Uromodulin, the most abundant protein in normal urine, is produced by cells lining the thick ascending limb (TAL) of the loop of Henle. Uromodulin regulates the activity of the potassium channel ROMK in TAL cells. Common variants in KCNJ1, the gene encoding ROMK, are associated with urinary levels of uromodulin in population studies. Here, we investigated the functional link between ROMK and uromodulin in Kcnj1 knock-out mouse models, in primary cultures of mouse TAL (mTAL) cells, and in patients with Bartter syndrome due to KCNJ1 mutations. Both global and kidney-specific Kcnj1 knock-out mice showed reduced urinary levels of uromodulin paralleled by increased levels in the kidney, compared to wild-type controls. Pharmacological inhibition and genetic deletion of ROMK in mTAL cells caused a reduction in apical uromodulin excretion, reflected by cellular accumulation. In contrast, NKCC2 inhibition showed no effect on uromodulin processing. Patients with Bartter syndrome type 2 showed reduced urinary uromodulin levels compared to age and gender matched controls. These results demonstrate that ROMK directly regulates processing and release of uromodulin by TAL cells, independently from NKCC2. They support the functional link between transport activity and uromodulin in the TAL, relevant for blood pressure control and urinary concentrating ability.
Background Apoptotic pathways in platelets are important for their survival and function. Platelet apoptosis may be involved in the pathogenesis of immune thrombocytopenia (ITP), an autoimmune-mediated disease. In contrast to the intrinsic apoptosis pathway, not much is known about the extrinsic pathway mechanisms in platelets. Objectives To investigate the expression of proteins involved in the extrinsic apoptosis pathway, including the death receptors, adaptor and regulator proteins in human platelets. To determine a possible trigger of the extrinsic apoptosis pathway in platelets. Methods To investigate the expression of key markers of the extrinsic pathway we used targeted immunofluorescence and flow cytometry assays. To study their expression and interaction we performed Western blotting and co-immunoprecipitation. Treated platelets with different apoptosis triggers were subjected to flow cytometry. Results We could identify the protein expression of the pro-apoptotic proteins TRADD (Tumor Necrosis Factor Receptor type 1- Associated DEATH Domain protein), TRAF2/5, (TNF Associated Factor) and DEDAF (Death Effector Domain- Associated Factor), FADD (Fas-Associated protein with death domain) as well as the anti-apoptotic proteins DJ-1 (Deglycase 1) and c-FLIP in human platelets. ABT-737 treatment induced a disruption in the co-localization of DJ-1 with FADD. Platelets treated with ABT-737 showed an activation in caspase-3 and -8. The exposure to TNF (Tumor Necrosis Factor), FasL (Fas ligand), and TWEAK or to plasma derived from ITP patients, did not lead to changes in caspase-3 and -8 activation in platelets. Conclusions Human platelets express some proteins of the extrinsic apoptosis pathway which can be modulated only by ABT-737 treatment. However so far, no other apoptosis trigger or interaction with an external receptor have been yet identified.
In this study we investigated if and how cannabinoid receptor stimulation regulates macrophageal differentiation, which is one of the key steps in the immune effector reaction. For that reason, we used a well established differentiation model system of human U937 myelocytic leukemia cells that differentiate along the monocyte/macrophage lineage upon stimulation with the phorbol ester PMA. Constant cannabinoid receptor (CB) stimulation was performed using WIN55212-2, a potent synthetic CB agonist. We found that WIN55212-2 inhibited CB1/2-receptor-dependent PMA-induced differentiation of human myelocytic U937 cells into the macrophageal phenotype, which was associated with impaired vimentin, ICAM-1 and CD11b expression. In the presence of WIN55212-2, cdc2 protein and mRNA expression was progressively enhanced and Tyr-15-phosporylation of cdc2 was reduced in differentiating U937 cells. Additionally, p21Waf1/Cip1 expression was up-regulated. PMA-induced apoptosis was not enhanced by WIN55212-2 and differentiation-associated c-jun expression was not altered. In conclusion, we suppose that WIN55212-2-induced signals interferes with cell-cycle-arrest-signaling in differentiating myelocytic cells and thus inhibits macrophageal differentiation. Thus, it is possible that the cannabinoid system is able to influence one of the key steps in the immune effector function, the monocytic-macrophageal differentiation by alteration of cell cycle control proteins cdc2 and p21, and is therefore representing a promising option for therapeutic intervention in exacerbated immune reactions.
