the use of command line interfaces. Methods: We have developed Histo-Cloud, a tool for segmentation of whole slide images (WSIs) that has an easy-to-use graphical user interface. This tool runs a state-of-the-art convolutional neural network (CNN) for segmentation of WSIs in the cloud and allows the extraction of features from segmented regions for further analysis. Results: By segmenting glomeruli, interstitial fibrosis and tubular atrophy, and vascular structures from renal and non-renal WSIs, we demonstrate the scalability, best practices for transfer learning, and effects of dataset variability. Finally, we demonstrate an application for animal model research, analyzing glomerular features in three murine models. Conclusions: Histo-Cloud is open source, accessible over the internet, and adaptable for segmentation of any histological structure regardless of stain.

PodoCount: A Robust, Fully Automated, Whole-Slide Podocyte Quantification Tool.

Introduction: Podocyte depletion is a histomorphologic indicator of glomerular injury and predicts clinical outcomes. Podocyte estimation methods or podometrics are semiquantitative, technically involved, and laborious. Implementation of high-throughput podometrics in experimental and clinical workflows necessitates an automated podometrics pipeline. Recognizing that computational image analysis offers a robust approach to study cell and tissue structure, we developed and validated PodoCount (a computational tool for automated podocyte quantification in immunohistochemically labeled tissues) using a diverse data set. Methods: Whole-slide images (WSIs) of tissues immunostained with a podocyte nuclear marker and periodic acid-Schiff counterstain were acquired. The data set consisted of murine whole kidney sections (n = 135) from 6 disease models and human kidney biopsy specimens from patients with diabetic nephropathy (DN) (n = 45). Within segmented glomeruli, podocytes were extracted and image analysis was applied to compute measures of podocyte depletion and nuclear morphometry. Computational performance evaluation and statistical testing were performed to validate podometric and associated image features. PodoCount was disbursed as an open-source, cloud-based computational tool. Results: PodoCount produced highly accurate podocyte quantification when benchmarked against existing methods. Podocyte nuclear profiles were identified with 0.98 accuracy and segmented with 0.85 sensitivity and 0.99 specificity. Errors in podocyte count were bounded by 1 podocyte per glomerulus. Podocyte-specific image features were found to be significant predictors of disease state, proteinuria, and clinical outcome. Conclusion: PodoCount offers high-performance podocyte quantitation in diverse murine disease models and in human kidney biopsy specimens. Resultant features offer significant correlation with associated metadata and outcome. Our cloud-based tool will provide end users with a standardized approach for automated podometrics from gigapixel-sized WSIs.

Empagliflozin Treatment Attenuates Hepatic Steatosis by Promoting White Adipose Expansion in Obese TallyHo Mice.

Sodium-glucose co-transporters (SGLTs) serve to reabsorb glucose in the kidney. Recently, these transporters, mainly SGLT2, have emerged as new therapeutic targets for patients with diabetes and kidney disease; by inhibiting glucose reabsorption, they promote glycosuria, weight loss, and improve glucose tolerance. They have also been linked to cardiac protection and mitigation of liver injury. However, to date, the mechanism(s) by which SGLT2 inhibition promotes systemic improvements is not fully appreciated. Using an obese TallyHo mouse model which recapitulates the human condition of diabetes and nonalcoholic fatty liver disease (NAFLD), we sought to determine how modulation of renal glucose handling impacts liver structure and function. Apart from an attenuation of hyperglycemia, Empagliflozin was found to decrease circulating triglycerides and lipid accumulation in the liver in male TallyHo mice. This correlated with lowered hepatic cholesterol esters. Using in vivo MRI analysis, we further determined that the reduction in hepatic steatosis in male TallyHo mice was associated with an increase in nuchal white fat indicative of "healthy adipose expansion". Notably, this whitening of the adipose came at the expense of brown adipose tissue. Collectively, these data indicate that the modulation of renal glucose handling has systemic effects and may be useful as a treatment option for NAFLD and steatohepatitis.

Endothelial Cell-Specific Molecule-1 Inhibits Albuminuria in Diabetic Mice.

