Thus, low toxicity of "morunga" extracts, competence of dispersion and plant maintenance, resistance to inhospitable environments, low cost and simple technology are some factors that convert *M. oleifera* an alternative to unpolluted drinking water and add it in programs to control disease-transmitting mosquitoes, especially in rural areas and developing countries, where access to drinking water is problematical and its accumulation in artificial containers com‐ monly found in and around human residences create an ideal site to lay eggs and breed larvae.

Aqueous (100-450 mg/k, oral) and methanolic (350-700 mg/kg, intraperitoneal) root extracts reduced locomotor activity of rats and the number of seizures induced by penicillin and strychnine [27, 103]. Aqueous extract also amplified rates of 5-HT and reduced levels of dopamine in the brain cortex, cerebellum and caudate nucleus and noradrenaline measure in the cerebral cortex [103]. Methanol extract produced CNS depression, decreases the mortality of strychnine-and leptazol-treated animals, increased the sleeping time, caused analgesia and potentiated morphine analgesic effects [27]. This sleepiness extension and anticonvulsant and

More recently, discoveries also showed that ethanol extract from *M. oleifera* leaves (250-2000 mg/kg) caused decreasing in rearing, grooming, head dips and locomotion of mice, enhanced learning and memory, increased anxiogenic effect and reduced convulsions induced by pentylenetetrazol, though it has no effect on picrotoxin and strychnine induced convulsion. In this event, it is possible that these activities are mediated through the enhancement of central inhibitory mechanism involving release γ-amino butyric acid (GABA) [104]. These findings

Other pharmacological activities of *M. oleifera* include the seed biological action upon *Plasmodium falciparum* [105], *Schistosoma mansoni* cercariae [106] and helminth eggs [53], diuretic activity [80] and spleen and thymus enlargement [32,74]; the leaves are purgative, antipyretic [18], immunomodulatory [107] and inhibit conversion of thyroxine (T4) in triiodo‐ thyronine (T3), with high likelihood to be employed in the treatment of hyperthyroidism [108]; the flowers are aphrodisiac [68], hepatoprotective [109] and antidiabetic [110]; the roots, carminative and anti-constipant [99] and stem barks possess antitumor activity and prevent splenomegaly [68]. This notable pharmacological potential suggests that the beneficial effect of the plant may be associated with individual or combined action of its constituents, such as

partially justified the traditional use of *M. oleifera* parts for the treatment of epilepsy.

repellent [70,98,102].

188 Pharmacology and Therapeutics

**4.5. Action on Central Nervous System (CNS)**

analgesic activities can be justified by the 5-HT brain rising.

phenols, aromatic isothiocyanates, flavonoids and sterols [39,102].

Plants have a variety of indispensable macro and micronutrients to feed heterotrophic organisms, including ruminants and monogastric animals such as sheeps, rats, mice and humans. However, side effects and aversions to vegetal substances as alkaloids, tannins, cyanogenic glycosides, terpenes, lectins and glucosinolates are habitual [111]. Thus, animals can identify tastes from sweet (carbohydrate, for example, an indication of calories) to the unpleasant taste of toxins. Among these, some present bitter flavor (alkaloids, saponins and cyanogenic glycosides), astringent (tannins) or offensive odors (terpenes). Dislikes can be wild (temporary) or strong (permanent) depending on the toxin dosages and how they affect the gut and central nervous system. These aversions hardly develop if toxins act gradually (days to weeks). Furthermore, toxins can activate the emetic center, causing nausea and vomiting [112]. Tropical seeds usually have high content of antinutritional factors, specially tannins and lectins [111].

#### **5.1. Leaves, flowers and roots**

*M. oleifera* leaves possess minor quantities of tannin (12 g/kg dry material), phytic acid (21 g/kg) and absence of trypsin, amylase inhibitors, lectins and glucosinolates, an aspect which encourages their consumption. Pods and stem have negligible amounts of tannin, but saponins and alkaloids are found in significant quantities in leaves and stem, respectively, though they should be considered non-toxic to ruminants [39].

Water extract of roots inhibit development of uterus and blastocyst implantation [113], indicating an abortifacient effect that interferes in estrogen and progesterone levels, modifying the normal physiology of the genital tract during the fertile period. Relatedly, Indian women frequently use leaf extracts as natural oral contraceptives [114].

Extracts from roots and flowers (200 mg/kg/day) were able to maintain transaminase (aspartate aminotransferase, AST; alanine aminotransferase, ALT) and bilirubin levels, protect against hepatotoxicity induced by acetaminophen toxic metabolites produced by P450 monooxygenase enzymes and presented slight acute toxicity, since it was found LD50 values of 1023 and 1078 mg/kg for root and 1047 and 1092 mg/kg for flowers extracts (ethanolic and aqueous extracts, respectively) [109] (Table 2).

However, root methanolic extracts (intraperitoneally and weekly doses greater than 46 mg/kg/day) produced hepatotoxicity and nephrotoxicity associated with hematological and plasma changes, particularly, AST, ALT, cholesterol, bilirubin, urea, proteins and causing leukocytosis and clotting time increasing [115]. Histological examinations in guinea pigs also propose toxicity of root methanolic extract (3.5, 4.6 and 7.0 mg/kg), whereas balloon degener‐ ation and micro and macrovesicular steatosis (in liver) and interstitial inflammation, tubular damage and amorphous eosinophilic materials (in kidneys) were seen, demonstrating reversible signals of histo-architectural distortions [116].

Reference [117] reported that acute and sub-cronic exposure to higher doses of aqueous leaf extracts (400 to 6400 mg/kg) revealed to be relatively safe for human and rodents, since any

[74]. *Ad libitum* intake of aqueous extract as the unique source of water in doses of 1300-1670mg/ kg/day for a month was also harmless and no change suggestive of toxicity was observed [32].

Safety and Efficacy of *Moringa oleifera* Lamarck (1785) — Therapeutic and Toxicological Properties

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191

In [120] verified that seeds orally administered for 5 days at 500 mg/kg/day protected against toxic arsenic effects and recovery physiological measures to normal values (hemoglobin, erythrocytes and levels of δ-aminolevulinic acid dehydratase and glutathione S-transferase), probably due to the arsenic tissue removal. Previously, [121] showed that oral administration of hydroalcoholic extract of *M. oleifera* fresh pods increased hepatic levels of cytochrome b5, cytochrome P450, glutathione peroxidase, catalase, reductase and S-transferase enzymes involved in reactions of Phases I and II responsible by detoxification of exogenous substances such as carcinogens and plant poisonous. These findings were corroborated by [122], who showed that seed hydroethanolic extract (1g/kg) avoided the development of hepatic fibrosis induced by carbon tetrachloride and reduced histopathological and biochemical characters of inflammatory necrosis on hepatocytes (cellular infiltration, fatty degeneration and levels of AST, ALT, myeloperoxidase, collagens and biomarkers of oxidative stress). These findings highlighted the chemopreventive properties that have been attributed to antioxidant com‐

Despite investigations have indicated absence of toxicity following oral consumption of the seed aqueous extract, reproducing the intake of treated water with clarifying agent [32,74,123], nutritional assessments reported that those growing rats fed during 10 days with a diet whose total protein content (10%) was replaced by seed flour and whose doses were 24-fold higher than the highest dose tested by [123], suffered from severe growth disorders, loss of appetite and weight, hyperplasia of the small and large intestine, liver, pancreas, kidneys, heart, stomach and atrophy of key organs like spleen and thymus, though protein digestibility is similar to the foodstuff presenting egg white [30]. The antinutritional compounds prevailing in mature seeds, mainly glucosinolates (65.5 mmol/g), phytic acid (41 g/kg) and lectins [30,39, 124,125] should be responsible for these effects. Phytates, when found in percentages between 1-6% and ingested for extended periods, they may reduce the bioavailability of minerals (Ca2+and Zn2+), starch and proteins in monogastric animals. Glucosinolates disturb growth and

Lectins, in turn, are proteins or glycoproteins with reversible binding sites to carbohydrates [111]. They interact with the intestinal mucosa and interfere with digestion and absorption of nutrients, reduce activity of amylase, establish stable complexes with trypsin/chymotrypsin [126], cause pancreatic hypertrophy [127] and decrease growth rate [30]. In fact, studies have emphasized the *M. oleifera* haemagglutinating activity and associated it with lectins detected

Additionally, weight gain reduction in sheep supplemented with 6 g/dia of *M. oleifera* delipidated seeds compared with animals feed with 4 g/day may be explicated by the abundant presence of cationic proteins with antimicrobial activity [38] and/or because of bitter taste [39]. Therefore, antibacterial activity of seeds should inhibit the animal growth, altering its intestinal

pounds in the seeds [68,72,121].

reproduction.

in the seeds [30,100,128].

flora and rates of fermentation efficiency.

**Table 2.** Lethal dose 50% (LD50) of *Moringa oleifera* extracts upon laboratory mammals.

mortality was detected when administered orally. These results are in according to [118], who documented that moringa leaf extracts are non-lethal at 2000 mg/kg and [104], whose publi‐ cation demonstrated that ethanol extract from moringa leaves were not toxic to mice and revealed a LD50 higher than 6.4 g/kg in oral acute toxicity studies. Nevertheless, i.p. injection presented 20% and 80% mortality in Wistar albino mice at doses of 1000 and 2000 mg/kg, with LD50 of 1585 mg/kg and acute administration at 3000mg/kg reduces urea and albumin levels, indicating liver and renal dysfunction [119] probably initiated by toxicants such as isothioci‐ nates and glycosides during biotransformation and corroborating those outcomes described by [115] and [118], whose mice presented biochemical alterations suggestive of renal damage. An opposing discovery to all previous researches divulged, for the first time, showed that *M. oleifera* has genotoxic potential at higher doses (3000 mg/kg), increasing significantly the number of polychromatic micronucleated erythrocytes derived from bone marrow of rodents (20.2 ± 4.0 cells/1000 cells) when compared to control (0.9% saline) [119].

#### **5.2. Seeds**

The best advantage of using *M. oleifera* seeds for water clarification is its low toxicity. The aqueous extract of seeds (400 mg/kg/day) caused no biochemical, histological and hematolog‐ ical alterations, while it increased albumin and HDL-C serum and reduced AST and ALT levels [74]. *Ad libitum* intake of aqueous extract as the unique source of water in doses of 1300-1670mg/ kg/day for a month was also harmless and no change suggestive of toxicity was observed [32].

In [120] verified that seeds orally administered for 5 days at 500 mg/kg/day protected against toxic arsenic effects and recovery physiological measures to normal values (hemoglobin, erythrocytes and levels of δ-aminolevulinic acid dehydratase and glutathione S-transferase), probably due to the arsenic tissue removal. Previously, [121] showed that oral administration of hydroalcoholic extract of *M. oleifera* fresh pods increased hepatic levels of cytochrome b5, cytochrome P450, glutathione peroxidase, catalase, reductase and S-transferase enzymes involved in reactions of Phases I and II responsible by detoxification of exogenous substances such as carcinogens and plant poisonous. These findings were corroborated by [122], who showed that seed hydroethanolic extract (1g/kg) avoided the development of hepatic fibrosis induced by carbon tetrachloride and reduced histopathological and biochemical characters of inflammatory necrosis on hepatocytes (cellular infiltration, fatty degeneration and levels of AST, ALT, myeloperoxidase, collagens and biomarkers of oxidative stress). These findings highlighted the chemopreventive properties that have been attributed to antioxidant com‐ pounds in the seeds [68,72,121].

