Matrix Factorization-based Technique for Drug Repurposing Predictions.

Classical drug design methodologies are hugely costly and time-consuming, with approximately 85% of the new proposed molecules failing in the first three phases of the FDA drug approval process. Thus, strategies to find alternative indications for already approved drugs that leverage computational methods are of crucial relevance. We previously demonstrated the efficacy of the Non-negative Matrix Tri-Factorization, a method that allows exploiting both data integration and machine learning, to infer novel indications for approved drugs. In this work, we present an innovative enhancement of the NMTF method that consists of a shortest-path evaluation of drug-protein pairs using the protein-to-protein interaction network. This approach allows inferring novel protein targets that were never considered as drug targets before, increasing the information fed to the NMTF method. Indeed, this novel advance enables the investigation of drug-centric predictions, simultaneously identifying therapeutic classes, protein targets and diseases associated with a particular drug. To test our methodology, we applied the NMTF and shortest-path enhancement methods to an outdated collection of data and compared the predictions against the most updated version, obtaining very good performance, with an Average Precision Score of 0.82. The data enhancement strategy allowed increasing the number of putative protein targets from 3,691 to 15,295, while the predictive performance of the method is slightly increased. Finally, we also validated our top-scored predictions according to the literature, finding relevant confirmation of predicted interactions between drugs and protein targets, as well as of predicted annotations between drugs and both therapeutic classes and diseases.

A multicentric European study testing the reproducibility of the WHO classification of endometrial hyperplasia with a proposal of a simplified working classification for biopsy and curettage specimens.

This study was designed to assess intraobserver and interobserver agreement in the diagnosis of 56 endometrial specimens by five European expert gynecologic pathologists using the WHO classification and to establish which histologic features are significantly associated with each classification category. The seven categories were simple hyperplasia, complex hyperplasia, atypical hyperplasia, well-differentiated adenocarcinoma, proliferative endometria, secretory endometria, and other. Slides were reviewed twice for diagnosis, with accompanying evaluation of a checklist of histologic features. These seven categories were eventually reduced to four and three for the purposes of data analysis. The four modified diagnostic categories consisted of hyperplasia (previously simple hyperplasia and complex hyperplasia), atypical hyperplasia, well-differentiated adenocarcinoma, and cyclical endometrium (previously proliferative, secretory, and other). The three diagnostic categories consisted of hyperplasia, endometrioid neoplasia (previously atypical hyperplasia and well-differentiated adenocarcinoma), and cyclical endometrium. Intraobserver and interobserver agreement was assessed using the percentage agreement and kappa statistics. The associations among the various histologic features and diagnoses was analyzed using multiple logistic regression to identify those features that were useful for distinguishing diagnostic categories. When using seven categories, kappa values ranged from 0.53 to 0.74 (percentage agreement, 61-79%) and from 0.33 to 0.59 (percentage agreement, 43-63%) for intraobserver and interobserver agreement, respectively. When using four categories, kappa values ranged from 0.68 to 0.73 (percentage agreement, 77-80%) and from 0.39 to 0.64 (percentage agreement, 54-73%) for intraobserver and interobserver agreement, respectively. When using three categories, kappa values ranged from 0.70 to 0.83 (percentage agreement, 80-89%) and from 0.55 to 0.73 (percentage agreement, 70-82%) for intraobserver and interobserver agreement, respectively. Data were analyzed in each diagnostic category. When using four or three diagnostic categories, the mean intraobserver and interobserver agreements varied less between categories and achieved higher values, with smaller 95% confidence intervals. The mean percentage agreement was lowest for complex hyperplasia and for atypical hyperplasia. For distinguishing cyclical endometrium versus hyperplasia, the useful histologic feature was glandular crowding. For hyperplasia versus atypical hyperplasia and for hyperplasia versus endometrioid neoplasia, the useful features were nuclear enlargement, nuclear pleomorphism, vesicular chromatin, and nucleoli, but of these, only nuclear pleomorphism achieved substantial mean intraobserver and interobserver agreements. For discriminating atypical hyperplasia from well-differentiated adenocarcinoma, the only useful feature was stromal alterations, which achieved only fair mean intraobserver and interobserver agreements. In summary, in endometrial biopsy or curettage specimens, the lack of agreement in the diagnoses of complex hyperplasia and atypical hyperplasia and the lack of reproducibility in the recognition of the histologic feature of stromal alterations to differentiate atypical hyperplasia from well-differentiated adenocarcinoma suggest that the histologic classification should be simplified by including a combined category for simple and complex hyperplasia, called hyperplasia, and a combined category for atypical hyperplasia and well-differentiated adenocarcinoma, called endometrioid neoplasia. Diagnoses of hyperplasia and endometrioid neoplasia are highly reproducible between observers from different institutions. Glandular crowding is the best histologic feature to differentiate cyclical endometrium from hyperplasia, whereas nuclear pleomorphism is the reproducible cytologic feature to differentiate hyperplasia from endometrioid neoplasia.

