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P Glycoprotein: A New Mechanism to Control Drug-Induced Nephrotoxicity

The role of P glycoprotein (P-gp) in kidney is now being explored, and under physiological conditions, this protein is thought to be an excretory pump of cationic xenobiotics and metabolites. Functionally, two different types of P-gp have been described, but only the class I has been related to drug transport, and its overexpression confers the multidrug resistance phenotype in tumoral cells. It has been proposed that P-gp is involved in the energy-dependent transport of substrates through the cell membrane (toxic metabolites, toxins, nutrients, ions, peptides, etc.) – like a ‘hydrophobic molecule vacuum cleaner’. Several physiological functions have been attributed to P-gp: defense against xenobiotic aggression and transmembrane transport of prenylcysteine methyl esters, removing these cytotoxic metabolites from cells. A variety of substrates ranging from chemotherapeutics to steroid hormones, antibiotics, and calcium channel blockers can be transported by P-gp, suggesting the possible involvement of this protein in other unknown functions. Results from our group and others have suggested that overexpression of P-gp in renal tubular and mesangial cells prevents pharmacological nephrotoxicity by cyclosporin A (CsA). On the other hand CsA, a substrate of the pump, could act as a blocker in tubular cells by competitive inhibition. One relevant aspect in kidney is the possible relationship between P-gp and protein kinase C. Several reports suggest that protein kinase C may play a role in inducing the P-gp overexpression in cells under xenobiotic pressure, through activation of the ras oncoprotein family. This could be mediated directly by angiotensin II as a ras activator. This way, the detoxicant function of P-gp against products of the ras catabolism could mediate their accumulation when the ‘vacuum cleaner’ function is blocked by CsA or tacrolimus, contributing to the initial development of fibroblastic activation that leads to interstitial fibrosis associated with nephrotoxicity by these immunosuppressor drugs. In conclusion, P-gp expression could be an important component of a complex detoxifying system in kidney against xenobiotics or regulating the traffic of endogenous metabolites responsible for the susceptibility of subjects to the development of nephrotoxicity against different drugs.

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.

Role of P-glycoprotein in chronic cyclosporine nephrotoxicity and its relationship to intrarenal angiotensin II deposits.

CYCLOSPORINE (CsA), among other actions in the kidney, induces P-glycoprotein (P-gp) overexpression in tubular cells. P-gp expels hydrophobic xenobiotics from the cytoplasm, including chemotherapeutic and immunosuppressive agents such as CsA, tacrolimus, and rapamycin. One of the main secondary effects of CsA treatment is nephrotoxicity. Morphologic findings of CsA nephrotoxicity include vacuolar isometric degeneration of kidney tubules, hyaline arteriopathy, and stripped and diffuse interstitial fibrosis. Experimental models in rats have shown that low sodium (LS) diets decrease renal blood flow and intensify histopathologic lesions of CsA nephrotoxicity, especially hyaline arteriopathy and stripped tubulointerstitial fibrosis, parallel with the upregulation of the renin-angiotensin system (RAS). In this context, some studies have found that blockers of angiotensin II receptors, as well as angiotensin II–converting enzyme inhibitors: (a) prevent the development of interstitial fibrosis in experimental models of chronic CsA nephrotoxicity; and (b) reduce the accumulation of TGF-b1 and proteins of the extracellular matrix in CsA-induced chronic nephropathy. Our aim was to analyze the alterations induced by CsA in P-gp expression in kidney tubular cells of Sprague–Dawley rats and to characterize the relationship of these changes with the angiotensin II deposits and chronic CsA nephrotoxicity lesions.

Effects of environmental stress on mRNA and protein expression levels of steroid 5α-Reductase isozymes in adult rat brain

Environmental stress conditions are important factors in human health and should be considered in the development of appropriate health policies, since they have been associated with psychological disorders and even with death. A link between stress and changes in 3alpha,5alpha-reduced neurosteroids has been reported. Steroid 5alpha-Reductase (5alpha-R) is the rate-limiting enzyme in the biosynthesis of 3alpha,5alpha-reduced neurosteroids. Using reverse transcription-polymerase chain reaction and immunohistochemistry, 5alpha-R isozymes (5alpha-R1 and 5alpha-R2) mRNA and protein levels were detected in prefrontal cortex of male and female rats after they underwent environmental stresses, i.e., excessive heat, artificial light, and the sensation of immobility in a small space, similar to those found in common workplace situations. Results showed significantly higher 5alpha-R2 mRNA and protein levels in environmentally-stressed versus control rats. Interestingly, a sexual dimorphism in 5alpha-R1 mRNA and protein levels was observed after environmental stress, with an increase in males and a decrease in females. This fact might explain gender differences in the incidence of some type of minor depression.

Bisphenol A Modifies the Regulation Exerted by Testosterone on 5α-Reductase Isozymes in Ventral Prostate of Adult Rats

The development, growth, and function of the prostate gland depend on androgen stimulation. The primary androgen in prostate is 5α-dihydrotestosterone (DHT) which is synthesized from circulating testosterone (T) through the action of 5α-reductase (5α-R). Although 5α-R occurs as five isozymes, only 5α-R1 and 5α-R2 are physiologically involved in steroidogenesis. The endocrine disruptor bisphenol A (BPA) alters sexual organs, including the prostate. Our previous findings indicated that BPA decreased the expression of 5α-R1 and 5α-R2 in rat prostate but also circulating T. Thus, it is unclear whether BPA exerts this effect on 5α-R isozymes by reducing circulating T or by any other mechanism. In this study, we examine the effects of short-term exposure to BPA at doses below 25 μg/Kg/d and above 300 μg/Kg/d of the TDI on mRNA levels of 5α-R1 and 5α-R2 in prostate of adult castrated rats supplemented with T to achieve constant circulating T levels. mRNA levels were measured by absolute quantitative RT-PCR, T levels by RIA, and DHT levels by ELISA. Our results indicated that in castrated rats treated with T BPA at the two doses studied significantly decreased the mRNA levels of both 5α-R isozymes in a dose-dependent manner without modifications in circulating T.

Expression of steroid 5α-reductase isozymes in prostate of adult rats after environmental stress.

The elevated incidence of prostate cancer and benign prostatic hypertrophy is a cause of increasing public health concern in the Western world. The normal and pathological growth of the prostate are both dependent on stimulation by dihydrotestosterone, which is synthesized from circulating testosterone by two 5α‐reductase (5α‐R) isozymes, 5α‐reductase type 1 (5α‐R1) and 5α‐reductase type 2 (5α‐R2). Both isozymes have been implicated in prostate disease. We used quantitative RT‐PCR and immunohistochemistry, respectively, to quantify mRNA and protein levels of 5α‐R isozymes in the ventral prostate of adult rats under environmental stress conditions analogous to those found in some common workplace situations, i.e. artificial light, excessive heat, and the sensation of immobility in a small space. Transcription and expression levels of both 5α‐R isozymes were significantly higher in environmentally stressed rats than in unstressed rats. Increased 5α‐R isozyme levels may play a role in the development or maintenance of prostate disease. Further research is warranted to explore these effects of environmental stress on human health and their implications for environmental and occupational health policies.