Background: Primary cilia regulation of renal function and blood pressure (BP) in health and disease is incompletely understood. The current study investigated the effect of nephron ciliary loss on renal physiology, blood pressure and ensuing cystogenesis. Methods: Mice underwent doxycycline (DOX)-inducible nephron-specific knockout (KO) of the Ift88 gene at 2 months of age using a Cre-LoxP strategy. Blood pressure, kidney function, and renal pathology were studied 2 and 9 months after DOX (Ift88 KO) or vehicle (control). Results: At 2 months post DOX, male, but not female, Ift88 KO, compared to sex-matched control, mice had reduced BP, enhanced salt-induced natriuresis, increased urinary nitrite/nitrate (NOx) excretion, and increased kidney NOS3 levels which localized to the outer medulla; the reductions in BP in male mice were prevented by L-NAME. At 9 months post DOX, male, but not female, Ift88 KO mice had polycystic kidneys, elevated BP and reduced urinary NOx excretion. No differences were observed in plasma renin concentration, plasma aldosterone, urine vasopressin or urine PGE2 between Ift88 KO and control mice at 2 or 9 months post DOX. Conclusions: Nephron cilia disruption in male, but not female, mice: 1) reduces BP prior to cyst formation; 2) increases NOx production that may account for the lower BP prior to cyst formation; and 3) induces polycystic kidneys that are associated with hypertension and reduced renal NO production.
Renal iron deposition correlates with elevated plasma ET‐1 in patients and mouse model of sickle cell disease (SCD). Additionally, excessive renal iron accumulation in sickle cell mice (HbSS) is ameliorated by ETA receptor antagonism. Thus, we hypothesized that ET‐1 via ETA receptor activity leads to dysfunctional renal iron trafficking, promoting iron accumulation in the kidney. We conducted time‐ and concentration‐dependent in vitro studies on mouse proximal tubule (PT) cells and observed that ET‐1/ ETA signaling promotes cellular iron uptake (by increasing transferrin receptor 1 expression) and inhibits cellular iron removal pathways (65% reduction of ferroportin‐1 (FPN‐1) and doubled hepcidin (Hamp) expression). In vivo studies demonstrated that altered expression of iron transporters in PT of male HbSS mice was consistent with iron accumulation phenotype. ETA receptor blockade attenuated expression of the cellular iron uptake mediator, DMT‐1, and also decreased renal iron accumulation (1.43±0.06 vs. 2.62±0.12; p=0.097) by preserving FPN‐1, reducing Hamp expression, and increasing urinary iron excretion (10.9±1.0 vs 6.4±1.2 μg/24h; p=0.010). To determine the long‐term effect of ET‐1 on chronic renal iron accumulation, we generated HbSS mice lacking ET‐1 in vascular endothelial cells mice (HbSS VEET KO; using Tie‐2 Cre). There were no differences in anemia status between 20 weeks old male HbSS VEET KO and flox mice. However, plasma iron concentration (117.3±4.3 vs. 103.9±4.3 μg/dl, p=0.04) and urinary iron excretion (10.4±1.9 vs. 7.4±1.1 μg/24h; p=0.059) were elevated and renal iron deposition attenuated (24.3±1.3 vs. 33.8±2.3; p=0.008) in HbSS VEET KO mice. To further elucidate ET‐1/ETA receptor‐mediated renal iron accumulation already observed with pharmacological and genetic approaches in HbSS mice, using PEPCK Cre we generated PT specific ETA receptor knockout (PT ETA KO) mice. At baseline, 16 weeks old male and female PT ETA KO mice presented with higher plasma iron concentration (1.0±0.2 vs. 0.5±0.1 μg/ml; p=0.03) and urinary iron excretion (4.5±0.7 vs. 1.7±0.5 μg/24h; p=0.002), whereas renal iron deposition remained unchanged (2.26±0.48 vs. 2.44±0.73 Kpix/ μm; p=0.85). To induce hemolysis with acute iron overload, mice were injected with phenylhydrazine (PhZ, 40 mg/kg, IP; 2 consecutive days) or saline. PhZ significantly reduced hemoglobin in both PT ETA KO and littermate controls when compared to saline injected mice. Moreover, PT ETA KO mice given PhZ had increased plasma iron concentration (4.4±0.3 vs. 3.0±0.5 μg/ml; p=0.03) and urinary iron excretion (5.6±0.7 vs. 3.4±0.7 μg/24h; p=0.05) and decreased renal iron accumulation (0.50±0.22 vs. 0.90±0.20; p=0.011) when compared with saline injected KO control mice. Together, these data demonstrate that ET‐1, via ETA receptor activity, contributes to renal iron accumulation in murine models of iron overload.