Despite investigations have indicated absence of toxicity following oral consumption of the seed aqueous extract, reproducing the intake of treated water with clarifying agent [32,74,123], nutritional assessments reported that those growing rats fed during 10 days with a diet whose total protein content (10%) was replaced by seed flour and whose doses were 24-fold higher than the highest dose tested by [123], suffered from severe growth disorders, loss of appetite and weight, hyperplasia of the small and large intestine, liver, pancreas, kidneys, heart, stomach and atrophy of key organs like spleen and thymus, though protein digestibility is similar to the foodstuff presenting egg white [30]. The antinutritional compounds prevailing in mature seeds, mainly glucosinolates (65.5 mmol/g), phytic acid (41 g/kg) and lectins [30,39, 124,125] should be responsible for these effects. Phytates, when found in percentages between 1-6% and ingested for extended periods, they may reduce the bioavailability of minerals (Ca2+and Zn2+), starch and proteins in monogastric animals. Glucosinolates disturb growth and reproduction.

mortality was detected when administered orally. These results are in according to [118], who documented that moringa leaf extracts are non-lethal at 2000 mg/kg and [104], whose publi‐ cation demonstrated that ethanol extract from moringa leaves were not toxic to mice and revealed a LD50 higher than 6.4 g/kg in oral acute toxicity studies. Nevertheless, i.p. injection presented 20% and 80% mortality in Wistar albino mice at doses of 1000 and 2000 mg/kg, with LD50 of 1585 mg/kg and acute administration at 3000mg/kg reduces urea and albumin levels, indicating liver and renal dysfunction [119] probably initiated by toxicants such as isothioci‐ nates and glycosides during biotransformation and corroborating those outcomes described by [115] and [118], whose mice presented biochemical alterations suggestive of renal damage. An opposing discovery to all previous researches divulged, for the first time, showed that *M. oleifera* has genotoxic potential at higher doses (3000 mg/kg), increasing significantly the number of polychromatic micronucleated erythrocytes derived from bone marrow of rodents

**LD50 value (mg/kg body weight)**

Seeds Aqueous 446.5 intraperitoneal [32]

Leaves Aqueous 1585 oral [119]

Flowers Aqueous 1092 intraperitoneal [108]

Root Aqueous 1078 intraperitoneal [108]

Stem Ethanolic > 5000 oral [75]

**Table 2.** Lethal dose 50% (LD50) of *Moringa oleifera* extracts upon laboratory mammals.

Ethanolic > 6400 oral [104]

Methanolic 7420 intraperitoneal [63]

Ethanolic 1047 intraperitoneal [108]

Ethanolic 1023 intraperitoneal [108]

Methanolic 223.6 intraperitoneal [116]

**Route of administration Reference**

> 2000 [117], [118], [119],

The best advantage of using *M. oleifera* seeds for water clarification is its low toxicity. The aqueous extract of seeds (400 mg/kg/day) caused no biochemical, histological and hematolog‐ ical alterations, while it increased albumin and HDL-C serum and reduced AST and ALT levels

(20.2 ± 4.0 cells/1000 cells) when compared to control (0.9% saline) [119].

**5.2. Seeds**

**Part of plant Extract**

190 Pharmacology and Therapeutics

Lectins, in turn, are proteins or glycoproteins with reversible binding sites to carbohydrates [111]. They interact with the intestinal mucosa and interfere with digestion and absorption of nutrients, reduce activity of amylase, establish stable complexes with trypsin/chymotrypsin [126], cause pancreatic hypertrophy [127] and decrease growth rate [30]. In fact, studies have emphasized the *M. oleifera* haemagglutinating activity and associated it with lectins detected in the seeds [30,100,128].

Additionally, weight gain reduction in sheep supplemented with 6 g/dia of *M. oleifera* delipidated seeds compared with animals feed with 4 g/day may be explicated by the abundant presence of cationic proteins with antimicrobial activity [38] and/or because of bitter taste [39]. Therefore, antibacterial activity of seeds should inhibit the animal growth, altering its intestinal flora and rates of fermentation efficiency.

The bitter taste in the seeds, important to provide its typical aroma, is alleviated by treatment [39], suggesting that its taste would not be a limiting factor for using them, since even cow's milk whose dairy cattle was treated with moringa meal did not reveal changes in quality [41]. Furthermore, it is known that most adverse effects are eliminated by suitable methods as washing, storage, drying and/or heating. For example, lectin biological properties are lost after protein denaturation by temperature and pH. Nevertheless, these techniques are expensive and prolonged cooking of seeds result in nutritional value reduction and loss of micronu‐ trients, specifically vitamins and minerals. The text [129] showed that roasted seeds promote formation of mutagenic compounds [4-(alpha-L-rhamnosyloxy)phenylacetonitrile, 4 hydrox‐ yphenylacetontrile and 4-hydroxyphenyl-acetamide). On the other hand, it was observed that moringa seed flour has post-treated proteins with good digestibility and absorption [39,41].

lógico) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brasil)

, Rivelilson Mendes de Freitas2

, Maria Carolina de Abreu3

1 Department of Biophysics and Physiology, Campus Ministro Petrônio Portella, Federal

2 Postgraduate Program in Pharmaceutical Sciences, Federal University of Piauí, Teresina,

3 Department of Biological Sciences, Campus Senador Helvídio Nunes de Barros, Federal

4 Faculty of Pharmacy, Center for Health Sciences, Universidade de Fortaleza, Fortaleza, CE,

5 Department of Physiology and Pharmacology, Faculty of Medicine, Federal University of

[1] Organización Mundial de La Salud (OMS). Estrategia de la OMS sobre medicina tra‐

[2] Butler MS. The role of natural product chemistry in drug discovery. Journal of Natu‐

[3] Ferreira PMP, Farias DF, Oliveira JTA, Carvalho AFU. *Moringa oleifera*: Bioactive compounds and nutritional potential. Revista de Nutrição 2008;21(4) 431-437.

[4] Magalhães HIF, Ferreira PMP, Moura ES, Torres MR, Alves APNN, Pessoa ODL, Costa-Lotufo LV, Moraes MO, Pessoa C. *In vitro* and *in vivo* antiproliferative activity of *Calotropis procera* stem extracts. Anais da Academia Brasileira de Ciências

[5] Ferreira PMP, Farias DF, Viana MP, Souza TM, Vasconcelos IM, Soares BM, Pessoa C, Costa-Lotufo LV, Moraes MO, Carvalho AFU. Study of the antiproliferative po‐

, Janiella Buenos Aires Pereira3

Safety and Efficacy of *Moringa oleifera* Lamarck (1785) — Therapeutic and Toxicological Properties

,

,

and Cláudia Pessoa5

, Nagilla Daniela de Jesus Costa3

http://dx.doi.org/10.5772/58627

,

193

Paulo Michel Pinheiro Ferreira1,2, Éverton José Ferreira de Araújo2

for the financial support.

Jurandy do Nascimento Silva2

Jaksilania Aires Forte Pinheiro4

Samara Ferreira de Carvalho Oliveira3

University of Piauí, Teresina, PI, Brazil

University of Piauí, Picos, PI, Brazil

dicional 2002-2005. Genebra: OMS; 2002.

ral Products 2004;67(12) 2141-2153.

2010;82(2) 407-416.

Ceará, Fortaleza, CE, Brazil

**Author details**

PI, Brazil

Brazil

**References**

Environmental assessments with seed water extract using the microcrustaceans *Artemia salina* and *Daphnia magna* showed LC50 values of 177.8 and 188.7 μg/mL, respectively [32,74]. The research [130], working with the *Scenedesmus obliquus* green algae, another aquatic organism, found LC50 of 207.5 and 287.5 mg/mL (methanolic and aqueous seed extracts, respectively). In addition, acute toxicity tests in mammals (*Mus musculus*) revealed a LD50 of 446.5 mg/kg body weight [32]. Thus, both studies with marine organisms as well as those performed in mice indicate low toxicity of moringa seed extracts [131,132].

In summary, results obtained by [30,32,74,104,116,117,119,123,133,] confirm that toxicity of *M. oleifera* depends on concentration, part of the plant used, and manner of preparation and routes of administration. Then, though the consumption of different parts of specie for various purposes has been widely accepted, it is important to note that intake without any pretreatment should be done carefully, since the specific adverse(s) factor(s) remains unclear whereas the presence of other unknown toxicants is uncertain. Additionally, little has been done to define, optimize and standardize conditions for their use and a few government programs encourage or disseminate such treatment for household water or determine its acceptability, sustainability, costs and effectiveness.

#### **6. Conclusion**

Relatively safe for human, *M. oleifera* is a worthy pharmacological and nutritional alternative, especially taking into account that technology requirements for leaves and seeds' flour production is cheap and simple, which benefits small farmers and the general population by providing an abundant food supply and bioactive substances.

#### **Acknowledgements**

We wish to thank the Brazilian Agencies Fundação de Amparo à Pesquisa do Estado do Piauí (FAPEPI), FUNCAP (Fundação Cearense de Apoio ao Desenvolvimento Científico de Tecno‐ lógico) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brasil) for the financial support.

#### **Author details**

The bitter taste in the seeds, important to provide its typical aroma, is alleviated by treatment [39], suggesting that its taste would not be a limiting factor for using them, since even cow's milk whose dairy cattle was treated with moringa meal did not reveal changes in quality [41]. Furthermore, it is known that most adverse effects are eliminated by suitable methods as washing, storage, drying and/or heating. For example, lectin biological properties are lost after protein denaturation by temperature and pH. Nevertheless, these techniques are expensive and prolonged cooking of seeds result in nutritional value reduction and loss of micronu‐ trients, specifically vitamins and minerals. The text [129] showed that roasted seeds promote formation of mutagenic compounds [4-(alpha-L-rhamnosyloxy)phenylacetonitrile, 4 hydrox‐ yphenylacetontrile and 4-hydroxyphenyl-acetamide). On the other hand, it was observed that moringa seed flour has post-treated proteins with good digestibility and absorption [39,41].

Environmental assessments with seed water extract using the microcrustaceans *Artemia salina* and *Daphnia magna* showed LC50 values of 177.8 and 188.7 μg/mL, respectively [32,74]. The research [130], working with the *Scenedesmus obliquus* green algae, another aquatic organism, found LC50 of 207.5 and 287.5 mg/mL (methanolic and aqueous seed extracts, respectively). In addition, acute toxicity tests in mammals (*Mus musculus*) revealed a LD50 of 446.5 mg/kg body weight [32]. Thus, both studies with marine organisms as well as those

In summary, results obtained by [30,32,74,104,116,117,119,123,133,] confirm that toxicity of *M. oleifera* depends on concentration, part of the plant used, and manner of preparation and routes of administration. Then, though the consumption of different parts of specie for various purposes has been widely accepted, it is important to note that intake without any pretreatment should be done carefully, since the specific adverse(s) factor(s) remains unclear whereas the presence of other unknown toxicants is uncertain. Additionally, little has been done to define, optimize and standardize conditions for their use and a few government programs encourage or disseminate such treatment for household water or determine its

Relatively safe for human, *M. oleifera* is a worthy pharmacological and nutritional alternative, especially taking into account that technology requirements for leaves and seeds' flour production is cheap and simple, which benefits small farmers and the general population by

We wish to thank the Brazilian Agencies Fundação de Amparo à Pesquisa do Estado do Piauí (FAPEPI), FUNCAP (Fundação Cearense de Apoio ao Desenvolvimento Científico de Tecno‐

performed in mice indicate low toxicity of moringa seed extracts [131,132].

acceptability, sustainability, costs and effectiveness.

providing an abundant food supply and bioactive substances.

