Key points
Increased 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.
Key points
Increased 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.
Lyophilization is a cost-effective method for biological specimen preservation but detailed tissue-specific reference protocols are still lacking. Moreover, data are limited on the long-term ...stability of proteins and nucleic acids in lyophilized samples.
Here, we offer lyophilization protocols for various rat and mouse tissues (kidney, heart, liver, lung, aorta, and skin) coupled with technical hints for optimal sample preparation. We demonstrate that lyophilized samples stored at 4 °C for 20 months can yield protein and RNA of similar quantity and quality to −80 °C storage, while phosphorylated proteins are preserved as well. Freeze-dried and subsequently pulverized samples can provide more consistent, more reliable data especially when investigating focal injuries, such as fibrosis. We developed a protocol for the concentration of biological solutions and achieved 20-times concentration in human peritoneal dialysis effluent solution which enables the previously unattainable detection of proteins in these samples. We established a method for water removal as well as accurate water content measurement of fecal samples, which can be valuable for gut metabolome analysis.
Taken together, lyophilization is a valuable tool for the preservation of biological samples with many advantages. We aim to draw attention to the wide range of possibilities offered by freeze drying in pre-clinical or basic research.
Cardiovascular disease (CVD) is two to five times more prevalent in diabetic patients and is the leading cause of death. Therefore, identification of novel therapeutic strategies that reduce the risk ...of CVD is a research priority. Clinical trials showed that reduction in the relative risk of heart failure by sodium-glucose cotransporter 2 inhibitors (SGLT2i) are partly beyond their glucose lowering effects, however, the molecular mechanisms are still elusive. Here we investigated the role of SGLT2i dapagliflozin (DAPA) in the prevention of diabetes-induced cardiovascular complications.
Type 1 diabetes was induced with streptozotocin (65 mg/bwkg, ip.) in adult, male Wistar rats. Following the onset of diabetes rats were treated for six weeks with DAPA (1 mg/bwkg/day, po.).
DAPA decreased blood glucose levels (D: 37±2.7 vs. D+DAPA: 18±5.6 mmol/L; p<0.05) and prevented metabolic decline. Aortic intima-media thickening was mitigated by DAPA. DAPA abolished cardiac hypertrophy, and myocardial damage. Cardiac inflammation and fibrosis were also moderated after DAPA treatment.
These data support the preventive and protective role of SGLT2i in diabetes-associated cardiovascular disease. SGLT2i may provide novel therapeutic strategy to hinder the development of cardiovascular diseases in type 1 diabetes, thereby improve the outcomes.
Primary open-angle glaucoma remains a global issue, lacking a definitive treatment. Increased intraocular pressure (IOP) is considered the primary risk factor of the disease and it can be caused by ...fibrotic-like changes in the trabecular meshwork (TM) such as increased tissue stiffness and outflow resistance. Previously, we demonstrated that the sigma-1 receptor (S1R) agonist fluvoxamine (FLU) has anti-fibrotic properties in the kidney and lung. In this study, the localization of the S1R in TM cells was determined, and the anti-fibrotic efficacy of FLU was examined in both mouse and human TM cells. Treatment with FLU reduced the F-actin rearrangement, inhibited cell proliferation and migration induced by the platelet-derived growth factor and decreased the levels of fibrotic proteins. The protective role of the S1R in fibrosis was confirmed by a more pronounced increase in alpha smooth muscle actin and F-actin bundle and clump formation in primary mouse S1R knockout TM cells. Furthermore, FLU demonstrated its protective effects by increasing the production of nitric oxide and facilitating the degradation of the extracellular matrix through the elevation of cathepsin K. These findings suggest that the S1R could be a novel target for the development of anti-fibrotic drugs and offer a new therapeutic approach for glaucoma.
Rationale
Depression is highly prevalent in diabetes (DM).
Brain-derived neurotrophic factor
(BDNF) which is mainly regulated by the endoplasmic reticulum chaperon sigma-1 receptor (S1R) plays a ...relevant role in the development of depression.