Immune thrombocytopenia (ITP) is defined as an autoimmune disease that leads to platelet clearance by macrophages. It is well known that auto-reactive B cells and CD4+ T-helper (Th) cells, and in particular their cytokines, have been associated with ITP. Cytokines and chemokines are key players in our immune response to recruit different cells, e.g. macrophages and monocytes, to respond to certain inflammatory signals. Specific cytokines (i.e. TNF-a, IFN-g) are known to induce apoptosis in nucleated blood cells. To understand the role of cytokines and chemokines in acute ITP, we studied their plasma levels in 10 pediatric ITP patients at diagnosis with a median platelet count of 3 x 109/L (range < 1 to 22 x 109/L) and compared them with controls: healthy children (n=9; platelet count > 150 x 109/L); and chemotherapy-induced thrombocytopenia patients (cTP; n=9; median platelet count of 12 x 109/L; range: 3-53 x 109/L). ITP patients fulfilled the criteria for acute ITP and presented with mild to moderate bleeding symptoms. The median age at diagnosis was 3.4 yrs (range: 1.6 - 6.5 yrs); blood samples were taken prior to treatment. Luminex technology was used to measure plasma levels of 42 cytokines and chemokines. Markers of platelet apoptosis - activated caspase-3, -8 and -9 - and of platelet activation - CD62P and CD63 expression and PAC-1 binding - were measured by flow cytometry. Distinct plasma cytokine/chemokine patterns were observed in ITP patients compared with controls. Significantly increased levels of the Th1 cell commitment cytokines TNF-α (p < 0.01) and IFN-g (p < 0.05), as well as of the Th2 cytokines IL-6 (p < 0.01), IL-10 (p < 0.01) and IL-13 (p < 0.05), were identified in ITP patients. We have previously shown that there is activation of platelet caspase-3, -8 and -9 at diagnosis in acute paediatric ITP patients compared with controls (Winkler et al, Br J Haematol 2012;156:508). In ITP patients, but not in controls, a negative correlation between eotaxin and caspase- 3 (r2 = 0.72), -8 (r2 = 0.76) and -9 (r2 = 0.53) activity were observed, as well as a negative correlations between GM-CSF and caspase-8 (r2 = 0.52) and -9 (r2 = 0.33). Furthermore, we found a correlation between IL-13 and platelet activation as measured by CD62P (r2 = 0.87) and CD63 (r2 = 0.67) expression in ITP patients but not in controls. In summary, increased plasma levels of the cytokines TNF-α, IFN-g, IL-6, IL-10, and IL-13 were observed in ITP patients at initial presentation, suggesting that these cytokines contribute to the pathogenesis of the disease. Since IL-6 and IFN-g are known to activate macrophages, while higher levels TNF-α, IL-10 and IL-13 in the plasma are signs for activated T cells, our findings are consistent with the current model of ITP, in which activated macrophages induce B and T cells to produce anti-platelet autoantibodies. Our goal is to study the interplay between the immune system and the reduction of platelet count in ITP and to further define apoptosis/activation-related pathways in ITP platelets. Furthermore, we showed a correlation in ITP patients between the chemokine eotaxin and GM-CSF with caspase-3, -8 and -9 activity, and a correlation between IL-13 and platelet activation. Our results imply, that apoptosis and platelet activation at diagnosis in ITP play a role in the development of ITP, but the underlying mechanisms are still unknown and needs to be further evaluated by increasing the patient cohort in our study. Disclosures No relevant conflicts of interest to declare.
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