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Environmental exposures to cadmium (Cd) are a major cause of human toxicity. The kidney is the most sensitive organ; however, the natures of injuries and of adaptive responses have not been adequately investigated, particularly in response to environmental relevant Cd concentrations. In this study, rats received a daily ip injection of low CdCl₂ dose (0.3 mg Cd/kg body mass) and killed at 1, 3, and 5 days of intoxication. Functional, ultrastructural, and biochemical observations were used to evaluate Cd effects. We show that Cd at such subtoxic doses does not affect the tubular functions nor does it induce apoptosis. Meanwhile, Cd accumulates within lysosomes of proximal convoluted tubule (PCT) cells where it triggers cell proliferation and autophagy. By developing an immunohistochemical assay, a punctate staining of light chain 3-II is prominent in Cd-intoxicated kidneys, as compared with control. We provide the evidence of a direct upregulation of autophagy by Cd using a PCT cell line. Compared with the other heavy metals, Cd is the most powerful inducer of endoplasmic reticulum stress and autophagy in PCT cells, in relation to the hypersensitivity of PCT cells. Altogether, these findings suggest that kidney cortex adapts to subtoxic Cd dose by activating autophagy, a housekeeping process that ensures the degradation of damaged proteins. Given that Cd is persistent within cytosol, it might damage proteins continuously and impair at long-term autophagy efficiency. We therefore propose the autophagy pathway as a new sensitive biomarker for renal injury even after exposure to subtoxic Cd doses.

Newborn rat distal cells express an apical Ca2+ channel activated by dihydropyridine drugs. Similarly, in Madin-Darby canine kidney (MDCK) cells, nifedipine increased Ca2+i in a concentration-dependent manner (IC50=4 μM) in fura-2-loaded cells. Response to nifedipine was abolished by EGTA, suggesting that it depends on extracellular calcium. Ca2+ channel antagonist isradipine and agonist BayK8644 increased Ca2+i indicating that this effect is related to the dihydropyridine group. Diltiazem (20 μM) and gadolinium (200 μM) decreased the nifedipine effect (62 and 43%, respectively). Lanthanum (100 μM) did not change the response. Valinomycin clamping of the membrane potential did not modify nifedipine-induced increment, indicating that it was unrelated to potassium fluxes. We performed whole cell clamp experiments in MDCK cells maintained at -50 mV with perfusion solution containing 10 mM CaCl2. Nifedipine (20 μM) induced an increase in current (1.2±0.3 nA), which was partially inhibited by Gd3+. No significant current was induced by nifedipine in the presence of 0.5 mM EGTA. To determine the effects of nifedipine on the membrane potential, we performed oxonol fluorescence experiments. The addition of nifedipine or Bay K8644 induced depolarization, highly dependent on external sodium. Nifedipine (20 μM) induced depolarization of 6.9±0.8 mV (n=21). EC50 to nifedipine was in the 10 μM range. We conclude that MDCK cells exhibit a dihydropyridine-activated cationic channel., (Copyright © 2011 S. Karger AG, Basel.)

In the adult rat, chronic cadmium intoxication induces nephropathy with Fanconi-like features. This result raises the question of whether intoxication of pregnant rats has any deleterious effects on renal function in their offspring. To test this hypothesis, we measured the renal function of 2- to 60-day-old postnatal offspring from female rats administered cadmium chloride by the oral route (0.5 mg.kg(-1).day(-1)) throughout their entire gestation. Investigations of rat offspring from contaminated pregnant rats showed the presence of cadmium in the kidney at gestational day 20. After birth, the cadmium kidney concentration increased from postnatal day 2 to day 60 (PND2 to PND60), presumably because of 1) milk contamination and 2) neonatal liver cadmium content release. Although the renal parameters (glomerular filtration, U/P inulin, and urinary excretion rate) were not significantly affected until PND45, renal failure appeared at PND60, as demonstrated by a dramatic decrease of the glomerular filtration rate associated with increased excretion of the main ions. In parallel, an immunofluorescence study of tight-junction protein expression of PND60 offspring from contaminated rats showed a disorganization of the tight-junction proteins claudin-2 and claudin-5, specifically expressed in the proximal tubule and glomerulus, respectively. In contrast, expression of a distal claudin protein, claudin-3, was not affected. In conclusion, in utero exposure of cadmium leads to toxic renal effects in adult offspring. These results suggest that contamination of pregnant rats is a serious and critical hazard for renal function of their offspring.

