Bismuth overdosing-induced reversible nephropathy in rats.

Overdosing of colloidal bismuth subcitrate (CBS), used to treat peptic ulcers and Helicobacter pylori infections, has been reported to result in serious, though reversible, nephrotoxicity in humans. However, little is known about the nature of the renal damage induced by bismuth (Bi), and no well-described experimental model exists. Single large oral CBS doses (0.75, 1.5, and 3.0 mmol Bi/kg) were administered to three groups of 20 female Wistar rats. A control group (n = 20) received only the vehicle. Standard kidney function parameters, urinary excretion of N-acetyl-beta-D-glucosaminidase (NAG) and the Bi content were monitored in blood, urine, liver, and kidneys for 14 days. A dose of 3.0 mmol Bi/kg, 100 times the daily therapeutic dose, caused kidney damage within 6 h as detected by proteinuria, glucosuria, and elevated plasma urea and plasma creatinine levels. The kidneys of all animals, except two that died, recovered functionally within 10 days. At a dose of 1.5 mmol Bi/kg, clinical parameters changed less and normalized within 48 h, whereas a dose of 0.75 mmol Bi/kg induced no changes. Histological evaluation revealed that the S3 tubular segment necrotized first with additional necrotization of the S1/S2 segment when more Bi was absorbed. The lesions were accompanied by interstitial infiltrates of CD45+ leukocytes. In summary, we developed a rat model for Bi-induced reversible nephropathy. A large single oral overdose of CBS administered to Wistar rats led to damage to the proximal tubule, especially in the last segment.

Identification and characterization of novel chromogranin B-derived peptides from porcine chromaffin granules by liquid chromatography/electrospray tandem MS.

Chromogranin B (CgB) is a regulated secretory protein that is stored in endocrine and neuroendocrine cells. It can be processed proteolytically to small peptide fragments. In the present study three proteolytic products of porcine CgB were obtained after size-exclusion, immunoaffinity, and reversed-phase chromatography, and then identified by electrospray tandem MS. One novel peptide was identified as S586-R602 (SR-17) and is phosphorylated at one or two serine residues. Another novel peptide H603-Q636 (HQ-34), with molecular mass 3815.56 Da, was found to be oxidized at the methionine residue. In addition, a secretolytin-like peptide fragment (KR-11), which is two amino acids shorter than the bovine secretolytin, was found. This is the first report that the C-terminal region of CgB, the homologue of human CCB, is proteolytically processed further into three small peptide fragments.

Antibodies to both ICAM-1 and LFA-1 do not protect the kidney against toxic (HgCl2) injury.

BACKGROUND: The role of inflammatory leukocytes in acute renal failure (ARF) remains controversial and appears largely uninvestigated in toxic (in contrast to ischemic) ARF. METHODS: Female Wistar rats were injected with monoclonal antibodies (mAbs) directed to both the leukocyte function-associated antigen 1 (LFA-1) and the intercellular adhesion molecule 1 (ICAM-1). Doses (6 mg/kg of each mAb) were given 24 hours prior to the induction of acute tubular necrosis (ATN) by mercuric chloride administration (2 mg/kg, subcutaneously, day 0) and subsequently every 48 hours. Control rats similarly received either control antibody (12 mg/kg) or vehicle prior to and following the induction of ATN. Renal function was also measured from male Lewis rats that were similarly treated with anti-adhesion antibodies during exposure to 30 minutes of unilateral renal ischemia. RESULTS: Injected antibodies were demonstrated on peripheral blood leukocytes (flow cytometrical detection of mouse anti-LFA-1) and on endothelium (immunohistochemical staining of mouse anti-ICAM-1) and were measured in serum (enzyme-linked immunosorbent assay). Macrophages and T cells were prominent in the kidney of control treatment rats after HgCl2 injection, but anti-adhesion treatment clearly had prevented their infiltration. Notwithstanding, renal tubular injury was equally pronounced in all mercuric chloride treatment groups and so was the decline in renal function (serum creatinine, proteinuria). Tubular epithelial cell proliferation seemed slightly less pronounced and delayed in anti-adhesion treated rats. Kidneys from ischemia exposed rats were, however, functionally protected by identical anti-ICAM-1/anti-LFA-1 treatment. CONCLUSION: Prevention of cellular infiltration by mAbs to LFA-1 and ICAM-1 has no effect on renal morphology, function, or regeneration following mercuric chloride-induced ARF in the rat. This result contrasts with the functional protection of the rat kidney to ischemia/reperfusion injury by virtue of an identical antibody treatment protocol. Resolving that controversy should bring better insight in fundamental processes underlying different types of ARF, and will be the subject of further study.

