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Resume La communaute diabetologique a pu s’etonner de voir evoluer (en 2016) les recommandations relatives au traitement par metformine dans l’insuffisance renale chronique en l’absence d’etude prospective publiee, et donc d’argumentation scientifique solide. Mais c’est desormais chose faite, et cet article relate la construction de cet element manquant. Et de nouveaux elements s’agissant de la relation entre le rein et la metformine : cette derniere s’avere nephroprotectrice !

Background Metformin is the first prescribed drug for hyperglycemia in type 2 diabetes mellitus. Mainly by activating AMPK pathway, this drug exerts various functions that among them protective effects are of the interest. Purpose Herein, we aimed to gather data about the protective impacts of metformin against various natural or chemical toxicities. Results An extensive search among PubMed, Scopus, and Google Scholar was conducted by keywords related to protection, toxicity, natural and chemical toxins and, metformin. Our literature review showed metformin alongside its anti-hyperglycemic effect has a wide range of anti-toxic effects against anti-tumour and routine drugs, natural and chemical toxins, herbicides and, heavy metals. Conclusion It is evident that metformin is a potent drug against the toxicity of a broad spectrum of natural, chemical toxic agents which is proved by a vast number of studies. Metformin mainly through AMPK axis can protect different organs against toxicities. Moreover, metformin preserves DNA integrity and can be an option for adjuvant therapy to ameliorate side effect of other therapeutics.

In the present study, we characterized and compared the mineral phase deposited in the aortic wall of two different frequently used chronic renal failure rat models of vascular calcification. Vascular calcification was induced in rats by either a 4-week adenine treatment followed by a 10-week high-phosphate diet or 5/6 nephrectomy followed by 6 weeks of 0.25 μ g/kg/day calcitriol treatment and a high-phosphate diet. Multi-element mapping for calcium and phosphate together with mineral identification was performed on several regions of aortic sections by means of synchrotron X-ray- μ -fluorescence and diffraction. Bulk calcium and magnesium content of the aorta was assessed using flame atomic absorption spectrometry. Based on the diffraction data the Von Kossa-positive precipitate in the aortic regions ( N =38) could be classified into three groups: (1) amorphous precipitate (absence of any diffraction peak pattern, N =12); (2) apatite ( N =16); (3) a combination of apatite and magnesium-containing whitlockite ( N =10). The occurrence of these precipitates differed significantly between the two models. Furthermore, the combination of apatite and whitlockite was exclusively found in the calcitriol-treated animals. These data indicate that in adenine/phosphate-induced uremia-related vascular calcification, apatite is the main component of the mineral phase. The presence of magnesium-containing whitlockite found in addition to apatite in the vitamin D-treated rats, has to be seen in view of the well-known vitamin D-stimulated gastrointestinal absorption of magnesium.

BACKGROUND During the past decade, the donor age of cadaveric renal allografts steadily increased. Because cerebrovascular injury is the main cause of death in this donor population, an increased prevalence of atherosclerotic lesions in the retrieved grafts could be anticipated. In a prospective study, we investigated the predictive value of morphologic lesions at implantation for the functional and morphologic outcome of cadaveric renal allografts at 1 1/2 years. METHODS In 50 consecutive adult recipients of a cadaveric renal allograft, under cyclosporine-based regimen, implantation biopsies and subsequent protocol biopsies at 18 months were performed, and morphometrically analyzed for the extent of glomerulosclerosis, interstitial fibrosis, and atherosclerosis. Risk factors were assessed at implantation and during the subsequent observation period of 18 months. Endpoints for this study were: the 24-hr creatinine clearance (normalized for body surface area) and the fractional interstitial volume at 1 1/2 years. RESULTS In multivariate analysis, fibrous intimal thickening at implantation (FIT) was the main determinant of the functional and morphologic outcome at 1 1/2 years. FIT represented a relative risk of 4.55 for interstitial fibrosis (95% CI=1.855-11.138), and 1.89 for impaired renal function (95% CI=1.185-3.007) at 1 1/2 years. FIT adversely affected fractional interstitial volume at 1 1/2 years (34.3 vs. 27.7%, P=0.004), as well as renal function (54 vs. 68 ml/min/1.73 m2, P=0.028). CONCLUSIONS Fibrous intimal thickening at implantation is a determinant risk factor for the functional and morphologic outcome of cadaveric renal allografts at 1 1/2 years.

In the present article, we show that flow cytometric immunodissection of cells immediately following their preparation from a tumor nephrectomy specimen is an accurate way of obtaining pure human primary cultures of proximal convoluted tubule origin, proximal straight tubule origin, distal tubular origin and/or collecting duct origin. By studying the expression of a panel of cell surface markers in these purified cultures, we could identify a number of markers that retain their lineage specificity in vitro. Using these appropriate stable markers, flow cytometry provides a simple yet accurate way of determining cell composition in previously unsorted (mixed type) tubular epithelial cultures in terms of proximal versus distal tubule/collecting duct subpopulations. Both subpopulations in mixed type cultures are shown to retain functional characteristics of their in vivo counterparts (glucose uptake, hormonal stimulation of adenylate cyclase) as well as cell type-specific response patterns (such as inducibility of cell adhesion and histocompatibility molecules), indicating the usefulness of studying cell responses in vitro in a cell-type-dependent way. Finally we illustrate that multi-parameter flow cytometry is a powerful tool for assessing constitutive characteristics of and/or responses by the distinct cell subpopulations present in mixed type cultures.

