Determination of glomerular filtration rate (GFR) in conscious mice is cumbersome for the experimenter and stressful for the animals. Here we report on a simple new technique allowing the transcutaneous measurement of GFR in conscious mice. This approach extends our previously developed technique for rats to mice. The technique relies on a miniaturized device equipped with an internal memory that permits the transcutaneous measurement of the elimination kinetics of the fluorescent renal marker FITC-sinistrin. This device is described and validated compared with FITC-sinistrin plasma clearance in healthy, unilaterally nephrectomized and pcy mice. In summary, we describe a technique allowing the measurement of renal function in freely moving mice independent of blood or urine sampling as well as of laboratory assays.

BACKGROUND AND PURPOSERecently, two phase-II trials demonstrated improved renal function in critically ill patients with sepsis-associated acute kidney injury treated with the enzyme alkaline phosphatase. Here, we elucidated the dual active effect on renal protection of alkaline phosphatase. EXPERIMENTAL APPROACHThe effect of human recombinant alkaline phosphatase (recAP) on LPS-induced renal injury was studied in Sprague-Dawley rats. Renal function was assessed by transcutaneous measurement of FITC-sinistrin elimination in freely moving, awake rats. The mechanism of action of recAP was further investigated in vitro using conditionally immortalized human proximal tubular epithelial cells (ciPTEC). KEY RESULTSIn vivo, LPS administration significantly prolonged FITC-sinistrin half-life and increased fractional urea excretion, which was prevented by recAP co-administration. Moreover, recAP prevented LPS-induced increase in proximal tubule injury marker, kidney injury molecule-1 expression and excretion. In vitro, LPS-induced production of TNF-α, IL-6 and IL-8 was significantly attenuated by recAP. This effect was linked to dephosphorylation, as enzymatically inactive recAP had no effect on LPS-induced cytokine production. RecAP-mediated protection resulted in increased adenosine levels through dephosphorylation of LPS-induced extracellular ADP and ATP. Also, recAP attenuated LPS-induced increased expression of adenosine A 2A receptor. However, the A 2A receptor antagonist ZM-241385 did not diminish the effects of recAP. CONCLUSIONS AND IMPLICATIONSThese results indicate that the ability of recAP to reduce renal inflammation may account for the beneficial effect observed in septic acute kidney injury patients, and that dephosphorylation of ATP and LPS are responsible for this protective effect.Abbreviations AKI, acute kidney injury; ciPTEC, conditionally immortalized proximal tubular epithelial cells; dLPS, detoxified LPS; IAP, intestinal alkaline phosphatase; KIM-1, kidney injury molecule-1; NGAL, neutrophil gelatinase-associated lipocalin; PBMCs, peripheral blood mononuclear cells; PFA, paraformaldehyde; recAP, human recombinant alkaline phosphatase; TLR4, toll-like receptor 4