Detecting Functional and Accessible Folate Receptor Expression in Cancer and Polycystic Kidneys.

Folate-based small molecule drug conjugates (SMDCs) are currently under development and have shown promising preclinical and clinical results against various cancers and polycystic kidney disease. Two requisites for response to a folate-based SMDC are (i) folate receptor alpha (FRα) protein is expressed in the diseased tissues, and (ii) FRα in those tissues is accessible and functionally competent to bind systemically administered SMDCs. Here we report on the development of a small molecule reporter conjugate (SMRC), called EC2220, which is composed of a folate ligand for FRα binding, a multilysine containing linker that can cross-link to FRα in the presence of formaldehyde fixation, and a small hapten (fluorescein) used for immunohistochemical detection. Data show that EC2220 produces a far greater IHC signal in FRα-positive tissues over that produced with EC17, a folate-fluorescein SMRC that is released from the formaldehyde-denatured FRα protein. Furthermore, the extent of the EC2220 IHC signal was proportional to the level of FRα expression. This EC2220-based assay was qualified both in vitro and in vivo using normal tissue, cancer tissue, and polycystic kidneys. Overall, EC2220 is a sensitive and effective reagent for evaluating functional and accessible receptor expression in vitro and in vivo.

Sheathless CE-MS based metabolic profiling of kidney tissue section samples from a mouse model of Polycystic Kidney Disease.

Capillary electrophoresis-mass spectrometry (CE-MS) using a sheathless porous tip interface emerged as an attractive tool in metabolomics thanks to its numerous advantages. One of the main advantages compared to the classical co-axial sheath liquid interface is the increased sensitivity, while maintaining the inherent properties of CE, such as a high separation efficiency and low sample consumption. Specially, the ability to perform nanoliter-based injections from only a few microliters of material in the sample vial makes sheathless CE-MS a well-suited and unique approach for highly sensitive metabolic profiling of limited sample amounts. Therefore, in this work, we demonstrate the utility of sheathless CE-MS for metabolic profiling of biomass-restricted samples, namely for 20 µm-thick tissue sections of kidney from a mouse model of polycystic kidney disease (PKD). The extraction method was designed in such a way to keep a minimum sample-volume in the injection vial, thereby still allowing multiple nanoliter injections for repeatability studies. The developed strategy enabled to differentiate between different stages of PKD and as well changes in a variety of different metabolites could be annotated over experimental groups. These metabolites include carnitine, glutamine, creatine, betaine and creatinine. Overall, this study shows the utility of sheathless CE-MS for biomass-limited metabolomics studies.

Cyclooxygenase product inhibition with acetylsalicylic acid slows disease progression in the Han:SPRD-Cy rat model of polycystic kidney disease.

Renal cyclooxygenase (COX) derived eicosanoids are elevated and lipoxygenase (LOX) products are reduced in the Han:SPRD-Cy rat model of polycystic kidney disease (PKD). Selective COX2 inhibition reduces kidney disease progression, but COX1 levels also are elevated in this model. Since the effect of reducing the products of both COX isoforms and the role of LOX products is not known, weanling normal and diseased Han:SPRD-cy littermates were given either low dose acetylsalicylic acid (ASA), nordihydroguaiaretic (NDGA) or no treatment for eight weeks. Renal eicosanoids were altered in the diseased compared to normal cortex, with COX products being higher and LOX products being lower. ASA reduced COX products, cyst growth and kidney water content, while NDGA reduced LOX products without altering disease progression or kidney function. Hence, a human equivalent ASA dose equal to less than one regular strength aspirin per day slowed disease progression, while further reduction of LOX products did not worsen disease progression.

[Polycystic kidney disease in a patient using lithium chronically]. / Yillardir Lityum Kullanan Bir Hastada Polikistik Böbrek Hastaligi

Lithium remains to be the gold standard in the treatment of mood disorders. This study presents a case treated with lithium for an extended period with a good response. Following an increase in creatinine levels, further investigation of renal dysfunction revealed polycystic kidney disease. Lithium was used prior to the diagnosis of polycystic kidney disease, resulting in the unique opportunity to examine the effects of lithium on kidneys with polycystic kidney disease. Within this context, this study also discusses the pharmacokinetics of lithium, and its possible relation to cyst formation in polycystic kidney disease.

Ex vivo modeling of chemical synergy in prenatal kidney cystogenesis.