But Nous menons une etude pilote dans le diabete de type 2 aux differents stades d’insuffisance renale (IR) pour savoir s’il est possible de poursuivre le traitement par metformine avec une posologie adaptee a la fonction renale. Nous presentons les resultats concernant les stades 1 a 4. Patients et methodes Il s’agit d’une etude ouverte des taux de metformine dans le plasma et les erythrocytes mesures au terme d’une semaine de traitement a dose progressive de metformine (mg/j): 500 ; 1 000 (500 matin et soir) ; 2 000 (1 000 matin et soir) ; et, pour le stade 1 d’IR seulement, 3 000 (1 000 matin et 2 000 soir). Chaque semaine de traitement est entrecoupee d’une semaine de « wash-out ». Les sujets inclus ont moins de 80 ans, une fonction renale stable depuis ≥ 3 mois, et un taux d’HbA1c ≥ 6,5 %. Les prelevements sanguins sont effectues 12 heures apres la derniere prise de metformine ; les valeurs sont comparees avec les tests de Kruskal-Wallis et de Mann-Whitney. La lactatemie est dosee dans les stades 3 et 4 d’IR. Resultats Quarante-neuf patients ont ete inclus, 42 ont termine l’etude (respectivement, 12, 9, 11, et 10 pour les stades 1 a 4 d’IR). Les concentrations plasmatiques de metformine augmentent progressivement avec la posologie, de facon significative dans les stades 1, 2, et 4 d’IR. Les taux erythrocytaires evoluent de facon comparable. La lactatemie est moderement augmentee (2,7 mmol/L) chez 1 patient de stade 3 et 1 autre de stade 4. Conclusion Une augmentation progressive des taux de metformine est objectivee avec l’augmentation de la posologie de metformine dans les stades 1 a 4 d’IR. La tolerance metabolique est bonne. Cette etude est a poursuivre au plus long cours avant d’envisager la poursuite de la metformine dans l’IR stable. Declaration d’interet Les auteurs declarent ne pas avoir d’interet direct ou indirect (financier ou en nature) avec un organisme prive, industriel ou commercial en relation avec le sujet presente.

The transient presence of infiltrated leukocytes in the kidney during acute renal failure as well as the location of these cells within the renal interstitium suggest their association with tubular injury and/or regeneration. To date, however, neither a positive nor a negative contribution of these cells to the pathophysiology of this disease could be unambiguously demonstrated. Ill-defined methods for identifying interstitial leukocytes have added to the controversy concerning the role of inflammatory cells in renal regeneration. The current literature survey presents a qualitative description of the renal interstitial accumulation of leukocytes as observed in some acute renal failure models, with special attention to those displaying acute tubular necrosis of particular nephron subsegments. We conclude that lethal or sublethal injury to renal tubular epithelial cells following toxic or ischemic insults leads to the manifestation of an interstitial mononuclear cell infiltrate. Whereas macrophages and T lymphocytes almost invariably take part, the former being the dominant cell population with respect to both magnitude and presence over time, polymorphonuclear cells seem to be significantly increased only in the case of pyelonephritis. Infiltrating cells have often been regarded rather harmful to the tissue, mainly due to the quite well understood injuring capacity of the latter. On the other hand, we speculate mononuclear leukocytes through their potential of producing different cytokines and growth factors (FGF, TGF-α, EGF-like, IL-2, etc.) might well play an initiating and mediating role in renal regeneration after acute tubular necrosis. Therefore, the role of infiltrating leukocytes in the injury/regeneration process during acute renal failure remains highly controversial and should be further elucidated.

An ECVAM task force on nephrotoxicity has been established to advise, in particular, on the follow-up to recommendations made in the ECVAM workshop report on nephrotoxicity testing in vitro. Since this workshop was held, in 1994, there have been several improvements in the techniques used. For example, the duration of renal slice viability, and the maintenance of functional activities in slices, have been improved by using dynamic incubation systems with higher oxygen tensions and more-appropriate cell culture media. Highly differentiated primary cultures of pig, human and rabbit proximal tubule cells have been established by using specific cell isolation procedures and/or selective culture media. To date, the most comparable phenotypic expression and transepithelial transport capacities to proximal tubules in vivo have been obtained with primary cultures of rabbit proximal tubule cells which are grown on bicompartmental supports; in this system, transepithelial substrate gradients are generated and the transepithelial transport of both organic anions and cations is highly active. This in vitro system has been selected by ECVAM for further evaluation and prevalidation. Industrial needs in the area of nephrotoxicity testing have been identified, and recommendations are made at the end of this report concerning possible future initiatives.

Background. The association between aluminium and dialysis encephalopathy and deterioration of the neurological state during desferrioxamine treatment of dialysis patients is well established. At present little is known about the speciation and the mechanisms underlying the element's neurotoxicity. Methods. Aluminium speciation was performed in cerebrospinal fluid samples of acutely aluminium-intoxicated dialysis patients using a recently developed high-performance liquid chromatographic/electrothermal atomic absorption spectrometric hybrid method. Results. Baseline cerebrospinal fluid aluminium levels of samples taken shortly after the intoxication were low but elevated (5.0 ± 2.0 μg/l, n=3) as compared to subjects with normal renal function (< 1 μg/l). In contrast to the situation noted in serum and to the iron speciation in cerebrospinal fluid, aluminium was not bound to transferrin but appeared as two distinct compounds, the main fraction eluting at the elution volume of aluminium-citrate/silicate. The second compound was not identified. Forty-four hours after desferrioxamine administration the cerebrospinal fluid aluminium levels had increased up to a concentration of 10.3 ± 2.5 μg/l (n =3). This was accompanied by a change in the speciation profile with aluminium appearing at the elution volume of aluminoxamine. Conclusions. Our findings may contribute to a better understanding of the neurotoxic effects of aluminium and its desferrioxamine chelate in dialysis patients.

