Fructose Metabolism and Disease Mechanisms: From Nutritional Excess to Obesity and Multiorgan Pathophysiology.

Key pointsIncreased activation of the renin‐angiotensin‐aldosterone system (RAAS) and elevated growth factor production are of crucial importance in the development of renal fibrosis leading to diabetic kidney disease.The aim of this study was to provide evidence for the antifibrotic potential of RAAS inhibitor (RAASi) treatment and to explore the exact mechanism of this protective effect.We found that RAASi ameliorate diabetes‐induced renal interstitial fibrosis and decrease profibrotic growth factor production.RAASi prevents fibrosis by acting directly on proximal tubular cells, and inhibits hyperglycaemia‐induced growth factor production and thereby fibroblast activation.These results suggest a novel therapeutic indication and potential of RAASi in the treatment of renal fibrosis.In diabetic kidney disease (DKD) increased activation of renin‐angiotensin‐aldosterone system (RAAS) contributes to renal fibrosis. Although RAAS inhibitors (RAASi) are the gold standard therapy in DKD, the mechanism of their antifibrotic effect is not yet clarified. Here we tested the antifibrotic and renoprotective action of RAASi in a rat model of streptozotocin‐induced DKD. In vitro studies on proximal tubular cells and renal fibroblasts were also performed to further clarify the signal transduction pathways that are directly altered by hyperglycaemia. After 5 weeks of diabetes, male Wistar rats were treated for two more weeks per os with the RAASi ramipril, losartan, spironolactone or eplerenone. Proximal tubular cells were cultured in normal or high glucose (HG) medium and treated with RAASi. Platelet‐derived growth factor (PDGF) or connective tissue growth factor (CTGF/CCN2)‐induced renal fibroblasts were also treated with various RAASi. In diabetic rats, reduced renal function and interstitial fibrosis were ameliorated and elevated renal profibrotic factors (TGFβ1, PDGF, CTGF/CCN2, MMP2, TIMP1) and alpha‐smooth muscle actin (αSMA) levels were decreased by RAASi. HG increased growth factor production of HK‐2 cells, which in turn induced activation and αSMA production of fibroblasts. RAASi decreased tubular PDGF and CTGF expression and reduced production of extracellular matrix (ECM) components in fibroblasts. In proximal tubular cells, hyperglycaemia‐induced growth factor production increased renal fibroblast transformation, contributing to the development of fibrosis. RAASi, even in non‐antihypertensive doses, decreased the production of profibrotic factors and directly prevented fibroblast activation. All these findings suggest a novel therapeutic role for RAASi in the treatment of renal fibrosis.

Fructose Takes a Toll† ‡

ABSTRACTCholera is caused by infection with Vibrio cholerae. This study aimed to investigate the pathophysiology of diarrhea caused by the V. cholerae O1 El Tor variant (EL), a major epidemic strain causing severe diarrhea in several regions. In the ligated ileal loop model of EL-induced diarrhea in the ICR mice, a cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor and a calcium-activated chloride channel (CaCC) inhibitor similarly inhibited intestinal fluid secretion. In addition, barrier disruption and NF-κB-mediated inflammatory responses, e.g., iNOS and COX-2 expression, were observed in the infected ileal loops. Interestingly, intestinal fluid secretion and barrier disruption were suppressed by NF-κB and COX-2 inhibitors, whereas an iNOS inhibitor suppressed barrier disruption without affecting fluid secretion. Furthermore, EP2 and EP4 PGE2 receptor antagonists ameliorated the fluid secretion in the infected ileal loops. The amount of cholera toxin (CT) produced in the ileal loops by the EL was ∼2.4-fold of the classical biotype. The CT transcription inhibitor virstatin, a toll-like receptor-4 (TLR-4) antibody and a CT antibody suppressed the EL-induced intestinal fluid secretion, barrier disruption and COX-2 expression. The CT at levels detected during EL infection induced mild intestinal barrier disruption without inducing inflammatory responses in mouse intestine. Collectively, this study indicates that CT-induced intestinal barrier disruption and subsequent TLR-4-NF-κB-mediated COX-2 expression are involved in the pathogenesis of EL-induced diarrhea and represent promising novel therapeutic targets of cholera.