Cyclic adenosine monophosphate (cAMP) drives genetic polycystic kidney disease (PKD) cystogenesis. Yet within certain PKD families, striking differences in disease severity exist between affected individuals, and genomic and/or environmental modifying factors have been evoked to explain these observations. We hypothesized that PKD cystogenesis is accentuated by an aberrant fetal milieu, specifically by glucocorticoids. The extent and nature of cystogenesis was assessed in explanted wild-type mouse embryonic metanephroi, using 8-Br-cAMP as a chemical to mimic genetic PKD and the glucocorticoid dexamethasone as the environmental modulator. Cysts and glomeruli were quantified by an observer blinded to culture conditions, and tubules were phenotyped using specific markers. Dexamethasone or 8-Br-cAMP applied on their own produced cysts predominantly arising in proximal tubules and descending limbs of loops of Henle. When applied together, however, dexamethasone over a wide concentration range synergized with 8-Br-cAMP to generate a more severe, glomerulocystic, phenotype; we note that prominent glomerular cysts have been reported in autosomal dominant PKD fetal kidneys. Our data support the idea that an adverse antenatal environment exacerbates renal cystogenesis.

Ang II induce kidney damage by recruiting inflammatory cells and up regulates PPAR gamma and Renin 1 gene: effect of ß carotene on chronic renal damage.

Antioxidants are widely used for prevention of diseases associated with oxidative stress and ischemic disorder. We investigated the hypothesis of antioxidants (α-tocopherol and ß-carotene) can suppress the renal disorder in apo E-/-mice. Renal damage induced by chronic infusion of Angiotensin II (Ang II) into 4 month old male apo E-/-mice. After that the mice were treated with diet enriched α tocopherol and ß carotene (800 mg/kg) for 150 days. Ang II treated kidney showed polycystic appearance with accumulation of clear fluid and constriction of renal artery and renal vein was noticed. Vacuolar/cystic degeneration as well as inflammatory reactions was noticed in the tubules/glomerulus of Ang II treated mice. ß carotene treated mice showed enormous numbers of regenerated tubules in the kidney and over expression of ICAM proteins in the regenerated tubules. CD 45.2, MAC 3 proteins were over expressed in the inflammatory cells infiltrated into the tubular region of Ang II treated kidney. Gene expression studies revealed up regulation of Renin 1 (Ren 1) and PPARγ genes in the kidney of Ang II treated animals, but the ß carotene treatment controlled the expression of these genes in the regenerated kidneys. ß carotene may have protective effective on chronic renal disorder. It may repress the inflammatory genes (Ren 1, PPARγ) to achieve the protective effect on Ang II induced renal damage.

Doxycycline accelerates renal cyst growth and fibrosis in the pcy/pcy mouse model of type 3 nephronophthisis, a form of recessive polycystic kidney disease.

Nephronophthisis belongs to a family of recessive cystic kidney diseases and may arise from mutations in multiple genes. In this report we have used a spontaneous mouse mutant of type 3 nephronophthisis to examine whether the doxycycline-inducible synthesis of Timp-2, a natural inhibitor of matrix metalloproteinases, can influence renal cyst growth in transgenic mice. Metalloproteinases may exert either a negative or a positive effect on the progression of cystic kidney disease, and we reasoned that this may be most effectively examined by using a natural inhibitor. Surprisingly, already the application of doxycycline, which also inhibits matrix metalloproteinases, accelerated renal cyst growth and led to increased renal fibrosis, an additional effect of Timp-2 was not detected. The positive effect of doxycycline on kidney size was not due to a non-specific "anabolic effect" but was specific for cystic kidneys because it was not observed in non-cystic kidneys. When looking for potential metabolic changes we noticed that the urine of control animals led to an increase in the calcium response of LLC-PK(1) cells, whereas the urine of doxycycline-treated mice showed the opposite effect and even antagonized the urine of control animals. Further experiments demonstrated that the urine of control animals contained a heat-labile, proteinase K-resistant substance which appears to be responsible for the induction of a calcium response in LLC-PK(1) cells. We conclude that doxycycline accelerates cyst growth possibly by the induction of a substance which lowers the intracellular calcium concentration. Our data also add a note of caution when interpreting phenotypes of animal models based upon the tet system.

Calcium, cyclic AMP, and MAP kinases: dysregulation in polycystic kidney disease.

