Toxicity, metabolism, and impact of mycotoxins on humans and animals.

According to the traditional model, steroid hormones bind to intracellular receptors and subsequently modulate transcription and protein synthesis, thus triggering genomic events finally responsible for delayed effects. Based upon similarities in molecular structure, specific receptors for steroids, vitamin D3 derivatives, thyroid hormone, retinoids, and a variety of orphan receptors are considered to represent a superfamily of steroid receptors. In addition, very rapid effects of steroids mainly affecting intracellular signaling have been widely recognized that are clearly incompatible with the genomic model. These rapid, nongenomic steroid actions are likely to be transmitted via specific membrane receptors. Evidence for nongenomic steroid effects and distinct receptors involved is presented for all steroid groups including related compounds like vitamin D3 and thyroid hormones. The physiological and clinical relevance of these rapid effects is still largely unclear, but their existence in vivo has been clearly shown in various settings including human studies. Drugs that specifically affect nongenomic steroid action may find applications in various clinical areas such as cardiovascular and central nervous disorders, electrolyte homeostasis, and infertility. In addition to a short description of genomic steroid action, this review pays particular attention to the current knowledge and important results on the mechanisms of nongenomic steroid action. The modes of action are discussed in relation to their potential physiological or pathophysiological relevance and with regard to a cross-talk between genomic and nongenomic responses.

Multiple Actions of Steroid Hormones—A Focus on Rapid, Nongenomic Effects

Ochratoxin A (OTA) is a ubiquitous mycotoxin produced by fungi of improperly stored food products. OTA is nephrotoxic and is suspected of being the main etiological agent responsible for human Balkan endemic nephropathy (BEN) and associated urinary tract tumours. Striking similarities between OTA-induced porcine nephropathy in pigs and BEN in humans are observed. International Agency for Research on Cancer (IARC) has classified OTA as a possible human carcinogen (group 2B). Currently, the mode of carcinogenic action by OTA is unknown. OTA is genotoxic following oxidative metabolism. This activity is thought to play a central role in OTA-mediated carcinogenesis and may be divided into direct (covalent DNA adduction) and indirect (oxidative DNA damage) mechanisms of action. Evidence for a direct mode of genotoxicity has been derived from the sensitive 32P-postlabelling assay. OTA facilitates guanine-specific DNA adducts in vitro and in rat and pig kidney orally dosed, one adduct comigrates with a synthetic carbon (C)-bonded C8-dG OTA adduct standard. In this paper, our current understanding of OTA toxicity and carcinogenicity are reviewed. The available evidence suggests that OTA is a genotoxic carcinogen by induction of oxidative DNA lesions coupled with direct DNA adducts via quinone formation. This mechanism of action should be used to establish acceptable intake levels of OTA from human food sources.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/mnfr.200790020

Ochratoxin A: An overview on toxicity and carcinogenicity in animals and humans

Betaine in human nutrition

The transport of amino acids in kidney and intestine is critical for the supply of amino acids to all tissues and the homeostasis of plasma amino acid levels. This is illustrated by a number of inherited disorders affecting amino acid transport in epithelial cells, such as cystinuria, lysinuric protein intolerance, Hartnup disorder, iminoglycinuria, dicarboxylic aminoaciduria, and some other less well-described disturbances of amino acid transport. The identification of most epithelial amino acid transporters over the past 15 years allows the definition of these disorders at the molecular level and provides a clear picture of the functional cooperation between transporters in the apical and basolateral membranes of mammalian epithelial cells. Transport of amino acids across the apical membrane not only makes use of sodium-dependent symporters, but also uses the proton-motive force and the gradient of other amino acids to efficiently absorb amino acids from the lumen. In the basolateral membrane, antiporters cooperate with facilitators to release amino acids without depleting cells of valuable nutrients. With very few exceptions, individual amino acids are transported by more than one transporter, providing backup capacity for absorption in the case of mutational inactivation of a transport system.

Amino acid transport across mammalian intestinal and renal epithelia.

The renal handling of amino acids and oligopeptides.

