LPS is an immunostimulatory component of Gram-negative bacteria. Acting on the immune system in a systemic fashion, LPS exposes the body to the hazard of septic shock. In this study we report that cysteine-rich secretory protein LCCL domain containing 2 (CRISPLD2/Crispld2; human and mouse/rat versions, respectively), expressed by multitissues and leukocytes, is a novel LPS-binding protein. As a serum protein, median CRISPLD2 concentrations in health volunteers and umbilical cord blood samples are 607 μg/ml and 290 μg/ml, respectively. Human peripheral blood granulocytes and mononuclear cells including monocytes, NK cells, and T cells spontaneously release CRISPLD2 (range, 0.2–0.9 μg/ml) and enhance CRISPLD2 secretion (range, 1.5–4.2 μg/ml) in response to stimulation of both LPS and humanized anti-human TLR4-IgA Ab in vitro. CRISPLD2 exhibits significant LPS binding affinity similar to that of soluble CD14, prevents LPS binding to target cells, reduces LPS-induced TNF-α and IL-6 production, and protects mice against endotoxin shock. In in vivo experiments, serum Crispld2 concentrations increased in response to a nontoxic dose of LPS and correlated negatively with LPS lethality, suggesting that CRISPLD2 serum concentrations not only are indicators of the degree of a body’s exposure to LPS but also reflect an individual’s LPS sensitivity.

ROMK1 channels are located in the apical membrane of the connecting tubule and cortical collecting duct and mediate the potassium secretion during normal dietary intake. We used a perforated whole-cell patch clamp to explore the effect of angiotensin II on these channels in HEK293 cells transfected with green fluorescent protein (GFP)-ROMK1. Angiotensin II inhibited ROMK1 channels in a dose-dependent manner, an effect abolished by losartan or by inhibition of protein kinase C. Furthermore, angiotensin II stimulated a protein kinase C-sensitive phosphorylation of tyrosine 416 within c-Src. Inhibition of protein tyrosine kinase attenuated the effect of angiotensin II. Western blot studies suggested that angiotensin II inhibited ROMK1 channels by enhancing its tyrosine phosphorylation, a notion supported by angiotensin II's failure to inhibit potassium channels in cells transfected with the ROMK1 tyrosine mutant (R1Y337A). However, angiotensin II restored the with-no-lysine kinase-4 (WNK4)-induced inhibition of R1Y337A in the presence of serum-glucocorticoids-induced kinase 1 (SGK1), which reversed the inhibitory effect of WNK4 on ROMK1. Moreover, protein tyrosine kinase inhibition abolished the angiotensin II-induced restoration of WNK4-mediated inhibition of ROMK1. Angiotensin II inhibited ROMK channels in the cortical collecting duct of rats on a low sodium diet, an effect blocked by protein tyrosine kinase inhibition. Thus, angiotensin II inhibits ROMK channels by two mechanisms: increasing tyrosine phosphorylation of the channel and synergizing the WNK4-induced inhibition. Hence, angiotensin II may have an important role in suppressing potassium secretion during volume depletion.

We previously demonstrated that K depletion inhibited ROMK-like small-conductance K channels (SK) in the cortical collecting duct (CCD) and that the effect was mediated by superoxide anions that stimulated Src family protein tyrosine kinase (PTK) and mitogen-activated protein kinase (MAPK) (51). However, because animals on a K-deficient diet had a severe hypokalemia, superoxide-dependent signaling may not regulate ROMK channels under physiological conditions with a normal plasma K concentration. In the present study, we used the patch-clamp technique and Western blot to examine the effect of a moderate K restriction on ROMK-like SK channels and the role of PTK and MAPK in regulating apical K channels in the CCD of animals on a low-K diet (LK; 0.1% K). Rats and mice fed a LK diet for 7 days had a normal plasma K concentration. However, a LK intake increased the expression of angiotensin II type 1 receptor in the kidney. Moreover, patch-clamp experiments demonstrated that LK intake decreased the probability finding SK channels and channel activity defined by NPo (a product of channel number and open probability) in the CCD of both rat and mouse kidneys. Also, LK intake significantly stimulated the production of superoxide anions in the renal cortex and outer medulla in both rats and mice and increased superoxide level in the rat CCD. Moreover, LK intake augments the phosphorylation of p38 and ERK MAPK, the expression of c-Src and tyrosine phosphorylation of ROMK channels. However, treatment of animals with tempol abolished the effect of LK intake on MAPK and c-Src and increased ROMK channel activity in comparing with those of nontreated rats on a LK diet. Inhibiting p38 and ERK with SB202190 and PD98059 significantly stimulated SK in the CCD in rats on a LK diet. In addition, inhibition of PTK with herbimycin A activated SK channels in the CCD from rats on a LK diet. We conclude that LK intake stimulates the generation of superoxide anion and related products and that MAPK and Src family PTK play a physiological role in inhibiting apical K channels in the principal cells in response to LK intake. Ca 2ϩ -activated BK channel; ROMK (Kiy1-1); renal K secretion; K homeostasis A DECREASE IN THE DIETARY K intake suppresses renal K secretion (41,43,53). K restriction-induced decrease in renal K secretion is achieved by both inhibition of apical ROMK-like small-conductance K channels (SK) and Ca 2ϩ -activated big conductance K channel (BK) channels in principal cell (PC) and by stimulating K absorption through K-H-ATPase in intercalated cell (IC) (57,58). Several studies demonstrated that K restriction inhibits ROMK-like SK channels by stimulating endocytosis (12,55,56). Although the mechanism by which K restriction inhibits ROMK channels is not completely understood, WNK (with-no-lysine kinase) and superoxide anions play an important role in mediating endocytosis (27, 51). WNK1, 3, and 4 have been shown to be expressed in the cortical collecting duct (CCD) and inhibit ROMK channels by stimulating a clathrin-dependent pat...