Low intracellular calcium, present in untreated polycystic kidney epithelia, results in a proliferative response to cyclic adenosine monophosphate. Treatment with a calcium channel blocker (CCB) caused exacerbation of autosomal dominant polycystic kidney disease in rats. Data regarding use of CCBs in human polycystic kidney disease (PKD) are limited and mixed. Thus, it is premature to extrapolate these findings to human PKD.

Calcium channel inhibition accelerates polycystic kidney disease progression in the Cy/+ rat.

In polycystic kidney disease, abnormal epithelial cell proliferation is the main factor leading to cyst formation and kidney enlargement. Cyclic AMP (cAMP) is mitogenic in cystic but antimitogenic in normal human kidney cells, which is due to reduced steady-state intracellular calcium levels in cystic compared to the normal cells. Inhibition of intracellular calcium entry with channel blockers, such as verapamil, induced cAMP-dependent cell proliferation in normal renal cells. To determine if calcium channel blockers have a similar effect on cell proliferation in vivo, Cy/+ rats, a model of dominant polycystic kidney disease, were treated with verapamil. Kidney weight and cyst index were elevated in verapamil-treated Cy/+ rats. This was associated with increased cell proliferation and apoptosis, elevated expression, and phosphorylation of B-Raf with stimulation of the mitogen-activated protein kinase MEK/ERK (mitogen-activated protein kinase kinase/extracellular-regulated kinase) pathway. Verapamil had no effect on kidney morphology or B-Raf stimulation in wild-type rats. We conclude that treatment of Cy/+ rats with calcium channel blockers increases activity of the B-Raf/MEK/ERK pathway accelerating cyst growth in the presence of endogenous cAMP, thus exacerbating renal cystic disease.

Kidney-specific inactivation of the Pkd1 gene induces rapid cyst formation in developing kidneys and a slow onset of disease in adult mice.

Autosomal dominant polycystic kidney disease, caused by mutations in the PKD1 gene, is characterized by progressive deterioration of kidney function due to the formation of thousands of cysts leading to kidney failure in mid-life or later. How cysts develop and grow is currently unknown, although extensive research revealed a plethora of cellular changes in cyst lining cells. We have constructed a tamoxifen-inducible, kidney epithelium-specific Pkd1-deletion mouse model. Upon administration of tamoxifen to these mice, a genomic fragment containing exons 2-11 of the Pkd1-gene is specifically deleted in the kidneys and cysts are formed. Interestingly, the timing of Pkd1-deletion has strong effects on the phenotype. At 1 month upon gene disruption, adult mice develop only a very mild cystic phenotype showing some small cysts and dilated tubules. Young mice, however, show massive cyst formation. In these mice, at the moment of gene disruption, cell proliferation takes place to elongate the nephron. Our data indicate that Pkd1 gene deficiency does not initiate sufficient autonomous cell proliferation leading to cyst formation and that additional stimuli are required. Furthermore, we show that one germ-line mutation of Pkd1 is already associated with increased proliferation.

Galectin-3 associates with the primary cilium and modulates cyst growth in congenital polycystic kidney disease.

Several lines of evidence implicate the beta-galactoside-binding lectin galectin-3 in development and pathological processes in renal collecting ducts: galectin-3 is expressed in the ureteric bud/collecting duct lineage during nephrogenesis, modulates collecting duct growth/differentiation in vitro, and is expressed in human autosomal recessive polycystic kidney disease in cyst epithelia, almost all of which arise from collecting ducts. Moreover, exogenous galectin-3 restricts growth of cysts generated by Madin-Darby canine kidney collecting duct-derived cells in three-dimensional culture in collagen. Using the cpk mouse model of recessively inherited polycystic kidney disease, we observed widespread galectin-3 mRNA and protein in cyst epithelia. Exogenous galectin-3 reduced cyst formation in suspension culture, and mice-null mutant for galectin-3 had more extensive renal cysts in vivo. Galectin-3 was also detected for the first time in the centrosome/primary cilium, which has been implicated in diverse polycystic kidney disease. Cilia structure/number appeared normal in galectin-3-null mutants. Finally, paclitaxel, a therapy that retards polycystic kidney disease in cpk mice, increased extracellular galectin-3, in which the lectin could potentially interact with cilia. These data raise the possibility that galectin-3 may act as a natural brake on cystogenesis in cpk mice, perhaps via ciliary roles.