In situ hybridization histochemistry quantification: automatic count on single cell in digital image.

It is extremely useful in investigations of the central nervous system (CNS) to measure mRNA expression in cells by in situ hybridization. However, this approach is limited by the difficulties of a reliable quantitative evaluation. In the present paper we describe a method for quantifying radioactive hybrids on individual cells by a silver grain count in digital images. Quantification is based on the real size and grey level of a single grain obtained by computerized microscope image analysis (IBAS 2, Kontron-Zeiss, PC 286). The program provides for automatic identification of cell area, the portion occupied by grains and their grey level. The number of grains per cell results from a mathematical function integrating these parameters with a 'density factor'. This factor is introduced to better estimate the number of grains when there is overlapping. The method was tested by measuring the expression of preproNPY (pp-NPY) mRNA in rat dentate gyrus and preproSomatostatin (pp-SOM) mRNA in frontal cerebral cortex of control and colchicine-treated rats. Colchicine did not modify the number of pp-SOM mRNA-positive cells but reduced the expression per cell. These results confirm the advantages of our method to quantify a wide range of silver grains (10-5000) and improves the sensitivity of in situ hybridization. With this support the in situ hybridization technique could be considered a real quantitative method for measuring small alterations in neuronal function.

A simple, automatic method for morphometric analysis of the left ventricle in rats with myocardial infarction.

Induction of acute myocardial infarction in the rat is an established model for studying effects of therapeutic interventions. Images of sections of the rat left ventricle, stained with nitroblue tetrazolium, were digitized and several parameters estimated by dedicated software on an image analyzer (IBAS 2.0). The method was tested on 7 rats with 48-hr-old myocardial infarction and 4 sham-operated controls. Infarct size can be evaluated by two largely used methods, based on area or on angular extension of the lesion. Results of the two methods are linearly correlated, but area calculations give values half of those obtained from angular extension. Five minutes were needed for a complete evaluation of a section of the left ventricle. Estimates of the parameters showed a relatively low between- and within-operator variability and a good correlation with a classic, but time-consuming, planimetric method. The method simultaneously measures infarct size and left ventricular geometry in the rat. The advantages over previous nonautomatic methods are simplicity, good reproducibility, and speed of execution, which make it particularly useful in the evaluation of drug effects.

Automatic quantitative evaluation of autoradiographic band films by computerized image analysis.

The present paper describes a new image processing method for automatic quantitative analysis of autoradiographic band films. It was developed in a specific image analysis environment (IBAS 2.0), but the algorithms and methods can be utilized elsewhere. The program is easy to use and presents some particularly useful features for evaluation of autoradiographic band films, such as the choice of whole film or single lane background determination; the possibility of evaluating bands with film scratch artifacts and the quantification in absolute terms or relative to reference values. The method was tested by comparison with laser-scanner densitometric quantifications of the same autoradiograms. The results show the full compatibility of the two methods and demonstrate the reliability and sensitivity of image analysis. The method can be used not only to evaluate autoradiographic band films, but to analyze any type of signal bands on other materials (e.g. electrophoresis gel, chromatographic paper, etc.).

Quality control of cervical cytology in high-risk women. PAPNET system compared with manual rescreening.

