Spiros Vamvakas

Cancer in End-Stage Renal Disease: Potential Factors Involved

Increased incidence of cancer at various sites is observed in patients with end-stage renal disease (ESRD). In particular, lymphomas and carcinomas of the kidney, prostate, liver and uterus show an enhanced prevalence in these subjects compared with the general population. A multitude of factors directly or indirectly associated with the renal disease and the treatment regimens may contribute to the increased tumor formation in these patients. Impaired function of the immune system and of DNA repair mechanisms as well as reduced antioxidant defense, accumulation of carcinogenic compounds partly due to reduced renal elimination as well as chronic infections and inflammations are found more frequently in patients with ESRD compared with the general population and may act in concert to accelerate malignant transformation and tumor formation.

On the role of DNA double-strand breaks in toxicity and carcinogenesis.

DNA double-strand breaks are associated with various endogenous processes, such as transcription, recombination, replication, and with the process of active cell death, which aims to eliminate cells. In addition, DNA double-strand breaks can be induced by irradiation, exposure to chemicals, increased formation of reactive oxygen species, and, indirectly, during repair of other types of DNA damage or as a consequence of extranuclear lesions. In addition to the neutral filter elution of DNA, the recently introduced pulsed-field gel electrophoresis is capable of determining DNA double-strand breaks with higher accuracy and sensitivity and is expected to increase our knowledge on the frequency and the role of DNA breakage. Parallel determination of parameters for cytotoxicity is necessary to elucidate the causal primary lesion. Although the repair of DNA double-strand breaks is a complex task, cells are capable of repairing--with or without errors and up to a certain extent--and surviving this DNA lesion. Gene translocations, rearrangements, amplifications, and deletions arising during repair and misrepair of double-strand breaks may contribute to cell transformation and tumor development.

Increased genomic damage in lymphocytes of patients before and after long-term maintenance hemodialysis therapy

This study investigates spontaneous genomic damage in peripheral lymphocytes of 19 patients with severe end-stage renal disease not enrolled onto a maintenance hemodialysis (MHD) program (creatinine level, 5.4 to 10.5 mg/dL) and 16 long-term MHD patients (111 to 282 months on MHD) and the possible association of genomic damage with the degree of renal insufficiency and duration of MHD. Genomic damage was assessed by evaluating the numbers of micronuclei (MN), which are cytoplasmic DNA-containing structures. The average number of MN in the control group of 23 healthy subjects was 15.3 +/- 4.7 MN/1,000 binucleate (BN) cells. The MN frequencies in the long-term MHD group were significantly greater (44.3 +/- 13.7 MN/1,000 BN) than the control frequencies. A significant increase in MN frequencies (28.2 +/- 9.4 MN/1,000 BN) was also seen in patients with advanced renal failure. The major findings of the study, high MN frequencies in long-term hemodialysis and advanced chronic renal failure patients, may result from decreased DNA repair previously shown and may contribute to the increased cancer incidence in these patients.

Biotransformation and Membrane Transport in Nephrotoxicity

The kidney is a frequent target organ for toxic effects of xenobiotics. In recent years, the molecular mechanisms responsible for the selective renal toxicity of many nephrotoxic xenobiotics have been elucidated. Accumulation by renal transport mechanisms, and thus aspects of renal physiology, plays an important role in the renal toxicity of some antibiotics, metals, and agents binding to low molecular weight proteins such as alpha(2u)-globulin. The accumulation by active transport of metabolites formed in other organs is involved in the kidney-specific toxicity of certain polyhaloalkanes, polyhaloalkenes, hydroquinones, and aminophenols. Other xenobiotics are selectively metabolized to reactive electrophiles by enzymes expressed in the kidney. This review summarizes the present knowledge on the mechanistic basis of target organ selectivity of these compounds.