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Renal fibrosis is a major pathological event in the development of diabetic nephropathy (DN). Baicalin is a flavonoid glycoside that possesses multiple pharmacological properties including anti-fibrotic activity. In the present study, the effects of baicalin on renal fibrosis along with related molecular basis were investigated in streptozotocin (STZ)-induced DN mouse model and high glucose (HG)-treated HK-2 human proximal tubule epithelial cell model. Renal injury was evaluated through blood urea nitrogen (BUN) and serum creatinine (Scr) levels and urine albumin creatine ratio (ACR). Renal fibrosis was assessed by type IV collagen (COLIV) and fibronectin (FN) protein expression and histopathologic analysis via Masson trichrome staining. Protein levels of COLIV, FN, NF-κB inhibitor alpha (IκBα), phosphorylated IκBα (p-IκBα), p65, phosphorylated p65 (p-p65), and toll-like receptor 4 (TLR4) were measured by western blot assay. MicroRNA-124 (miR-124) and TLR4 mRNA levels were detected by RT-qPCR assay. The interaction of miR-124 and TLR4 was examined by bioinformatics analysis, luciferase reporter assay, and RIP assay. Baicalin or miR-124 attenuated renal injury and fibrosis in STZ-induced DN mice. Baicalin inhibited the increase of COLIV and FN expression induced by HG through upregulating miR-124 in HK-2 cells. TLR4 was a target of miR-124. MiR-124 inhibited TLR4/NF-κB pathway activation and the inactivation of the NF-κB pathway hindered COLIV and FN expression in HG-stimulated HK-2 cells. Baicalin prevented renal fibrosis by increasing miR-124 and inactivating downstream TLR4/NF-κB pathway in DN, hinting the pivotal values of baicalin and miR-124 in the management of DN and renal fibrosis.

Tissue fibrosis and chronic inflammation are common causes of progressive organ damage, including progressive renal disease, leading to loss of physiological functions. Recently, it was shown that Toll-like receptor 2 (TLR2) is expressed in the kidney and activated by endogenous danger signals. The expression and function of TLR2 during renal fibrosis and chronic inflammation has however not yet been elucidated. Therefore, we studied TLR2 expression in human and murine progressive renal diseases and explored its role by inducing obstructive nephropathy in TLR2−/− or TLR2+/+ mice. We found that TLR2 is markedly upregulated on tubular and tubulointerstitial cells in patients with chronic renal injury. In mice with obstructive nephropathy, renal injury was associated with a marked upregulation and change in distribution of TLR2 and upregulation of murine TLR2 danger ligands Gp96, biglycan, and HMGB1. Notably, TLR2 enhanced inflammation as reflected by a significantly reduced influx of neutrophils and production of chemokines and TGF-β in kidneys of TLR2−/− mice compared with TLR2+/+ animals. Although, the obstructed kidneys of TLR2−/− mice had less interstitial myofibroblasts in the later phase of obstructive nephropathy, tubular injury and renal matrix accumulation was similar in both mouse strains. Together, these data demonstrate that TLR2 can initiate renal inflammation during progressive renal injury and that the absence of TLR2 does not affect the development of chronic renal injury and fibrosis.

Mitochondria were first described in 1840 as bioblasts, elementary organisms responsible for vital cellular functions, but were subsequently named mitochondria, from the Greek names mitos (thread) and chondros (granule), which describes their appearance during spermatogenesis.1 Their discovery generated substantial interest given their structure resembling bacteria, which led in subsequent years to important scientific discoveries positioning mitochondria as the energy powerhouse of the cell. The unique architecture of mitochondria, consisting of 2 membranes (outer and inner) and compartments (intermembrane space and matrix), is crucial for their vital functions. Mitochondria serve not only as primary sources of cellular energy, but also modulate several cellular processes, including oxidative phosphorylation, calcium homeostasis, thermogenesis, oxygen sensing, proliferation, and apoptosis.2 Therefore, mitochondrial injury and dysfunction might be implicated in the pathogenesis of several diseases. Hypertension accounts for nearly 30% of patients reaching end-stage renal disease.3 Renal injury secondary to hypertension or to ischemia associated with renovascular hypertension (distal to renal artery stenosis) may have significant and detrimental effect on health outcomes. Studies have highlighted several deleterious pathways, including inflammation, oxidative stress, and fibrosis that are activated in the hypertensive kidney, eliciting functional decline.4,5 However, the precise molecular mechanisms responsible for renal injury have not been fully elucidated. Over the past few years, increasing evidence has established the experimental foundations linking mitochondrial alterations to hypertensive renal injury (Table). Mitochondriopathies, abnormalities of energy metabolism secondary to sporadic or inherited mutations in nuclear or mitochondrial DNA (mtDNA) genes, may contribute to the development and progression of hypertension and its complications. In addition, several studies have reported mitochondrial damage and dysfunction consequent to hypertensive renal disease. View this table: Table. Evidence of Renal Mitochondrial Damage in Models of Hypertension and Antihypertensive Treatment Importantly, hypertensive-induced renal injury is characterized by activation of several deleterious pathways, including oxidative stress, renin–angiotensin–aldosterone …