**6. Conclusion**

192 Pharmacology and Therapeutics

**Acknowledgements**

Paulo Michel Pinheiro Ferreira1,2, Éverton José Ferreira de Araújo2 , Jurandy do Nascimento Silva2 , Rivelilson Mendes de Freitas2 , Nagilla Daniela de Jesus Costa3 , Samara Ferreira de Carvalho Oliveira3 , Janiella Buenos Aires Pereira3 , Jaksilania Aires Forte Pinheiro4 , Maria Carolina de Abreu3 and Cláudia Pessoa5

1 Department of Biophysics and Physiology, Campus Ministro Petrônio Portella, Federal University of Piauí, Teresina, PI, Brazil

2 Postgraduate Program in Pharmaceutical Sciences, Federal University of Piauí, Teresina, PI, Brazil

3 Department of Biological Sciences, Campus Senador Helvídio Nunes de Barros, Federal University of Piauí, Picos, PI, Brazil

4 Faculty of Pharmacy, Center for Health Sciences, Universidade de Fortaleza, Fortaleza, CE, Brazil

5 Department of Physiology and Pharmacology, Faculty of Medicine, Federal University of Ceará, Fortaleza, CE, Brazil

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**Chapter 7**

**Anticalcineurinics: Role of Mitochondrial Transition Pore**

Cyclosporin A (CyA) and tacrolimus (TAC) are calcineurin phosphatase inhibitors which are currently used in immunosuppression therapy for solid organ and hematopoietic stem cell transplantation to prevent and treat allograft rejection, and for autoimmune disease treatment [1]. Although drugs are not structurally related (Figure 1, Materials and Methods), they have identical cellular and molecular actions, including nephrotoxicity as a major side effect [2-4].

It is known that CyA and TAC may cause acute as well as chronic tubulointerstitial nephrop‐ athy characterized by tubular atrophy, loss of tubular cells and interstitial fibrosis [5], although the mechanisms of immunosuppression-associated renal damage are not fully understood [6, 7]. In any case, it is generally agreed that acute haemodynamic effects of CyA may be mediated by an imbalance of different vasoconstrictors and vasodilators, including renin-angiotensin system and nitric oxide among others [1,3]. Histologically, vacuolization, calcification and necrosis of tubular epithelial cells are characteristics, and endothelial cell swelling with variable degrees of hyalinosis. Abnormalities similar to those are found with TAC [4,8]. For chronic CyA nephrotoxicity, different studies have suggested a toxic effect of the drug on afferent arterioles and tubular epithelial cells, vasoconstriction and endothelial injury leading

> © 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**on Nephrotoxicity**

Alberto Lázaro Fernández, Blanca Humanes Sánchez, Juan Carlos Jado Rodríguez,

José Antonio Lázaro Manero,

Manuela Castilla Barba,

http://dx.doi.org/10.5772/57286

**1. Introduction**

Ana María Torres Redondo, Sonia Camaño Páez,

Marina Mojena Sánchez and Alberto Tejedor Jorge

Additional information is available at the end of the chapter

## **Anticalcineurinics: Role of Mitochondrial Transition Pore on Nephrotoxicity**

Alberto Lázaro Fernández, Blanca Humanes Sánchez, Juan Carlos Jado Rodríguez, Ana María Torres Redondo, Sonia Camaño Páez, José Antonio Lázaro Manero, Manuela Castilla Barba, Marina Mojena Sánchez and Alberto Tejedor Jorge

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/57286

**1. Introduction**

Cyclosporin A (CyA) and tacrolimus (TAC) are calcineurin phosphatase inhibitors which are currently used in immunosuppression therapy for solid organ and hematopoietic stem cell transplantation to prevent and treat allograft rejection, and for autoimmune disease treatment [1]. Although drugs are not structurally related (Figure 1, Materials and Methods), they have identical cellular and molecular actions, including nephrotoxicity as a major side effect [2-4].

It is known that CyA and TAC may cause acute as well as chronic tubulointerstitial nephrop‐ athy characterized by tubular atrophy, loss of tubular cells and interstitial fibrosis [5], although the mechanisms of immunosuppression-associated renal damage are not fully understood [6, 7]. In any case, it is generally agreed that acute haemodynamic effects of CyA may be mediated by an imbalance of different vasoconstrictors and vasodilators, including renin-angiotensin system and nitric oxide among others [1,3]. Histologically, vacuolization, calcification and necrosis of tubular epithelial cells are characteristics, and endothelial cell swelling with variable degrees of hyalinosis. Abnormalities similar to those are found with TAC [4,8]. For chronic CyA nephrotoxicity, different studies have suggested a toxic effect of the drug on afferent arterioles and tubular epithelial cells, vasoconstriction and endothelial injury leading

to ischemia, as well as a direct toxic effect of CyA on tubular epithelium [5,9]. Moreover, apoptosis has been clearly evidenced in tubular and interstitial cells in patients, animals and cell culture models [9]. We have described that loss of mitochondrial potential usually precedes caspase activation and endonuclease mediated DNA laddering when proximal tubule cells are incubated in the presence of increasing doses of CyA [1,7]. Similar effects are found in TACinduced nephrotoxicity [7, 10]. Mitochondrial depolarisation, caspase activation, cytosolic nucleosomal formation, and other morphological features of apoptosis induction are com‐ monly found with both CyA and TAC in proximal tubule cells [1, 7]. Transforming growth factor-β (TGF-β), hypoxia and oxidative stress have also been considered to be important mediators of CyA and TAC-induced renal injury [6, 7]. Significantly, although both drugs have similar effects, TAC is recognized to induce fewer haemodynamic and fibrogenic alterations at equivalent doses of CyA [11], and studies have shown that the incidence of acute rejection is reduced by TAC use over CyA [12].

pore only closes again if intramitochondrial free calcium is reduced. However, no clear explanations have been offered about how mitochondria may release calcium without causing the death of the cell. Although there should be a physiological role for MPT pore transitory opening, many authors agree that a role of MPT pore in healthy cells has not been established, and it only plays a role in pathophysiological conditions [26, 28, 30], such as necrotic and apoptotic cell death, ischemia/reperfusion injury, heart failure and other cardiovascular or neurodegenerative diseases [26,31,32]. In opinion of Martin Crompton, a possible physiolog‐ ical function of MPT pore may be to establish contact between mitochondria in the formation of mitochondrial networks [28]. It is established that mitochondria can form tight intermito‐ chondrial junctions, allowing the thus-conjugated mitochondria to operate as a bioenergetics continuum permitting efficient energy transfer between different parts of the cells [33]. Other authors have also hypothesized about other possible physiological roles of MPT pore, as the regulation of protein import, matrix volume and pH, cristae remodeling, redox equilibrium

Anticalcineurinics: Role of Mitochondrial Transition Pore on Nephrotoxicity

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209

In any case, it is accepted that MPT pore formation is considered a prelude of mitochondria triggered apoptosis; prolonged times of MPT pore activity are accompanied by loss of transmembrane mitochondrial potential [36, 37] and release of proapoptotic mitochondrial proteins as cytochrome c [38], SMAC/Diablo [39, 40], HtrA2/Omi [41], apoptosis inducing factor (AIF) [42] or endonuclease G [43]. Although there is no agreement as to whether these proapoptotic mediators leave the mitochondria through the MPT pore or by other pathways [44-46], or whether the leak takes place after or before pore opening [27], protein implicated in apoptotic balance Bcl-2, Bcl-XL, Bax and Bid [47-48] are implicated on MPT pore activity regulation. Agents capable to keep MPT pore timely opened as nitric oxide [49], free oxygen radicals [50, 51] or Bax [52] are proapoptotic. Those who block or interfere with MPT pore

It is currently known that CyA is to date, the most specific inhibitor of the MPT pore by inhibiting the peptidylprolyl cistrans-isomerase activity of cyclophilin D [53, 54], and is considered as antiapoptotic on most cellular systems [26, 28]. On the other hand, no TAC binding proteins able to modify MPT pore activity have been reported to date. Therefore, MPT pore mediation on the nephrotoxicity of calcineurin inhibitors has always been considered a

In this chapter we provide some insights into the proximal tubule mitochondria modifications that take place during CyA and TAC induced nephrotoxicity. We explore the potential implication of MPT modulation on cell protection, acute renal failure and mitochondrial

CyA (Sandimmun Neoral®) was obtained from Novartis Farmacéutica S.A. (Spain) and TAC (Prograf®)fromFujisawaS.A.(Spain).Their chemical structuresarerepresentedinFigure1.The

and control of metabolism [31, 34, 35].

activity are antiapoptotic.

ammoniogenesis.

**2.1. Drugs**

non-directly related phenomenon.

**2. Materials and methods**

Although mitochondrial mediation in the damage is generally accepted to be common to both immunosuppressors, the exact mechanism has not been fully investigated. Several authors have found a decreased mitochondrial respiration with CyA, mediated by a reduction in complex II activity, with stage 3 and 4 activity reductions [13-15]. Others describe a preferential inhibition of respiration by CyA when substrates of complex I are used [16]. TAC also inhibits the succinate supported state 3 respiration [13]. ATP depletion is a key factor in cell death associated with renal failure; therefore phosphorylation has also been studied as a potential target for CyA induced nephrotoxicity. CyA induced inhibition of ATP synthesis ranges from 12% [17] to 40% [18], and may be partially prevented by calcium antagonists [19]. Like CyA, TAC inhibits net ATP uptake [13]. However, the most intriguing finding regarding mitochon‐ drial mediation in CyA and TAC induced apoptosis is the relationship of both immunosup‐ pressors with the permeability transition pore.

The mitochondrial permeability transition (MPT) pore is an unstable structure that mitochon‐ dria reversibly assemble in their inner membrane under several physiological and nonphysiological conditions, mainly cell calcium overload. When assembled, inner and outer membranes physically touch each other at specific points; when assembled and opened, it allows non selective permeation of ions and solutes up to 1.5 kDa [20, 21], dragged by a water movement from cytosol into mitochondria resulting in mitochondria swelling that may be measured [22]. MPT pore assembly includes cyclophilin-dependent conformational changes in adenine nucleotide translocase (ANT), located in the inner membrane and in a voltage dependent anion channel (VDAC). Cyclophilin D, benzodiazepine receptor, creatine kinase, hexokinase, cytochrome c and Bax protein are also involved in MPT pore formation and regulation [23-25]. The exact molecular structure of the MPT pore is still controversial, as it is the MPT pore relation to energy transfer and apoptosis, and for these reasons the physiological function of the MPT pore is not well established [23, 26].

It is generally agreed that MPT pore opening is triggered by a sudden rise in mitochondrial matrix free calcium concentration, representing the third of three sequential phases of mitochondrial accumulation: respiratory chain acceleration, extramitochondrial calcium buffering and finally activation of the MPT pore [27]. Transitory openings are necessary to prevent mitochondrial calcium overload, by allowing calcium excesses release [26, 28, 29]. MPT pore only closes again if intramitochondrial free calcium is reduced. However, no clear explanations have been offered about how mitochondria may release calcium without causing the death of the cell. Although there should be a physiological role for MPT pore transitory opening, many authors agree that a role of MPT pore in healthy cells has not been established, and it only plays a role in pathophysiological conditions [26, 28, 30], such as necrotic and apoptotic cell death, ischemia/reperfusion injury, heart failure and other cardiovascular or neurodegenerative diseases [26,31,32]. In opinion of Martin Crompton, a possible physiolog‐ ical function of MPT pore may be to establish contact between mitochondria in the formation of mitochondrial networks [28]. It is established that mitochondria can form tight intermito‐ chondrial junctions, allowing the thus-conjugated mitochondria to operate as a bioenergetics continuum permitting efficient energy transfer between different parts of the cells [33]. Other authors have also hypothesized about other possible physiological roles of MPT pore, as the regulation of protein import, matrix volume and pH, cristae remodeling, redox equilibrium and control of metabolism [31, 34, 35].

In any case, it is accepted that MPT pore formation is considered a prelude of mitochondria triggered apoptosis; prolonged times of MPT pore activity are accompanied by loss of transmembrane mitochondrial potential [36, 37] and release of proapoptotic mitochondrial proteins as cytochrome c [38], SMAC/Diablo [39, 40], HtrA2/Omi [41], apoptosis inducing factor (AIF) [42] or endonuclease G [43]. Although there is no agreement as to whether these proapoptotic mediators leave the mitochondria through the MPT pore or by other pathways [44-46], or whether the leak takes place after or before pore opening [27], protein implicated in apoptotic balance Bcl-2, Bcl-XL, Bax and Bid [47-48] are implicated on MPT pore activity regulation. Agents capable to keep MPT pore timely opened as nitric oxide [49], free oxygen radicals [50, 51] or Bax [52] are proapoptotic. Those who block or interfere with MPT pore activity are antiapoptotic.