Neutrophils and acute ischemia-reperfusion injury.

Leukocyte infiltration in response to I/R injury is a well-known but poorly understood phenomenon. The contribution of neutrophils in this process is still controversial. Despite numerous data, little is known about exact numbers of infiltrating neutrophils. The role of monocytes/macrophages in this process is even more unclear. The role neutrophils in the kidney and other organs was reviewed. The variability in models and methods for neutrophil quantification were examined, along with carrying out a critical overview of depletion and anti-adhesion approaches. Nevertheless, the exact role attributed to neutrophils in the I/R process remains unclear.

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 markets 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 gamma-glutamyl transferase (gamma-GT)], the S3 segment-specific marker intestinal type alkaline phosphatase (iAP) and an S1S2 marker (TN20-antigen), originally proposed as a surface marker for interstitial fibroblasts. Segmental (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 (S1 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.

Cholesterol feeding accentuates the cyclosporine-induced elevation of renal plasminogen activator inhibitor type 1.

Long-term cyclosporine (CsA) therapy is accompanied by the occurrence of hypercholesterolemia and renal interstitial fibrosis. The present study investigates the effect of dietary cholesterol on CsA-induced lipid disturbances in the rat and on CsA nephrotoxicity. Since plasminogen activator inhibitor type 1 (PAI-1) is a major inhibitor of matrix degradation and elevated plasma PAI-1 levels are reported to be associated with increased low-density lipoprotein (LDL) cholesterol, PAI-1 was examined in the kidneys of rats fed a sodium-deficient diet, with or without cholesterol. After nine weeks, both diet groups were subdivided into a CsA-treated group and a vehicle-treated group. Although cholesterol feeding significantly aggravated CsA-induced renal function impairment, CsA-induced histological lesions were comparable in both diet groups. Cholesterol feeding significantly decreased high-density lipoprotein (HDL) cholesterol irrespective of the treatment, while CsA treatment significantly elevated serum triglycerides irrespective of the diet. Cholesterol feeding alone did not increase the number of infiltrating cells in the renal interstitium. In contrast, in both diet groups CsA treatment caused a significant influx of macrophages, while combined treatment with CsA and cholesterol additionally elevated the number of T-helper cells in the cortex. In all rats, PAI-1 immunostaining was found mainly in intracellular vesicles (lysosomes) in proximal tubules, which stained most intensely in fibrotic areas of kidneys from CsA-treated rats. Cholesterol feeding enhanced the CsA-induced elevation of renal PAI-1 immunostaining to a significant level. These results show that, although serum creatinine, PAI-1 staining and T cell influx were significantly increased in the cholesterol-fed CsA-treated group compared to the other groups, renal CsA-induced histological lesions were not influenced by cholesterol feeding after short-term (3 weeks) CsA administration. To what extent the more pronounced proximal tubular PAI-1 (inhibitor of matrix degradation) immunostaining in fibrotic areas in the cortex of cholesterol-fed CsA-treated rats contributes to the progression of CsA-induced renal fibrosis remains to be determined.

Prevalence and clinical expression of HCV-genotypes in haemodialysis-patients of two geographically remote countries: Belgium and Saudi-Arabia.