Objectives
We studied the dose-dependent efficacy of S1R agonist fluvoxamine (FLU) in the prevention of DM-induced depression and investigated the significance of the S1R-BDNF pathway.
Methods
We used streptozotocin to induce DM in adult male rats that were treated for 2 weeks
p.o.
with either different doses of FLU (2 or 20 mg/bwkg) or FLU + S1R antagonist NE100 (1 mg/bwkg) or vehicle. Healthy controls were also enrolled. Metabolic, behaviour, and neuroendocrine changes were determined, and S1R and BDNF levels were measured in the different brain regions.
Results
In DM rats, immobility time was increased, adrenal glands were enlarged, and thymuses were involuted. FLU in 20 mg/bwkg, but not in 2 mg/bwkg dosage, ameliorated depression-like behaviour. S1R and BDNF protein levels were decreased in DM, while FLU induced SIR-BDNF production. NE100 suspended all effects of FLU.
Conclusions
We suggest that disturbed S1R-BDNF signaling in the brain plays a relevant role in DM-induced depression. The activation of this cascade serves as an additional target in the prevention of DM-associated depression.
Phagocytosis of naturally dying cells usually blocks inflammatory reactions in host cells. We have recently observed that clearance of cells dying through autophagy leads to a pro-inflammatory ...response in human macrophages. Investigating this response further, we found that during engulfment of MCF-7 or 293T cells undergoing autophagic death, but not apoptotic or anoikic ones, caspase-1 was activated and IL-1β was processed, then secreted in a MyD88-independent manner. Autophagic dying cells were capable of preventing some LPS-induced pro-inflammatory responses, such as TNFα, IL-6 and IL-8 induction, but synergized with LPS for IL-1β production. Caspase-1 inhibition prevented macrophage IL-1β release triggered by the dying cells and also other pro-inflammatory cytokines which were not formed in the presence of IL-1 receptor antagonist anakinra either. IL-1β secretion was also observed using calreticulin knock down or necrostatin treated autophagic MCF-7 cells and it required phagocytosis of the dying cells which led to ATP secretion from macrophages. Blocking K
+
efflux during phagocytosis, the presence of apyrase, adding an antagonist of the P2X7 receptor or silencing the NOD-like receptor protein NALP3 inhibited IL-1β secretion. These data suggest that during phagocytosis of autophagic dying cells ATP, acting through its receptor, initiates K
+
efflux, inflammasome activation and secretion of IL-1β, which initiates further pro-inflammatory events. Thus, autophagic death of malignant cells and their clearance may lead to immunogenic response.
The trabecular meshwork (TM) route is the principal outflow egress of the aqueous humor. Actin cytoskeletal remodeling in the TM and extracellular matrix (ECM) deposition increase TM stiffness, ...outflow resistance, and elevate intraocular pressure (IOP). These alterations are strongly linked to transforming growth factor-β2 (TGFβ2), a known profibrotic cytokine that is markedly elevated in the aqueous humor of glaucomatous eyes. Sigma-1 receptor (S1R) has been shown to have neuroprotective effects in the retina, but data are lacking about its role in the TM. In this study, we identified the presence of S1R in mouse TM tissue and investigated the effect of an S1R agonist fluvoxamine (FLU) on TGFβ2-induced human TM cells regarding cell proliferation; ECM-related functions, including F-actin reorganization; and the accumulation of ECM elements. TGFβ2 increased the proliferation, cytoskeletal remodeling, and protein levels of fibronectin, collagen type IV, and connective tissue growth factor, and decreased the level of matrix metalloproteinase-2. Most importantly, FLU reversed all these effects of TGFβ2, suggesting that S1R agonists could be potential candidates for preserving TM function and thus maintaining normal IOP.