Immunodissection of the human proximal nephron: Flow sorting of S1S2S3, S1S2 and S3 proximal tubular cells. We report on the use of several proximal tubular cell (PTC) surface markers and corresponding antibodies in fluorescence-activated cell sorting (FACS), and their ability to identify and flow sort cells of defined proximal tubular origin (S1S2S3) or of defined proximal subsegmental origin (S1S2 only/S3 only). We tested monoclonal/polyclonal antibodies directed against five different surface peptidases [leucine aminopeptidase (LAP), neutral endopeptidase 24.11 (NEP), dipeptidyl peptidase IV (DPPIV), aminopeptidase A (APA) and γ-glutamyl transferase (γ-GT)], the S3 segment-specific marker intestinal type alkaline phosphatase (LAP) and an S1S2 marker (TN20-antigen), originally proposed as a surface marker for interstitial fibroblasts. Seg-mental (proximal tubular vs. distal tubular) and proximal subsegmental (S1S2 vs. S3) expression of all five surface peptidases and TN20 antigen were first assessed by comparing immunohistochemical staining on normal human kidney tissue with staining for well-known segment-specific differentiation markers (intestinal type alkaline phosphatase, Tamm-Horsfall protein) on adjacent sections. All five peptidases were found to be expressed to a certain degree in all subsegments (SI S2 and S3) of the proximal nephron, whereas expression was never seen in the more distal parts of the nephron. Flow cytometry was performed on cells obtained following gradient purification of collagenase-digested human renal tissue. Labeling cells for expression of LAP, NEP or DPPIV resulted in high yields of specifically labeled PTC (S1S2S3 origin). Labeling with anti-LAP resulted in the clearest distinction between positive and negative cell subpopulations, and therefore LAP was considered the best PTC marker for use in FACS. iAP histochemical staining on sorted cells showed that flow sorting with monoclonal antibody (moAb) 250 (anti-intestinal type alkaline phosphatase) allowed sorting of S3 cells with > 90% purity. Likewise, moAb TN20 enabled us to obtain a highly purified S1S2 population as confirmed by the absence of iAP on sorted cells.

(1997). Urinary Biomarkers to Detect Significant Effects of Environmental and Occupational Exposure to Nephrotoxins. IV. Current Information on Interpreting the Health Implications of Tests. Renal Failure: Vol. 19, No. 4, pp. 553-566.

Background. The occurrence of analgesic nephropathy (AN) among renal replacement therapy patients in former Czechoslovakia is not known. Previous surveys were not based on representative samples and lacked uniform criteria for diagnosing the disease. Methods. Incidence of AN in former Czechoslovakia was investigated in patients commencing renal replacement therapy in 24 (1/3 of all) dialysis centres from 1 January to 31 December 1992. Patients showing an unclear renal diagnosis (« = 149) were investigated with an interview and renal imaging techniques. The diagnosis of AN was withheld or rejected on the base of recently published diagnostic criteria demonstrating that a decreased renal mass of both kidneys combined with bumpy contours and/or papillary calcifications had a high performance for diagnosing AN (Nephrol Dial Transplant 1992; 7: 479-486). Results. Based on the renal imaging criteria, AN was diagnosed in 30 of 328 registered patients, resulting in an AN incidence of 9.1% while the EDTA data only mentioned an incidence of 4.8% (period 1986-1989). The products most commonly abused were analgesic mixtures containing two analgesic substances combined with caffeine and/or codeine. Conclusions. AN was found to be a common disease in the Czech and Slovak Republics. The disease was diagnosed using reliable renal imaging criteria.

Cytosolic glutathione S-transferase (GST) (EC 2.5.1.18) isoenzymes of dog kidney and MDCK (an established dog renal cell line) were purified and studied. Specific GST activity was 248 and 317 nmol/min/mg protein, for dog and MDCK, respectively. Cytosolic GST was only partially purified by glutathione affinity chromatography, a substantial amount (43% and 84% for dog kidney and MDCK, respectively) of the GST activity was found in the flow-through fraction. Affinity bound GST was separated into 6 and 3 isoenzymes by anionic chromatofocusing for dog and MDCK, respectively. Flow-through GST was purified by gel filtration, anion exchange chromatography and anionic chromatofocusing showing only one GST isoenzyme, with distinct features from the affinity bound GST, for both dog and MDCK. The isoenzymes were characterized by their kinetic properties, subunit composition, specific substrates and inhibitors and immunoblot. The major dog GSTs (DII, DIV and DVI) correspond to the MDCK isoenzymes (MI, MII and MIII). Comparable pI values, a comparable affinity towards GSH and comparable sensitivitive towards the inhibitors N-ethylmaleimide (NEM), triphenyltin chloride, cibacron blue and hematin were observed for the corresponding isoenzymes: DII and MI, DIV and MII, DVI and MIII. Co-electrophoresis showed that the subunit composition was identical for DII and MI, and for DIV and MII. Inhibitor and substrate sensitivities showed that the affinity bound GSTs belong to class pi and mu, the presence of class pi was confirmed by immunoblot analysis. One homodimeric GST isoenzyme was observed in the dog kidney and MDCK flow-through. Both dog and MDCK isoenzyme have a nearly neutral pI, a high affinity towards CDNB and an equal sensitivity towards triphenyltin chloride, cibacron blue and hematin. However, based on inhibitor studies and immunoblot, this isoenzyme could not be attributed to an identified GST class. The overall isoenzyme pattern of dog and MDCK affinity bound and flow through GST is comparable. The dog and MDCK affinity bound GSTs have similar characteristics and all belong to class mu or pi.