Hepatitis C virus is the leading cause of acute and chronic liver disease in hemodialysis patients. There are at least six major HCV-genotypes, with a well documented geographical distribution in the general population. Moreover, HCV-genotype is one of the major determinants of the therapeutic response to Interferon Alpha in affected patients. Since the therapeutic outcome in HCV-positive hemodialysis patients, especially with regard to the different HCV-genotypes, is of interest, a multicentre epidemiologic study was performed in HCV-antibody positive hemodialysis patients of two geographically remote countries, i.e. in Flanders (Belgium) and in Saudi-Arabia. 184 chronic hemodialysis patients, with a positive second or third generation Elisa assay for HCV, were tested for HCV-viremia and HCV-genotype, using a 5' untranslated region (UR) nested PCR for the detection of HCV-RNA and subsequently type-specific probes to hybridize with HCV-RNA (Inno-Lipa). Additionally, clinical data were collected by means of a standardized questionnaire, thoroughly completed by the nephrologist in charge of each respective patient. Viremia was present in 79% of the patients (146 out of 184). The prevalence of HCV-genotypes differed significantly between Belgian and Saudi-Arabian dialysis-patients. In Belgian dialysis patients HCV-genotype 1b was most prevalent (i.e. 62%), while in Saudi-Arabian patients HCV-genotypes 4, 1b, and la were present in respectively 36,4%, 31,7%, and 25,8% of the HCV-PCR positive patients. Although there were significant differences between Belgian and Saudi-Arabian dialysis patients, no clinical data showed any significant correlation with the HCV-genotype. Transaminases, determined over a six months period, showed normal average values. Doubling of the transaminases, in at least one out of six measurements over a six monthly period, occurred only in 14% (alanine aminotransferase, ALT) and 10% (aspartate aminotransferase, AST) of the patients. In Belgian dialysis patients, HCV-genotype 4 (or HCV-genotype 5) significantly correlated with a more recent start of dialysis treatment. We conclude that there is a significant different geographical prevalence of HCV-genotypes in HCV-affected hemodialysis patients. None of the different HCV-genotypes shows any particular clinical expression. Transaminases are not a sensitive marker for ongoing HCV-replication in hemodialysis patients. In Belgian dialysis patients, a changing pattern of HCV-infection is suggested, with an increasing prevalence of HCV-genotype 4 (or HCV-genotype 5) in more recent years. These data suggest possible implications for the therapeutic strategy in dialysis patients.

Evidence of differential renal dysfunctions during exercise in men.

Post-exercise proteinuria is a common phenomenon in healthy subjects. Previous studies have used albumin (Alb) and beta 2-microglobulin (beta 2-m) molecules as representatives of high- and low-molecular-weight proteins. Recently, more specific markers of the human kidney proximal tubule have been used to identify the precise site of alterations. Active male subjects underwent two strenuous runs, one 400-m run and one 3000-m run. Urine was collected from the subjects before and after each event. Total protein (TP), Alb, alpha 1-microglobulin (alpha 1-m), beta 2-m, intestinal alkaline phosphatase (IAP), tissue-nonspecific alkaline phosphatase (TNAP) and N-acetyl-beta-D-glucosaminidase (NAG) were determined for each sample. The short-distance run (400 m) resulted in the largest increases (P < or = 0.05) in TP (31-fold), Alb (100-fold) and beta 2-m (164-fold) as compared to the long-distance run (3000-m). The alpha 1-m excretion rates were increased to a lesser extent by the exercises. The IAP activity was slightly increased (+90%) by the 400-m run while the TNAP and NAG activities showed a 6.8-fold and a 3.6-fold increase, respectively, after this event. Smaller increases were recorded for the long-distance run (P = 0.05). To conclude, the present investigation showed that: (1) post-exercise proteinuria is related to the absolute intensity of exercise; (2) the impairment of protein reabsorption is revealed better by changes in Alb and beta 2-m; (3) changes in TNAP and NAG activities could reveal biochemical modifications that occur in the proximal tubule, particularly at the S1-S2 segment.

Selective depletion of CD8-positive leukocytes does not alter mercuric chloride induced acute renal failure in the rat.