Discrimination between genotoxicity and cytotoxicity for the induction of DNA double-strand breaks in cells treated with aldehydes and diepoxides

The time-dependent dose-response relationships for the induction of DNA double-strand breaks (DSB) assessed by pulsed-field gel electrophoresis (PFGE) and for viability (evaluated by the MTT cytotoxicity test) were investigated in order to discriminate between genotoxic and cytotoxic mechanisms of DNA fragmentation. Cultured human lung epithelial cells (A549) were treated (i) with the aldehydes formaldehyde or glutaraldehyde and (ii) with the DNA-DNA interstrand crosslinkers melphalan, diepoxybutane or diepoxyoctane. Induction of DSB by formaldehyde and glutaraldehyde was seen only after cell viability was reduced to less than about 60% of the control values, indicating that DSB were the consequence of extragenomic damage and viability loss. Melphalan, diepoxybutane and diepoxyoctane induced DSB by a genotoxic mode with concentrations that did not affect cell survival: 8 h after treatment initiation both heat-labile crosslinks and DSB could be detected. Cells were not able to repair the crosslinks induced by diepoxybutane, the crosslinker with the shortest chain length. In contrast, with melphalan and diepoxyoctane, which have a longer crosslinking property considerable repair of crosslinks was observed. The molecular size distribution of the produced DNA fragments supported this mechanistic distinction. The DNA fragments generated by diepoxides were initially large, their concentration decreasing monotonously from 7 Mbp to less than 1 Mbp and were converted to smaller fragments by 72 h in the course of cell death. In contrast, DNA fragments induced by formaldehyde peaked below 1 Mbp, implicating activation of DNA-degrading enzymes.

High-Glucose Media Enhance the Responsiveness of Tubular Cells to Growth Promoters: Effect on Lysosomal Cathepsins and Protein Degradation

Nephromegaly is a prominent feature of diabetic nephropathy and predominantly reflects increased renal tubule mass, mostly due to hypertrophy. To elucidate pathogenetic factors involved, we studied the effects of high glucose (HG) alone, and in combination with hormones/growth promoters: angiotensin II (10–7 M); parathyroid hormone (10–7 M); insulin-like growth factor-1 (10–7 M), or transforming growth factor-β1 (TGF-β1, 10 ng/ml) in a renal cell line (LLC-PK1) with many characteristics of the proximal tubule. Activities of lysosomal cathepsins (B, L+B and H) and the protein turnover were investigated. Exposure to HG (25 mM) for up to 48 h increased cellular protein content, due to enhanced protein synthesis, while protein degradation rate and cathepsin activities tended to lower values. Hyperosmotic mechanisms of glucose action were excluded, since these effects were not induced by mannitol. In normoglycemic conditions only TGF-β1 decreased cathepsin activities and protein degradation rate significantly. However, in HG media all applied hormones/growth factors significantly lowered the protein degradation rate, as well as lysosomal cathepsin activities. The enhanced responsiveness could contribute to the impaired protein turnover, with consequent hypertrophy of the tubulointerstitium in diabetic nephropathy.

Nephrotoxic effects of bacterial ribonucleases in the isolated perfused rat kidney

Alterations of the renal function in the isolated perfused rat kidney system after application of two bacterial RNases, Bacillus intermedius RNase (binase) and ribonuclease produced by Bacillus amyloliquefaciens (barnase), were investigated with two different treatment regimens in comparison with catalytically inactive derivates of the enzymes, photooxidated at the active site His101 binase and inactive mutant His102Gln barnase. For the in vitro approach the test enzymes were dissolved in the perfusion media and applied to the kidney after removal from the animal. Alternatively, the test ribonucleases were administered to rats in vivo and the renal effects were assessed in the isolated perfused rat kidney 1 and 6 h after treatment. In the in vitro regimen both active enzymes induced time- and concentration-dependent nephrotoxicity reflected in enhancement of urinary protein excretion, decline of glucose reabsorption, increase of gamma-glutamyltranspeptidase and alkaline phosphatase activities in urine. In vivo administration of active binase induced functional impairment of the isolated perfused organ in a similar way. None of the inactive RNases in both regimens and at all concentrations tested altered any renal parameter. The results suggest that RNA degradation may be involved in the nephrotoxic effects of bacillar RNases.

S-(1,2-dichlorovinyl)-L-cysteine-induced dedifferentiation and p53 gene mutations in LLC-PK1 cells: a comparative investigation with S-(2-chloroethyl)cysteine, potassium bromate, cis-platinum and styrene oxide.