The Gating of Polycystin Signaling Complex

Mutations in either polycystin-2 (PC2) or polycystin-1 (PC1) proteins cause severe, potentially lethal, kidney disorders (autosomal dominant polycystic kidney disease, ADPKD) and multiple extrarenal disease phenotypes. PC2, a member of the transient receptor potential channel superfamily and PC1, an orphan membrane receptor of largely unknown function, are thought to be part of a common signalling pathway. Here, I show that co-assembly of full-length PC1 with PC2 forms an ion channel signalling complex in which PC1 regulates PC2 channel gating through a structural rearrangement of the polycystin complex (Delmas et al., 2004a). These polycystin complexes function either as a receptor-cation channel or as a G-protein-coupled receptor. Thus, PC1 acts as a prototypical membrane receptor that regulates G-proteins and plasmalemmal PC2, a bimodal mechanism that may account for the multifunctional roles of polycystin proteins in various cell types. Genetic alteration of polycystin proteins such as those occurring in kidney diseases may impede polycystin signalling, thereby providing a likely mechanistic explanation to the pathogenesis of ADPKD. Our proposed mechanism may also be paradigmatic for the function of polycystin orthologues and other polycystin-related proteins in a variety of nonrenal cell types, including sperm, muscle cells and sensory neurons.

The effect of melatonin treatment regimen on mammary adenocarcinoma development in HER-2/neu transgenic mice.

The effect of various regimens of treatment with melatonin on the development of mammary tumors in HER2/neu transgenic mice was investigated. Female HER-2/neu mice starting from the age of 2 months were kept under standard light/dark regimen and as given melatonin with tap water (20 mg/l) during the night time 5 times monthly (interrupted treatments) or constantly to natural death. Intact mice served as controls. Treatment with melatonin slowed down age-related disturbances in estrous function most in the group exposed to interrupted treatment with the hormone. Constant treatment with melatonin decreased incidence and size of mammary adenocarcinomas, and incidence of lung metastases, compared to controls. The number of mice bearing 4 and more tumors was reduced in the group with constant melatonin treatment. Interrupted treatment with melatonin promote mammary carcinogenesis in HER-2/neu transgenic mice. The data demonstrate the regimen-dependent inhibitory effect of melatonin on the development of spontaneous mammary tumors in HER-2/neu mice but not on overall survival with implication about the likely cause of the effect. Polycystic kidney disease is common in this transgenic line. Adverse effect of melatonin on the life span in our study may be unique to the transgenic model used and may not be relevant to the suppressive effect of melatonin in delay of mammary cancer.

Decreased sulfotransferase SULT1C2 gene expression in DPT-induced polycystic kidney.

BACKGROUND: The pathogenesis of polycystic kidney disease (PKD) remains unclear despite the identification of the genes responsible for hereditary PKD. In this study, we investigated the alteration of gene expressions in an acquired PKD model induced by 2-amino-4,5-diphenylthiazole (DPT) using the differential display method. METHODS: Kidney mRNA from a Sprague-Dawley rat fed with 1% DPT for 4 days and from a control rat was compared by the RT-PCR differential display method. Differentially expressed bands were re-amplified and subcloned. Using these subclones as probes, the changes in gene expressions were confirmed by Northern blot analysis. Subsequently, mouse kidney cDNA library was screened. RESULTS: The isolated 1.5-kb cDNA contained an open reading frame encoding 296 amino acids, which shared 94.3% identity with rat SULT1C2 sulfotransferase, and was considered to be its mouse ortholog (GenBank Accession No. AY005469). Mouse SULT1C2 mRNA was abundant in the kidney and stomach among normal mouse tissues. The expression of SULT1C2 mRNA was decreased in the rat kidney after DPT feeding but not in the stomach. Mouse SULT1C2 was expressed successfully using pET plasmid vector and E. coli. The recombinant 34-kD protein was capable of catalyzing the sulfation of p-nitrophenol at a Km of 3.1 mmol/L, by utilizing 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as the sulfate donor. CONCLUSIONS: Although the physiological substrate and function of SULT1C2 have yet to be elucidated, its down-regulation could be involved in the cystic changes of tubules by decreasing the sulfation of the tubular basement membrane components.

Enhancing effects of 2-amino-4,5-diphenylthiazole-induced polycystic kidneys on renal carcinogenesis in rats treated with dimethylnitrosamine.

