Heavy metals such as cadmium (Cd), mercury (Hg), lead (Pb), chromium (Cr) and platinum (Pt) are a major environmental and occupational hazard. Unfortunately, these non-essential elements are toxic at very low doses and non-biodegradable with a very long biological half-life. Thus, exposure to heavy metals is potentially harmful. Because of its ability to reabsorb and accumulate divalent metals, the kidney is the first target organ of heavy metal toxicity. The extent of renal damage by heavy metals depends on the nature, the dose, route and duration of exposure. Both acute and chronic intoxication have been demonstrated to cause nephropathies, with various levels of severity ranging from tubular dysfunctions like acquired Fanconi syndrome to severe renal failure leading occasionally to death. Very varied pathways are involved in uptake of heavy metals by the epithelium, depending on the form (free or bound) of the metal and the segment of the nephron where reabsorption occurs (proximal tubule, loop of Henle, distal tubule and terminal segments). In this review, we address the putative uptake pathways involved along the nephron, the mechanisms of intracellular sequestration and detoxification and the nephropathies caused by heavy metals. We also tackle the question of the possible therapeutic means of decreasing the toxic effect of heavy metals by increasing their urinary excretion without affecting the renal uptake of essential trace elements. We have chosen to focus mainly on Cd, Hg and Pb and on in vivo studies.

The eukaryotic initiation factor 5A (eIF5A), which is highly conserved throughout evolution, has the unique characteristic of post-translational activation through hypusination. This modification is catalyzed by two enzymatic steps involving deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). Notably, eIF5A may be involved in regulating the lifespan of during long-term hypoxia. Therefore, we investigated the possibility of a link between eIF5A hypusination and cellular resistance to hypoxia/anoxia. Pharmacologic targeting of DHPS by1-guanyl-1,7-diaminoheptane (GC7) or RNA interference-mediated inhibition of DHPS or DOHH induced tolerance to anoxia in immortalized mouse renal proximal cells. Furthermore, GC7 treatment of cells reversibly induced a metabolic shift toward glycolysis as well as mitochondrial remodeling and led to downregulated expression and activity of respiratory chain complexes, features characteristic of mitochondrial silencing. GC7 treatment also attenuated anoxia-induced generation of reactive oxygen species in these cells and in normoxic conditions, decreased the mitochondrial oxygen consumption rate of cultured cells and mice. In rats, intraperitoneal injection of GC7 substantially reduced renal levels of hypusinated eIF5A and protected against ischemia-reperfusion-induced renal injury. Finally, in the preclinical pig kidney transplant model, intravenous injection of GC7 before kidney removal significantly improved graft function recovery and late graft function and reduced interstitial fibrosis after transplant. This unconventional signaling pathway offers an innovative therapeutic target for treating hypoxic-ischemic human diseases and organ transplantation.

The functional properties and the pharmacological profile of the recently cloned cDNA colonic P-ATPase alpha subunit (Crowson, M.S., and Shull, G.E. (1992) J. Biol. Chem. 267, 13740-13748) were investigated by using the Xenopus oocyte expression system. Xenopus oocytes were injected with alpha subunit cRNAs from Bufo marinus bladder or rat distal colon and/or with beta subunit cRNA from B. marinus bladder. Two days after injection, K+ uptake was measured by using 86 Rb+ as a K+ surrogate, and pH measurements were performed by means of ion-selective microelectrodes. Co-injection of alpha and beta subunit cRNAs led to a large increase in 86Rb+ uptake, an intracellular alkalinization, and an extracellular medium acidification, as compared to alpha or beta injection alone. These results indicate that the colonic P-ATPase alpha subunit, like the bladder alpha subunit, acts as a functional H+,K+-ATPase, and that co-expression of alpha and beta subunits is required for the function. External K+ activation of the 86Rb+ uptake had a K1/2 of approximately 440 microM for the bladder isoform (consistent with the previously reported value (Jaisser, F., Horisberger, J.D., Geering, K., and Rossier, B.C. (1993) J. Cell. Biol. 123, 1421-1431) and a K1/2 of approximately 730 microM for the colonic isoform. Sch28080 was ineffective to reduce 86Rb+ uptake whereas ouabain inhibited the activity expressed from rat colon alpha subunit with a Ki of 970 microM when measured at the Vmax of the enzyme. We conclude that, when expressed in Xenopus oocytes, the rat colon P-ATPase alpha subunit encodes a ouabain-sensitive H+,K+-ATPase.