P-selectin (Psel) is an adhesion molecule expressed on platelets and endothelial cells, which interacts with its ligand PSGL-1 on leukocytes and a PSGL-1-like molecule on sickle red blood cells (RBCs). Psel mediates the formation of the multicellular aggregates that promote vaso-occlusive crisis (VOC) and acute painful episodes in sickle cell disease (SCD). Crizanlizumab, a monoclonal antibody blocking Psel, reduces the frequency of VOC in SCD patients. In SCD mice, Psel contributes to heme-induced microvascular stasis, and thrombus and platelet-neutrophil aggregate formation in the lung and liver. Unexpectedly, Psel deficiency promotes liver senescence in a mouse model of SCD, suggesting a possible detrimental effect of Psel inhibition. In this study, we further investigated the long-term effects of Psel deficiency on thromboinflammation and end-organ damage in murine SCD. We used Townes wild type (AA) and sickle (SS) mice (n=12-27) that either express Psel (Psel +/+) or lack Psel (Psel -/-) at 11-12 months of age. Kidney damage was evaluated by histology and urine analysis. Heart function was determined using echocardiography. Complete blood counts and plasma biomarkers of thrombin generation (thrombin anti-thrombin [TAT] complexes) and inflammation (interleukin [IL]-6) were also analyzed. In addition, in a pilot experiment, we analyzed the effect of Psel deficiency on experimental venous thrombosis in SS mice. SS Psel +/+ mice exhibited renal damage, urinary concentrating defect, and reduced creatinine clearance, which was not improved in SS Psel -/- mice. Psel deficiency attenuated glomerular (GLM) congestion (0.17±0.05 vs 0.39±0.06, p<0.05) but failed to prevent GLM injury, as evidenced by similar extent of GLM sclerosis, hypertrophy and podocyte loss in SS mice. Interestingly, only SS P-sel -/- mice presented withdistinct differences in GLM structure, demonstrated by extensive hypercellularity, mesangial expansion and mesangiolysis. We also observed tubular injury concomitant with brush border loss and interstitial fibrosis in SS mice, regardless of Psel expression. Additionally, there was a significant reduction in CD3 + T cells (5.0±1.2 vs 9.5±1.6 cells/field; p<0.05) and macrophages (1.0±0.1 vs 1.6±0.2 % of area; p<0.05) infiltration in the kidney cortex of SS Psel -/- compared to SS Psel +/+ mice. This corresponded to a greater renal iron accumulation in SS Psel -/- mice (59.9±4.2 vs 48.2±3.2 Mpix/μm; p<0.05). We observed cardiac hypertrophy (elevated heart weight to tibia length ratio) in SS mice, regardless of Psel expression. Echocardiography of left ventricle (LV) revealed that SS mice had increased LV mass and LV internal diameter with no change in ejection fraction or fractional shortening compared to AA mice. None of these parameters were affected by Psel expression. Furthermore, hypertrophy observed in other organs (kidney, liver, lung and spleen) of SS Psel +/+ mice was also not affected by Psel deficiency. Consistent with previous studies, SS Psel +/+ mice had elevated plasma levels of TAT and IL-6 compared to AA Psel +/+ mice. Psel deficiency significantly reduced IL-6 (47±15 vs 20±10 ng/mL, P<0.05), but had no effect on elevated plasma TAT levels in SS mice. Despite the lack of effect of Psel deficiency on systemic thrombin generation in SS mice, femoral vein thrombi formed after electrolytic injury exhibited a strong trend in reduced fibrin and platelet content compared to SS Psel+/+ mice (n=5 per group). As expected, SS Psel +/+ mice exhibited anemia as shown by reduced RBC number, hemoglobin (Hb) and hematocrit (Hct) compared to AA Psel +/+ animals. Interestingly, these parameters were further decreased in SS Psel -/- mice (p<0.01 for all); RBC (5.6±0.2 vs 4.4±0.4 x 10 6/mL), Hg (6.7±0.2 vs 5.7±0.2 g/dL) and Hct (25.1±1.1 vs 19.7±1.1%). This was accompanied by a significant increase in mean corpuscular Hb concentration (26.9±0.5 vs 29.2±0.5 g/dL p< 0.01) but no changes in RBC mean corpuscular volume in SS Psel -/-mice compared to SS Psel +/+ mice. Our data suggest that despite improving acute vascular pathologies, including VOC and thromboinflammation, long term deficiency of Psel does not prevent end-organ damage. If fact, it may contribute to organ dysfunction and enhanced anemia. More mechanistic studies are needed to better understand the long-term effects of anti-Psel treatment in SCD patients. Disclosures Reeves: Incyte Corporation: Honoraria; Takeda: Honoraria; Bristol-Myers Squibb: Speakers Bureau; Pharma Essentia: Consultancy, Honoraria. Sundd: CSL Behring Inc: Research Funding; Bayer: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.
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