Dok-2 adaptor protein regulates the shear-dependent adhesive function of platelet integrin αIIbß3 in mice.

The Dok proteins are a family of adaptor molecules that have a well defined role in regulating cellular migration, immune responses, and tumor progression. Previous studies have demonstrated that Doks-1 to 3 are expressed in platelets and that Dok-2 is tyrosine-phosphorylated downstream of integrin αIIbß3, raising the possibility that it participates in integrin αIIbß3 outside-in signaling. We demonstrate that Dok-2 in platelets is primarily phosphorylated by Lyn kinase. Moreover, deficiency of Dok-2 leads to dysregulated integrin αIIbß3-dependent cytosolic calcium flux and phosphatidylinositol(3,4)P2 accumulation. Although agonist-induced integrin αIIbß3 affinity regulation was unaltered in Dok-2(-/-) platelets, Dok-2 deficiency was associated with a shear-dependent increase in integrin αIIbß3 adhesive function, resulting in enhanced platelet-fibrinogen and platelet-platelet adhesive interactions under flow. This increase in adhesion was restricted to discoid platelets and involved the shear-dependent regulation of membrane tethers. Dok-2 deficiency was associated with an increased rate of platelet aggregate formation on thrombogenic surfaces, leading to accelerated thrombus growth in vivo. Overall, this study defines an important role for Dok-2 in regulating biomechanical adhesive function of discoid platelets. Moreover, they define a previously unrecognized prothrombotic mechanism that is not detected by conventional platelet function assays.

Stability of tissue PO2 in the face of altered perfusion: a phenomenon specific to the renal cortex and independent of resting renal oxygen consumption.

1. Oxygen tension (PO(2)) in renal cortical tissue can remain relatively constant when renal blood flow changes in the physiological range, even when changes in renal oxygen delivery (DO(2)) and oxygen consumption (VO(2)) are mismatched. In the current study, we examined whether this also occurs in the renal medulla and skeletal muscle, or if it is an unusual property of the renal cortex. We also examined the potential for dysfunction of the mechanisms underlying this phenomenon to contribute to kidney hypoxia in disease states associated with increased renal VO(2) . 2. In both the kidney and hindlimb of pentobarbitone anaesthetized rabbits, whole organ blood flow was reduced by intra-arterial infusion of angiotensin-II and increased by acetylcholine infusion. In the kidney, this was carried out before and during renal arterial infusion of the mitochondrial uncoupler, 2,4-dinitrophenol (DNP), or its vehicle. 3. Angiotensin-II reduced renal (-34%) and hindlimb (-25%) DO(2) , whereas acetylcholine increased renal (+38%) and hindlimb (+66%) DO(2) . However, neither renal nor hindlimb VO(2) were altered. Tissue PO(2) varied with local perfusion in the renal medulla and biceps femoris, but not the renal cortex. DNP increased renal VO(2) (+38%) and reduced cortical tissue PO(2) (-44%), but both still remained stable during subsequent infusion of angiotensin-II and acetylcholine. 4. We conclude that maintenance of tissue PO(2) in the face of mismatched changes in local perfusion and VO(2) is an unusual property of the renal cortex. The underlying mechanisms remain unknown, but our current findings suggest they are not compromised when resting renal VO(2) is increased.

Factors that render the kidney susceptible to tissue hypoxia in hypoxemia.

To better understand what makes the kidney susceptible to tissue hypoxia, we compared, in the rabbit kidney and hindlimb, the ability of feedback mechanisms governing oxygen consumption (Vo(2)) and oxygen delivery (Do(2)) to attenuate tissue hypoxia during hypoxemia. In the kidney (cortex and medulla) and hindlimb (biceps femoris muscle), we determined responses of whole organ blood flow and Vo(2), and local perfusion and tissue Po(2), to reductions in Do(2) mediated by graded systemic hypoxemia. Progressive hypoxemia reduced tissue Po(2) similarly in the renal cortex, renal medulla, and biceps femoris. Falls in tissue Po(2) could be detected when arterial oxygen content was reduced by as little as 4-8%. Vo(2) remained stable during progressive hypoxemia, only tending to fall once arterial oxygen content was reduced by 55% for the kidney or 42% for the hindlimb. Even then, the fall in renal Vo(2) could be accounted for by reduced oxygen demand for sodium transport rather than limited oxygen availability. Hindlimb blood flow and local biceps femoris perfusion increased progressively during graded hypoxia. In contrast, neither total renal blood flow nor cortical or medullary perfusion was altered by hypoxemia. Our data suggest that the absence in the kidney of hyperemic responses to hypoxia, and the insensitivity of renal Vo(2) to limited oxygen availability, contribute to kidney hypoxia during hypoxemia. The susceptibility of the kidney to tissue hypoxia, even in relatively mild hypoxemia, may have important implications for the progression of kidney disease, particularly in patients at high altitude or with chronic obstructive pulmonary disease.

Local maximum oxygen disappearance rate has limited utility as a measure of local renal tissue oxygen consumption.

INTRODUCTION: Methods for measurement of local oxygen consumption (VO2) are required to allow development of treatments for kidney disease that target kidney oxygen dysregulation. In anaesthetized rabbits, we determined whether local oxygen disappearance rate (ODR) during complete renal ischaemia reflects tissue VO2 in the kidney and in hindlimb skeletal muscle (biceps femoris). METHODS: Whole kidney VO2 was determined under conditions employed to alter oxygen consumption. The ureter was ligated to reduce VO2 (n=6) or the mitochondrial uncoupler 2,4-dinitrophenol was administered to increase VO2 (n=6). An additional 10 rabbits were studied which received neither treatment. Immediately following VO2 measurements, oxygen partial pressure (PO2) was measured, over the first 60s after abrupt cardiac arrest, using fluorescence optodes and in a subset of experiments (n=6), Clark electrodes. Parallel experiments were performed in hindlimb skeletal muscle (biceps femoris). RESULTS: ODR in the renal cortex and medulla, and in biceps femoris, was linear during the first approximately 15s after cardiac arrest (r(2) approximately 0.98). Using fluorescence optodes, maximum ODR averaged across all 22 rabbits in which the kidney was studied was -0.75+/-0.09 and -0.60+/-0.06 mm Hg/s respectively in the renal cortex and medulla. Maximum ODR averaged across all 10 rabbits in which the biceps femoris was studied was -0.30+/-0.06 mm Hg/s. ODR increased at greater initial PO2 only in the renal cortex. ODR in neither the renal medulla nor biceps femoris varied with whole organ VO2, although renal cortical ODR normalized for initial PO2 was significantly correlated with whole organ VO2 (r(2)=0.19). Maximum ODR obtained by Clark electrode was approximately four-fold greater than that obtained by fluorescence optode. DISCUSSION: Because ODR correlates poorly with whole organ VO2, it likely has limited utility as a measure of local VO2.