The Selective A3AR Antagonist LJ-1888 Ameliorates UUO-Induced Tubulointerstitial Fibrosis

Maternal Exposure to High Fructose and Offspring Health.

Background/Aims:There has been controversy about the role of Toll-like receptor 2 (TLR2) in renal injury following ureteric obstruction. Although inhibition of the renin angiotensin system (RAS) reduces TLR2 expression in mice, the exact relationship between TLR2 and RAS is not known. The aim of this study was to determine whether the RAS modulates TLR2.Methods:We used 8-week-old male wild type (WT) and TLR2-knockout (KO) mice on a C57Bl/6 background. Unilateral ureteral obstruction (UUO) was induced by complete ligation of the left ureter. Angiotensin (Ang) II (1,000 ng/kg/min) and the direct renin inhibitor aliskiren (25 mg/kg/day) were administrated to mice using an osmotic minipump. Molecular and histologic evaluations were performed.Results:Ang II infusion increased mRNA expression of TLR2 in WT mouse kidneys (p < 0.05). The expression of renin mRNA in TLR2-KO UUO kidneys was significantly higher than that in WT UUO kidneys (p < 0.05). There were no differences in tissue injury score or mRNA expression of monocyte chemotactic protein 1 (MCP-1), osteopontin (OPN), or transforming growth factor β (TGF-β) between TLR2-KO UUO and WT UUO kidneys. However, aliskiren decreased the tissue injury score and mRNA expression of TLR2, MCP-1, OPN, and TGF-β in WT UUO kidneys (p < 0.05). Aliskiren-treated TLR2-KO UUO kidneys showed less kidney injury than aliskiren-treated WT UUO kidneys.Conclusions:TLR2 deletion induced activation of the RAS in UUO kidneys. Moreover, inhibition of both RAS and TLR2 had an additive ameliorative effect on UUO injury of the kidney.

The renin-angiotensin system blockade does not prevent renal interstitial fibrosis induced by aristolochic acids

IntroductionThis study aimed to discuss the use of hydroxysafflor yellow A (HSYA) to improve renal fibrosis induced by diabetes and its relative mechanisms, as well as to evaluate the level of renal histopathology and fibrosis in different rat groups.Material and methodsSprague-Dawley (SD) rats (n = 27) were divided into the normal control (NC), diabetes mellitus (DM), and HSYA groups. The miRNA-140-5p mRNA, blood glucose (BG), 24-h urine protein (UP), total cholesterol (TC), triglyceride (TG), total anti-oxidant capacity (T-AOC), malondialdehyde (MDA), interleukin-6 (IL-6), and tumour necrosis factor-α (TNF-α) were measured in different groups. Moreover, the relative protein expression was measured via immunohistochemistry (IHC) assay. In the in vitro cell experiment, we discussed the effects of miRNA-140-5p on renal fibrosis induced by diabetes. Toll-like receptor 4 (TLR4), nuclear factor B (p65) (NF-κB(p65)), NOD-like receptor protein 3 (NLRP3), Notch2, and collagen IV (Col-IV) protein expression were evaluated using western blotting in the different cell groups. We evaluated the important protein expressions and NF-κB(p65) nuclear volume by cellular immunofluorescence. A correlation between miRNA-140-5p and TLR4 was found by dual-luciferase reporter assay.ResultsIn the in vivo study, HSYA improved diabetes-induced renal fibrosis. The severity of fibrosis significantly decreased after treatment with HSYA. In the HSYA group, the miRNA-140-5p mRNA, BG, 24-h UP, TC, TG, T-AOC, MDA, IL-6, and TNF-α significantly improved, as well as the TLR4, NF-κB(p65), NLRP3, Notch2, and Col-IV proteins. In the in vitro experiment, miRNA-140-5p was significantly decreased after treatment with HSYA in diabetes-induced renal fibrosis.ConclusionsHSYA improved diabetes-induced renal fibrosis by regulating miRNA-140-5p.