This study investigates the effect in the rat of chronic CdCl2 intoxication (500 μg Cd2+/kg, daily ip injection for 5 days) on renal function and the changes in tight junction proteins claudin-2, claudin-3, and claudin-5 present in rat kidney. We also studied the effect of coadministration of ZnCl2 (500 μg Zn2+/kg) during chronic CdCl2 intoxication. Our results indicate that 1) most of the filtered Cd2+ is reabsorbed within the kidney; 2) chronic Cd2+ intoxication can induce a change in renal handling of ions without altering glomerular filtration rate; 3) a delayed nephropathy, showing Fanconi-like features, appears more than 5 days after the end of CdCl2 exposure; 4) epithelial integrity is altered by chronic Cd2+ intoxication affecting the expression and localization of claudin tight junction proteins; and 5) cotreatment with Zn2+ protects against the renal toxic effects of Cd2+, preventing altered claudin expression and inhibiting apoptosis. In conclusion, these results show that Cd2+ toxicity and cellular toxic mechanisms are complex, probably affecting both membrane transporters and tight junction proteins. Finally, Zn2+ supplementation may provide a basis for future treatments.

The Na,K-ATPase is composed of two subunits, alpha and beta, and each subunit consists of multiple isoforms. In the case of alpha, four isoforms, alpha1, alpha2, alpha3, and alpha4 are present in mammalian cells. The distribution of these isoforms is tissue- and developmental-specific, suggesting that they may play specific roles, either during development or coupled to specific physiological processes. In order to understand the functional properties of each of these isoforms, we are using gene targeting, where animals are produced lacking either one copy or both copies of the corresponding gene or have a modified gene. To date, we have produced animals lacking the alpha1 and alpha2 isoform genes. Animals lacking both copies of the alpha1 isoform gene are not viable, while animals lacking both copies of the alpha2 isoform gene make it to birth, but are either born dead or die very soon after. In the case of animals lacking one copy of the alpha1 or alpha2 isoform gene, the animals survive and appear healthy. Heart and EDL muscle from animals lacking one copy of the alpha2 isoform exhibit an increase in force of contraction, while there is reduced force of contraction in both muscles from animals lacking one copy of the alpha1 isoform gene. These studies indicate that the alpha1 and alpha2 isoforms carry out different physiological roles. The alpha2 isoform appears to be involved in regulating Ca(2+) transients involved in muscle contraction, while the alpha1 isoform probably plays a more generalized role. While we have not yet knocked out the alpha3 or alpha4 isoform genes, studies to date indicate that the alpha4 isoform is necessary to maintain sperm motility. It is thus possible that the alpha2, alpha3, and alpha4 isoforms are involved in specialized functions of various tissues, helping to explain their tissue- and developmental-specific regulation.

Barbier, O., G. Jacquillet, M. Tauc, P. Poujeol, and M. Cougnon. Acute study of interaction among cadmium, calcium, and zinc transport along the rat nephron in vivo. Am J Physiol Renal Physiol 287: F1067-F1075, 2004. First published July 27, 2004 doi:10.1152/ajprenal.00120.2004.-This study investigates the effect in rats of acute CdCl2 (5 M) intoxication on renal function and characterizes the transport of Ca 2ϩ , Cd 2ϩ , and Zn 2ϩ in the proximal tubule (PT), loop of Henle (LH), and terminal segments of the nephron (DT) using whole kidney clearance and nephron microinjection techniques. Acute Cd 2ϩ injection resulted in renal losses of Na ϩ , K ϩ , Ca 2ϩ , Mg 2ϩ , PO 4 Ϫ2 , and water, but the glomerular filtration rate remained stable. 45 Ca microinjections showed that Ca 2ϩ permeability in the DT was strongly inhibited by Cd 2ϩ (20 M), Gd 3ϩ (100 M), and La 3ϩ (1 mM), whereas nifedipine (20 M) had no effect. 109 Cd and 65 Zn 2ϩ microinjections showed that each segment of nephron was permeable to these metals. In the PT, 95% of injected amounts of 109 Cd were taken up. 109 Cd fluxes were inhibited by Gd 3ϩ (90 M), Co 2ϩ (100 M), and Fe 2ϩ (100 M) in all nephron segments. Bumetanide (50 M) only inhibited 109 Cd fluxes in LH; Zn 2ϩ (50 and 500 M) inhibited transport of 109 Cd in DT. In conclusion, these results indicate that 1) the renal effects of acute Cd 2ϩ intoxication are suggestive of proximal tubulopathy; 2) Cd 2ϩ inhibits Ca 2ϩ reabsorption possibly through the epithelial Ca 2ϩ channel in the DT, and this blockade could account for the hypercalciuria associated with Cd 2ϩ intoxication; 3) the PT is the major site of Cd 2ϩ reabsorption; 4) the paracellular pathway and DMT1 could be involved in Cd 2ϩ reabsorption along the LH; 5) DMT1 may be one of the major transporters of Cd 2ϩ in the DT; and 6) Zn 2ϩ is taken up along each part of the nephron and its transport in the terminal segments could occur via DMT1. heavy metals; epithelial calcium channel; divalent metal transporter 1; kidney CADMIUM (CD 2ϩ ) IS ONE OF THE most commonly found toxic metals present in our environment. The major sources of exposure to Cd 2ϩ are contaminated food and water, tobacco, and industrial fumes and dusts (16). Cd 2ϩ accumulates in the body, and chronic exposure causes severe nephrotoxicity in humans (16) and animals (2, 4). The renal dysfunction may be due to proximal tubular damage affecting the passive paracellular pathway (14, 27) and decreasing active transcellular ion transport (30). With the use of in vitro models, deleterious effects of Cd 2ϩ have been described on several solute transporters, such as stretch-activated ion channels (24), the epithelial Ca 2ϩ channel (ECaC) transporter (32), the NaPi-II transporter (19), the Na/glucose transporter (1), and the NaSi-1 transporter (25). These acute effects of Cd 2ϩ suggest the involvement of ion transporters in Cd 2ϩ -induced nephropathy. Therefore, the question arises as to whether these transporters are affected in vivo after Cd 2ϩ exposure. To answer this question,...