OBJECTIVE: To compare the effectiveness of the PAPNET System with conventional rescreening of negative cervical smears in a high-risk population. STUDY DESIGN: Three thousand ninety-seven negative cervical smears from women with past history of cervical abnormalities were rescreened manually and with the PAPNET System. There were two reviews of PAPNET images: the first by two cytotechnologists with limited exposure to the instrument, and the second, limited to smears with discrepant diagnoses, by an expert in the use of the system. The remaining discrepant smears were submitted to a blinded microscopic review by a third party. The a priori consensus diagnosis was arbitrarily established when the result of two of the three reviews--manual, PAPNET and the independent third review--were concordant. The results of rescreening were compared with available biopsies. RESULTS: On manual rescreening of the 3,097 smears, 2,901 (93.66%) were reported as negative and 170 (5.49%) as abnormal. On the first PAPNET review, 2,938 (94.87%) were reported as negative and 150 (4.84%) as abnormal. There were 144 smears with discrepant diagnoses. After the second PAPNET review of these discrepant smears, the agreement between manual and PAPNET rescreening rose from 94.27% to 95.58%. A final, blinded review of 89 residual discrepant smears was used to establish consensus diagnoses. The diagnoses made by PAPNET-assisted rescreening agreed much better with the consensus diagnoses than did manual rescreening (Kappa = .61 vs. Kappa = -.32, P < .001). When compared with the results of 50 available biopsies, PAPNET-assisted rescreening also had a somewhat lower false negative rate (sensitivity 58.82% vs. 41.18%, P = .17) and a statistically significant lower false positive rate (specificity 63.64% vs. 36.36%, P = .01). CONCLUSION: PAPNET-assisted rescreening, when carried out by an experienced person, is more efficient than manual rescreening.

Quantitative morphology and shape classification of neurons by computerized image analysis.

We describe a new image processing method for semiautomatic quantitative analysis of neuronal morphology. It has been developed in a specific image analysis environment (IBAS 2.0), but the algorithms and the methods can be employed elsewhere. The program is versatile and allows the analysis of histological preparations of different quality on the basis of different levels of evaluation and image extraction. Some significant algorithms have been implemented (i.e. one for multiple focus image acquisition and one for automatic cell body shape recognition and classification). A wide set of specific morphological parameters has been defined to allow a better mathematical characterization of neuronal morphology as regards both dendrite trees and cell bodies. Cell bodies' shapes can be classified automatically, defining different neuronal populations. This is done by evaluating the number of main dendrites and perikarya shapes through a multi-valued-decision-tree based method, tested on somatostatin-positive cells in mouse brain. The methods presented have been applied to analysis of neurons, but they can well be used for any quantitative morphological study of other cell populations.

Web architecture for the remote browsing and analysis of distributed medical images and data.

To provide easy retrieval, integration and evaluation of multimodal medical images and data in a web browser environment, distributed application technologies and Java programming were used to develop a client-server architecture based on software agents. The server side manages secure connections and queries to heterogeneous remote databases and file systems containing patient personal and clinical data. The client side is a Java applet running in a web browser and providing a friendly medical user interface to perform queries on patient and medical test data and integrate and visualize properly the various query results. A set of tools based on Java Advanced Imaging API enables to process and analyze the retrieved bioimages, and quantify their features in different regions of interest. The platform-independence Java technology makes the developed prototype easy to be managed in a centralized form and provided in each site where an intranet or internet connection can be located. Giving the healthcare providers effective tools for browsing, querying, visualizing and evaluating comprehensively medical images and records in all locations where they can need them - e.g. emergency, operating theaters, ward, or even outpatient clinics- the implemented prototype represents an important aid in providing more efficient diagnoses and medical treatments.

Remote access to anatomical information: an integration between semantic knowledge and visual data.

A novel internet-based application is presented which provides access to anatomy knowledge through symbolic modality expressed by keywords taken from controlled or non-controlled terminology. The system is based on a database where anatomical concepts have been organized into a hierarchical framework. Along with term queries that allow retrieving concepts containing or exactly matching the used keyword, the system also provides semantic access to anatomical information. Queries can be setup, which retrieve concepts relying to a particular meaning and sharing a particular relationship. Moreover, the application has the capability to refine the search of the terms by querying the UMLS knowledge server. Anatomical image data have been integrated by using Visible Human Dataset. A set of these images has been indexed according to our anatomical classification and is used inside the application. The system has been implemented through Java client-server technology and works within standard Internet browsers.

A three-layer multidatabase system for functional statistical analyses of microarray experiment results.