It is currently known that CyA is to date, the most specific inhibitor of the MPT pore by inhibiting the peptidylprolyl cistrans-isomerase activity of cyclophilin D [53, 54], and is considered as antiapoptotic on most cellular systems [26, 28]. On the other hand, no TAC binding proteins able to modify MPT pore activity have been reported to date. Therefore, MPT pore mediation on the nephrotoxicity of calcineurin inhibitors has always been considered a non-directly related phenomenon.

In this chapter we provide some insights into the proximal tubule mitochondria modifications that take place during CyA and TAC induced nephrotoxicity. We explore the potential implication of MPT modulation on cell protection, acute renal failure and mitochondrial ammoniogenesis.

#### **2. Materials and methods**

#### **2.1. Drugs**

to ischemia, as well as a direct toxic effect of CyA on tubular epithelium [5,9]. Moreover, apoptosis has been clearly evidenced in tubular and interstitial cells in patients, animals and cell culture models [9]. We have described that loss of mitochondrial potential usually precedes caspase activation and endonuclease mediated DNA laddering when proximal tubule cells are incubated in the presence of increasing doses of CyA [1,7]. Similar effects are found in TACinduced nephrotoxicity [7, 10]. Mitochondrial depolarisation, caspase activation, cytosolic nucleosomal formation, and other morphological features of apoptosis induction are com‐ monly found with both CyA and TAC in proximal tubule cells [1, 7]. Transforming growth factor-β (TGF-β), hypoxia and oxidative stress have also been considered to be important mediators of CyA and TAC-induced renal injury [6, 7]. Significantly, although both drugs have similar effects, TAC is recognized to induce fewer haemodynamic and fibrogenic alterations at equivalent doses of CyA [11], and studies have shown that the incidence of acute rejection

Although mitochondrial mediation in the damage is generally accepted to be common to both immunosuppressors, the exact mechanism has not been fully investigated. Several authors have found a decreased mitochondrial respiration with CyA, mediated by a reduction in complex II activity, with stage 3 and 4 activity reductions [13-15]. Others describe a preferential inhibition of respiration by CyA when substrates of complex I are used [16]. TAC also inhibits the succinate supported state 3 respiration [13]. ATP depletion is a key factor in cell death associated with renal failure; therefore phosphorylation has also been studied as a potential target for CyA induced nephrotoxicity. CyA induced inhibition of ATP synthesis ranges from 12% [17] to 40% [18], and may be partially prevented by calcium antagonists [19]. Like CyA, TAC inhibits net ATP uptake [13]. However, the most intriguing finding regarding mitochon‐ drial mediation in CyA and TAC induced apoptosis is the relationship of both immunosup‐

The mitochondrial permeability transition (MPT) pore is an unstable structure that mitochon‐ dria reversibly assemble in their inner membrane under several physiological and nonphysiological conditions, mainly cell calcium overload. When assembled, inner and outer membranes physically touch each other at specific points; when assembled and opened, it allows non selective permeation of ions and solutes up to 1.5 kDa [20, 21], dragged by a water movement from cytosol into mitochondria resulting in mitochondria swelling that may be measured [22]. MPT pore assembly includes cyclophilin-dependent conformational changes in adenine nucleotide translocase (ANT), located in the inner membrane and in a voltage dependent anion channel (VDAC). Cyclophilin D, benzodiazepine receptor, creatine kinase, hexokinase, cytochrome c and Bax protein are also involved in MPT pore formation and regulation [23-25]. The exact molecular structure of the MPT pore is still controversial, as it is the MPT pore relation to energy transfer and apoptosis, and for these reasons the physiological

It is generally agreed that MPT pore opening is triggered by a sudden rise in mitochondrial matrix free calcium concentration, representing the third of three sequential phases of mitochondrial accumulation: respiratory chain acceleration, extramitochondrial calcium buffering and finally activation of the MPT pore [27]. Transitory openings are necessary to prevent mitochondrial calcium overload, by allowing calcium excesses release [26, 28, 29]. MPT

is reduced by TAC use over CyA [12].

208 Pharmacology and Therapeutics

pressors with the permeability transition pore.

function of the MPT pore is not well established [23, 26].

CyA (Sandimmun Neoral®) was obtained from Novartis Farmacéutica S.A. (Spain) and TAC (Prograf®)fromFujisawaS.A.(Spain).Their chemical structuresarerepresentedinFigure1.The concentration used for both drugs were similar to the pharmacologically active recommend‐ ed plasma level, and they were selected from our previous studies and experience [1, 7]. Cell cultures were exposed to different increasing concentrations of CyA (1-1000 ng/ml) and TAC (50-500 ng/ml), although most of the experiments were performed with 1000 ng/ml (CyA) and 50 ng/ml (TAC) based on the proven effectiveness of the drug to induce nephrotoxicity [1].

#### **2.2. Experimental animals**

All experiments were performed on miniature swine genetically selected to be isogenic for three loci of the major histocompatibility locus [55]. They were 3 months old and weighed 31.3 ± 0.7 kg (mean ± SEM). Isogenic minipigs were housed under controlled conditions and the study was approved by the Institutional Board for Animal Experiments. Animals were handled at all times according to the applicable legal regulations in RD 1201/2005, of 10 October, on the protection of animals used for experimentation and other scientific purposes.

#### **2.3. Isolation and primary culture of renal proximal tubule epithelial cells**

Proximal tubule suspensions were obtained as described [56]. The kidneys were aseptically removed from the animals and placed in a sterilized beaker containing Ham´s F-12 culture medium, pH 7.4. Briefly, renal cortex was sliced with a Staddie-Riggs microtome, and incubated with 0.6 mg/ml of collagenase A (Boehringer Mannheim, Germany) in Ham's F-12 medium for 30 minutes at 37 °C. Digested tissue was then filtered through a 250 μm metal mesh, washed three times in Ham's F-12 medium and centrifuged at 20,000 x g for 30 minutes in a 45% (v/v) Percoll gradient. The last band contains pure proximal tubules.

Proximal tubules were recovered from the deepest fraction, washed, and resuspended in culture medium (1:1 Ham´s F-12:DMEN supplemented with 25 mM hepes, 3.7 mg/mL sodium bicarbonate, 2.5 mM glutamine, 1% non-essential aminoacids, 100 U/mL penicillin, 100 μg/ml streptomycin, 5.10-8 M hydrocortisone, 5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/mL sodium selenite and 2% fetal bovine serum), at a final concentration of 0.66 mg/mL, and plated on plastic culture dishes (60 mm) [57]. The culture was incubated at 37 °C in a 95% air/5% CO2 atmosphere. The medium was renewed on the fourth day and every two days thereafter.

**2.5. Mitochondrial oxygen consumption**

a molecular weight of 804.018 g/mol.

content of oxygen was calculated using the following equation:

mmHg and the vapour water pressure is 46.6 mmHg.

State 3 and state 4 respiration was measured with a Clark-type electrode (YSI incorporat‐ ed, Yellow Springs, Ohio, USA) in a metacrilate chamber at 37 °C equipped with magnet‐ ic stirring in the presence of 5 mM succinate before (state 4) and after (state 3) the addition of 2 mM ADP and 10 μM atractilosyde. Mitochondria were suspended in continuously stirred assay medium, maintained at 37°C at a final mitochondrial protein concentration of 0.40 ± 0.02 mg/ml. In parallel experiments, CyA 1μg/ml and TAC 50 ng/ml were added 30 minutes prior to addition of effectors or simultaneously with then (no preincubation). The

**Figure 1.** Formula of the nephrotoxic compounds. A and B represent the molecular structure of cyclosporin A; cyclic endecapeptide with chemical formula C62H111N11O12 and a molecular weight of 1202.16 g/mol. C and D represent the molecular structure of tacrolimus; macrolide whose empirical formula in its monohydrate form is C44H69NO12, and with

where 0.21 is the O2% in the air, 0.024 is the O2 solubility coefficient, 22.4 is the molar volume of an ideal gas (L/mol). The cuvette volume is 807 μl. Atmospheric pressure was 708.9 ± 1.96

O mol = 0.21 x 0.024 x V x P -P /22.4 <sup>2</sup> ( ) cuvette atm H2O ( ) (1)

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211

CyA or TAC were added to cell cultures as specified in every experiment.

#### **2.4. Isolation of renal cortical mitochondria**

The renal cortex was rapidly removed and placed into 100 mL of ice-cold isolation buffer (300 mM mannitol, 1 mM EGTA, 10 mM TRIS HCl, 1 mM PO4H2K, 1.74 mg/mL phenylmethylsul‐ fonyl fluoride (PMSF), 0.2% BSA, pH 7.4 and gassed with N2) and homogenized. The homo‐ genate was centrifuged at 1075 x g for 10 minutes at 4 °C. The supernatant was centrifuged at 8635 x g for 10 minutes. The resulting pellet was washed twice at 8635 x g for 10 minutes and resuspended in isolation buffer to 32.5 ± 1.97 mg of protein/mL.

All experiments with isolated mitochondria are carried out in assay medium (300 mM mannitol, 10 mM TRIS HCl, 1mM PO4H2K, pH 7.4).

Anticalcineurinics: Role of Mitochondrial Transition Pore on Nephrotoxicity http://dx.doi.org/10.5772/57286 211

**Figure 1.** Formula of the nephrotoxic compounds. A and B represent the molecular structure of cyclosporin A; cyclic endecapeptide with chemical formula C62H111N11O12 and a molecular weight of 1202.16 g/mol. C and D represent the molecular structure of tacrolimus; macrolide whose empirical formula in its monohydrate form is C44H69NO12, and with a molecular weight of 804.018 g/mol.

#### **2.5. Mitochondrial oxygen consumption**

concentration used for both drugs were similar to the pharmacologically active recommend‐ ed plasma level, and they were selected from our previous studies and experience [1, 7]. Cell cultures were exposed to different increasing concentrations of CyA (1-1000 ng/ml) and TAC (50-500 ng/ml), although most of the experiments were performed with 1000 ng/ml (CyA) and 50 ng/ml (TAC) based on the proven effectiveness of the drug to induce nephrotoxicity [1].

All experiments were performed on miniature swine genetically selected to be isogenic for three loci of the major histocompatibility locus [55]. They were 3 months old and weighed 31.3 ± 0.7 kg (mean ± SEM). Isogenic minipigs were housed under controlled conditions and the study was approved by the Institutional Board for Animal Experiments. Animals were handled at all times according to the applicable legal regulations in RD 1201/2005, of 10 October, on the protection of animals used for experimentation and other scientific purposes.

Proximal tubule suspensions were obtained as described [56]. The kidneys were aseptically removed from the animals and placed in a sterilized beaker containing Ham´s F-12 culture medium, pH 7.4. Briefly, renal cortex was sliced with a Staddie-Riggs microtome, and incubated with 0.6 mg/ml of collagenase A (Boehringer Mannheim, Germany) in Ham's F-12 medium for 30 minutes at 37 °C. Digested tissue was then filtered through a 250 μm metal mesh, washed three times in Ham's F-12 medium and centrifuged at 20,000 x g for 30 minutes

Proximal tubules were recovered from the deepest fraction, washed, and resuspended in culture medium (1:1 Ham´s F-12:DMEN supplemented with 25 mM hepes, 3.7 mg/mL sodium bicarbonate, 2.5 mM glutamine, 1% non-essential aminoacids, 100 U/mL penicillin, 100 μg/ml streptomycin, 5.10-8 M hydrocortisone, 5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/mL sodium selenite and 2% fetal bovine serum), at a final concentration of 0.66 mg/mL, and plated on plastic culture dishes (60 mm) [57]. The culture was incubated at 37 °C in a 95% air/5% CO2 atmosphere. The medium was renewed on the fourth day and every two days thereafter.