A number of considerations suggest that trace element disturbances might occur in dialysed patients. These must at least in part be ascribed to the dialysis treatment itself during which these constituents may either be transferred to or removed from the patient. Tap water must be considered as the main source of dialysate trace metal contamination. These can adequately be removed during water treatment provided that, in addition to softening and deionization, reverse osmosis is available. However, even in the presence of the latter devices the possibility of serious contamination of the dialysis fluids leading to either chronic or acute intoxications still exists. The addition of chemical concentrates may also contribute to the increased concentrations of a number of trace metals. The toxic effects of aluminium in dialysis patients are well known and at the present time the element is still responsible for the greater part of trace metal-related problems in dialysis patients. Hence, the need for regular monitoring of aluminium cannot be ruled out at present. Strategies for diagnosis and treatment of aluminium overload have been updated. Recent studies demonstrated the efficacy of low desferrioxamine doses in diagnosis and treatment of aluminium overload, and optimal schedules for administration of the chelator and duration of treatment have been presented. Recently, in an epidemiological survey serum silicon concentrations in dialysis patients were found to be increased up to 100-fold compared to subjects with normal renal function. Moreover, it was noted that silicon concentrations in the dialysis population differ from one centre to another and that increased levels are due to either the use of silicon-contaminated dialysis fluids or an increased oral intake of the element originating from a high silicon content in the drinking water. Besides aluminium and silicon, a transfer towards the patients during dialysis has also been reported for a number of other elements including copper, zinc, nickel, strontium and chromium. The possible consequences of dialysate contamination with these elements will briefly be dealt with in the present paper. In contrast to trace metal accumulation, removal of trace metals during dialysis may at least in part contribute to the relative deficiency of particular essential elements. Selenium deficiency has repeatedly been observed. In view of the element's well-known essential role in glutathion peroxidase activity and the association of its deficiency with the development of some malignant diseases, further studies on the clinical impact of decreased serum selenium in dialysis patients are worthwhile. In conclusion, trace metal dialysate contamination/ depletion may contribute to the disturbed trace element concentration in dialysis patients. Aluminium accumulation is still an important problem in clinical nephrology. The clinical importance of the accumulation/ deficiency of trace elements other than aluminium is not yet fully understood and deserves further investigation.

Transferrin saturated with Al3+ subjected to isoelectric focusing (IEF) in a pH gradient can be separated into four fractions, representing the apotransferrin, transferrin with aluminum at the metal binding site in the C- or N-terminal lobe, or both. The electrophoretic mobilities of these four fractions are identical to those of the iron-transferrin counterparts. Simultaneous binding of aluminum and iron to transferrin can also be demonstrated. The decreased saturation after IEF indicates that the affinity of transferrin for aluminum is low compared with its affinity for iron. This effect is particularly evident when bicarbonate is used as the synergistic anion in the loading procedure. In contrast, loading of transferrin with aluminum in the presence of oxalate produces a di-aluminum-transferrin complex that is stable during IEF.

Rationnel La metformine n'a jamais ete etudiee de facon prospective dans les differents stades d'insuffisance renale (IR). Patients et Methodes L'etude (N° EudraCT 2012-001207-20) a consiste a traiter par metformine 12 sujets diabetiques de type 2 par stade d'IR (de 1 a 5, soit de « normal » a « insuffisance terminale », avec une subdivision pour le stade 3 en 3a et 3b), avec le plan experimental suivant : un traitement par metformine en 3 (stades 2 a 5) ou 4 (stade 1) phases de 1 semaine intercalees de 1 semaine de wash-out, aux doses de 500, 1 000, 2 000 et (pour le stade 1 seulement) 3 000mg/ j. Ont ete dosees au terme de chaque phase : la metforminemie plasmatique et erythrocytaire et, pour les stades 3 a 5, la lactatemie. Resultats Quatre-vingt-quatre patients ont ete etudies, dont 63 ont termine l'etude, avec un changement de stade d'IR pour 11 d'entre eux. L'âge moyen dans les differents stades d'IR etait de 52,4 a 63,6 ans. Sont rapportees seulement ici les metforminemies plasmatiques pour les doses de metformine de 500 et de 2 000mg/j : stade 1, 0,23±0,11 et 0,73±0,27mg/L ; stade 2, 0,46±0,26 et 0,84±0,62mg/L ; stade 3a, 0,86±0,54 et 1,31±0,90mg/L ; stade 3b, 0,97±0,52 et 2,07±1,03mg/L ; stade 4, 1,44±1,13 et 3,09±1,58mg/L ; et stade 5 ( n =6), 1,38±0,57 et 4,73±1,73mg/L. Les valeurs les plus elevees de metforminemie (jusqu'a 7,2mg/L) n'etaient pas associees a une hyperlactatemie (> 2,5mmol/l). Conclusions Une augmentation dose-dependante des metforminemies est observee, conduisant a une accumulation dans l'insuffisance renale severe. Cette observation justifie pleinement l'ajustement de la dose de metformine a la fonction renale. L'etude est actuellement poursuivie dans le plus long terme (4 mois) dans les stades 3 et 4 d'IR, cette fois avec une dose ajustee fixe. Travail realise avec le soutien de Merck Serono.