Background: Diabetic kidney disease (DKD) is the most common cause of kidney failure in the world, and novel predictive biomarkers and molecular mechanisms of disease are needed. Endothelial cell-specific molecule-1 (Esm-1) is a secreted proteoglycan that attenuates inflammation. We previously identified that a glomerular deficiency of Esm-1 associates with more pronounced albuminuria and glomerular inflammation in DKD-susceptible relative to DKD-resistant mice, but its contribution to DKD remains unexplored. Methods: Using hydrodynamic tail-vein injection, we overexpress Esm-1 in DKD-susceptible DBA/2 mice and delete Esm-1 in DKD-resistant C57BL/6 mice to study the contribution of Esm-1 to DKD. We analyze clinical indices of DKD, leukocyte infiltration, podocytopenia, and extracellular matrix production. We also study transcriptomic changes to assess potential mechanisms of Esm-1 in glomeruli. Results: In DKD-susceptible mice, Esm-1 inversely correlates with albuminuria and glomerular leukocyte infiltration. We show that overexpression of Esm-1 reduces albuminuria and diabetes-induced podocyte injury, independent of changes in leukocyte infiltration. Using a complementary approach, we find that constitutive deletion of Esm-1 in DKD-resistant mice modestly increases the degree of diabetes-induced albuminuria versus wild-type controls. By glomerular RNAseq, we identify that Esm-1 attenuates expression of kidney disease-promoting and interferon (IFN)-related genes, including Ackr2 and Cxcl11. Conclusions: We demonstrate that, in DKD-susceptible mice, Esm-1 protects against diabetes-induced albuminuria and podocytopathy, possibly through select IFN signaling. Companion studies in patients with diabetes suggest a role of Esm-1 in human DKD.

Nuclear Receptors and Transcription Factors in Obesity-Related Kidney Disease.

Both obesity and chronic kidney disease are increasingly common causes of morbidity and mortality worldwide. Although obesity often co-exists with diabetes and hypertension, it has become clear over the past several decades that obesity is an independent cause of chronic kidney disease, termed obesity-related glomerulopathy. This review defines the attributes of obesity-related glomerulopathy and describes potential pharmacologic interventions. Interventions discussed include peroxisome proliferator-activated receptors, the farnesoid X receptor, the Takeda G-protein-coupled receptor 5, and the vitamin D receptor.

Sacubitril/valsartan treatment has differential effects in modulating diabetic kidney disease in db/db mice and KKAy mice compared with valsartan treatment.

Although renin-angiotensin blockade has shown beneficial outcomes in patients with diabetes, renal injury progresses in most of these patients. Therefore, there remains a need for new therapeutic targets in diabetic kidney disease. Enhancement of vasoactive peptides, such as natriuretic peptides, via neprilysin inhibition, has been a new approach. A first-in-class drug, sacubitril/valsartan (Sac/Val), a combination of the angiotensin II receptor blocker Val and neprilysin inhibitor prodrug Sac, has been shown to be more effective than renin-angiotensin blockade alone in the treatment of heart failure with reduced ejection fraction. In this study, we tested the effects of Sac/Val in diabetic kidney disease. We administered Sac/Val or Val to two type 2 diabetes mouse models, db/db mice or KKAy mice. After 3 mo of treatment, Sac/Val attenuated the progression of proteinuria, glomerulosclerosis, and podocyte loss in both models of diabetic mice. Val shared a similar improvement but to a lesser degree in some parameters compared with Sac/Val. Sac/Val but not Val decreased the blood glucose level in KKAy mice. Sac/Val exerted renal protection through coordinated effects on antioxidative stress and anti-inflammation. In both diabetic models, we revealed a new mechanism to cause inflammation, self-DNA-activated cGMP-AMP synthase-stimulator of interferon genes (cGAS-STING) signaling, which was activated in diabetic kidneys and prevented by Sac/Val or Val treatment. The present data suggest that Sac/Val has sufficient therapeutical potential to counter the pathophysiological effects of diabetic kidney disease, and its effectiveness could be better than Val alone.NEW & NOTEWORTHY The first-in-class drug sacubitril/valsartan, a combination of the angiotensin II receptor blocker valsartan and neprilysin inhibitor sacubitril, was tested for its effects in diabetic kidney disease using db/db mice and KKAy mice. We found that Sac/Val has sufficient therapeutical potential to counter the pathophysiological effects of diabetic kidney disease. We further revealed a new mechanism to cause inflammation, self-DNA-activated cGAS-STING signaling, which was activated in diabetic kidneys and prevented by sacubitril/valsartan or valsartan treatment.

Bile acid sequestration reverses liver injury and prevents progression of nonalcoholic steatohepatitis in Western diet-fed mice.