The renal cortex was rapidly removed and placed into 100 mL of ice-cold isolation buffer (300 mM mannitol, 1 mM EGTA, 10 mM TRIS HCl, 1 mM PO4H2K, 1.74 mg/mL phenylmethylsul‐ fonyl fluoride (PMSF), 0.2% BSA, pH 7.4 and gassed with N2) and homogenized. The homo‐ genate was centrifuged at 1075 x g for 10 minutes at 4 °C. The supernatant was centrifuged at 8635 x g for 10 minutes. The resulting pellet was washed twice at 8635 x g for 10 minutes and

All experiments with isolated mitochondria are carried out in assay medium (300 mM

**2.3. Isolation and primary culture of renal proximal tubule epithelial cells**

in a 45% (v/v) Percoll gradient. The last band contains pure proximal tubules.

CyA or TAC were added to cell cultures as specified in every experiment.

resuspended in isolation buffer to 32.5 ± 1.97 mg of protein/mL.

mannitol, 10 mM TRIS HCl, 1mM PO4H2K, pH 7.4).

**2.4. Isolation of renal cortical mitochondria**

**2.2. Experimental animals**

210 Pharmacology and Therapeutics

State 3 and state 4 respiration was measured with a Clark-type electrode (YSI incorporat‐ ed, Yellow Springs, Ohio, USA) in a metacrilate chamber at 37 °C equipped with magnet‐ ic stirring in the presence of 5 mM succinate before (state 4) and after (state 3) the addition of 2 mM ADP and 10 μM atractilosyde. Mitochondria were suspended in continuously stirred assay medium, maintained at 37°C at a final mitochondrial protein concentration of 0.40 ± 0.02 mg/ml. In parallel experiments, CyA 1μg/ml and TAC 50 ng/ml were added 30 minutes prior to addition of effectors or simultaneously with then (no preincubation). The content of oxygen was calculated using the following equation:

$$\text{CO}\_2\text{(mol)} = 0.21 \ge 0.024 \ge \text{V}\_{\text{cuvette}} \ge \left(\text{P}\_{\text{atm}} \text{-P}\_{\text{H2O}}\right) / 22.4 \tag{1}$$

where 0.21 is the O2% in the air, 0.024 is the O2 solubility coefficient, 22.4 is the molar volume of an ideal gas (L/mol). The cuvette volume is 807 μl. Atmospheric pressure was 708.9 ± 1.96 mmHg and the vapour water pressure is 46.6 mmHg.

#### **2.6. Calcium uptake**

One mL of mitochondrial suspension was incubated with 5 μM fura-2, AM (Molecular Probes) for 30 minutes at 4 °C, washed three times at 8635 x g for 10 minutes and resuspended in isolation buffer at 4°C in a 1:1 proportion.

Fura-2 fluorescence was monitored with a SLM Aminco 8000 fluorometer at 37°C in stirred cuvettes, by exciting it at 340 nm (λex1) and 380 nm (λex2) and rationing the fluorescence intensities detected at 510 nm (λem). Mitochondrial matrix [Ca2+] was determined using the following equation [58]:

$$\left[\mathbf{C}\mathbf{a}^{2+}\right] = \mathbf{K}\_d \mathbf{Q} \left( (\mathbf{R} \cdot \mathbf{R}\_{\min}) / (\mathbf{R}\_{\max} \cdot \mathbf{R}) \right) \tag{2}$$

glutamate. Determinations are based on following reactions of glutaminase and glutamate

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++ NADH

Release of cythochrome c from mitochondria into cytosol was measured using Western blot analysis. RPTECs treated for 48 h with 1 μg/ml CyA or 50 ng/ml TAC were harvested, washed once with ice-cold PBS, and gently lysed for 10 minutes in ice with 90 μl of lysis buffer (250 mM sucrose, 80 mM KCl, 500 μg/ml digitonin, 1 mM dithiothreitol, 0.1 mM PMSF, and protease inhibitors in PBS). Cell lysates were centrifuged at 12,000 x g at 4°C for 5 minutes to obtain the supernatants (cytosolic extract free of mitochondria) and the pellets (fractions containing the mitochondria), which were resuspended in 90 μl of lysis buffer. Equal amounts of protein were loaded (30 μg in each lane) and electrophoresed on 15% polyacrylamide gels as previously

Rabbit polyclonal antibody anti-cytochrome c (Santa Cruz Biotechnology, Inc, CA, USA) was

The membranes were also probed with goat polyclonal antibody against a peptide of voltagedependent anion-selective channel 1 of human origin (VDAC-1, 1:500; Santa Cruz Biotechnol‐ ogy, Inc.) for mitochondrial fractions and monoclonal anti-tubulin Clone B-5-1-2 Mouse Ascites Fluid (mouse IgG1 isotype) antibody (1:10,000; Sigma-Aldrich) for cytosolic fractions as internal controls for the technique. Proteins were visualized with the enhanced chemilu‐ minescence detection system (ECL, GE Healthcare, Little Chalfont, Buckinghamshire, UK).

After CyA or TAC treatment, cell number was determined with a computer coupled video‐

Flow cytometry was performed on supernatant and monolayer harvested cells after dilution

with PBS-Tween 0.5% (15 minutes at room temperature) and incubation with 40 μg/mL RNAse and 250 μg/mL propidium iodide (Sigma, St Louis, USA) at room temperature for 45 minutes. A minimum of 10,000 cells per sample were acquired in a FACScan equipped with a single argon-ion laser (Becton Dickinson, San Jose, CA), using standard Lysis II software. A gate was set on the basis of forward and scatter characteristics before fluorescence (FL2) was analysed.

Caspase 3 and caspase 9 activities were determined in RPTECs using the caspase 3 inhibitor, DEVD-FMK conjugated to FITC (FITC-DEVD-FMK) and the caspase 9 inhibitor LEHD-FMK conjugated to FITC (FITC-LEHD-FMK) as a fluorescent markers respectively, following the protocols of the CaspGLOWTM Fluorescein Active Caspase-3 and Caspase-9 Staining Kit from

cells/mL in 2% paraformaldehyde/PBS (30 minutes, 4°C, pH 7.4), permeabilization

chosen at

**2.11. Apoptosis studies: Flow cytometry, microscopy and caspase activity assay**

microscopy system. Every count was assessed on 21 sectors images of 0.0775 mm2

dehydrogenase, respectively [60]:

Glutamate +NAD→Ketoglutarate + NH4

random from three different culture dishes.

+

Glutamine→Glutamate + NH4

**2.10. Cytochrome c distribution**

described [56].

used at 1:500.

in 2x106

where R represents the fluorescence intensity ratio F (340 nm)/ F (380 nm), Rmin and Rmax requires taking fluorescence measurements for the completely ion-free (EGTA 2 mM) and ionsaturated (CaCl2 5 mM) indicators. Q is the ratio of Fmin to Fmax at 380 nm. Kd is the dissociation constant of fura-2 for calcium (0.14 μM).

#### **2.7. Mitochondrial swelling: MPT pore**

Ca2+-induced mitochondrial swelling was determined spectrophotometrically as described by Beavis and Col. [59], adapted to our experimental conditions, by using authors equation:

$$\text{Mitochandi volume } \left(\text{\textquotedblleft/mg\right)} = \left[\left(1/\text{Abs}\_{\text{S20 nm}} \cdot 0.1832\right) \ge \left[\text{Protein}\right]\right] / 0.06\tag{3}$$

where 0.1832 is the theoretician inverse of absorbance for an infinite protein concentration, and 0.06 is a proportionality constant established by the authors [59]. Mitochondria were suspended in 3 mL of continuously stirred assay medium, maintained at 37°C at a final mitochondrial protein concentration of 0.54 ± 0.1 mg/ml. Swelling was initiated by the addition of succinate to the sample cuvette, and the absorbance changes at 520 nm were monitored with an Uvikon 930 spectrophotometer.

A mitochondrial basal volume of 3.56 ± 0.06 μl/mg protein was obtained.

#### **2.8. Analysis of mitochondrial transmembrane potential by fluorescence microscopy**

Mitochondrial transmembrane potential in intact renal proximal tubular epithelial cells (RPTECs) was followed by cellular distribution of Rhodamine. Cells were incubated at 37°C for 15 minutes in the presence of 10 mM Rhodamine 123 (Sigma) and washed twice in PBS. The cells were then observed with a fluorescence microscope Olympus IX70 (wide-band cube U-MWG; λex: 510-550, λem: >590, λdic: 570).

#### **2.9. Cellular glutamine metabolism**

Glutamine uptake was determined on proximal tubules following spectrophotometrically the NADH production at 340 nm. Glutamine uptake was determined by difference with the glutamate. Determinations are based on following reactions of glutaminase and glutamate dehydrogenase, respectively [60]:

Glutamine→Glutamate + NH4 +

**2.6. Calcium uptake**

212 Pharmacology and Therapeutics

following equation [58]:

isolation buffer at 4°C in a 1:1 proportion.

constant of fura-2 for calcium (0.14 μM).

**2.7. Mitochondrial swelling: MPT pore**

an Uvikon 930 spectrophotometer.

U-MWG; λex: 510-550, λem: >590, λdic: 570).

**2.9. Cellular glutamine metabolism**

One mL of mitochondrial suspension was incubated with 5 μM fura-2, AM (Molecular Probes) for 30 minutes at 4 °C, washed three times at 8635 x g for 10 minutes and resuspended in

Fura-2 fluorescence was monitored with a SLM Aminco 8000 fluorometer at 37°C in stirred cuvettes, by exciting it at 340 nm (λex1) and 380 nm (λex2) and rationing the fluorescence intensities detected at 510 nm (λem). Mitochondrial matrix [Ca2+] was determined using the

where R represents the fluorescence intensity ratio F (340 nm)/ F (380 nm), Rmin and Rmax requires taking fluorescence measurements for the completely ion-free (EGTA 2 mM) and ionsaturated (CaCl2 5 mM) indicators. Q is the ratio of Fmin to Fmax at 380 nm. Kd is the dissociation

Ca2+-induced mitochondrial swelling was determined spectrophotometrically as described by Beavis and Col. [59], adapted to our experimental conditions, by using authors equation:

where 0.1832 is the theoretician inverse of absorbance for an infinite protein concentration, and 0.06 is a proportionality constant established by the authors [59]. Mitochondria were suspended in 3 mL of continuously stirred assay medium, maintained at 37°C at a final mitochondrial protein concentration of 0.54 ± 0.1 mg/ml. Swelling was initiated by the addition of succinate to the sample cuvette, and the absorbance changes at 520 nm were monitored with

**2.8. Analysis of mitochondrial transmembrane potential by fluorescence microscopy**

Mitochondrial transmembrane potential in intact renal proximal tubular epithelial cells (RPTECs) was followed by cellular distribution of Rhodamine. Cells were incubated at 37°C for 15 minutes in the presence of 10 mM Rhodamine 123 (Sigma) and washed twice in PBS. The cells were then observed with a fluorescence microscope Olympus IX70 (wide-band cube

Glutamine uptake was determined on proximal tubules following spectrophotometrically the NADH production at 340 nm. Glutamine uptake was determined by difference with the

A mitochondrial basal volume of 3.56 ± 0.06 μl/mg protein was obtained.

Mitochondrial volume µl/mg = 1/Abs - 0.1832 x Protein /0.06 ( ) é ù ( 520 nm ) [ ] ë û (3)

ë û (2)

(( ) ( )) 2+ Ca =K Q R-R / R -R <sup>d</sup> min max é ù

Glutamate +NAD→Ketoglutarate + NH4 ++ NADH

#### **2.10. Cytochrome c distribution**

Release of cythochrome c from mitochondria into cytosol was measured using Western blot analysis. RPTECs treated for 48 h with 1 μg/ml CyA or 50 ng/ml TAC were harvested, washed once with ice-cold PBS, and gently lysed for 10 minutes in ice with 90 μl of lysis buffer (250 mM sucrose, 80 mM KCl, 500 μg/ml digitonin, 1 mM dithiothreitol, 0.1 mM PMSF, and protease inhibitors in PBS). Cell lysates were centrifuged at 12,000 x g at 4°C for 5 minutes to obtain the supernatants (cytosolic extract free of mitochondria) and the pellets (fractions containing the mitochondria), which were resuspended in 90 μl of lysis buffer. Equal amounts of protein were loaded (30 μg in each lane) and electrophoresed on 15% polyacrylamide gels as previously described [56].