A quantitative metal-free high-performance liquid chromatographic/electrothermal atomic absorption spectrometric (HPLC/ETAAS) technique allowing an adequate separation of the proteins and of the inorganic/organic metal species of interest was developed. A silica-based scavenger was placed proximal to the injection valve retaining any remaining Al and Fe originating from buffer solutions and recipients. ETAAS instrumental conditions and matrix modifiers were carefully selected to eliminate interferences occurring due to the salt gradient elution. The postelution Al recovery (143-1428 micrograms L-1; 100-microL sample volume) was found to be 100.1 +/- 15.3% (N = 50) whereas that of Fe was 97.3 +/- 2.0% (N = 20; 442-2653 micrograms L-1). The protein recovery varied between 95 and 105%. The HPLC/ETAAS intraassay CV (N = 3) was 8.0% for Al (eluted amount of Al bound to transferrin (Tf), 7.3 ng) whereas that of Fe was 1.4% (eluted amount of Fe bound to Tf, 124.8 ng). The intraassay CV of the HPLC Tf determination was 1.6%. The proposed method is sensitive and accurate, allowing for the first time quantitative evaluation of the competition between Al and Fe for protein binding at clinical relevant concentrations. The technique will also be used for studying the protein binding of elements other than Al and Fe.

We developed a precise and accurate graphite furnace atomic absorption spectrometric method for the direct determination of zinc in serum. Serum samples are analyzed after 20-fold dilution with water of ultrapure analytical grade. No other reagent is used, from the moment of sampling until measurement. During atomization, the argon flow is kept at 150 mL/min instead of gas stop, to decrease the sensitivity and thus allow lower dilution ratios. Zinc concentrations are determined against a serum-matched calibration curve. Graphite tubes are uncoated and no L'vov platform is used. Between-run CVs were 5.9%, 3.5%, and 1.9% for serum zinc concentrations of 0.93, 1.15, and 1.43 mg/L, respectively. The characteristic mass was 9 pg, and the detection limit (meanblank + 3SDblank) was 0.060 mg/L.

Human intestinal alkaline phosphatase (IAP) can be released by the enterocyte into duodenal fluid as a mixture of three isoforms. A proportion of the enzyme is associated with triple-layered membrane vesicles (vesicular IAP). Although, occasionally, free hydrophilic IAP dimers are present, the remaining enzyme usually consists of a mixture of hydrophobic IAP dimers and more complex hydrophobic IAP structures of larger size, both entities being identified as "intestinal variant" alkaline phosphatase (VAR IAP). The hydrophobicity of VAR IAP stems exclusively from its attached glycosyl-phosphatidylinositol (GPI) anchor. Both vesicular IAP and VAR IAP are converted to hydrophilic enzyme upon removal of the GPI tail by phospholipase D (PLD) present in duodenal fluid. The IAP released into the vascular bed consists mainly of VAR IAP; vesicular IAP is absent. The enzyme characteristics of VAR IAP partially purified from duodenal fluid and from serum are identical. In plasma, VAR IAP appears to associate with (lipo)protein complexes and is thus protected from further degradation by plasma PLD. Such complex formation may explain why, in the serum of a healthy reference population, VAR IAP was more abundant than hydrophilic dimeric IAP.

Early treatment of patients with malignant disease and liver or bone metastasis may increase their survival time. We have used the activity patterns of liver and bone isoenzymes of alkaline phosphatase (ALP), separated by agarose gel electrophoresis, to detect early metastasis. We studied ALP isoenzyme patterns in a background population of 101 patients with no evidence of any disease that might influence this pattern; a healthy reference population (n = 330); and the following three groups of patients: 143 with malignant disease, 47 with nonmalignant liver disease, and 22 with nonmalignant bone disease. Cutoff and predictive values of liver ALP, high-molecular-mass (high-M(r)) ALP, and bone ALP were established for detecting liver and bone metastasis. The positive predictive value of liver and high-M(r) ALP was higher than that of total ALP in detecting liver metastasis, but liver and high-M(r) ALP did not enable us to differentiate between malignant and nonmalignant liver disease. Total ALP activity was of slightly more value than liver and high-M(r) ALP in enabling us to rule out liver metastasis. From bone ALP activity we could not distinguish between nonmalignant bone disease and bone metastasis. The negative predictive value of bone ALP in the diagnosis of bone metastasis was low, but its positive predictive value was high and superior to that of total ALP.

Using laser microprobe mass analysis (LAMMA), the composition of 'micro-calcifications (dystrophic type)' or 'metastatic calcifications', previously described in rats given high doses of cyclosporin, have been identified. Female Wistar rats given 12.5, 25 or 50 mg kg-1 per day of cyclosporin in olive oil by gastric gavage developed intrarenal calcifications detected by silver nitrate staining (von Kossa) in the outer medulla after two weeks. The calcifications stained red with periodic acid-Schiff reagent and appeared to be intraluminal microliths with concentric laminations. They could be identified by LAMMA as calcium phosphate (hydroxyapatite), indistinguishable from undecalcified bone. These studies indicate that intraluminal obstruction by calcium phosphate microliths, similar to that seen with magnesium depletion or high phosphate diets, may contribute to renal damage in rats given high-dose cyclosporin. LAMMA appears to be an appropriate technique for identifying both the elemental and organic moieities of intrarenal calcium deposits.

Even in specific risk groups, the relation between exposure to organic solvents and chronic renal diseases remains controversial. Thus, in a collaborative European study, we assessed the renal effects of occupational exposure to perchloroethylene (PCE) in dry-cleaners compared with matched controls who were simultaneously examined. Single high and low molecular weight proteins, kidney-derived antigens and enzymes, and prostanoids were measured in urine. beta 2-microglobulin, creatinine, laminin fragments, and anti-glomerular basement membrane antibodies were also measured in serum. A canonical function based on 23 such variables correctly classified 93% of individuals as either PCE-exposed or controls; with 13 markers, group membership was identified in 87% of subjects. Increased high molecular weight protein in urine was frequently (17/50 vs 1/50, p less than 0.0001) associated with tubular alterations. Changes were consistent with diffuse abnormalities along the nephron in workers exposed to low levels of PCE (median 15 parts per million). Generalised membrane disturbances might account for the increased release of laminin fragments, fibronectin, and glycosaminoglycans, for high molecular weight proteinuria, and for the increased shedding of epithelial membrane components from tubular cells with different location along the nephron (brush-border antigens and Tamm-Horsfall glycoprotein). These findings of early renal changes indicate that solvent-exposed subjects, especially dry-cleaners, need to be monitored for the possible development of chronic renal diseases.

