Background Sickle cell disease (SCD) is characterized by chronic hemolytic anemia, vaso‐occlusive crises, chronic inflammation, and activation of coagulation. The clinical complications such as painful crisis, stroke, pulmonary hypertension, nephropathy and venous thromboembolism lead to cumulative organ damage and premature death. High molecular weight kininogen (HK) is a central cofactor for the kallikrein‐kinin and intrinsic coagulation pathways, which contributes to both coagulation and inflammation. Objective We hypothesize that HK contributes to the hypercoagulable and pro‐inflammatory state that causes end‐organ damage and early mortality in sickle mice. Methods We evaluated the role of HK in the Townes mouse model of SCD. Results/Conclusions We found elevated plasma levels of cleaved HK in sickle patients compared to healthy controls, suggesting ongoing HK activation in SCD. We used bone marrow transplantation to generate wild type and sickle cell mice on a HK‐deficient background. We found that short‐term HK deficiency attenuated thrombin generation and inflammation in sickle mice at steady state, which was independent of bradykinin signaling. Moreover, long‐term HK deficiency attenuates kidney injury, reduces chronic inflammation, and ultimately improves survival of sickle mice.
Progressive iron accumulation and renal impairment are prominent in both patients and mouse models of sickle cell disease (SCD). Endothelin A receptor (ETA) antagonism prevents this iron accumulation phenotype and reduces renal iron deposition in proximal tubules of SCD mice. To better understand the mechanisms of iron metabolism in the kidney and the role of ETA receptor in iron chelation and transport, we studied renal iron handling in a non-sickle cell iron overload model, heme oxygenase-1 (Hmox-1-/-) knockout mice. We found that Hmox-1-/- mice had elevated plasma endothelin-1 (ET-1), cortical ET-1 mRNA expression, and renal iron content compared to Hmox-1+/+ controls. The ETA receptor antagonist, ambrisentan, attenuated renal iron deposition, without any changes to anemia status in Hmox-1-/- mice. This was accompanied by reduced urinary iron excretion. Finally, ambrisentan had an important iron recycling effect by increasing expression of cellular iron exporter, ferroportin-1 (FPN-1) and circulating total iron levels in Hmox-1-/- mice. These findings suggest the ET-1/ETA signaling pathway contributes to in renal iron trafficking in a murine model of iron overload.
We recently reported that there are > 400 proteins in the inner medullary collecting duct that are post‐translationally lysine acetylated (ac‐K). Although historically, ac‐K proteins were thought to be predominantly histone proteins in the nucleus, we now know that all cellular compartments are enriched with ac‐K proteins. Pathway analyses determined that ac‐K proteins are enriched in various physiological processes such as glycolysis and vasopressin‐mediated water reabsorption. We recently identified the basolateral water channel, aquaporin‐3 (AQP3), as undergoing lysine acetylation (AQP3 K282). Thus, the purpose of this study was to determine if ac‐K AQP3 affects water permeability. We developed antibodies to specifically detected ac‐K AQP3 or total AQP3. In both male and female mice, ac‐K AQP3 was expressed in the basolateral membrane of the cortical and outer medullary collecting duct. However, following 24 h of water deprivation, ac‐K AQP3 was also found in the inner medullary collecting duct. Next, we developed AQP3 K point mutation plasmids, with K282Q representing an acetylated mimic, and K282 representing a deacetylated mimic. These constructs were stably expressed in vasopressin‐responsive mouse cortical collecting duct cells (AQP3mpkCCDs). Using the cell volume sensitive dye, calcein, water permeability of these mutant cells was determined following an osmotic stimulus. We found the acetylated mimic (K282Q) had the highest water permeability followed by the AQP3 wild type K282 cells and the deacetylated mimic (K282R). Finally, using CRISPR/CAS we engineered whole body point mutation mice. The K282Q mutant mice and littermate controls (K282) all developed normally. As adults, K282Q mice presented with elevated plasma osmolality (307 ± 2 mOsm/Kg H2O vs 298 ± 4 mOsm/kg H2O, P = 0.04) at baseline. Following 3 days of hydration with 5% sucrose water, K282Q produced twice as much urine as controls during their active period (2.16 ± 0.5 ml/12h vs 0.88 ± 0.2, P = 0.03). Moreover, when challenged with a 2 ml sterile water i.p., K282Q mice excreted majority of the water load within the first 3 h, while controls reached peak urine flow after 3 h. Finally, we quantified AQP2 (the apical water channel) and AQP3 mRNA expression and protein abundance. AQP2, AQP3 and AQP4 mRNA expression was similar between control and mutant mice. Surprisingly, K282Q mutant mice presented with significantly less AQP2 and AQP3 protein abundance compared to controls. Thus, mutating K282 in vivo resulted in a loss of AQP3 (a potentially AQP2) stability, and highlights the importance of this lysine residue. To conclude, lysine acetylation of AQP3 is a novel posttranslational modification that may regulate AQP function and water permeability in the collecting duct. Support or Funding Information K01DK105038, R03DK120503, the University of Alabama at Birmingham Pittman Scholarship, NIH P30 DK074038
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