Rabbit polyclonal antibody anti-cytochrome c (Santa Cruz Biotechnology, Inc, CA, USA) was used at 1:500.

The membranes were also probed with goat polyclonal antibody against a peptide of voltagedependent anion-selective channel 1 of human origin (VDAC-1, 1:500; Santa Cruz Biotechnol‐ ogy, Inc.) for mitochondrial fractions and monoclonal anti-tubulin Clone B-5-1-2 Mouse Ascites Fluid (mouse IgG1 isotype) antibody (1:10,000; Sigma-Aldrich) for cytosolic fractions as internal controls for the technique. Proteins were visualized with the enhanced chemilu‐ minescence detection system (ECL, GE Healthcare, Little Chalfont, Buckinghamshire, UK).

#### **2.11. Apoptosis studies: Flow cytometry, microscopy and caspase activity assay**

After CyA or TAC treatment, cell number was determined with a computer coupled video‐ microscopy system. Every count was assessed on 21 sectors images of 0.0775 mm2 chosen at random from three different culture dishes.

Flow cytometry was performed on supernatant and monolayer harvested cells after dilution in 2x106 cells/mL in 2% paraformaldehyde/PBS (30 minutes, 4°C, pH 7.4), permeabilization with PBS-Tween 0.5% (15 minutes at room temperature) and incubation with 40 μg/mL RNAse and 250 μg/mL propidium iodide (Sigma, St Louis, USA) at room temperature for 45 minutes. A minimum of 10,000 cells per sample were acquired in a FACScan equipped with a single argon-ion laser (Becton Dickinson, San Jose, CA), using standard Lysis II software. A gate was set on the basis of forward and scatter characteristics before fluorescence (FL2) was analysed.

Caspase 3 and caspase 9 activities were determined in RPTECs using the caspase 3 inhibitor, DEVD-FMK conjugated to FITC (FITC-DEVD-FMK) and the caspase 9 inhibitor LEHD-FMK conjugated to FITC (FITC-LEHD-FMK) as a fluorescent markers respectively, following the protocols of the CaspGLOWTM Fluorescein Active Caspase-3 and Caspase-9 Staining Kit from BioVision, Inc., (Milpitas, CA, USA). Activated caspases detection was examined with the 20X PL-APO 0.7-numerical aperture objective of a Leica-SP2 confocal microscope (Leica Micro‐ systems, Heidelberg, Germany). Different intensity measurements were assessed with the Leica Confocal Software LCS-1537 (Leica Microsystems).

induction with both treatments. Table 1 shows the dose dependence exhibited by the apoptosis

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**Figure 3.** Effect of Cyclosporin A (CyA) and Tacrolimus (TAC) on apoptosis of pig kidney proximal tubule epithelial cells as assessed by flow cytometry analysis (FACScan). 3-D histograms of the cell flow cytometry in either control cultures or treated with 1 µg/mL CyA or 50 ng/mL TAC for 8 days. The hypodiploid population is greater in the CyA and TAC

> **% hypodiploid cells (CyA)**

**(TAC)**

0 28.55 ± 2.9 28.55 ± 2.9

100 33.08 ± 3.8

1000 42.32 ± 5.4\*

When suspensions of fura-2 AM loaded mitochondria were incubated in the presence of 21% oxygen and 5 mM succinate, but in the absence of ADP, an increase in intramitochondrial calcium concentration is observed (Figure 4A), paralleling an 8-fold increase in oxygen consumption (Table 2). Subsequent addition of 2 mM of ADP significantly increases the rate of oxygen consumption (Table 2) and drastically decreases intramitochondrial calcium concentration, although not reaching baseline levels (Figure 4A). This reduction in mitochon‐ drial calcium when adding ADP is accompanied by a net calcium extrusion from mitochondria, as can be seen in Figure 4B where the same maneuver was repeated with non fura-2 loaded mitochondria suspended in the presence of the non-permanent acid form of fura-2. ADP

**DOSE (ng/mL) % hypodiploid cells**

50 35.87 ± 5.8

500 44.42 ± 5.9\*

**Table 1.** Apoptosis assessed by flow cytometry analysis.

Values are means ± SEM. \*p<0.05 vs.dose 0 ng/ml. TAC, tacrolimus; CyA, cyclosporin A.

**3.2. Effect of CyA and TAC on intramitochondrial free calcium**

induced by the two drugs.

treated cells.

#### **2.12. Statistics**

Pooled data are presented as mean ± SEM. Comparison between treatment groups were done by one or two ways factorial ANOVA. Least significant differences were computed for between-level comparisons. A *p* value <0.05 was considered to be statistically significant.

#### **3. Results**

#### **3.1. Effect of CyA and TAC on primary growth of the renal proximal tubule cells**

When primary cultures of pig RPTECs are exposed to increasing CyA or TAC concentrations, a dose dependent growth inhibition is produced, even with concentrations as low as CyA 1 μg/mL or 50 ng/mL. Figure 2 shows the growth dynamics of cultures in the presence of CyA 1 μg/mL and TAC 50 ng/mL, concentrations near to the peak plasma levels reached with oral administration of these two drugs. Both CyA and TAC inhibit growth from day 6, which impedes the culture confluence from day 8 (Figure 2).

**Figure 2.**  Culture cells were incubated with 1 µg/mL CyA or 50 ng/mL TAC. Viable cell number was determined by cell counting in a phase-contrast inverted microscope. Values shown are the mean ± SEM of cell counts per cm2. \*p<0.05 CyA vs. control; †p<0.05 TAC vs. control.

Figure 3 gives a 3-D histogram of the cell flow cytometry in cultures treated with CyA or TAC for 8 days. A distinct hypodiploid population can be observed, indicative of apoptosis induction with both treatments. Table 1 shows the dose dependence exhibited by the apoptosis induced by the two drugs.

**Figure 3.** Effect of Cyclosporin A (CyA) and Tacrolimus (TAC) on apoptosis of pig kidney proximal tubule epithelial cells as assessed by flow cytometry analysis (FACScan). 3-D histograms of the cell flow cytometry in either control cultures or treated with 1 µg/mL CyA or 50 ng/mL TAC for 8 days. The hypodiploid population is greater in the CyA and TAC treated cells.

Values are means ± SEM. \*p<0.05 vs.dose 0 ng/ml. TAC, tacrolimus; CyA, cyclosporin A.

**Table 1.** Apoptosis assessed by flow cytometry analysis.

BioVision, Inc., (Milpitas, CA, USA). Activated caspases detection was examined with the 20X PL-APO 0.7-numerical aperture objective of a Leica-SP2 confocal microscope (Leica Micro‐ systems, Heidelberg, Germany). Different intensity measurements were assessed with the

Pooled data are presented as mean ± SEM. Comparison between treatment groups were done by one or two ways factorial ANOVA. Least significant differences were computed for between-level comparisons. A *p* value <0.05 was considered to be statistically significant.

When primary cultures of pig RPTECs are exposed to increasing CyA or TAC concentrations, a dose dependent growth inhibition is produced, even with concentrations as low as CyA 1 μg/mL or 50 ng/mL. Figure 2 shows the growth dynamics of cultures in the presence of CyA 1 μg/mL and TAC 50 ng/mL, concentrations near to the peak plasma levels reached with oral administration of these two drugs. Both CyA and TAC inhibit growth from day 6, which

**Figure 2.**  Culture cells were incubated with 1 µg/mL CyA or 50 ng/mL TAC. Viable cell number was determined by cell counting in a phase-contrast inverted microscope. Values shown are the mean ± SEM of cell counts per cm2. \*p<0.05

Figure 3 gives a 3-D histogram of the cell flow cytometry in cultures treated with CyA or TAC for 8 days. A distinct hypodiploid population can be observed, indicative of apoptosis

**3.1. Effect of CyA and TAC on primary growth of the renal proximal tubule cells**

Leica Confocal Software LCS-1537 (Leica Microsystems).

impedes the culture confluence from day 8 (Figure 2).

CyA vs. control; †p<0.05 TAC vs. control.

**2.12. Statistics**

214 Pharmacology and Therapeutics

**3. Results**

#### **3.2. Effect of CyA and TAC on intramitochondrial free calcium**

When suspensions of fura-2 AM loaded mitochondria were incubated in the presence of 21% oxygen and 5 mM succinate, but in the absence of ADP, an increase in intramitochondrial calcium concentration is observed (Figure 4A), paralleling an 8-fold increase in oxygen consumption (Table 2). Subsequent addition of 2 mM of ADP significantly increases the rate of oxygen consumption (Table 2) and drastically decreases intramitochondrial calcium concentration, although not reaching baseline levels (Figure 4A). This reduction in mitochon‐ drial calcium when adding ADP is accompanied by a net calcium extrusion from mitochondria, as can be seen in Figure 4B where the same maneuver was repeated with non fura-2 loaded mitochondria suspended in the presence of the non-permanent acid form of fura-2. ADP addition is followed by an increase in extramitochondrial free calcium, but there is a net difference between the initial free calcium mitochondria uptake when succcinate was added, and free calcium release after ADP addition.

**Groups Control CyA 1 µg/mL**

"succinate line"; &p<0.05 vs. "succinate + ADP line". SUCC, succinate.

rescence was monitored with a fluorometer at 37°C.

was 0,2 μM (as determined in Figure 4B).

SUCC+ ADP + Atractilosyde

**(NP)**

**CyA 1µg/mL (30 min)**

2130 ± 151& 2218 ± 322& 2188 ± 177& 2114 ± 167& 2090 ± 135&

Basal 341 ± 44 309 ± 39 541 ± 234 378 ± 36 469 ± 161 SUCC 2587 ± 126† 3290 ± 514\* 2929 ± 286† 2918 ± 149\* 2810 ± 240\* SUCC + ADP 3093 ± 187# 3675 ± 386\* 3381 ± 163 3537 ± 335\* 3440 ± 63#

Mitochondria were preincubated with both drugs 30 minutes prior the conducting experiment or simultaneously (NP: No preincubation).Values are means ± SEM, n=22 \*p<0.05 vs. "control" column; †p<0.05 vs. "basal line"; #p<0.05 vs.

**Figure 5.** Cyclosporin A (CyA) and tacrolimus (TAC) increase intramitochondrial calcium concentration. In 5 mM succi‐ nate (SUCC) presence, 1 µg/mL CyA and 50 ng/mL TAC induce an increase in isolated mitochondria. 2 mM ADP causes the outflow of most of the calcium accumulated. Mitochondrial suspension was incubated with 5 µM fura-2 and fluo‐

MPT pore opening was studied following mitochondrial electrochemical gradient restoration after respiratory chain activation (succinate addition), and subsequent mitochondrial calcium uptake. No extra calcium was added to the extramitochondrial medium, where free calcium

Figure 6 shows the temporary relationship between the changes in mitochondrial calcium influx, expressed as the first derivate of mitochondrial calcium concentration against time, and the change in mitochondrial volume expressed as the first derivate of the mitochondrial

**3.3. Effect of CyA and TAC on mitochondrial permeability transition pore**

**Table 2.** Mitochondrial oxygen consumption (µmol/g\*h) induced by cyclosporin A (CyA) and tacrolimus (TAC).

**TAC 50 ng/mL (NP)**

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**TAC 50ng/mL (30 min)**

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In contrast, if fura-2 loaded mitochondria are first incubated with ADP 2 mM, no changes in mitochondrial calcium occur (Figure 4A). Subsequent addition of 5 mM succinate caused a delayed and lower increase in mitochondrial calcium, reaching a final intramitochondrial concentration similar to the one observed in the previously described setting (Figure 4A). Atractilosyde addition blocks mitochondrial calcium rise (data not shown) and reduce O2 consumption in every condition.