The process of injury and regeneration in different models of acute renal failure is accompanied by the transient interstitial accumulation of mononuclear leukocytes. The relationship between these accumulated cells and the onset and progression of the regeneration process resulting in the complete functional and morphological recovery is still a matter of debate. In this process cell subsets may either be selectively important or combine to a communicative network, signalling the cells at the site of injury. In a first trial to investigate this hypothesis, the CD8-positive subset of leukocytes, consisting mainly of cytotoxic and suppressor T lymphocytes and to a lesser extent of natural killer cells, was depleted in vivo in rats by means of a monoclonal antibody directed against CD8. Although the depletion obtained evidently prevented the infiltration of these cells into the renal interstitium, it could not influence neither the development nor the resolution of renal insufficiency in response to mercuric chloride administration as compared with control animals who had received an irrelevant isotype-matched monoclonal antibody. The extent of renal damage was unaffected as were onset and duration of renal epithelial cell proliferation. Consequently, these data do not support a major role for the CD8-positive cell subset per se in the development of acute nephrotoxic injury and subsequent regeneration.

Isolation and characterisation of the class alpha, mu and pi glutathione transferases in LLC-PK1 and pig kidney.

Glutathione S-transferase (GST) isoenzymes from pig kidney cortex and LLC-PK1 (an established cell line derived from the pig proximal tubule) were purified by affinity chromatography, anionic and cationic chromatofocusing. Purification revealed nine isoenzymes in the pig kidney cortex and five isoenzymes in the LLC-PK1 cell line. SDS-polyacrylamide gel electrophoresis showed that the pig kidney cortex isoenzymes were homo- or heterodimeric; LLC-PK1 isoenzymes, however, were homodimeric. Isoenzymes from pig and LLC-PK1 showed a higher affinity towards glutathione. The isoenzymes were further characterised and divided into the different GST classes by studying specific inhibitors, specific substrates and immunological properties. Pig GSTs belong to class alpha, mu and pi. The GSTs in LLC-PK1 cells, on the other hand, belong to class pi and mu. The isoenzyme pattern in LLC-PK1 cells indicates the dedifferentiation of this particular cell line compared with the pig kidney cortex.

Inflammatory cells in renal regeneration.

Much research has been performed to gain better insight into the regeneration process, responsible for the functional and morphological recovery after acute renal failure (ARF). Many investigators focused on endogenously produced polypeptide growth factors as the major mediators of tubular epithelial cell proliferation. However, arguments contradicting this hypothesis have recently gained more support. Indeed, the early decrease of renal epidermal growth factor (EGF) and insulinlike growth factor-1 (IGF-1) in different experimental models of ARF has been frequently shown at both the mRNA and protein level, while other growth factors could not be shown to increase. Moreover, the inaccessibility of the upregulated receptors for endogenously produced growth factors has encouraged research to seek alternative origins for the signals inducing renal regeneration. The accumulation of mononuclear leukocytes in the renal interstitium is a striking observation in renal failure. Where the interstitial disease, recognized by the persistent interstitial accumulation of leukocytes, is a better predictor of chronic renal failure and developing fibrosis, ARF distinguishes itself by the disappearance of the infiltrate when regeneration is complete. The existence of a regenerative potential provided by the network of inflammatory mononuclear leukocytes is supported by studies on tissue repair in different fields. This review discusses the infiltrating network of mononuclear leukocytes as a major participant in the regeneration process after acute renal failure, including the approach which can be followed to investigate this hypothesis.

Vimentin expression and distal tubular damage in the rat kidney.

UNLABELLED: Renal damage and repair was investigated in Wistar rats after administering 40 or 80 mmol lithium chloride/kg dry food during 3 or 7 weeks. Serum creatinine levels remained normal. Light microscopic signs of injury were confined to the cortical and outer medullary collecting ducts (CDs) and to the distal convoluted tubules (DCTs; 39-97% of the cross-sections contained necrotic cells); no lesions were found in proximal tubules and thick ascending limbs (TALs). Nevertheless, the urinary excretion of N-acetyl-beta-D-glucosaminidase was increased in the high-dose group, but epidermal growth factor immunostaining in the TALs and DCTs was unchanged. Simultaneously, increased cell proliferation in the CDs and also the DCTs was accompanied by the appearance of vimentin immunostaining predominantly in the basal cell pole; the number of vimentin-positive cells amounted to 72% in the CDs of the high dose group after 3 weeks. In addition, in all lithium-treated animals, the interstitium throughout the entire kidney, but mainly around injured distal segments, displayed increased cell proliferation and leukocyte infiltration (antibody OX-1 positive); 7-25% of these were macrophages (antibody ED-1 positive). Collagen I and III and laminin staining patterns were not altered. IN CONCLUSION: (1) LiCl-induced damage and regeneration confined to the DCTs and CDs is accompanied by the expression of vimentin, possibly in response to an increased requirement for cell spreading and motility after epithelial desquamation, and (2) interstitial cell proliferation and leukocyte infiltration, largely confined around the injured nephron segments, may constitute early signs of the development of lithium-induced interstitial nephropathy.