New high-throughput technologies available for the post-genomic era, such as the microarrays, produce lists of hundreds of genes candidate regulated, or with particular expression profiles, in the conditions under study. These lists need to be biologically interpreted to gain a better knowledge of the pathophysiological phenomena involved. To this aim, several biological annotations are available within heterogeneous and widely distributed databases. Although numerous tools have been developed for annotating lists of genes, most of them do not provide methods for evaluating the relevance of the annotations provided, or for estimating the functional bias introduced by the gene set on the array used to identify the considered gene list. To reach this goal, we developed GFINDer, Genome Function INtegrated Discover, a Web-accessible three-layer multidatabase system that automatically provides large-scale lists of user-classified genes with functional profiles biologically characterizing the different gene classes in the list. GFINDer automatically retrieves annotations of several functional categories from different sources, identifies the categories enriched in each class of a user-classified gene list, and calculates statistical significance values for each category. It also enables to functionally classify genes according to user-selected criteria and to analyse the resulting classifications, aiding in better biologically interpreting microarray experiment results.

Java-based browsing, visualization and processing of heterogeneous medical data from remote repositories.

The actual development of distributed information technologies and Java programming enables employing them also in the medical arena to support the retrieval, integration and evaluation of heterogeneous data and multimodal images in a web browser environment. With this aim, we used them to implement a client-server architecture based on software agents. The client side is a Java applet running in a web browser and providing a friendly medical user interface to browse and visualize different patient and medical test data, integrating them properly. The server side manages secure connections and queries to heterogeneous remote databases and file systems containing patient personal and clinical data. Based on the Java Advanced Imaging API, processing and analysis tools were developed to support the evaluation of remotely retrieved bioimages through the quantification of their features in different regions of interest. The Java platform-independence allows the centralized management of the implemented prototype and its deployment to each site where an intranet or internet connection is available. Giving healthcare providers effective support for comprehensively browsing, visualizing and evaluating medical images and records located in different remote repositories, the developed prototype can represent an important aid in providing more efficient diagnoses and medical treatments.

GAAS: gene array analyzer software for management, analysis and visualization of gene expression data.

SUMMARY: GAAS, Gene Array Analyzer Software supports multi-user efficient management and suitable analyses of large amounts of gene expression data across replicated experiments. Its management framework handles input data generated by different technologies. A multi-user environment allows each user to store his/her own data visualization scheme, analysis parameters used, values and formats of the output data. The analysis engine performs: background and spot quality evaluation, data normalization, differential gene expression analyses in single and multiple replica experiments. Results of expression profiles can be interactively navigated through graphical interfaces and stored into output databases.

Automatic quantitation of cell colonies on petri dishes by computerized image analysis.

Clonogenic assay is one of the most sensitive assays, widely used to evaluate the effects of antineoplastic agentsin vitro. A computer program was developed on an IBAS 2.0 Image Analysis System for automated quantiation of cell colonies and clone area on Petri dishes. The sensitivity of the clonogenic assay can be greatly increased by evaluating the mean area of the clones. The program gives an objective, accurate and fast evaluation of large samples. It is simple to use and offers a high degree of flexibility. Special algorithms and techniques have been implemented for good quantitation of both connected and well-separated colonies and to reduce the background noise and the general error rate. The principles and solutions presented are applicable to any other image analysis system.

Automatic quantification of liver fibrosis: design and validation of a new image analysis method: comparison with semi-quantitative indexes of fibrosis.

BACKGROUND/AIMS: Liver fibrosis is one of the most important and characteristic histologic alterations in progressive and chronic liver diseases. Thus, in both clinical and experimental practice, it is fundamental to have a reliable and objective method for its precise quantification. Several semi-quantitative scoring systems have been described. All are time-consuming and produce partially subjective fibrosis evaluations that are not very precise. This paper describes the design and validation of an original image analysis-based application, FibroQuant, for automatically and rapidly quantifying perisinusoidal, perivenular and portal-periportal and septal fibrosis and portal-periportal and septal morphology in liver histologic specimens. METHODS: The implemented image-processing algorithms automatically segment interstitial fibrosis areas, while extraction of portal-periportal and septal region is carried out with an automatic algorithm and a simple interactive step. For validation, all automatically extracted areas were also manually segmented and quantified. RESULTS: Statistical analysis showed significant intra- and interoperator variability in manual segmentation of all areas. Automatic quantifications did not significantly differ from mean manual evaluations of the same areas. Comparison of our image analysis quantifications with staging histologic evaluations of liver fibrosis showed significant correlations (Spearman's, 0.72

Liver fibrosis assessment with semiquantitative indexes and image analysis quantification in sustained-responder and non-responder interferon-treated patients with chronic hepatitis C.