In the presence of 1 μg/mL of CyA in the assay medium, mitochondrial calcium increases further when 5 mM of succinate is added, always in the absence of ADP (Figure 5). Simulta‐ neously oxygen consumption increase is almost a 50% higher than observed in non-treated mitochondria (Table 2).

**Figure 4.** Succinate and ADP effects on mitochondrial calcium concentration. **A.)** The addition of 5 mM succinate (SUCC) to mitochondria in suspension induces an increase in the intramitochondrial calcium concentration. This calci‐ um increase is inhibited by 2 mM ADP. **B.**) Decrease of the extramitochondrial calcium concentration induced by 5 mM SUCC. Mitochondrial suspension was incubated with 5 µM fura-2 and fluorescence was monitored with a fluorometer at 37°C.

The presence of TAC 50 mg/ml also causes a significantly greater increase in intramitochon‐ drial calcium concentration and oxygen consumption when mitochondria begin succinate oxidation (Figure 5, Table 2).

In both cases, ADP addition significantly increases oxygen consumption (Table 2) and simultaneously the outflow of most of the calcium accumulated with or without CyA and TAC treatment (Figure 5). After ADP addition a stable intramitochondrial free calcium concentra‐ tion is reached indicative of a new influx /efflux balance establishment. However, after 50-100 seconds a spontaneous increase in mitochondrial calcium concentration can be observed, although to a lesser extent. Again this new increase in mitochondrial calcium is higher and happens earlier with TAC than CyA.

addition is followed by an increase in extramitochondrial free calcium, but there is a net difference between the initial free calcium mitochondria uptake when succcinate was added,

In contrast, if fura-2 loaded mitochondria are first incubated with ADP 2 mM, no changes in mitochondrial calcium occur (Figure 4A). Subsequent addition of 5 mM succinate caused a delayed and lower increase in mitochondrial calcium, reaching a final intramitochondrial concentration similar to the one observed in the previously described setting (Figure 4A). Atractilosyde addition blocks mitochondrial calcium rise (data not shown) and reduce O2

In the presence of 1 μg/mL of CyA in the assay medium, mitochondrial calcium increases further when 5 mM of succinate is added, always in the absence of ADP (Figure 5). Simulta‐ neously oxygen consumption increase is almost a 50% higher than observed in non-treated

**Figure 4.** Succinate and ADP effects on mitochondrial calcium concentration. **A.)** The addition of 5 mM succinate (SUCC) to mitochondria in suspension induces an increase in the intramitochondrial calcium concentration. This calci‐ um increase is inhibited by 2 mM ADP. **B.**) Decrease of the extramitochondrial calcium concentration induced by 5 mM SUCC. Mitochondrial suspension was incubated with 5 µM fura-2 and fluorescence was monitored with a fluorometer

The presence of TAC 50 mg/ml also causes a significantly greater increase in intramitochon‐ drial calcium concentration and oxygen consumption when mitochondria begin succinate

In both cases, ADP addition significantly increases oxygen consumption (Table 2) and simultaneously the outflow of most of the calcium accumulated with or without CyA and TAC treatment (Figure 5). After ADP addition a stable intramitochondrial free calcium concentra‐ tion is reached indicative of a new influx /efflux balance establishment. However, after 50-100 seconds a spontaneous increase in mitochondrial calcium concentration can be observed, although to a lesser extent. Again this new increase in mitochondrial calcium is higher and

and free calcium release after ADP addition.

consumption in every condition.

mitochondria (Table 2).

216 Pharmacology and Therapeutics

at 37°C.

oxidation (Figure 5, Table 2).

happens earlier with TAC than CyA.

Mitochondria were preincubated with both drugs 30 minutes prior the conducting experiment or simultaneously (NP: No preincubation).Values are means ± SEM, n=22 \*p<0.05 vs. "control" column; †p<0.05 vs. "basal line"; #p<0.05 vs. "succinate line"; &p<0.05 vs. "succinate + ADP line". SUCC, succinate.

**Table 2.** Mitochondrial oxygen consumption (µmol/g\*h) induced by cyclosporin A (CyA) and tacrolimus (TAC).

**Figure 5.** Cyclosporin A (CyA) and tacrolimus (TAC) increase intramitochondrial calcium concentration. In 5 mM succi‐ nate (SUCC) presence, 1 µg/mL CyA and 50 ng/mL TAC induce an increase in isolated mitochondria. 2 mM ADP causes the outflow of most of the calcium accumulated. Mitochondrial suspension was incubated with 5 µM fura-2 and fluo‐ rescence was monitored with a fluorometer at 37°C.

#### **3.3. Effect of CyA and TAC on mitochondrial permeability transition pore**

MPT pore opening was studied following mitochondrial electrochemical gradient restoration after respiratory chain activation (succinate addition), and subsequent mitochondrial calcium uptake. No extra calcium was added to the extramitochondrial medium, where free calcium was 0,2 μM (as determined in Figure 4B).

Figure 6 shows the temporary relationship between the changes in mitochondrial calcium influx, expressed as the first derivate of mitochondrial calcium concentration against time, and the change in mitochondrial volume expressed as the first derivate of the mitochondrial volume versus time. Net calcium influx reaches a maximum of 7.3 ± 1.1 nM/s at 17.5 ± 0.9 seconds after addition of 5 mM succinate (Table 3).

**Groups**

520 nm.

MPT pore opening (Table 4).

increase in intramitochondrial free calcium (Figure 9).

**Mitochondrial Basal Inflow (µL/mg prot.min)**

Values are means ±SEM. \*p<0.05 vs. control. NS: non-significant. CyA, cyclosporin A, TAC, tacrolimus.

**Table 4.** Characteristics of MPT pore induction after oxidation of succinate 5mM.

Control (n=21) 0.057 ± 0.009 0.432 ± 0.055 16.0 ± 1.5 CyA 1 µg/mL (n=17) 0.042 ± 0.007NS 0.235 ± 0.018\* 21.1 ± 1.9\* TAC 50 ng/mL (n=20) 0.062 ± 0.013NS 0.405 ± 0.052NS 16.1 ± 1.5NS

**Figure 7.** Inhibition of volume influx with well-known blocking of the mitochondrial permeability transition (MPT) pore. (**A**) Volume influx induced by 5 mM succinate (SUCC) is inhibited by 1 µM Ruthenium Red (RR), 1 mM potassium cyanide (CNK) and 2 mM ADP. (**B**) Close of the MPT pore and volume efflux induced by 2 mM ADP addition to a sus‐ pension of mitochondria, at different time point. Mitochondrial swelling was determined spectrophotometrically at

When CyA is present, 5 mM succinate addition to a mitochondria suspension causes a significantly reduced inflow through the MPT pore. The mitochondrial swelling observed in the presence of CyA is approximately half of that observed under control conditions (Table 4). It also takes significantly more time to reach its maximum (Table 4) and has an earlier closure (Figure 8A). Same results were obtained using the Met-Ile CyA, an analogue without anticalcineurin activity (is devoid of immunosuppressive activity), but still capable of binding mitochondrial cyclophilin D (Figure 8A). However TAC did not modify overall kinetics of

CyA-induced increase in mitochondrial calcium is not followed by a proportional opening of MPT pore, due to CyA dependent inhibition of MPT pore activation. TAC-induced increase in mitochondrial calcium is followed by the expected opening of MPT pore. To verify that volume inflow could be dragging calcium chelating anions into mitochondria, we studied again CyA and TAC effects on mitochondrial calcium in the presence of increasing doses of extramitochondrial phosphate, keeping constant the extramitochondrial calcium concentra‐ tion. By increasing phosphate availability, the pore opening is followed by a much modest

**Maximal Inflow (µL/mg prot.min)**

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**Time to Maximal Inflow (seconds)**

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Values are means ± SEM. \*p<0.05 vs.control. Cya, cyclosporin A; TAC, tacrolimus. NS, non-significant.

**Table 3.** Characteristics of calcium influx induction after oxidation of succinate 5 mM.

**Figure 6.** Temporary relationship between the changes in mitochondrial calcium influx and the change in mitochon‐ drial volume. The addition of 5 mM succinate (SUCC) cause a net calcium influx (**A**) and at the same time a gradual change in mitochondrial volume occurs (**B**). Ca2+-induced mitochondrial swelling was determined spectrophotometri‐ cally at 520 nm.

This influx subsequently decreases reaching a steady state when equilibrates with efflux (1st derivate = 0) (Figure 6A). A simultaneous gradual change in mitochondrial volume occurs, with a maximum value of 0.432 ± 0.055 μl/mg mitochondrial protein\*min at 16.0 ± 1.5 seconds (Table 4). The influx volume then decreases to baseline values (Figure 6B).

The characteristics of this volume influx correspond to those of the MPT pore. As can be seen in Figure 7A, if the oxidation of the succinate is blocked by potassium cyanide (CNK), or if calcium inflow is impeded by ruthenium red, or if the mitochondria are pre-incubated with 2 mM of ADP, change in mitochondrial volume is impeded.

ADP immediately blocks pore formation and could induce pore closing at any time during the process. This can be seen in Figure 7B where the addition of ADP at different time points to a suspension of mitochondria with the pore previously formed and open, closes the pore at any time during the process, causing a volume efflux (1st derivate <0).

Values are means ±SEM. \*p<0.05 vs. control. NS: non-significant. CyA, cyclosporin A, TAC, tacrolimus.

volume versus time. Net calcium influx reaches a maximum of 7.3 ± 1.1 nM/s at 17.5 ± 0.9

Control (n=11) 0.019 ± 0.011 7.3 ± 1.1 17.5 ± 0.9 CyA 1 µg/mL (n=4) 0.057 ± 0.015NS 17.5 ± 2.7\* 16.5 ± 0.9NS TAC 50 ng/mL (n=4) 0.006 ± 0.008NS 88.8 ± 5.41\* 17.5 ± 0.9NS

**Figure 6.** Temporary relationship between the changes in mitochondrial calcium influx and the change in mitochon‐ drial volume. The addition of 5 mM succinate (SUCC) cause a net calcium influx (**A**) and at the same time a gradual

This influx subsequently decreases reaching a steady state when equilibrates with efflux (1st derivate = 0) (Figure 6A). A simultaneous gradual change in mitochondrial volume occurs, with a maximum value of 0.432 ± 0.055 μl/mg mitochondrial protein\*min at 16.0 ± 1.5 seconds

The characteristics of this volume influx correspond to those of the MPT pore. As can be seen in Figure 7A, if the oxidation of the succinate is blocked by potassium cyanide (CNK), or if calcium inflow is impeded by ruthenium red, or if the mitochondria are pre-incubated with 2

ADP immediately blocks pore formation and could induce pore closing at any time during the process. This can be seen in Figure 7B where the addition of ADP at different time points to a suspension of mitochondria with the pore previously formed and open, closes the pore at any

(Table 4). The influx volume then decreases to baseline values (Figure 6B).

mM of ADP, change in mitochondrial volume is impeded.

time during the process, causing a volume efflux (1st derivate <0).

**Maximal Calcium Influx (nM/s)**

**Time to Maximal Calcium Influx (seconds)**

**Basal Calcium Influx (nM/s)**

Values are means ± SEM. \*p<0.05 vs.control. Cya, cyclosporin A; TAC, tacrolimus. NS, non-significant.

**Table 3.** Characteristics of calcium influx induction after oxidation of succinate 5 mM.

seconds after addition of 5 mM succinate (Table 3).

**Groups**

218 Pharmacology and Therapeutics

change in mitochondrial volume occurs (**B**). Ca2+

cally at 520 nm.