The cytosolic glutathione S-transferase isoenzymes in the dog kidney cortex as compared with the corresponding MDCK renal cell line.

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 sensitivities 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.

Purification of circulating liver plasma membrane fragments using a monoclonal antileucine aminopeptidase antibody.

Membrane-bound liver alkaline phosphatase (Mem-LiALP, EC 3.1.3.1) is a high-molecular-mass liver alkaline phosphatase (ALP) present in metastatic, infiltrative and cholestatic liver disease. Shedding of hepatocyte plasma membrane fragments (LiPMF) is thought to be responsible for the appearance of Mem-LiALP in the circulation. Several other membrane-bound enzymes, such as gamma-glutamyltransferase (gamma-GT), leucine aminopeptidase (LAP), and 5'-nucleotidase (5'-Nu) are present in the membrane of the shedded LiPMF. By means of immunohistochemical and immunoassay procedures, we presently show that AD-1, a specific monoclonal antibody originally produced against Mem-LiALP, reacts with LAP, a constituent of the human liver plasma membrane. Using AD-1 as an immunosorbant, we isolated circulating LiPMF from cholestatic sera to a high level of purity and separated it from other high-molecular-mass material, such as liver ALP or similar lipoprotein-X complexes. These purified membrane fragments retained their biochemical characteristics. Glycosyl-phosphatidylinositol anchor bearing liver ALP (Anch-LiALP) could be released from the LiPMF by Triton X-100. Whereas ALP was released upon treatment of AD-1 purified LiPMF with phospholipase C, phospholipase D only cleaved the glycosyl-phosphatidylinositol anchor following detergent solubilization of the enzyme. Serum LiPMF from patients with different kinds of cholestatic liver disease were bound onto AD-1 coated nitrocellulose disks and the activity of four membrane-bound enzymes (LAP, ALP, 5'Nu, gamma-GT) was analyzed. A considerable interindividual variation of enzyme activities was observed, suggesting some heterogeneity in the membrane composition of these fragments.

Cytotoxicity of mercury compounds in LLC-PK1, MDCK and human proximal tubular cells.

Six mercury compounds [HgCl2 (MC), Hg(CH3COO)2 (MA), Hg(NO3)2 (MN), C2H5HgSC6H4COONa (EMT), C6H5HgOCOCH3 (PMA) and CH3CIHg (MMC)] were studied using two kidney cell lines (MDCK and LLC-PK1), primary cultures of human proximal tubular cells (hPTC) and nonrenal cell lines (SAOS and Hep G2). Cell damage was measured with four different tests: neutral red uptake, mitochondrial dehydrogenase activity (MTT conversion), thymidine incorporation and protein content. Relative toxicity was established by the determination of the concentration of test compound inducing a 50% reduction of the parameter considered (EC50 value). Two groups could be distinguished: PMA, EMT and MMC are one order of magnitude more toxic than MC, MN and MA. Cellular uptake was measured by the HPLC-hybrid generation AAS after 24 hours treatment with 1.5 microM MC, MMC, PMA or EMT in MDCK cells, revealing Hg concentrations of 42.8 +/- 2.5 ng/mg protein for MC, 596.9 +/- 87.8 ng/mg protein for MMC, 269.8 +/- 75.7 ng/mg protein for PMA and of 115.9 +/- 25.2 ng/mg protein for EMT. Cytotoxicity was positively correlated with cellular uptake. The effect of the cellular GSH content on the toxicity of mercury was studied using the GSH synthesis inhibitor L-buthionine sulfoximine (BSO). In all cases an enhanced cytotoxicity was observed after BSO treatment. 2-Oxo-4-thiazolidine carboxylic acid (OTC) was used as a substrate for the GSH synthesis. Although OTC did not enhance the GSH content, the cytotoxicity of MC, MN and MA decreased significantly, no changes were observed for the other mercurials.(ABSTRACT TRUNCATED AT 250 WORDS)