Madin-Darby canine kidney (MDCK) cells originate from the renal collecting duct and consist of different cell subtypes. We cloned two MDCK cell subtypes denominated as C7 and C11 with different morphology and different function. The two clones maintained their functional differences after cloning. C7 monolayers exhibit a high transepithelial resistance (Rte = 5648 +/- 206 omega.cm2, n = 20) and secrete K+ (delta K+ = 1.31 +/- 0.08 mmol/l, n = 10) into the apical medium. C11 monolayers display a low Rte (330 +/- 52 omega.cm2, n = 20) and secrete Cl- (delta Cl- = 16.9 +/- 1.8 mmol/l, n = 10) into the apical medium. Aldosterone (1 mumol/l) stimulates K+ secretion (delta K+ of 3.58 +/- 0.11 mmol/l, n = 7) in C7 cells and H+ secretion in C11 cells (delta pH = 0.060 +/- 0.007, n = 10). Aldosterone-induced stimulation of K+ secretion is inhibited by apical application of amiloride (1 mumol/l). cAMP stimulates H+ secretion in C11 cells (delta pH = -0.068 +/- 0.004, n = 10). Furthermore, C7 cells are peanut-lectin(PNA)-negative and exhibit an intracellular pH of 7.39 +/- 0.05 (n = 7), whereas C11 cells maintain intracellular pH at 7.16 +/- 0.05 (n = 8) and a major fraction of cells is PNA positive. We conclude that we have cloned two subtypes of MDCK cells which stably express different functional characteristics. The C7 subtype resembles principal cells (PC) of the renal collecting duct, whereas the C11 subtype resembles intercalated cells (ICC) of the renal collecting duct.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of two MDCK-cell subtypes as a model system to study principal cell and intercalated cell properties.

There is increasing evidence for an additional acute, nongenomic action of the mineralocorticoid hormone aldosterone on renal epithelial cells, leading to a two-step model of mineralocorticoid action on electrolyte excretion. We investigated the acute effect of aldosterone on intracellular free Ca2+ and on intracellular pH in an aldosterone-sensitive Madin-Darby canine kidney cell clone. Within seconds of application of aldosterone, but not of the glucocorticoid hydrocortisone, there was a 3-fold sustained increase of intracellular Ca2+ at a half-maximal concentration of 10(-10) mol/liter. Omission of extracellular Ca2+ prevented this hormone response. In the presence of extracellular Ca2+ aldosterone led to intracellular alkalinization. The Na+/H+ exchange inhibitor ethyl-isopropanol-amiloride (EIPA) prevented the aldosterone-induced alkalinization but not the aldosterone-induced increase of intracellular Ca2+. Omission of extracellular Ca2+ also prevented aldosterone-induced alkalinization. Instead, aldosterone led to a Zn(2+)-dependent intracellular acidification in the presence of EIPA, indicative of an increase of plasma membrane proton conductance. Under control conditions, Zn2+ prevented the aldosterone-induced alkalinization completely. We conclude that aldosterone stimulated net-entry of Ca2+ from the extracellular compartment and a plasma membrane H+ conductance as prerequisites for the stimulation of plasma membrane Na+/H+ exchange which in turn modulates K+ channel acitivity. It is probable that the aldosterone-sensitive H+ conductance maintains Na+/H+ exchange activity by providing an acidic environment in the vicinity of the exchanger. Thus, genomic action of aldosterone determines cellular transport equipment, whereas the nongenomic action regulates transporter activity that requires responses within seconds or minutes, which explains the rapid effects on electrolyte excretion.

Rapid activation of Na+/H+ exchange by aldosterone in renal epithelial cells requires Ca2+ and stimulation of a plasma membrane proton conductance.

Receptor-mediated endocytosis is an essential mechanism for the transport of a variety of macromolecules into cells as well as across epithelia (Mukherjee et al. 1997). Besides transport of macromolecules, endocytosis is also involved in antigen presentation, maintenance of cell polarity and regulation of cell-surface receptor expression. The endocytic mechanisms underlying receptor-mediated endocytosis can be roughly subdivided into two types: (i) endocytosis via clathrin-coated pits and (ii) non-clathrin-mediated endocytosis, consisting mainly of caveolae-mediated endocytosis (Mukherjee et al. 1997; Schmid, 1997). Clathrin-mediated endocytosis is the best characterised endocytic mechanism and is the predominant pathway for macromolecule uptake along epithelia (Mukherjee et al. 1997; Schmid, 1997; Marshansky et al. 1997; Christensen et al. 1998). One example of clathrin-mediated endocytosis is the uptake of filtered serum albumin across the apical membrane of renal proximal tubular cells (Gekle et al. 1997; Gekle, 1998; Christensen et al. 1998). In the present study we used this model to study receptor-mediated endocytosis. Receptors undergoing clathrin-mediated endocytosis are concentrated in coated pits and subsequently delivered to the early endosomal compartment by endocytic vesicles (Mukherjee et al. 1997; Schmid, 1997). In sorting endosomes, internalised receptors and ligands are directed either to recycling endosomes or to the late endosomal compartment and further on to the lysosomes, where they undergo degradation. Serum albumin, for example, is directed mainly to lysosomes (Cui et al. 1996; Czekay et al. 1997; Christensen et al. 1998).