Exposure of cultured renal (LLC-PK1) cells for 7 weeks to non-cytotoxic concentrations of S-(1,2-dichlorovinyl)-L-cysteine had resulted in the induction of morphologically and biochemically dedifferentiated clones, which retained their altered properties after removal of the chemical. In this study we investigated by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis and direct sequencing if S-(1,2-dichlorovinyl)-L-cysteine-induced LLC-PK1 clones display mutations in the p53 gene in comparison with wild-type clones. In addition, the characteristics of S-(1,2-dichlorovinyl)-L-cysteine-induced clones were compared with clones induced by carcinogens/metabolites of carcinogens with different mechanisms of action: (i) The potent alkylating agent and bacterial mutagen chloroethylcysteine, the key metabolite of the carcinogen dichloroethane; (ii) potassium bromate, a nephrocarcinogen inducing reactive oxygen species, which give rise to the formation of 8OHdG and DNA strand-breaks; (iii) cis-platinum, a bifunctional cross-linking agent and strand-break inducer and (iv) styrene oxide, the main intermediate metabolite of styrene, an epoxide whose carcinogenicity is thought to be based on cytotoxicity. Three essential markers of the physiological integrity and renal tubule origin of the wild-type LLC-PK1 cells were disrupted in all chemical-derived clones: (i) the polarisation of the plasma membrane into a luminal and basolateral part; (ii) the sodium-dependent glucose uptake and (iii) the pH-dependent ammonia production. Compared with the wild-type clones, poly(ADP-ribosyl)ation, a posttranslational modification of nuclear proteins, was clearly increased in clones induced by S-(1,2-dichlorovinyl)-L-cysteine, potassium bromate and cis-platinum. These clones displayed also band shifts of p53 exon 7, indicating mutations, which were confirmed by sequencing: a double mutation consisting of a base substitution followed by one base insertion in the case of S-(1,2-dichlorovinyl)-L-cysteine and potassium bromate and a base substitution in the case of cis-platinum. The base insertions both lead to the formation of the stop codon UGA resulting in loss of protein function.

Induction of dedifferentiated clones of LLC-PK1 cells upon long-term exposure to dichlorovinylcysteine

Dichlorovinylcysteine (DCVC), the key metabolite of the nephrotoxic and nephrocarcinogenic chemicals, trichloroethylene and dichloroacetylene, exerts potent acute cellular toxicity in LLC-PK1 cells (Vamvakas S., Bittner, D., Dekant, W. and Anders, M.W. (1992). Events that precede and that follow S-(1,2-dichlorovinyl)-L-cysteine-induced release of mitochondrial Ca2+ and their association with cytotoxicity to renal cells. Biochem. Pharmacol. 44, 1131-1138). In the present study we investigated whether long-term exposure of LLC-PK1 cells to low, non-cytotoxic concentrations of DCVC results in stable morphological and biochemical dedifferentiation. After 7 weeks exposure to 1 and 5 microM DCVC, morphologically changed single cells were picked under the microscope and cultured in absence of DCVC for 4-8 weeks. In contrast to the physiological cuboidal shape of untreated LLC-PK1 cells, the clones derived from long-term exposure to DCVC consisted of elongated, spindle-shaped cells tending to form irregular borders. Moreover, glucose uptake, pH-dependent ammonia production and dome formation, important indicators of the renal tubule origin of the LLC-PK1 cells, were severely impaired in the clones. In addition to the loss of membrane polarity, the clones exhibited altered composition of the nuclear matrix and intermediate filament proteins by two-dimensional gel electrophoresis, increased poly(ADP-ribosyl)ation of nuclear proteins and enhanced expression of c-fos. The induction of dedifferentiated LLC-PK1 clones with stable characteristics upon long-term exposure to the nephrocarcinogen DCVC may represent a useful in vitro model to study biochemical alterations involved in chronic renal toxicity and carcinogenicity.

Poly(ADP-Ribosyl)ation and Nuclear Matrix/Intermediate Filament Proteins in Renal Carcinogenesis

Extragenomic changes play a crucial role in the regulation of gene expression and consequently in cell proliferation and differentiation. Hence, changes in cellular components other than the genome may influence — promote or inhibit — the process of malignant transformation. Among these extragenomic cellular components, the histories and the proteins of the nuclear matrix, consisting mainly of the nuclear pore complex and the nuclear lamins, are directly involved in the regulation of gene expression. Posttranslational modification of these proteins may have a direct impact on the three-dimensional organization of the genome and hence, on the availability of specific DNA sequences for repair, replication, or transcription.