In chronic kidney disease, the activation of the renin-angiotensin-aldosterone system (RAAS) and renal inflammation stimulates renal fibrosis and the progression to end-stage renal disease. The low levels of vitamin D receptor (VDR) and its activators (VDRAs) contribute to worsen secondary hyperparathyroidism and renal fibrosis. The 7/8 nephrectomy model of experimental chronic renal failure (CRF) was used to examine the anti-fibrotic effects of treatment with two VDRAs, paricalcitol and calcitriol, at equivalent doses (3/1 dose ratio) during 4 weeks. CRF increased the activation of the RAAS, renal inflammation and interstitial fibrosis. Paricalcitol treatment reduced renal collagen I and renal interstitial fibrosis by decreasing the activation of the RAAS through renal changes in renin, angiotensin receptor 1 (ATR1) and ATR2 mRNAs levels and renal inflammation by decreasing renal inflammatory leucocytes (CD45), a desintegrin and metaloproteinase mRNA, transforming growth factor beta mRNA and protein, and maintaining E-cadherin mRNA levels. Calcitriol showed similar trends without significant changes in most of these biomarkers. Paricalcitol effectively attenuated the renal interstitial fibrosis induced by CRF through a combination of inhibitory actions on the RAAS, inflammation and epithelial/mesenchymal transition.

Toll-like receptors 2 (TLR2) and 4 (TLR4) are present in the plasma membrane of skeletal muscle cells where their functions remain incompletely resolved. They can bind various extracellular ligands, such as FSL-1, lipopolysaccharide (LPS) and/or palmitic acid (PA). We have investigated the link between PA, TLR2/4 and ribosomal S6 kinase 1 (S6K1) in C2C12 myotubes. Incubation with agonists of either TLR2 or TLR4, and with a high concentration of PA, increased S6K1 phosphorylation. Canonical upstream kinases of S6K1, protein kinase B (PKB) and mammalian target of rapamycin complex 1 (mTORC1), were regulated in the opposite way by PA, indicating that these kinases were probably not involved. By using the SB202190 inhibitor, p38 MAPK (mitogen-activated protein kinase) was found to be a key mediator of PA-induced phosphorylation of S6K1. Downregulation of either tlr2 or tlr4 gene expression by small interfering RNAs prevented the activation of both p38 MAPK and S6K1 by FSL-1, LPS or PA. Thus TLR2 and TLR4 agonists can increase the level of S6K1 phosphorylation in a p38 MAPK-dependent way in C2C12 myotubes. As PA induced the same intracellular signalling, a novel atypical pathway for PA is induced at the cellular membrane level and results in a higher phosphorylation state of S6K1.

Severe acute pancreatitis (SAP)-induced intestinal bacterial translocation and enterogenic infection are among the leading causes of mortality in patients. However, the mechanisms by which SAP disrupted the intestinal barrier and led to bacterial translocation remained unclear. Therefore, we employed multi-omics analysis including microbiome, metabolome, epigenome, transcriptome, and mass cytometry (CyTOF) to identify potential targets, followed by functional validation using transgenic mice. The integrated multi-omics analysis primarily indicated overgrowth of intestinal flagellated bacteria, upregulation of intestinal Toll-like receptor 5 (TLR5) and acute inflammatory response, and increased infiltration of intestinal high-expressing TLR5 lamina propria dendritic cells (TLR5hi LPDC) after SAP. Subsequently, intestinal flagellin-TLR5 signaling was activated after SAP. Intestinal barrier disruption, bacterial translocation, and helper T cells (Th) differentiation imbalance caused by SAP were alleviated in TLR5 knocked out (Tlr5 −/−) or conditionally knocked out on LPDC (Tlr5 ΔDC) mice. However, TLR5 conditional knockout on intestinal epithelial cells (Tlr5 ΔIEC) failed to improve SAP-induced bacterial translocation. Moreover, depletion of LPDC and regulatory T cells (Treg) ameliorated bacterial translocation after SAP. Our findings identify TLR5 on LPDC as a potential novel target for preventing or treating intestinal bacterial translocation caused by SAP.