The effects of the polycystic kidney environment on dimethylnitrosamine (DMN)-induced renal carcinogenesis were investigated in rats. In Experiment 1, male Wistar rats were given 25 or 10 ppm DMN in their drinking water and simultaneously administered 1% 2-amino-4,5-diphenylthiazole (DPT) in the diet for 30 weeks. DPT-induced polycystic kidney was associated with a significant increase in the number of renal cell tumors and incidence of mesenchymal tumors in the 25 ppm DMN + DPT group and the incidence of atypical tubules in the 10 ppm DMN + DPT group. PCNA labeling indices of cystic renal tubules in DPT-treated rats were significantly higher than for corresponding noncystic tubules. In Experiment 2, PCNA indices of renal tubules in 10 ppm + DPT rats and immunohistochemically CYP2E1-positive renal tubules in DPT-treated rats were demonstrated to be significantly increased on day 14. CYP2E1 mRNA expression in the kidneys of DPT-treated rats showed a fivefold increase over constitutive levels. The results thus indicate that DPT induction of polycystic kidneys enhances DMN-induced renal carcinogenesis in rats, with DPT-induced elevated cell proliferation and CYP2E1 expression in renal tubules as possible underlying mechanisms.

[Chemical-induced polycyst with renal tumor and expression of 8-OHdG in kidney tissue].

OBJECTIVE: To induce the rat model of polycyst with renal tumor and investigate the expression of 8-OHdG in the kidney tissues. METHODS: The rat model of polycyst with renal tumor, similar to human acquired cystic disease of the kidney (ACDK), was induced by oral administration of 2-amino-4, 5-diphenylthiazole (DPT) and N-nitrosomorpholine (NNM). Immunohistochemical method (LSAB) was used to assay the expression of 8-hydroxydeoxyguanosine (8-OHdG) in the rat kidney tissue. RESULTS: Three of 10 rats in the NNM group had renal solid adenomatous lesions. Bilateral polycysts were observed in all 9 rats of the DPT/NNM group. Seven of the 9 rats had cystic multistage renal tumor. In the DPT/NNM group, 4 rats had cystic adenomatous lesions, but none in the other groups showed this lesion. In the model, adenomatous lesions derived from polycysts in the rats were closely consistent to human ACDK in morphology. The significant expression of 8-OHdG was found on renal tubular cell, cystic epithelial cell, stromal cell and tumor cell in all rats of the NNM and DPT/NNM group. CONCLUSION: A rat model of polycysts with renal tumor, similar to human ACDK, induced by DPT and NNM provides evidences for further study on pathogenic mechanism in human ACDK with renal cell carcinoma. The expression of 8-OHdG, a DNA damage marker, in the renal tissues of rat model might help to explain the mechanism of cysticogenesis and carcinogenesis in human ACDK.

A rat model of chemical-induced polycystic kidney disease with multistage tumors.

Diphenylthiazole (DPT) induces polycystic kidney disease in the rat which serves as a model of human acquired cystic disease of the kidney. However, DPT administration alone does not produce neoplastic changes in renal cysts. We examined the effect of N-nitrosomorpholine (NNM), a carcinogen, in rats bearing DPT-induced renal cysts. Forty Sprague-Dawley rats were divided into four groups: DPT/NNM, DPT, NNM, and nontreated groups. DPT was administered throughout the experimental period, and NNM was given from weeks 4 to 7 after the start of the experiment. The rats were sampled from weeks 39 to 48, and histopathological examinations of the excised kidneys were performed. Multiple cystic changes were observed in all the DPT-treated rats in both DPT and DPT/NNM groups which were absent in almost all other rats. Solid adenomatous lesions were observed in the NNM-treated rats: in 7 of 9 and in 3 of 10 rats in the DPT/NNM and NNM groups, respectively. Cystic adenomatous lesions were found in 4 of 9 rats in the DPT/NNM group exclusively and not in the other groups. Combined DPT and NNM administration to rats produced an animal model showing neoplastic changes in renal cysts resembling microscopically renal cancer lesions in human acquired cystic disease of the kidney (on hematoxylin and eosin staining).

Evaluation of apoptosis and cell proliferation in experimentally induced renal cysts.