BACKGROUND/AIMS: The effect of interferon on the reduction of liver fibrosis is controversial. We aimed to compare semiquantitative methods with a quantitative digital image analysis system to assess liver fibrosis in biopsies from patients with chronic hepatitis and different responses to interferon. METHODS: We studied 98 liver biopsies with chronic hepatitis C before and after recombinant interferon alfa-2 treatment, using conventional histological assessment, grading of histological activity, scoring/staging of fibrosis (Knodell and Scheuer), and quantification of fibrosis with image analysis (FibroQuant). RESULTS: Sustained-responders to interferon showed a significant reduction in histological lesions and in their Knodell and Scheuer activity indexes. The semiquantitative systems showed no reduction in fibrosis. The FibroQuant application showed a significant reduction in porto-periportal and septal areas among sustained-responders (P < 0.001) and non-responders (P < 0.05), and in porto-periportal and septal fibrosis areas only in sustained-responders (P < 0.001), whereas the percentage of fibrosis increased in non-responders (P < 0.001). CONCLUSIONS: The Scheuer system is useful for the daily evaluation of fibrosis, but the FibroQuant application provides more objective data on the anti-fibrogenic effects of interferon, which include a reduction in the porto-periportal area in sustained-responders and non-responders, accompanied by a reduction in the area of fibrosis only when the viral replication has ceased.

Quantification of intramural calcification in coronary intravascular ultrasound images with automated image analysis.

BACKGROUND: Recent studies have documented the utility of intravascular ultrasonography in quantifying coronary morphologic characteristics and determining an appropriate intervention. Unfortunately, its potential for quantifying lesion calcification is limited by subjective evaluation and manual tracing. The aim of this study was to develop an objective automated method for quantifying calcification in intracoronary images with digital image analysis. METHODS: Images of human coronary arteries acquired with a 30 MHz intracoronary ultrasound catheter were evaluated with digital image analysis and compared with manual tracings. Calcifications were automatically identified as highly echogenic regions detected by global thresholding within sectors of acoustic shadowing defined as regions devoid of texture. RESULTS: The mean percentage agreement, sensitivity, and specificity of detecting calcification in 1-degree sectors of calcified vessels were 82%, 73%, and 87%, respectively. Similar results were obtained in noncalcified images. CONCLUSION: The accuracy of this automated technique was comparable to interoperator and intraoperator variability in manually tracing calcification.

Effects of a DA2/alpha2 agonist and a beta1-blocker in combination with an ACE inhibitor on adrenergic activity and left ventricular remodeling in an experimental model of left ventricular dysfunction after coronary artery occlusion.

Renin-angiotensin-aldosterone and sympathetic nervous systems overactivity play a major role in worsening the extent of heart failure. Attenuation of neurohumoral activation with angiotensin-converting enzyme (ACE) inhibitors and beta-blockers has proven beneficial in congestive heart failure. Because ACE inhibition is a recommended treatment for heart failure, this study was designed to test the effects on neurohumoral activation, hemodynamics, and left ventricular (LV) volume of the combination of an ACE inhibitor (delapril) with a DA2-dopaminergic receptor/alpha2-adrenoceptor agonist (CHF-1024) or a beta1-adrenoceptor antagonist (metoprolol) after a moderate to large myocardial infarction (MI) in rats. MI was induced by left coronary artery ligation in 134 rats, and six were not operated on. After 2 months, the animals with ECG evidence of MI were treated for 1 more month with CHF- 1024, 0.33 mg/kg/day or with metoprolol (10 mg/kg/day), delivered through implanted osmotic minipumps, in addition to delapril (6 mg/kg/day) in the drinking water. Daily urinary excretion of norepinephrine (NE) and circulating concentration were measured. Hemodynamic variables were measured, and three-dimensional morphometric analysis was done on the diastole-arrested hearts to quantify infarct size and LV geometry. In conscious animals, delapril alone or with CHF-1024 or metropolol did not modify heart rate or systolic blood pressure. Both combination treatments, however, significantly reduced heart rate in anesthetized animals compared with the group receiving vehicle. Infarct size was not different between treatments, averaging 20-22% of LV volume. The threefold increase of LV chamber volume in infarcted rats was significantly attenuated by delapril alone or with CHF-1024 or metoprolol (-37 to -44%, p<0.05). Treatment with a combination of the ACEi and CHF-1024 tended to normalize the shape of the LV cavity. Urinary NE excretion was unaffected by delapril alone but was reduced by the addition of CHF-1024 or metoprolol. In conclusion, 1 month of treatment with doses of delapril having no hemodynamic effect, reduced LV volume in a model of chronic heart failure. When CHF-1024 or metoprolol was given with delapril, sympathetic activation decreased with no unwanted effects, such as excessive hypotension.

