Modulation of Enzyme-Catalyzed Synthesis of Prostaglandins by Components Contained in Kidney Microsomal Preparations

In the kidney, prostaglandins formed by cyclooxygenase 1 and 2 (COX-1 and COX-2) play an important role in regulating renal blood flow. In the present study, we report our observations regarding a unique modulatory effect of renal microsomal preparation on COX-1/2-mediated formation of major prostaglandin (PG) products in vitro. We found that microsomes prepared from pig and rat kidneys had a dual stimulatory–inhibitory effect on the formation of certain PG products catalyzed by COX-1 and COX-2. At lower concentrations, kidney microsomes stimulated the formation of certain PG products, whereas at higher concentrations, their presence inhibited the formation. Presence of kidney microsomes consistently increased the Km values of the COX-1/2-mediated reactions, while the Vmax might be increased or decreased depending on stimulation or inhibition observed. Experimental evidence was presented to show that a protein component present in the pig kidney microsomes was primarily responsible for the activation of the enzyme-catalyzed arachidonic acid metabolism leading to the formation of certain PG products.

The Role of Interleukin-6 (IL-6) in the Systemic Inflammatory Response in Xenograft Recipients and in Pig Kidney Xenograft Failure

BackgroundIn pig-to-baboon transplantation models, there is increasing evidence of systemic inflammation in xenograft recipients (SIXR) associated with pig xenograft failure. We evaluated the relationship between systemic inflammatory factors and pig kidney xenograft failure.MethodsBaboons received kidney transplants from genetically engineered pigs (n=9), and received an anti-CD40mAb-based (n=4) or conventional (n=5) immunosuppressive regimen. The pig kidney grafts were monitored by measurements of serum creatinine, serum amyloid A (SAA), white blood cell (WBC) and platelet counts, plasma fibrinogen, and pro-inflammatory cytokines (baboon and pig IL-6, TNF-α, IL-1β).ResultsSix baboons were euthanized or died from rejection, and 3 were euthanized for infection. Changes in serum creatinine correlated with those of SAA (r=0.56, p<0.01). Serum baboon IL-6 was increased significantly on day 1 after transplantation and at euthanasia (both p<0.05) and correlated with serum creatinine and SAA (r=0.59, p<0.001, r=0.58, p<0.01; respectively). but no difference was observed between rejection and infection. Levels of serum pig IL-6, TNF-α, IL-1β were also significantly increased on day 1 and at euthanasia, and serum pig IL-6 and IL-1β correlated with serum creatinine and SAA. The level of serum baboon IL-6 correlated with the expression of IL-6 and amyloid A in the baboon liver (r=0.93, p<0.01, r=0.79, p<0.05; respectively).ConclusionEarly upregulation of SAA and serum IL-6 may indicate the development of rejection or infection, and are associated with impaired kidney graft function. Detection and prevention of systemic inflammation may be required to prevent pig kidney xenograft failure after xenotransplantation.

The beryllium fluoride form of the Na+,K+-ATPase and the intermediates of the functional cycle

The Na+,K+-ATPase generates electrochemical gradients of Na+ and K+ across the plasma membrane. Here, we describe a 4.0 Å resolution crystal structure of the pig kidney Na+,K+-ATPase stabilized by beryllium fluoride (denoted E2-BeFx). The structure shows high resemblance to the E2P phosphoenzyme obtained by phosphorylation from inorganic phosphate (Pi) and stabilised by cardiotonic steroids, and reveals a Mg2+ bound near the ion binding site II. Anomalous Fourier analysis of the crystals soaked in Rb+ (K+ congener) followed by a low resolution rigid-body refinement (6.9-7.5 Å) revealed pre-occlusion transitions leading to activation of the desphosphorylation reaction. Mg2+ location indicates a site of an initial K+ recognition and acceptance upon binding to the outward-open E2P state after Na+ release. Despite the overall structural resemblance to the Pi-induced E2P phosphoform, BeFx inhibited enzyme is able to binds both ADP/ATP and ions, the features that relate E2-BeFx complex to an intermediate of the functional cycle of the Na+,K+-ATPase prior E2P.