Time course of growth factor expression in mercuric chloride acute renal failure.

BACKGROUND: Renal EGF expression decreases in varying models of acute renal failure (ARF). We found previously that the loss of distal tubular EGF during gentamicin ARF is strongest in the cortex, where proximal tubular injury was most severe. To gain more insight into the mechanism underlying this apparent anatomical association, renal growth factor expression was investigated during mercuric chloride ARF, in which proximal tubular injury is most severe in the outer stripe of the outer medulla (OSOM). METHODS: Endogenous renal growth factor expression was investigated by RNA hybridization and by immunohistochemistry in a rat model of mercuric chloride ARF. In addition we determined temporal and spatial profiles of tubular injury, cell proliferation, and mononuclear cell infiltration during the 3-week observation period. RESULTS: Serum creatinine values were maximal 2 days after treatment and were again normalized at day 6. Tubular injury was most severe in the PST and maximal at day 2. Cell proliferation was also higher in the PST and maximal at day 4. Three weeks after treatment, normal renal morphology was restored. Increased numbers of mononuclear cells appeared transiently in the renal interstitium from day 1 on. Most of these cells were macrophages and T lymphocytes; macrophages surrounded preferentially the severely injured PST in the OSOM. In analogy to gentamicin ARF, renal EGF and IGF-I gene expression were decreased early in the setting of mercuric chloride ARF. The decrease in distal tubular EGF staining was most pronounced in the OSOM, i.e. the anatomical area where mercuric-chloride-induced proximal tubular injury was most severe. CONCLUSIONS: Renal EGF and IGF-I gene expression decreases strongly during mercuric chloride ARF. The spatial association between the initial decrease of distal tubular EGF expression and the zone of major proximal tubular injury could originate from metabolic alterations secondary to oxygen starvation. A possible role of mononuclear cells remains to be determined.

Human intestinal versus tissue-nonspecific alkaline phosphatase as complementary urinary markers for the proximal tubule.

The availability of early biological markers of renal damage is important for the identification of risk factors and for starting therapeutic intervention in the reversible phase of renal pathology. The usefulness of such markers relies upon their capacity to detect alterations in distinct nephron segments. Using specific monoclonal antibodies against the intestinal isoenzyme of alkaline phosphatase (IAP) and against the tissue-nonspecific isoenzyme (TNAP), we demonstrated that IAP expression in the human kidney is restricted to the straight part of the proximal tubule (the S3 segment), whereas TNAP is expressed mainly in the proximal convoluted tubule (the S1 and S2 segments) but also in the S3 segment. This complementarity opens perspectives for IAP and TNAP as distinct proximal tubular markers, particularly for IAP, since there are no other markers available that are specific for the S3 segment. Based on these monoclonal antibodies, specific and easy to use enzyme-antigen immunoassay (EAIA) procedures were developed to detect IAP and TNAP in human urine samples. The detection limits are below the lowest enzyme activities found in the urine of normal subjects, the intra- and inter-assay variability is low, the analytical recovery approaches 100%, and EAIA enzyme activity values correlate with ELISA immunoreactivity values. Furthermore, easy urine sample preconditioning allows antigen preservation over an extended time period at 4 degrees and -80 degrees C. Using these assays, it could be demonstrated in more than 20 occupationally and environmentally exposed cohorts and clinical patient groups that urinary IAP is indeed a marker of early alterations in the S3 segment, and that it behaves largely independently from urinary TNAP.(ABSTRACT TRUNCATED AT 250 WORDS)