Neutrophil extracellular trap (NET) ejected from activated dying neutrophils is a highly ordered structure of DNA and selected proteins capable to eliminate pathogenic microorganisms. Biochemical ...determinants of the non-randomly formed stable NETs have not been revealed so far. Studying the formation of human NETs we have observed that polyamines were incorporated into the NET. Inhibition of myeloperoxidase, which is essential for NET formation and can generate reactive chlorinated polyamines through hypochlorous acid, decreased polyamine incorporation. Addition of exogenous primary amines that similarly to polyamines inhibit reactions catalyzed by the protein cross-linker transglutaminases (TGases) has similar effect. Proteomic analysis of the highly reproducible pattern of NET components revealed cross-linking of NET proteins through chlorinated polyamines and ɛ(γ-glutamyl)lysine as well as bis-γ-glutamyl polyamine bonds catalyzed by the TGases detected in neutrophils. Competitive inhibition of protein cross-linking by monoamines disturbed the cross-linking pattern of NET proteins, which resulted in the loss of the ordered structure of the NET and significantly reduced capacity to trap bacteria. Our findings provide explanation of how NETs are formed in a reproducible and ordered manner to efficiently neutralize microorganisms at the first defense line of the innate immune system.
Neutrophils represent the most abundant cell type in peripheral blood and exhibit a remarkably brief (6–8 h) half‐life in circulation. The fundamental role of these professional phagocytes has been ...established in acute inflammation, based on their potential to both initiate and receive inflammatory signals. Furthermore, neutrophils also take part in maintaining chronic inflammatory processes, such as in various autoimmune diseases. Here, we demonstrate that human autologous apoptotic neutrophils are readily engulfed by immature monocyte‐derived dendritic cells (moDCs) with similar efficiency as allogeneic apoptotic neutrophils Majai G et al. (2010) J Leukoc Biol 88, 981–991. Interestingly, in contrast to the allogeneic system, exposure of moDCs to autologous apoptotic neutrophils inhibits LPS + IFN‐γ‐induced production of inflammatory cytokines in a phagocytosis‐independent manner. Autologous apoptotic neutrophil‐primed DCs are able to modulate T‐cell responses by inducing the generation of IFN‐γ‐secreting cells while hampering that of IL‐17A‐producing cells. Our observations indicate that capture of autologous apoptotic neutrophils by immature DCs may impede further neutrophil‐mediated phagocytosis and tissue damage, and allow increased clearance of dying cells by macrophages.
Immature human monocyte‐derived dendritic cells (moDCs) are able to engulf apoptotic autologous or allogeneic neutrophils in the same manner. Exposure of moDCs to autologous apoptotic neutrophils inhibits secretion of inflammatory cytokines parallel with an impact on T‐cell response by inhibiting IL‐17A and increasing IFN‐γ production. These contribute to the maintenance of a steady‐state condition of the immune system.
GCs are powerful anti-inflammatory compounds inhibiting inflammatory cell recruitment and production of proinflammatory cytokines. We have recently found that DCs, the key players of T cell priming ...and polarization, respond to allogeneic apoptotic neutrophils with proinflammatory cytokine release and Th1 cell activation. Here, we show that monocyte-derived human DCs develop their capacity to engulf apoptotic cells by up-regulating a set of apoptophagocytic genes. This gene expression pattern was reprogrammed when differentiation took place in the presence of the synthetic GC Dex, which increased the expression of phagocytosis receptors MERTK and CD14, the bridging molecule C1QA, DNASE2, and ADORA3. The increased phagocytosis was attenuated by the addition of ADORA3 antagonist and could not be observed when bone marrow-derived DCs of ADORA3 KO mice were treated with Dex. The GC-treated human DCs loaded with allogeneic apoptotic neutrophils secreted, in response to LPS and IFN-γ, the inflammatory cytokine TNF-α. Furthermore, the Dex-treated DCs could activate autologous T lymphocytes toward Th1 effector cells, and this was enhanced by their exposure to allogeneic apoptotic neutrophils.
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