Urinary intestinal alkaline phosphatase (EC 3.1.3.1; IAP) is a marker of the S3 segment of the human kidney proximal tubule. An accurate enzyme-antigen immunoassay (EAIA) with a high-affinity specific monoclonal antibody (IAP250) developed for this marker has a detection limit below the lowest IAP activity found in urine samples of normal subjects. The intra- and interassay CVs were less than 5%. Mean analytical recovery of pure IAP added to urine was 102% (SD 6%), and the EAIA results correlated well with immunoreactivity (measured by a sandwich ELISA), suggesting that the EAIA detected all of the IAP in urine. In healthy individuals (ages 20-80 years) the IAP concentrations, expressed as urinary creatinine ratios, ranged from 0.1 to 2.0 U/g (5-95 percentiles) without major differences related to sex and age. Workers exposed to mercury, which affects the S3 segment, showed an increased IAP elimination; abusers of analgesics, which affect more distal parts of the nephron, did not. As opposed to currently measured markers, the EAIA offers easy, accurate, and precise measurement of early alterations in the S3 segment.

Mammalian species of large stature have larger kidneys with larger nephrons than smaller animals, although this relationship is not linear. In order to investigate whether in animals of different sizes within the same species similar differences exist, a study in mongrel dogs was undertaken. In animals weighing between 9 and 42 kg with kidney weights from 25 to 79 g the area and perimeter of the glomeruli and the cortical width were measured. The number of glomeruli per unit area was counted and corrected for the size of the glomeruli. The results show that larger dogs have larger kidneys and that this enlargement is due to the increase in size of the nephrons and not in their number.

We investigated the pharmacokinetics of desferrioxamine and its chelated compounds aluminoxamine and ferrioxamine in normal volunteers and haemodialysis patients with and without iron overload. Desferrioxamine was administered in a single dose of 30 mg per kg body-weight was a 30-min infusion to five healthy volunteers and to 20 haemodialysis patients (five patients without haemosiderosis and 15 patients with haemosiderosis). The interdialytic half-life of ferrioxamine was 2.2 h in normal volunteers, 13.3 h in dialysis patients without haemosiderosis, and 24.6 h in patients with haemosiderosis. There was no interdialytic elimination of aluminoxamine. In a second study, seven dialysis patients received 5, 10, and 20 mg per kg body-weight desferrioxamine in a random order with a time interval of 2 weeks. The peak serum concentrations after these doses were 4.1 +/- 2.9, 6.4 +/- 2.9, and 10.7 +/- 7.1 mumol/l for ferrioxamine and 2.8 +/- 1.5, 3.1 +/- 1.5, and 4.2 +/- 1.7 mumol/l for aluminoxamine. Thus, a 4-fold increase in desferrioxamine dosage resulted in a 2.7-fold increase in peak ferrioxamine levels and in only a 1.5-fold increase in peak aluminoxamine levels. We conclude that dialysis patients, especially those with haemosiderosis, are exposed to persistently elevated ferrioxamine levels. Weekly doses of 5-10 mg/kg of desferrioxamine would be sufficient for aluminium chelation therapy.

Direct measurement of lead and cadmium in blood and urine by electrothermal atomic absorption spectrometry with deuterium background correction (D2-AAS) is prone to severe matrix and spectral interferences. We overcame these effects by coating the L'vov platform with ammonium molybdate, reducing the atomization time, introducing a post-atomization cooling step, carefully selecting ashing and atomization temperatures, and using an appropriate procedure for matrix modification. To determine Pb and Cd in blood and urine, we used matrix-matched calibration curves. With the proposed procedure for sample preparation, both Pb and Cd in whole blood can be determined in the same diluted sample. Results obtained by D2-AAS correlate closely with those by Zeeman-corrected AAS. Detection limits (mean blank + 3 SDblank) for Pb in urine and blood were 4 micrograms/L. For cadmium, the detection limits were 0.4 and 0.1 micrograms/L for urine and blood analysis, respectively. Between-run CVs were less than 5.0%.

We describe a direct-calibration method for determining aluminum in bone and soft tissues by graphite furnace atomic absorption spectrometry. Aluminum in bone was determined in uncoated tubes, whereas the analytical performance in soft tissue analysis was markedly improved by using pyrolytically coated tubes with a L'vov platform and added calcium. The analytical imprecision (CV) was less than or equal to 5.4% for aluminium concentrations between 12 and 98 micrograms/L, and analytical recoveries of added analyte varied between 94.5% and 103.0%. Detection limits (2 SD of determinations of the zero standard) were 7 and 6 pg for bone and soft tissues, respectively. Characteristic masses (amounts of Al yielding an absorbance signal of 0.0044 A) were 9 and 8 pg, respectively.