This study investigates the effect in the rat of chronic CdCl2 intoxication (500 microg Cd2+/kg, daily i.p. 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 microg 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.

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.

86Rb+ efflux was measured on polarized Madin-Darby canine kidney cells under A23187 or ATP stimulation. This efflux, inhibited by barium, Leiurus quinquestriatus hebraeus venom and charybdotoxin was attributed to the stimulation of Ca(++)-activated maxi K+ channels. Snake venom from Dendroaspis polylepis did not alter the stimulation as well as did apamine. ATP was effective on both the apical and basolateral membranes and the Ca(++)-activated maxi K+ channels were predominantly found on the basolateral membrane. This study presents the physiological evidence that dendrotoxin is ineffective on the epithelial Ca(++)-activated maxi K+ channel present in MDCK cells.

The effects of Leiurus quinquestriatus hebraeus (LQH) venom, mamba venom, Buthus tamulus (BT) venom, purified apamin and synthetic charybdotoxin on the membrane hyperpolarization induced by extracellular ATP were examined in Madin-Darby canine kidney cells. For this we used a membrane potential probe (bisoxonol) to determine the potential variations. The relation between bisoxonal fluorescence and membrane potential was established by treating Madin-Darby canine kidney cells suspended in solutions containing various external sodium concentrations with gramicidin. Extracellular ATP induced a rapid hyperpolarization that was blocked by LQH venom and synthetic charybdotoxin. BT venom also blocked the response but at a much higher concentration than that of LQH. Mamba venom (Dendroaspis polylepis) and apamin did not modify the ATP-induced hyperpolarization. We concluded that the ATP induced hyperpolarization was due to the augmentation of the potassium conductance probably through Ca(2+)-activated K+ channels sensitive to charybdotoxin but not to mamba venom. The interaction previously described between charybdotoxin and dendrotoxin (the main toxin of mamba venom) was not observed in our case.

The mouse strain C57BL is characterized by a high urinary fractional excretion of taurine. Hypertaurinuric [tau(-)] C57BL mice and three normal taurine 'excretors' [tau(+)]-Swiss, A/J, and C3H-were compared. Clearance experiments showed that fractional excretion of taurine was 4-8 times higher in C57BL relative to the other strains. Transport studies performed in brush border membrane vesicles, isolated from C57BL and C3H mouse renal cortex, showed that D-glucose uptake was similar in both strains while the taurine uptake appeared to be faster in tau(+) than in tau(-) mice. This difference was observed only in the presence of an inwardly directed NaCl gradient. The comparison of kinetic parameters showed a significant difference of apparent Km values: 90.8 +/- 11.4 microM in tau(+) mice vs. 129.5 +/- 17.1 microM in tau(-) mice. Jmax were not significantly different in both strains. These observations suggest a lower affinity of the taurine brush border membrane carrier in the hypertaurinuric strain as compared to the normotaurinuric mice. It is concluded that, in addition to a possible alteration of peritubular taurine efflux described by Rozen et al. (1983), a defect of taurine transport at the luminal membrane cannot be excluded and could account for the hypertaurinuria of the C57BL mice.

Changes in kidney function were studied in anesthetized female WAG/Rij rats 6, 18, and 30 mo old. The growth in kidney size measured between 6 and 18 mo and 18 and 30 mo was due to enlargement of the glomeruli and lengthening of the proximal tubules, without significant changes in the number of nephrons. Glomerular filtration per gram kidney was unaffected between 6 and 18 mo but diminished 27% between 18 and 30 mo. This reduction was accompanied by an intrarenal redistribution of individual filtration rates for the superficial and deep nephrons. Fractional excretion of sodium, potassium, calcium, and magnesium declined sharply between 6 and 18 mo but did not change further between 18 and 30 mo, whereas the fractional excretion of amino acids and glucose remained constant between 6 and 18 mo but rose in the 30-mo group. Taken together, these observations indicate that, in the rat, kidney aging cannot be reduced to a loss in the number of functional nephrons but is the result of several differential and specific processes.