Nonalcoholic fatty liver disease is a rapidly rising problem in the 21st century and is a leading cause of chronic liver disease that can lead to end-stage liver diseases, including cirrhosis and hepatocellular cancer. Despite this rising epidemic, no pharmacological treatment has yet been established to treat this disease. The rapidly increasing prevalence of nonalcoholic fatty liver disease and its aggressive form, nonalcoholic steatohepatitis (NASH), requires novel therapeutic approaches to prevent disease progression. Alterations in microbiome dynamics and dysbiosis play an important role in liver disease and may represent targetable pathways to treat liver disorders. Improving microbiome properties or restoring normal bile acid metabolism may prevent or slow the progression of liver diseases such as NASH. Importantly, aberrant systemic circulation of bile acids can greatly disrupt metabolic homeostasis. Bile acid sequestrants are orally administered polymers that bind bile acids in the intestine, forming nonabsorbable complexes. Bile acid sequestrants interrupt intestinal reabsorption of bile acids, decreasing their circulating levels. We determined that treatment with the bile acid sequestrant sevelamer reversed the liver injury and prevented the progression of NASH, including steatosis, inflammation, and fibrosis in a Western diet-induced NASH mouse model. Metabolomics and microbiome analysis revealed that this beneficial effect is associated with changes in the microbiota population and bile acid composition, including reversing microbiota complexity in cecum by increasing Lactobacillus and decreased Desulfovibrio The net effect of these changes was improvement in liver function and markers of liver injury and the positive effects of reversal of insulin resistance.

The Sodium-Glucose Cotransporter 2 Inhibitor Dapagliflozin Prevents Renal and Liver Disease in Western Diet Induced Obesity Mice.

Obesity and obesity related kidney and liver disease have become more prevalent over the past few decades, especially in the western world. Sodium-glucose cotransporter 2 (SGLT2) inhibitors are a new class of antidiabetic agents with promising effects on cardiovascular and renal function. Given SGLT2 inhibitors exert both anti-diabetic and anti-obesity effects by promoting urinary excretion of glucose and subsequent caloric loss, we investigated the effect of the highly selective renal SGLT2 inhibitor dapagliflozin in mice with Western diet (WD) induced obesity. Low fat (LF) diet or WD-fed male C57BL/6J mice were treated with dapagliflozin for 26 weeks. Dapagliflozin attenuated the WD-mediated increases in body weight, plasma glucose and plasma triglycerides. Treatment with dapagliflozin prevented podocyte injury, glomerular pathology and renal fibrosis determined by second harmonic generation (SHG), nephrin, synaptopodin, collagen IV, and fibronectin immunofluorescence microscopy. Oil Red O staining showed dapagliflozin also decreased renal lipid accumulation associated with decreased SREBP-1c mRNA abundance. Moreover, renal inflammation and oxidative stress were lower in the dapagliflozin-treated WD-fed mice than in the untreated WD-fed mice. In addition, dapagliflozin decreased serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), hepatic lipid accumulation as determined by H&E and Oil Red O staining, and Coherent Anti-Stokes Raman Scattering (CARS) microscopy, and hepatic fibrosis as determined by picrosirius red (PSR) staining and TPE-SHG microscopy in WD-fed mice. Thus, our study demonstrated that the co-administration of the SGLT2 inhibitor dapagliflozin attenuates renal and liver disease during WD feeding of mice.

FXR/TGR5 Dual Agonist Prevents Progression of Nephropathy in Diabetes and Obesity.

Bile acids are ligands for the nuclear hormone receptor farnesoid X receptor (FXR) and the G protein-coupled receptor TGR5. We have shown that FXR and TGR5 have renoprotective roles in diabetes- and obesity-related kidney disease. Here, we determined whether these effects are mediated through differential or synergistic signaling pathways. We administered the FXR/TGR5 dual agonist INT-767 to DBA/2J mice with streptozotocin-induced diabetes, db/db mice with type 2 diabetes, and C57BL/6J mice with high-fat diet-induced obesity. We also examined the individual effects of the selective FXR agonist obeticholic acid (OCA) and the TGR5 agonist INT-777 in diabetic mice. The FXR agonist OCA and the TGR5 agonist INT-777 modulated distinct renal signaling pathways involved in the pathogenesis and treatment of diabetic nephropathy. Treatment of diabetic DBA/2J and db/db mice with the dual FXR/TGR5 agonist INT-767 improved proteinuria and prevented podocyte injury, mesangial expansion, and tubulointerstitial fibrosis. INT-767 exerted coordinated effects on multiple pathways, including stimulation of a signaling cascade involving AMP-activated protein kinase, sirtuin 1, PGC-1α, sirtuin 3, estrogen-related receptor-α, and Nrf-1; inhibition of endoplasmic reticulum stress; and inhibition of enhanced renal fatty acid and cholesterol metabolism. Additionally, in mice with diet-induced obesity, INT-767 prevented mitochondrial dysfunction and oxidative stress determined by fluorescence lifetime imaging of NADH and kidney fibrosis determined by second harmonic imaging microscopy. These results identify the renal signaling pathways regulated by FXR and TGR5, which may be promising targets for the treatment of nephropathy in diabetes and obesity.

Serelaxin improves cardiac and renal function in DOCA-salt hypertensive rats.