A heteroconjugate (HC) was synthesized between OKT3 and monoclonal antibody (MAb) 7E8, which specifically reacts with the tumor marker placental alkaline phosphatase (PLAP). Similarly to OKT3, in vitro, the HC induced a dose-dependent proliferation response of human peripheral-blood mononuclear cells (PBMCs) and, in concert with rIL-2, it progressively activated T cells over a 4-day period. In co-cultures of continuously activated PBMCs and MO4 tumor cells (non-MHC-restricted mouse fibroblasts transfected with the cDNA for PLAP), the HC (25 ng/ml), again acting in concert with rIL-2, induced specific lysis of the MO4 cells. This process occurred progressively over 2 to 3 days and was monitored from the release in the supernatant fluid of cellular 3H-L-leucine, but also from analyses involving the remaining non-lysed cancer cells, i.e., by estimates of their protein content, by measurements of their viability, and most accurately by determinations of their PLAP content. Antibody 7E8 by itself induced a weak tumor-cell lysis (ADCC), potentiated by the addition of rIL-2. However, after 7 days of PBMC-preactivation with the HC and rIL-2, antibody 7E8 no longer mediated any ADCC, whereas the HC-dependent lysis was further potentiated. The observed proliferation of T cells and development of cytotoxicity at low concentrations of HC and rIL-2 support the idea that a moderate but continuous T-cell activation combined with T-cell targeting is sufficient for the induction of progressive and efficient tumor-cell lysis.

Introduction - biology of renal tubulo-interstitial cells, M.E. De Broe and M.F. Helbert tissue culture of human renal epithelial cells using a defined serum-free growth formulation, D.A. Sens, C.J. Detrisac et al isolation, culture and characterization of human renal proximal tubule and collecting duct cells, A.L. Trifillis flow cytometric immunodissection of the human nephron in vivo and in vitro, M.J.F. Herbert, S. Dauwe and M.E. De Broe renal interstitial fibroblast culture, C. Grupp and G.A. Muller immortalized kidney cells derived from transgenic mice harbouring L-type pyruvate kinase and vimentin promoters, A. Vandewalle modulation of cell differentiation in perfusion culture, W.W. Minuth, P. Steiner et al renal tubular cells cultured from genetically modified animals, G. Friedlander, I. Runembert et al antisense and kidney cell research, M.B. Ganz protein overload activates proximal tubular cells to release vasoactive and inflammatory mediators, C. Zoja, A. Benigni and G. Remuzzi tubular epithelial cells - a critical cell type in the regulation of renal inflammatory processes, C. Van Kooten, M.R. Daha and L.A. van Es cytokine-stimulated nitric oxide production in the human renal proximal tubule and its modulation by natriuretic peptides - a novel immunomodulatory mechanism? P.K. Chatterjee, G.M. Hawksworth and J.S. MacLay decreased degradation of collagen and fibronectin following exposure of proximal cells to glucose, A.O. Phillips, K. Morrisey et al hypoxia-induced changes in extracellular matrix metabolism in renal cells, J.T. Norman, C. Orphanides et al fibrogenic effects of cyclosporin A on the tubulointerstitium - role of cytokines and growth factors, D.W. Johnson, H.J. Saunders et al.

Placental alkaline phosphatase (placental ALP, PLAP) and germ-cell ALP (GCAP, also known as placental-like ALP), expressed in gonadal cancer tissues, are potential tumor markers. Four monoclonal antibodies, raised against PLAP and recognizing different epitopes, were selected to study the influence of the following variables on the accuracy of PLAP and GCAP measurement: phenotype, molecular form, and glycation pattern of PLAP and GCAP; incubation temperature; and interferences by serum during immunobinding. Nine GCAP phenotypes were identified, interacting with each antibody at a lower affinity than was seen for the more common PLAP phenotypes. Antibody affinity is higher for the free hydrophilic dimeric forms of PLAP and GCAP, and is not influenced by the degree of glycation. In serum or tissue extracts, measurement of PLAP or GCAP is most nearly accurate when immunoincubations are performed at 37 degrees C, with use of antibodies 327 and 7E8, respectively. In addition, correct measurements are achieved only when, during immunobinding, serum is incubated with an equal volume of deoxycholate (9 g/L final concentration).

We separated isoenzymes of alkaline phosphatase (ALP; EC 3.1.3.1) in 1383 sera of normal individuals (ages 4-65 years) by agarose electrophoresis with the Isopal system (Analis). As expected, the predominant isoenzyme in children was of bone origin, and almost all (99%) of the children had low activities of a second bone fraction, "bone variant" ALP. The "bone variant" disappeared after age 17 in girls and after age 20 in boys. The highest (median) bone ALP activity was reached at age 9 to 10 in girls and at age 13 to 14 in boys, followed by a gradual decline in girls and a steep decline in boys. During adulthood, activity of the bone fraction was constant and no significant differences were observed between sexes, neither for bone nor for liver ALP activity. The latter remained almost unchanged throughout life. We observed no high-Mr ALP activity in children, whereas sera from 60% of the adults contained low activities of high-Mr ALP. Intestinal ALP (soluble form) and "intestinal variant" ALP (hydrophobic form) were frequently present, in 21% and 37% of all samples, respectively. No significant differences were observed between age groups and sexes for the intestinal isoenzymes.

Arterial media calcification is often considered a cell-regulated process resembling intramembranous bone formation, implying a conversion of vascular tissue into a bone-like structure without a cartilage intermediate. In this study, we examined the association of chondrocyte-specific marker expression with media calcification in arterial samples derived from rats with chronic renal failure (CRF) and from human transplant donors. CRF was induced in rats with a diet supplemented with adenine. Vascular calcification was evaluated histomorphometrically on Von Kossa-stained sections and the expression of the chondrocyte markers sox9 and collagen II with the osteogenic marker core-binding factor alpha1 (cbfa1) was determined immunohistochemically. Media calcification was detected in more than half of the rats with CRF. In over half of the rats with severe media calcification, a typical cartilage matrix was found by morphology. All of the animals with severe calcification showed the presence of chondrocyte-like cells expressing the markers sox9, collagen II, and cbfa1. Human aorta specimens showing mild to moderate media calcification also showed sox9, collagen II, and cbfa1 expression. The presence of chondrocytes in association with calcification of the media in aortas of rats with CRF mimics endochondral bone formation. The relevance of this association is further demonstrated by the chondrogenic conversion of medial smooth muscle cells in the human aorta.