Chloride channels play an essential role in a variety of physiological functions and in human diseases. Historically, the field of chloride channels has long been neglected owing to the lack of powerful selective pharmacological agents that are needed to overcome the technical challenge of characterizing the molecular identities of these channels. Recently, members of the LRRC8 family have been shown to be essential for generating the volume-regulated anion channel (VRAC) current, a chloride conductance that governs the regulatory volume decrease (RVD) process. The inhibitory effects of six commonly used chloride channel inhibitors on VRAC/LRRC8-mediated chloride transport were tested in wild-type HEK-293 cells expressing LRRC8 proteins and devoid of other types of chloride channels (CFTR and ANO1/2). We explored the effectiveness of the inhibitors using the patch-clamp whole-cell approach and fluorescence-based quantification of cellular volume changes during hypotonic challenge. Both DCPIB and NFA inhibited VRAC current in a whole-cell configuration, with IC50 values of 5 ± 1 μM and 55 ± 2 μM, respectively. Surprisingly, GlyH-101 and PPQ-102, two CFTR inhibitors, also inhibited VRAC conductance at concentrations in the range of their current use, with IC50 values of 10 ± 1 μM and 20 ± 1 μM, respectively. T16Ainh-A01, a so-called specific inhibitor of calcium-activated Cl- conductance, blocked the chloride current triggered by hypo-osmotic challenge, with an IC50 of 6 ± 1 μM. Moreover, RVD following hypotonic challenge was dramatically reduced by these inhibitors. CFTRinh-172 was the only inhibitor that had almost no effect on VRAC/LRRC8-mediated chloride conductance. All inhibitors tested except CFTRinh-172 inhibited VRAC/LRRC8-mediated chloride conductance and cellular volume changes during hypotonic challenge. These results shed light on the apparent lack of chloride channel inhibitors specificity and raise the question of how these inhibitors actually block chloride conductances.

We previously have demonstrated that the colonic P-ATPase ␣ subunit cDNA encodes an H,K-ATPase when expressed in Xenopus laevis oocytes. Besides its high level of amino acid homology (75%) with the Na,K-ATPase, the colonic H,K-ATPase also shares a common pharmacological profile with Na,K-ATPase, because both are ouabain-sensitive and Sch 28080-insensitive. These features raise the possibility that an unrecognized property of the colonic H,K-ATPase would be Na ؉ translocation. To test this hypothesis, ionselective microelectrodes were used to measure the intracellular Na ؉ activity of X. laevis oocytes expressing various combinations of P-ATPase subunits. The results show that expression in oocytes of the colonic H,K-ATPase affects intracellular Na ؉ homeostasis in a way similar to the expression of the Bufo marinus Na,K-ATPase; intracellular Na ؉ activity is lower in oocytes expressing the colonic H,K-ATPase or the B. marinus Na,K-ATPase than in oocytes expressing the gastric H,K-ATPase or a ␤ subunit alone. In oocytes expressing the colonic H,K-ATPase, the decrease in intracellular Na ؉ activity persists when diffusive Na ؉ inf lux is enhanced by functional expression of the amiloride-sensitive epithelial Na ؉ channel, suggesting that the decrease is related to increased active Na ؉ eff lux. The Na ؉ decrease depends on the presence of K ؉ in the external medium and is inhibited by 2 mM ouabain, a concentration that inhibits the colonic H,KATPase. These data are consistent with the hypothesis that the colonic H,K-ATPase may transport Na ؉ , acting as an (Na,H),K-ATPase. Despite its molecular and functional characterization, the physiological role of the colonic (Na,H),KATPase in colonic and renal ion homeostasis remains to be elucidated.