We have demonstrated that the kidney plays an important role in iron balance and that metabolically significant reabsorption of this ion occurs in the loop of Henle and the collecting ducts [Wareing M, Ferguson CJ, Green R, Riccardi D, and Smith CP. J Physiol (Lond) 524: 581-586, 2000]. To test the possibility that the divalent metal transporter DMT1 (Gunshin H, Mackenzie B, Berger UV, Gunshin Y, Romero MF, Boron WF, Nussberger S, Gollan JL, and Hediger MA. Nature 388: 482-488, 1997) could represent the apical route for iron entry in the kidney, we raised and affinity-purified an anti-DMT-1 polyclonal antibody and determined DMT-1 distribution in rat kidney by Western analysis, immunofluorescence, and confocal microscopy. The strongest DMT1-specific (i.e., peptide-protectable) immunoreactivity was found in the collecting ducts, in both principal and intercalated cells. Thick ascending limbs of Henle's loop and, more intensely, distal convoluted tubules exhibited apical immunostaining. Considerable intracellular DMT-1 immunoreactivity was seen throughout the nephron, particularly in S3 segments. The described distribution of DMT-1 protein is in agreement with our previous identification of nephron sites of iron reabsorption, suggesting that DMT-1 provides the molecular mechanism for apical iron entry in the distal nephron but not in the proximal tubule. Basolateral iron exit may be facilitated by a different system.

The balance between the concentrations of free ionized Ca 2؉ and bicarbonate in pancreatic juice is of critical importance in preventing the formation of calcium carbonate stones. How the pancreas regulates the ionic composition and the level of Ca 2؉ saturation in an alkaline environment such as the pancreatic juice is not known. Because of the tight cause-effect relationship between Ca 2؉ concentration and lithogenicity, and because hypercalcemia is proposed as an etiologic factor for several pancreatic diseases, we have investigated whether pancreatic tissues express a Ca 2؉ -sensing receptor (CaR) similar to that recently identified in parathyroid tissue. Using reverse transcriptase-polymerase chain reaction and immunofluorescence microscopy, we demonstrate the presence of a CaR-like molecule in rat pancreatic acinar cells, pancreatic ducts, and islets of Langerhans. Functional studies, in which intracellular free Ca 2؉ concentration was measured in isolated acinar cells and interlobular ducts, show that both cell types are responsive to the CaR agonist gadolinium (Gd 3؉ ) and to changes in extracellular Ca 2؉ concentration. We also assessed the effects of CaR stimulation on physiological HCO 3 ؊ secretion from ducts by making measurements of intracellular pH. Luminal Gd 3؉ is a potent stimulus for HCO 3 ؊ secretion, being equally as effective as raising intracellular cAMP with forskolin. These results suggest that the CaR in the exocrine pancreas monitors the Ca 2؉ concentration in the pancreatic juice, and might therefore be involved in regulating the level of Ca 2؉ in the lumen, both under basal conditions and during hormonal stimulation. The failure of this mechanism might lead to pancreatic stone formation and even to pancreatitis.

In vivo microinjections of 55FeCl3 were made to assess renal iron (Fe2+/3+) transport in the anaesthetized rat. Following microinjection into proximal convoluted tubules (PCTs), 18·5 ± 2·9 % (mean ± s.e.m., n= 11) of the 55Fe was recovered in the urine. This recovery was not dependent on the injection site indicating that iron is not reabsorbed across the surface convolutions of the proximal tubule. Following microinjection into distal convoluted tubules (DCTs) 46·1 ± 6·1 % (n= 8) of the injected 55Fe was recovered. Taken together the recovery data from the PCT and DCT microinjection studies indicate that the transport of iron occurs in the loop of Henle (LH) and collecting duct system. In vivo luminal microperfusion was used to examine iron transport by the LH in more detail. In tubules perfused with 7 μmol l−155FeCl3, 52·7 ± 8·3 % (n= 8) of the perfused 55Fe was recovered in the collected fluid, indicating significant iron reabsorption in the LH. Addition of copper (Cu2+ as 7 μmol l−1 CuSO4), manganese (Mn2+ as 7 μmol l−1 MnSO4) or zinc (Zn2+ as 7 μmol l−1 ZnSO4) to the perfusate did not affect reabsorption of water, Na+ or K+, but increased recovery of 55Fe to 83·5 ± 6·8 % (n= 8, P < 0·04), 75·8 ± 5·9 (n= 6, not significant, n.s.) and 67·9 ± 3·8; (n= 9, n.s.), respectively. Thus, iron transport in the LH can be reduced by the addition of copper or manganese to the luminal perfusate suggesting that these ions may compete with iron for a common transport pathway. However, this pathway may not be shared by zinc.

Both the acinar and ductal cells of the pancreas secrete a near-isotonic fluid and may thus be sites of aquaporin (AQP) water channel expression. Northern blot analysis of mRNA from whole rat pancreas revealed high levels of AQP1 and AQP8 expression, whereas lower levels of AQP4 and AQP5 expression were just detectable by RT-PCR Southern blot analysis. Immunohistochemistry showed that AQP1 is localized in the microvasculature, whereas AQP8 is confined to the apical pole of the acinar cells. No labeling of acinar, ductal, or vascular tissue was detected with antibodies to AQP2-7. With immunoelectron microscopy, AQP8 labeling was observed not only at the apical membrane of the acinar cells but also among small intracellular vesicles in the subapical cytoplasm, suggesting that there may be regulated trafficking of AQP8 to the apical plasma membrane. To evaluate the contribution of AQPs to the membrane water permeability, video microscopy was used to measure the swelling of acinar cells in response to hypotonic stress. Osmotic water permeability was reduced by 90% following exposure to Hg(2+). Since AQP8 is confined to the apical membrane, the marked effect of Hg(2+) suggests that other water channels may be expressed in the basolateral membrane.

Divalent metal transporter1 (DMT1; also known as DCT1 or NRAMP2) is an important component of the cellular machinery responsible for dietary iron absorption in the duodenum. DMT1 is also highly expressed in the kidney where it has been suggested to play a role in urinary iron handling. In this study, we determined the effect on renal DMT1 expression of feeding an iron-restricted diet (50 mg/kg) or an iron-enriched diet (5 g/kg) for 4 wk and measured urinary and fecal iron excretion rates. Feeding the low-iron diet caused a reduction in serum iron concentration and fecal iron output rate with an increase in renal DMT1 expression. Feeding an ironenriched diet had the converse effect. Therefore, DMT1 expression in the kidney is sensitive to dietary iron intake, and the level of expression is inversely related to the dietary iron content. Changes in DMT1 expression occurred intracellularly in the proximal tubule and in the apical membrane and subapical region of the distal convoluted tubule. Increased DMT1 expression was accompanied by a decrease in urinary iron excretion rate and vice versa when DMT1 expression was reduced. Together, these findings suggest that modulation of renal DMT1 expression may influence renal iron excretion rate. serum iron level; kidney; iron regulatory protein; SLC11A2; NRAMP2 IRON IS AN ESSENTIAL metal for life because it is a key constituent of a family of fundamental proteins, which includes hemoglobin, cytochromes, and NADH-coenzyme Q reductase. Maintaining the correct balance of iron is paramount to health because iron deficiency or excess results in morbidity and mortality. The molecular characterization of membrane-bound iron transporter proteins, in particular divalent metal transporter1 (DMT1; 9), also known as DCT1 (14) or NRAMP2 (13), has shed new light on some of the mechanisms of body iron homeostasis. DMT1 is the product of the