**Figure 7.** Inhibition of volume influx with well-known blocking of the mitochondrial permeability transition (MPT) pore. (**A**) Volume influx induced by 5 mM succinate (SUCC) is inhibited by 1 µM Ruthenium Red (RR), 1 mM potassium cyanide (CNK) and 2 mM ADP. (**B**) Close of the MPT pore and volume efflux induced by 2 mM ADP addition to a sus‐ pension of mitochondria, at different time point. Mitochondrial swelling was determined spectrophotometrically at 520 nm.

When CyA is present, 5 mM succinate addition to a mitochondria suspension causes a significantly reduced inflow through the MPT pore. The mitochondrial swelling observed in the presence of CyA is approximately half of that observed under control conditions (Table 4). It also takes significantly more time to reach its maximum (Table 4) and has an earlier closure (Figure 8A). Same results were obtained using the Met-Ile CyA, an analogue without anticalcineurin activity (is devoid of immunosuppressive activity), but still capable of binding mitochondrial cyclophilin D (Figure 8A). However TAC did not modify overall kinetics of MPT pore opening (Table 4).

CyA-induced increase in mitochondrial calcium is not followed by a proportional opening of MPT pore, due to CyA dependent inhibition of MPT pore activation. TAC-induced increase in mitochondrial calcium is followed by the expected opening of MPT pore. To verify that volume inflow could be dragging calcium chelating anions into mitochondria, we studied again CyA and TAC effects on mitochondrial calcium in the presence of increasing doses of extramitochondrial phosphate, keeping constant the extramitochondrial calcium concentra‐ tion. By increasing phosphate availability, the pore opening is followed by a much modest increase in intramitochondrial free calcium (Figure 9).

mine 123, which only stains the mitochondria when the mitochondrial potential is intact, diffuses out of the mitochondria and stains all the cytosol when the mitochondria is depolar‐

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221

**Figure 9.** Effect of extramitochondrial phosphate on intramitochondrial calcium. When increasing extramitochondrial phosphate, the increase of intramitochondrial calcium induced by 5 mM succinate (SUCC) is much smaller so much in the presence of 1 µg/mL cyclosporin A (**A**) like of 50 ng/mL tacrolimus (**B**). Mitochondrial suspension was incubated

**Figure 10.** Early and delayed mitochondrial permeability transition pore opening by cyclosporin A (CyA) and tacroli‐ mus (TAC). Incubation of a mitochondrial solution with 1 µg/mL CyA or 50 ng/mL TAC during at least 30 minutes pro‐ duced a second increase in mitochondrial volume. Succinate-induced mitochondrial swelling was determined

with 5 µM fura-2 and fluorescence was monitored with a fluorometer at 37°C.

spectrophotometrically at 520 nm.

ised in the presence of CyA or TAC.

**Figure 8.** Effect of Cyclosporin A (CyA) on mitochondrial swelling and glutamine consumption. (**A**) In 1 µg/mL CyA presence as well as on Met-Ile CyA analogue, the mitochondrial swelling induced by 5 mM succinate (SUCC) is re‐ duced. Mitochondrial swelling was determined spectrophotometrically at 520 nm. (**B**) CyA inhibited intramitochondri‐ al phosphate-dependent glutaminase activity at 1 ng/mL and 100 ng/mL. Glutamine consumption was determined spectrophotometrically following the NADH production at 340 nm.\*p<0.05 vs. control.

MPT pore opening inhibition by CyA will reduce the acute influx of phosphate into the mitochondria, causing free calcium to increase. This finding was indirectly supported when mitochondrial phosphate-dependent glutaminase (a selective enzyme of the proximal tubule mitochondria) activity was studied in intact proximal cells. CyA but not TAC (data not shown) inhibited mitochondrial phosphate-dependent glutaminase activity (Figure 8B).

#### **3.4. Early and delayed MPT pore opening: role in CyA and TAC-induced apoptosis**

Following succinate oxidation, opening and closure of MPT pore may be observed during the first minutes. However, if incubation with CyA or TAC is prolonged for 30 minutes more, a second increase in mitochondrial volume is produced (Figure 10) that is independent of any change on oxygen consumption rate. This second spontaneous swelling has some of the MPT characteristic: it can be inhibited by ruthenium red and by ADP. However, in contrast to that observed with the first opening of the pore, this time the change does not appear to be reversible, it is maintained over time and the presence of CyA does not impede or reduce it. This second opening is not observed in control mitochondria.

#### **3.5. Mitochondrial potential and cytochrome c: apoptosis induction**

Despite the fact that CyA is capable of a premature closing of the pore activated by succinate oxidation in the absence of ADP, and that TAC does not modify the usual response of the pore to such mechanism, both CyA and TAC cause the spontaneous, delayed opening of the transition pore, loss of cytochrome c to the cytosol (Figure 11B), and the gradual collapse of the mitochondrial potential gradient. In Figure 11A the mitochondrial potential dye, Rhoda‐ mine 123, which only stains the mitochondria when the mitochondrial potential is intact, diffuses out of the mitochondria and stains all the cytosol when the mitochondria is depolar‐ ised in the presence of CyA or TAC.

**Figure 9.** Effect of extramitochondrial phosphate on intramitochondrial calcium. When increasing extramitochondrial phosphate, the increase of intramitochondrial calcium induced by 5 mM succinate (SUCC) is much smaller so much in the presence of 1 µg/mL cyclosporin A (**A**) like of 50 ng/mL tacrolimus (**B**). Mitochondrial suspension was incubated with 5 µM fura-2 and fluorescence was monitored with a fluorometer at 37°C.

MPT pore opening inhibition by CyA will reduce the acute influx of phosphate into the mitochondria, causing free calcium to increase. This finding was indirectly supported when mitochondrial phosphate-dependent glutaminase (a selective enzyme of the proximal tubule mitochondria) activity was studied in intact proximal cells. CyA but not TAC (data not shown)

**Figure 8.** Effect of Cyclosporin A (CyA) on mitochondrial swelling and glutamine consumption. (**A**) In 1 µg/mL CyA presence as well as on Met-Ile CyA analogue, the mitochondrial swelling induced by 5 mM succinate (SUCC) is re‐ duced. Mitochondrial swelling was determined spectrophotometrically at 520 nm. (**B**) CyA inhibited intramitochondri‐ al phosphate-dependent glutaminase activity at 1 ng/mL and 100 ng/mL. Glutamine consumption was determined

Following succinate oxidation, opening and closure of MPT pore may be observed during the first minutes. However, if incubation with CyA or TAC is prolonged for 30 minutes more, a second increase in mitochondrial volume is produced (Figure 10) that is independent of any change on oxygen consumption rate. This second spontaneous swelling has some of the MPT characteristic: it can be inhibited by ruthenium red and by ADP. However, in contrast to that observed with the first opening of the pore, this time the change does not appear to be reversible, it is maintained over time and the presence of CyA does not impede or reduce it.

Despite the fact that CyA is capable of a premature closing of the pore activated by succinate oxidation in the absence of ADP, and that TAC does not modify the usual response of the pore to such mechanism, both CyA and TAC cause the spontaneous, delayed opening of the transition pore, loss of cytochrome c to the cytosol (Figure 11B), and the gradual collapse of the mitochondrial potential gradient. In Figure 11A the mitochondrial potential dye, Rhoda‐

inhibited mitochondrial phosphate-dependent glutaminase activity (Figure 8B).

spectrophotometrically following the NADH production at 340 nm.\*p<0.05 vs. control.

220 Pharmacology and Therapeutics

This second opening is not observed in control mitochondria.

**3.5. Mitochondrial potential and cytochrome c: apoptosis induction**

**3.4. Early and delayed MPT pore opening: role in CyA and TAC-induced apoptosis**

**Figure 10.** Early and delayed mitochondrial permeability transition pore opening by cyclosporin A (CyA) and tacroli‐ mus (TAC). Incubation of a mitochondrial solution with 1 µg/mL CyA or 50 ng/mL TAC during at least 30 minutes pro‐ duced a second increase in mitochondrial volume. Succinate-induced mitochondrial swelling was determined spectrophotometrically at 520 nm.

**a.** The electrochemical gradient has been created,

physiological conditions.

treated mitochondria [24, 27, 62].

**b.** There is extramitochondrial calcium in significant concentrations.

Both conditions take place during kidney trasnplantation when organ is removed from the cold storage and grafted in a donor. In our *in vitro* model, we induced the electrochemical gradient by adding O2 and succinate to our mitochondria suspensions. Most of the papers previously published on isolated mitochondria, use also extramitochondrial additions of calcium to study the MPT pore. However, this maneuver mimics too closely a near to death cell condition, and makes it difficult to extrapolate conclusions to MPT pore activity under

Anticalcineurinics: Role of Mitochondrial Transition Pore on Nephrotoxicity

http://dx.doi.org/10.5772/57286

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We observe the same inhibition of CyA permeates mitochondria and binds cyclophilin D, sparing it from calcium activation, and MPT pore assembly is barely activated. Imported free calcium rises inside the matrix and reaches values well over control values. We observed the same reduction in MPT pore activity with CyA analogous able to bind cyclophilin but unable to block calcineurin. Other authors reported an increased capacity of calcium buffering of CyA-

CyA is considered as an antiapoptotic factor because of this inhibition of MPT pore activity [26, 27, 63-65]. However, CyA does not inhibit calcium entry into mitrochondria. Crompton proposed MPT pore opening as the mechanism to prevent matrix free calcium accumulation and hypothesized that matrix calcium would be extruded through the pore, creating calcium currents until other cell calcium extrusion mechanisms would finally eject calcium out of the cell [24, 28]. Our results do not support such hypothesis. During pore opening, matrix calcium inflow stops, probably because VDAC is recruited to form the pore itself, but no calcium outflow was observed. Instead, based on our studies at different extramitochondrial phosphate concentrations, it is possible that water entry into mitochondria through the pore will have a "solvent drag" effect on phosphate or another anions able to reach the matrix, combine with free calcium, precipitate it as calcium phosphate granules, and release cyclophilin allowing the pore to close. If this mechanism is true, mitochondria will act as "calcium cleaner", trapping extramitochondrial calcium in order to keep cell calcium low, but with a limited capacity. As mitochondria gets older, more and more microcalcifications would be observed inside matrix and free calcium will progressively rise until opening again the pore, this time in an irreversible way as there is not a chance for free calcium to be precipitated any longer. Different indirect observations support this hypothesis from the early descriptions of calcium stores discovered in mitochondria of damaged tissues, to the mitochondria microcalcifications considered one of the hallmarks of CyA toxicity [66]. Interestingly, as CyA reduces influx into proximal tubule isolated mitochondria, we observe a dose-dependent inhibition of phosphate dependent glutaminase activity, an enzyme that is critical in proximal tubule ammoniagenesis extremely dependent on phosphate supply. It is possible that MPT pore inhibition by CyA is related to the reduced ammoniagenesis described with CyA in clinical and experimental settings.

When CyA or CyA analogues bind cyclophilin matrix free calcium rises faster than in control mitochondria. Although initially there is a transient reduced and delayed MPT pore opening, with time and calcium accumulation overwhelms CyA dependent cyclophilin inhibition, and

**Figure 11.** Mitochondrial depolarisation and cytochrome c release induced by Cyclosporin A (CyA) and Tacrolimus (TAC). (**A**) Renal proximal tubular epithelial cells (RPTECs) were treated during 48 hours with 1 µg/mL CyA or 50 ng/mL TAC and incubated with 10 mM Rhodamine 123. Cells were then observed with a fluorescence microscope. Rhodamine 123 diffuses out of the mitochondria and stains all the cytosol when the mitochondria are depolarised. (**B**) Western blot analysis of cytochrome c of cytosolic and mitochondrial fractions of RPTECs treated with CyA and TAC for 48 hours. Voltage-dependent anion-selective channel (VDAC1) and α-tubulin antibodies were used as internal loading controls for mitochondrial and cytosolic fractions respectively.

#### **4. Discussion and conclusion**