Inactivation of a non-canonical gp130 signaling arm attenuates chronic systemic inflammation and multimorbidity induced by a high-fat diet.

Interleukin-6 (IL-6) is a major pro-inflammatory cytokine for which the levels in plasma demonstrate a robust correlation with age and body mass index (BMI) as part of the senescence-associated secretory phenotype. IL-6 cytokines also play a crucial role in metabolic homeostasis and regenerative processes, primarily via the canonical STAT3 pathway. Thus, selective modulation of IL-6 signaling may offer a unique opportunity for therapeutic interventions. Recently, we discovered that a non-canonical signaling pathway downstream of tyrosine (Y) 814 within the intracellular domain of gp130, the IL-6 co-receptor, is responsible for the recruitment and activation of SRC family of kinases (SFK). Mice with constitutive genetic inactivation of gp130 Y814 (F814 mice) show accelerated resolution of inflammatory response and superior regenerative outcomes in skin wound healing and posttraumatic models of osteoarthritis. The current study was designed to explore if selective genetic or pharmacological inhibition of the non-canonical gp130-Y814/SFK signaling reduces systemic chronic inflammation and multimorbidity in a high-fat diet (HFD)-induced model of accelerated aging. F814 mice showed significantly reduced inflammatory response to HFD in adipose and liver tissue, with significantly reduced levels of systemic inflammation compared to wild type mice. F814 mice were also protected from HFD-induced bone loss and cartilage degeneration. Pharmacological inhibition of gp130-Y814/SFK in mice on HFD mirrored the effects observed in F814 mice on HFD; furthermore, this pharmacological treatment also demonstrated a marked increase in physical activity levels and protective effects against inflammation-associated suppression of neurogenesis in the brain tissue compared to the control group. These findings suggest that selective inhibition of SFK signaling downstream of gp130 receptor represents a promising strategy to alleviate systemic chronic inflammation. Increased degenerative changes and tissue senescence are inevitable in obese and aged organisms, but we demonstrated that the systemic response and inflammation-associated multi-morbidity can be therapeutically mitigated.

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.

Clinicopathologic and Proteomic Analysis of Amyloidomas Involving the Ocular Surface and Adnexa.

OBJECTIVES: Ocular amyloidoma is a rare disorder characterized by deposition of insoluble proteinaceous fibrils in the extracellular space of the ocular adnexa. This study details the clinicopathologic features and proteomic characteristics of periocular amyloid deposition. METHODS: Specimens (1991-2020) were retrieved and reviewed. All available H&E slides and special stains were reviewed. Proteomic analysis was performed using immunohistochemistry (IHC) for IgG, IgG4, IgA, IgD, IgM, CD20, CD3, CD138, and κ/λ, as well as chromatography-electrospray tandem mass spectrometry on formalin-fixed, paraffin-embedded tissue. RESULTS: There were 14 patients (7 men, 7 women). The depositions involved eyelid (n = 3), conjunctiva (n = 8), and orbit (n = 3). All patients were adults with a median age at diagnosis of 56 (range, 39-88) years. The deposits were predominantly λ light chain restricted (n = 6) and mixed light chains (n = 2), and one case was κ predominant. Two of the cases with a mixture of κ and λ light chains had an excess of transthyretin by mass spectrometry. Four of the cases did not have adequate material for proteomic subtyping. CONCLUSIONS: Amyloidomas involving ocular adnexa contain a variety of amyloid-related and immunoglobulin-associated peptides. The λ light chain predominates as in other body sites, but mixed patterns and rarely κ light chain restriction may be encountered.

Microvascular inflammation in renal allograft biopsies assessed by endothelial and leukocyte co-immunostain: a retrospective study on reproducibility and clinical/prognostic correlates.

The most prominent histologic lesion in antibody-mediated rejection is microvascular inflammation (MVI); however, its recognition and scoring can be challenging and poorly reproducible between pathologists. We developed a dual immunohistochemical (IHC)-stain (anti-CD34/anti-CD45 for endothelium/leukocytes) as ancillary tool to improve on the semi-quantitative Banff scores and allow quantification of MVI. We examined the relationship between CD34-CD45 IHC-based quantitative MVI score (the inflamed peritubular capillary ratio, iptcr) and renal-graft failure or donor-specific antibodies (DSA) strength at the time of biopsy. Quantitative iptcr score was significantly associated with renal graft failure (hazard ratio 1.81, per 1 SD-unit [0.13 points] of iptcr-increase; P = 0.026) and predicted the presence and strength of DSA (ordinal odds ratio: 2.42; P = 0.005; 75 biopsies/60 kidney transplant recipients; 30 HLA- and/or ABO-incompatible). Next, we assessed inter-pathologist agreement for ptc score and ptc extent (focal/diffuse) using CD34-CD45 IHC as compared to conventional stain. Compared to conventional stain, CD34-CD45 IHC significantly increased inter-pathologist agreement on ptc score severity and extent (κ-coefficient from 0.52-0.80 and 0.46-0.68, respectively, P < 0.001). Our findings show that CD34-CD45 IHC improves reproducibility of MVI scoring and facilitates MVI quantification and introduction of a dual anti-CD34/CD45 has the potential to improve recognition of MVI ahead of DSA results.

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.

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.

G Protein-Coupled Bile Acid Receptor TGR5 Activation Inhibits Kidney Disease in Obesity and Diabetes.

Obesity and diabetes mellitus are the leading causes of renal disease. In this study, we determined the regulation and role of the G protein-coupled bile acid receptor TGR5, previously shown to be regulated by high glucose and/or fatty acids, in obesity-related glomerulopathy (ORG) and diabetic nephropathy (DN). Treatment of diabetic db/db mice with the selective TGR5 agonist INT-777 decreased proteinuria, podocyte injury, mesangial expansion, fibrosis, and CD68 macrophage infiltration in the kidney. INT-777 also induced renal expression of master regulators of mitochondrial biogenesis, inhibitors of oxidative stress, and inducers of fatty acid ß-oxidation, including sirtuin 1 (SIRT1), sirtuin 3 (SIRT3), and Nrf-1. Increased activity of SIRT3 was evidenced by normalization of the increased acetylation of mitochondrial superoxide dismutase 2 (SOD2) and isocitrate dehydrogenase 2 (IDH2) observed in untreated db/db mice. Accordingly, INT-777 decreased mitochondrial H2O2 generation and increased the activity of SOD2, which associated with decreased urinary levels of H2O2 and thiobarbituric acid reactive substances. Furthermore, INT-777 decreased renal lipid accumulation. INT-777 also prevented kidney disease in mice with diet-induced obesity. In human podocytes cultured with high glucose, INT-777 induced mitochondrial biogenesis, decreased oxidative stress, and increased fatty acid ß-oxidation. Compared with normal kidney biopsy specimens, kidney specimens from patients with established ORG or DN expressed significantly less TGR5 mRNA, and levels inversely correlated with disease progression. Our results indicate that TGR5 activation induces mitochondrial biogenesis and prevents renal oxidative stress and lipid accumulation, establishing a role for TGR5 in inhibiting kidney disease in obesity and diabetes.