An important process along the endocytic pathway is the acidification of endosomal compartments (Mellman et al. 1986; Gruenberg & Maxfield, 1995; Mukherjee et al. 1997). Adequate acidification is a crucial process because endosomal pH interferes, for example, with ligand-receptor dissociation, vesicle trafficking, endosomal fusion events, recycling to the plasma membrane and COP-coat formation (Mellman et al. 1986; Gekle et al. 1995a, 1996a; Papkonstanti et al. 1996; Storrie & Desjardins, 1996; Mukherjee et al. 1997). Acidification is accomplished, at least in part, by the vacuole-type H+-ATPase which works in parallel with a counterion conductance, in order to limit the formation of an endosomal-positive membrane potential (Rybak et al. 1997). In most cases the counterion conductance consists of Cl− channels (Mellman et al. 1986; Gekle et al. 1995a; Steinmeyer et al. 1995; Marshansky & Vinay, 1996). In proximal tubular cells CLC-5-type Cl− channels play an important role in counterion conductance (Steinmeyer et al. 1995; Devuyst et al. 1998). Recently, evidence was presented for the involvement of Na+−H+ exchange (NHE), especially via isoform 3 (NHE3), in endosomal acidification (Kapus et al. 1994; Marshansky & Vinay, 1996; D'Souza et al. 1998). Na+−H+ exchangers are ubiquitous plasma membrane transport proteins involved in cellular pH homeostasis and volume regulation. NHE3 seems to cycle between the plasma membrane and the early endosomal compartment, contributing on its way to endosomal acidification (Janecki et al. 1998; Kurashima et al. 1998).

In the present study we used a cell line derived from opossum renal proximal tubule (OK cells) which shows a well-characterised apical endocytic uptake activity for albumin as well as apical expression of NHE3, but no basolateral expression of NHE (Noel et al. 1996; Gekle et al. 1997; Brunskill et al. 1998). Renal proximal tubular albumin reabsorption is of major importance because it prevents the loss of valuable amino acids, but at the same time it can induce tubulointerstitial inflammation and fibrosis (Burton & Harris, 1996; Jerums et al. 1997; Gekle, 1998). In this study we determined the contribution of NHE to the regulation of endosomal pH and to reabsorptive albumin endocytosis in renal proximal tubular cells. In addition we performed experiments in order to estimate the Na+ gradient across the endosomal membrane which is of importance for NHE activity. Our data show that NHE3 is an important determinant of receptor-mediated endocytosis in renal proximal tubular cells due to its contribution to endosomal acidification and its role in cytosolic pH homeostasis.

Inhibition of Na+‐H+ exchange impairs receptor‐mediated albumin endocytosis in renal proximal tubule‐derived epithelial cells from opossum

To test the apoptotic potential of the nephrotoxic mycotoxin ochratoxin A (OTA), we exposed human proximal tubule-derived cells (IHKE cells) for various times to OTA concentrations close to those occurring during dietary exposure (from 2 to 100 nmol/L) and investigated caspase 3 activation, chromatin condensation, and DNA fragmentation. OTA induced a time- and concentration-dependent activation of caspase 3: concentrations as low as 5 nmol/L OTA caused a slight but significant increase in caspase 3 activity after 7 days of OTA exposure. Exposure to 10 nmol/L OTA for 72 or 24 h led to a significantly increased activity of caspase 3 in human proximal tubule-derived cells. Radical scavengers such as N-acetylcysteine had no effect on OTA-induced caspase 3 activation. Chelation of intracellular calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethylester) (BAPTA-AM) also showed no effect. Exposure to 30 nmol/L or more OTA led to DNA fragmentation and chromatin condensation in IHKE cells. Cultured renal epithelial MDCK-C7 and MDCK-C11 or OK cells also showed increased caspase 3 activity after OTA exposure. We conclude that exposure to low OTA concentrations can lead to direct or indirect caspase 3 activation and subsequently to apoptosis in cultured human proximal tubule cells and in other renal epithelial cell lines of different origins.

https://link.springer.com/content/pdf/10.1023/A:1007662101880.pdf

Stefan Silbernagl

Papers

The renal handling of amino acids and oligopeptides.

Characterization of two MDCK-cell subtypes as a model system to study principal cell and intercalated cell properties.

Rapid activation of Na+/H+ exchange by aldosterone in renal epithelial cells requires Ca2+ and stimulation of a plasma membrane proton conductance.

Inhibition of Na+‐H+ exchange impairs receptor‐mediated albumin endocytosis in renal proximal tubule‐derived epithelial cells from opossum

The nephrotoxin ochratoxin A induces apoptosis in cultured human proximal tubule cells.