The appearance of lanthanum in liver cells as a result of the injection of lanthanum chloride into rats is investigated by advanced transmission electron microscopy techniques, including electron energy loss spectroscopy and high-resolution transmission electron microscopy. It is demonstrated that the lysosomes contain large amounts of lanthanum appearing in a granular form with particle dimensions between 5 and 25 nm, whereas no lanthanum could be detected in other surrounding cellular components.

Bisphosphonates are inhibitors of bone resorption that are widely used to treat osteoporosis. Price and colleagues demonstrate that ibandronate suppressed the development of uremia-related vascular calcification in rats. These findings extend the link between bone remodeling and vascular calcification to the context of chronic renal failure, opening perspectives toward novel therapeutic strategies.

(serum creatinine 1.1 mg/dl, weight 56 kg) and an oral the development of a particular type of chronic tubulotreatment with 5-ASA (PentasaB) was started at a interstitial nephritis, characterized by an important dose of 3×500 mg/day. Five months later his calcucellular infiltration of the interstitium with macrolated creatinine clearance was still 84 ml/min. He conphages, T-cells, and also B-cells. Furthermore, after tinued to take the same medication during the stopping the drug, there is improvement of the renal subsequent 23 months, and at that time in the context function, but not in all cases [7,8]. Indeed in several of a general malaise he was found to have a severe instances, particularly those in which there is a delayed renal failure, necessitating haemodialysis treatment diagnosis of renal damage, recovery of renal function (Figure 1). The 5-ASA treatment was stopped and does not occur. Instead, several of those patients before starting the haemodialysis treatment, a renal needed some form of renal replacement therapy [8]. biopsy was performed, which showed oedema and An important aspect of this type of toxic nephropathy severe cellular infiltration of the interstitium with is the documented persistence of inflammation of the T-cells, macrophages, and B-cells; the glomeruli were renal interstitium even several months after stopping normal. After 8 months of haemodialysis treatment the drug intake [9]. the renal function improved, allowing cessation of this That 5-ASA seems to be implicated in the generatreatment. However, the serum creatinine remained tion–development–maintenance of this particular reacelevated (between 5.0 and 6.0 mg/dl) and a second tion at the level of the kidney is supported by the renal biopsy was performed, again showing a severe number of case reports in recent literature of patients oedema and cellular infiltration of the interstitium with with inflammatory bowel disease using 5-ASA as the T-cells, macrophages, and B-cells, with signs of fibrosis; only medication, the improvement, at least partial, of the glomeruli were again normal (Figure 2). It was the impaired renal function upon stopping the drug, decided to start a course with methylprednisolone, and a worsening after resuming 5-ASA use [1]. which was tapered o and stopped after 6 weeks, since Furthermore, the molecular structure of 5-ASA is very there was no e ect on the serum creatinine. The close to that of salicylic acid, phenacetin, and aminopatient’s glomerular filtration rate 4 years after the phenol, drugs with a well-documented nephrotoxic start of the colitis ulcerosa was less than 20 ml/min. potential. The clinical importance of this case report consists in However, renal manifestations of chronic inflamthe association of a severe chronic tubulointerstitial matory bowel disease are well recognized. The most nephritis with the start of 5-ASA treatment in a patient frequent renal complications are oxalate stones and with chronic inflammatory bowel disease, the absence their consequences such as pyelonephritis, hydroof substantial recovery of the renal function more than nephrosis, and in the long term amyloidosis. As for 1 year after stopping the treatment, and the developmany drugs, reversible acute interstitial nephritis has ment of severe fibrotic lesions as shown in the second been described [5]. Also glomerulonephritis, showing renal biopsy. a heterogenous expression, has been associated with Over the last decade 5-ASA has replaced sulphasalachronic inflammatory bowel disease [10]. zine as first-line therapy for mild to moderate active Recently we conducted a limited retrospective study inflammatory bowel disease. In the past few years, to obtain further insight into the frequency of this

The most important ways of misconduct in clinical scientific research are falsifying results, plagiarize and over-interpretation. Motives are prestige, money, pressure of time and conflict of interests. The "publish or perish" phenomenon and the sometimes difficult attainable deadline play an important role. Furthermore, there is a "gray-zone" in which clinical scientific researchers are influenced by the pharmaceutical industry, leading to the writing of tendentious publications. Few data are available concerning the frequency of misconduct. In general, one may say that the phenomenon is underestimated. The number of professional organizations for the detection of misconduct and to start a balanced investigation procedure of this phenomenon is limited. An exception is "Office of Research Integrity" in the US that does pioneering work in the field. Some of the better journals in the profession are making serious efforts to detect misconduct (e.g. New England Journal of Medicine, British Medical Journal). To prevent misconduct the following measures can be considered: education concerning the topic in the faculties of medicine, biomedical sciences and pharmaceutics sciences; extension of the competences of the ethical committees; realistic financing of research and the establishing of a "clinical scientific fund", without any relation with the pharmaceutical industry, for the support of self-generated clinical project after 'peer review'. Finally, the questions can be raised if the Royal Academy of Medicine (Belgium) could not play a role in this important matter.