Cell proliferation and apoptosis in renal cysts induced by streptozotocin, alloxan and ferric-nitrilotriacetate were investigated in rats. In the kidneys of all treated animals dilated tubules at the cortico-medullary region, large cysts, glomerular cysts and tubular dilation in the medullary area were found. Both cell proliferation and apoptosis were increased in the epithelium of the non-dilated tubules, in the mesangial and interstitial cells. Cells lining the dilated tubules or cysts demonstrated apoptosis but their proliferating activity was low. By calculating the proliferation-apoptosis ratio we found that alloxan did not change the balance between the two mechanisms. Meanwhile streptozotocin resulted in an increased apoptosis and ferric-nitrilotriacetate in an increased cell proliferation. p53 expression might be responsible for the uncontrolled proliferation in rats treated with ferric-nitrilotriacetate as this oncoprotein was diffusely present in tubular cell nuclei. The observed apoptosis seemed to be independent of bcl-2 oncoprotein expression. We assume that the initial factor in such cystogenesis should be a cellular injury due to direct toxic or to the diabetogenic effect of the drugs. The latter is more likely as all the animals were hyperglycemic and insulin treatment following administration of streptozotocin prevented the morphologic changes.

Renal cystic disease in tuberous sclerosis: role of the polycystic kidney disease 1 gene.

Tuberous sclerosis is an autosomal dominant trait characterized by the development of hamartomatous growths in many organs. Renal cysts are also a frequent manifestation. Major genes for tuberous sclerosis and autosomal dominant polycystic kidney disease, TSC2 and PKD1, respectively, lie adjacent to each other at chromosome 16p13.3, suggesting a role for PKD1 in the etiology of renal cystic disease in tuberous sclerosis. We studied 27 unrelated patients with tuberous sclerosis and renal cystic disease. Clinical histories and radiographic features were reviewed, and renal function was assessed. We sought mutations at the TSC2 and PKD1 loci, using pulsed field- and conventional-gel electrophoresis and FISH. Twenty-two patients had contiguous deletions of TSC2 and PKD1. In 17 patients with constitutional deletions, cystic disease was severe, with early renal insufficiency. One patient with deletion of TSC2 and of only the 3' UTR of PKD1 had few cysts. Four patients were somatic mosaics; the severity of their cystic disease varied considerably. Mosaicism and mild cystic disease also were demonstrated in parents of 3 of the constitutionally deleted patients. Five patients without contiguous deletions had relatively mild cystic disease, 3 of whom had gross rearrangements of TSC2 and 2 in whom no mutation was identified. Significant renal cystic disease in tuberous sclerosis usually reflects mutational involvement of the PKD1 gene, and mosaicism for large deletions of TSC2 and PKD1 is a frequent phenomenon.

Experimental model of renal tumors in polycystic kidneys: effects of long-term 2-amino-4,5-diphenylthiazole administration in rats treated with N-ethyl-N-hydroxyethylnitrosamine.

We previously reported that treatment of Fischer-344 rats with 2-amino-4,5-diphenylthiazole (DPT) results in renal cystic changes. The present study was undertaken to examine the effects of long-term DPT treatment after initiation of kidney carcinogenesis with N-ethyl-N-hydroxyethylnitrosoamine (EHEN) in Wistar rats. One hundred forty-four 6-wk-old male Wistar rats were divided into 6 equal receiving groups: 1000 ppm EHEN or normal tap water for 2 wk followed by 1.06% DPT or basal diet for the subsequent 14 or 30 wk. Controls were maintained without treatment throughout. Subgroups of 6 animals from each group were sacrificed after 8, 16, 24, and 32 wk for histopathological assessment of lesion development in the kidneys and liver. Animals treated with DPT first developed cystic changes of the kidneys (primarily at the corticomedullary border) after 8 wk of treatment, and these changes progressed with time thereafter. In the groups in which DPT treatment was discontinued after 14 wk, cysts then gradually decreased in size. All tumors detected in the kidneys were histopathologically diagnosed as renal cell adenomas. The tumor multiplicity after 32 wk of treatment was significantly higher in Group I, receiving EHEN + DPT for 30 wk (6.33 +/- 4.46), and Group III, receiving EHEN + DPT for 14 wk (3.83 +/- 1.57), than in Group V, EHEN alone (1.00 +/- 0.58) (p < 0.05). Renal cell tumors within cysts were only seen in Groups I and III. The general bromodeoxyuridine labeling indices for the kidneys at week 32 were significantly higher in Group I (55.94 +/- 21.08 cells/mm2) and Group III (53.75 +/- 12.38 cells/mm2) than in Group V (22.38 +/- 6.98 cells/mm2) (p < 0.05). In conclusion, DPT caused cystic changes in rat kidneys, which, however, gradually decreased in size after the treatment was discontinued, suggesting a reversible nature. DPT clearly also promotes renal tumor development after EHEN initiation, and this effect persists, to a certain extent, even after the insult is removed.