Role of intrarenal endothelin 1, endothelin 3, and angiotensin II expression in chronic cyclosporin A nephrotoxicity in rats.

Endothelin 1 (Et1) is widely expressed in the kidney and is related to several functions and to pathological conditions with progression towards sclerosis. The function of endothelin 3 (Et3) at the renal level is debatable, but it could have an important regulatory function in the reabsorption of water through its action on tubular type B receptors. Angiotensin II has recently been implicated as the principal factor responsible for the progression of interstitial fibrosis induced by cyclosporin A (CsA). We investigated this relationship in vivo and analyzed the modifications induced by CsA toxicity in Sprague-Dawley rats treated with 25 mg/kg/day of CsA for 28 and 56 days. Immunohistochemical methods and molecular analysis were used to study the expression of Et1 and Et3 and immunohistochemistry alone to determine the intrarenal expression of angiotensin II. Rats treated with CsA developed chronic nephrotoxicity lesions; semiquantitative analyses of hyaline arteriolopathy revealed that the passage of time affected the extent of this lesion and led to the diminution of the total glomerular area. Immunohistochemical results showed that chronic CsA treatment induced moderate secretion of Et1 and Et3 at tubular and glomerular levels and that the local expression of angiotensin II in the treatment groups was more evident than in control animals. Besides, the mRNA levels of preproEt3 showed a dramatic increase from 28 days after CsA treatment (control group 0.07+/-0.11 vs. CsA group 0.48+/-0.11, p<0.01), while the mRNA levels of preproEt1 increased from 56 days (control group 0.15+/-0.05 vs. CsA group 0.34+/-0.09, p< 0.05). At 28 days, renal lesions correlated strongly with the mRNA levels of Et3 (r>0.50, p<0.01). However, at 56 days, the key finding was the strong correlation of the most important analytical, histological, and immunohistochemical parameters of CsA nephrotoxicity with Et1 mRNA levels (r>0.50, p<0.01). These results support the hypothesis that the clinical and morphological phenomena linked with CsA nephrotoxicity are related to hypersecretion of endothelins and local expression of angiotensin II in the outer medulla and medullary rays; Et3 and angiotensin II are the first to act, followed subsequently by Et1.

Chronic cyclosporin A nephrotoxicity, P-glycoprotein overexpression, and relationships with intrarenal angiotensin II deposits.

P-glycoprotein (P-gp) expels hydrophobic substances from the cell, including chemotherapeutic agents and immunosuppressants such as cyclosporin A (CsA) and FK506. Exposure of cultured renal tubular cells to CsA induces P-gp overexpression in cell membranes. Angiotensin II has recently been implicated as the principal factor responsible for progression of interstitial fibrosis induced by CsA. To investigate the in vivo relationships between histological lesions, P-gp overexpression, and intrarenal angiotensin II deposits, we developed a model of chronic CsA toxicity in Sprague-Dawley rats treated with 25 mg/kg/day CsA for 28 and 56 days and fed either a standard maintenance diet or a low-salt diet. Immunohistochemical methods were used to study the expression of P-gp in renal tubular cells and the appearance of intrarenal angiotensin II deposits. Rats treated with CsA developed chronic nephrotoxicity lesions that were more evident in the group fed the low-salt diet. Treatment with CsA induced overexpression of P-gp in tubular cells of the kidney that increased with time. We found that immunohistochemical expression of P-gp was slightly more severe in rats fed a low-salt diet. Intrarenal deposits of angiotensin II were more evident in rats treated with CsA; these deposits also increased with time. This finding was also more relevant in rats given the low-salt diet. The up-regulation of P-gp was inversely related to the incidence of hyaline arteriopathy (r = -0.65; P < 0.05), periglomerular (r = -0.58; P < 0.05) and peritubular fibrosis (r = -0.63; P < 0.05), and intrarenal angiotensin H deposits in animals with severe signs of nephrotoxicity (r = -0.65; P < 0.05). These results support the hypothesis that the role of P-gp as a detoxicant in renal cells may be related to mechanisms that control the cytoplasmic removal of both toxic metabolites from CsA and those originating from the catabolism of signal transduction proteins (methylcysteine esters), which are produced as a result of ras activation in presence of angiotensin II.