By use of immunodepletion studies, we characterized four monoclonal antibodies reactive with rabbit brush-border (BB) as specific for aminopeptidase N (AP), dipeptidylpeptidase IV (DPPIV), neutral endopeptidase (EP), and angiotensin-converting enzyme (ACE), and we used these antibodies for immunohistochemical detection of these four hydrolases. Expression within the kidney was studied by light and electron microscopy. All four hydrolases are expressed on the various segments of the proximal tubule. In addition, EP and DPPIV are detectable on visceral epithelial cells of the glomerulus and AP on the cells of Bowman's capsule. Outside the kidney, the four hydrolases are expressed within the digestive and genital tracts, where AP, EP, and DPPIV predominate on epithelial structures, whereas ACE is essentially located in vascular structures. The latter localization is also characteristic of ACE in the other organs studied, where clear-cut systematic distribution of the other hydrolases was often difficult to demonstrate. In addition, AP, DPPIV, and EP were detected on lymphoid cells. As compared to reports of data obtained essentially by enzymatic or immunoradiometric assays, these observations suggest considerable interspecies variations of extrarenal expression of the major BB hydrolases. This should be taken into account in attempting to define a general physiological role for a given enzyme.

Several studies have suggested that clonidine, guanfacine and rilmenidine decrease systemic blood pressure by stimulating central alpha 2-adrenergic receptors. However, we have shown that these molecules interact not only with alpha 2-adrenergic but also a new type of "non catecholamine" receptor in rabbit and human renal proximal tubules. This receptor, which we have called the imidazoline-guanidium receptor site (IGRS) seems to be pharmacologically, biochemically and fractionally distinct from alpha 2-adrenergic receptors. In order to determine the relative affinity of rilmenidine for these two types of receptor, we studied its capacity to inhibit the liaison of (H3)-idazoxan, a ligand with a high affinity for the IGRS, and of (H3)-rauwolscine, a ligand selective for alpha 2-adrenergic receptors in the rabbit kidney. The results based on the apparent constants of inhibition (Ki) of the two radioligands [231 +/- 34 nM for (H3)-idazoxan and 2440 +/- 322 nM for (H3)-rauwolscine] showed that the selectivity of rilmenidine was 10 times greater for IGRS than for alpha 2-adrenergic receptors. This preferential activity on IGRS was confirmed by studies of the influx of Na22 into isolated renal proximal tubule cells of the rabbit. They showed that rilmenidine, in contrast to catecholamines, inhibited the transport of Na22 into the renal cells. In conclusion, the data from our studies shows that rilmenidine interacts with renal IGRS and inhibits cellular transport of sodium by a mechanism other than the stimulation of alpha 2-adrenergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

In control non diuretic (ND) and in salt-loaded (SL) rats, both the microsphere technique and the 14C ferrocyanide infusion technique were used to determine the distribution of microspheres in single glomeruli and the SNGFR of the corresponding nephrons. A sample of microspheres with a diameter averaging 11.0 +/- 2.6 mu SD was selected from a 15 +/- 5 mu unlabelled batch. In each rat, three million of these microspheres were injected through the left carotid artery. The microspheres were directly counted under microscopic observation in the glomeruli of the nephrons which were microdissected to determine the SNGFR value. The number of microspheres per glomerulus for a given kidney generally varied from 0 to 8 and was independent to the SNGFR value. The diameter of the microspheres trapped was constant in all the animals. The mean number of microspheres for superificial (S) and juxtamedullary (JM) nephrons was, (ND); S = 1.99 +/- 0.48 SE., n = 5; JM = 3.02 +/- 0.51 SE, n = 5; P less than 0.02, (SL): S = 3.75 +/- 0.53 SE, n = 6; JM = 2.86 +/- 0.33 SE, n = 6; P less than 0.05. This distribution was directly related to that of SNGFR in ND rats (S = 39.0 +/- 6.1 SD and JM = 49.5 +/- 10.3 nl/min) but not in SL rats (S = 50.9 +/- 6.1 and JM = 66.9 +/- 10.0 nl/min). In conclusion, the microsphere technique described in the present paper, appears more suitable for investigating single glomerular blood flow since the number and the size of the microspheres trapped are directly determined at the level of the glomerulus.