Serelaxin, a recombinant form of the naturally occurring peptide hormone relaxin-2, is a pleiotropic vasodilating hormone that has been studied in patients with acute heart failure. In this study, the effects of serelaxin on cardiac and renal function, fibrosis, inflammation and lipid accumulation were studied in DOCA-salt treated rats. Uninephrectomized rats were assigned to two groups: controls provided with normal drinking water and DOCA provided with DOCA pellets and sodium chloride drinking water. After 4 weeks, the DOCA-salt rats were randomly selected and implanted with osmotic minipumps delivering vehicle or serelaxin for another 4 weeks. Treatment with serelaxin prevented cardiac and renal dysfunction in DOCA-salt rats. Serelaxin prevented cardiac and renal fibrosis, as determined by Picrosirius Red staining and Second Harmonic Generation (SHG) Microscopy. Treatment of DOCA-salt rats with serelaxin decreased renal inflammation, including the expression of TGF-ß, NFκB, MCP-1, IL-1, IL-6, ICAM-1, VCAM-1 and CD68 macrophages. Serelaxin also decreased lipid accumulation in kidney in part by decreasing SREBP-1c, SREBP-2, ChREBP, FATP1, HMGCoAR, and LDL receptor, and increasing Acox1 and ABCA1. In summary, serelaxin reversed DOCA-salt induced cardiac and renal dysfunction.

SGLT2 Protein Expression Is Increased in Human Diabetic Nephropathy: SGLT2 PROTEIN INHIBITION DECREASES RENAL LIPID ACCUMULATION, INFLAMMATION, AND THE DEVELOPMENT OF NEPHROPATHY IN DIABETIC MICE.

There is very limited human renal sodium gradient-dependent glucose transporter protein (SGLT2) mRNA and protein expression data reported in the literature. The first aim of this study was to determine SGLT2 mRNA and protein levels in human and animal models of diabetic nephropathy. We have found that the expression of SGLT2 mRNA and protein is increased in renal biopsies from human subjects with diabetic nephropathy. This is in contrast to db-db mice that had no changes in renal SGLT2 protein expression. Furthermore, the effect of SGLT2 inhibition on renal lipid content and inflammation is not known. The second aim of this study was to determine the potential mechanisms of beneficial effects of SGLT2 inhibition in the progression of diabetic renal disease. We treated db/db mice with a selective SGLT2 inhibitor JNJ 39933673. We found that SGLT2 inhibition caused marked decreases in systolic blood pressure, kidney weight/body weight ratio, urinary albumin, and urinary thiobarbituric acid-reacting substances. SGLT2 inhibition prevented renal lipid accumulation via inhibition of carbohydrate-responsive element-binding protein-ß, pyruvate kinase L, SCD-1, and DGAT1, key transcriptional factors and enzymes that mediate fatty acid and triglyceride synthesis. SGLT2 inhibition also prevented inflammation via inhibition of CD68 macrophage accumulation and expression of p65, TLR4, MCP-1, and osteopontin. These effects were associated with reduced mesangial expansion, accumulation of the extracellular matrix proteins fibronectin and type IV collagen, and loss of podocyte markers WT1 and synaptopodin, as determined by immunofluorescence microscopy. In summary, our study showed that SGLT2 inhibition modulates renal lipid metabolism and inflammation and prevents the development of nephropathy in db/db mice.

Intestinal Depletion of NaPi-IIb/Slc34a2 in Mice: Renal and Hormonal Adaptation.

The Na(+) -dependent phosphate-cotransporter NaPi-IIb (SLC34A2) is widely expressed, with intestine, lung, and testis among the organs with highest levels of mRNA abundance. In mice, the intestinal expression of NaPi-IIb is restricted to the ileum, where the cotransporter localizes specifically at the brush border membrane (BBM) and mediates the active transport of inorganic phosphate (Pi). Constitutive full ablation of NaPi-IIb is embryonically lethal whereas the global but inducible removal of the transporter in young mice leads to intestinal loss of Pi and lung calcifications. Here we report the generation of a constitutive but intestinal-specific NaPi-IIb/Slc34a2-deficient mouse model. Constitutive intestinal ablation of NaPi-IIb results in viable pups with normal growth. Homozygous mice are characterized by fecal wasting of Pi and complete absence of Na/Pi cotransport activity in BBM vesicles (BBMVs) isolated from ileum. In contrast, the urinary excretion of Pi is reduced in these animals. The plasma levels of Pi are similar in wild-type and NaPi-IIb-deficient mice. In females, the reduced phosphaturia associates with higher expression of NaPi-IIa and higher Na/Pi cotransport activity in renal BBMVs, as well as with reduced plasma levels of intact FGF-23. A similar trend is found in males. Thus, NaPi-IIb is the only luminal Na(+) -dependent Pi transporter in the murine ileum and its absence is fully compensated for in adult females by a mechanism involving the bone-kidney axis. The contribution of this mechanism to the adaptive response is less apparent in adult males.