Proximal tubule overexpression of a locally acting IGF isoform, Igf-1Ea, increases inflammation after ischemic injury
Introduction
Acute kidney failure is characterised by a rapid loss of renal function resulting in retention of waste products that are normally excreted by the kidneys. Ischemia reperfusion injury (IR) is one of the main clinical causes of acute kidney injury [1]. Tubular necrosis and interstitial inflammatory cell infiltration along with apoptosis are characteristic pathologic changes of acute kidney injury [2]. Depending on the severity and the duration of the insult, tubular damage may recover although a critical number of surviving cells is required for structural integrity. Interstitial infiltrating leukocytes that are attracted and activated by chemokines are key mediators in the pathogenesis of tubular necrosis. Resolution of inflammatory signals is necessary for repair as prolonged inflammation can lead to chronic kidney disease.
The effects of insulin-like growth factors (IGFs) on the kidney have been studied using transgenic and knockout mouse models. Constitutional over-expression of the canonical Igf-1 gene, encoding the circulating form of the growth factor, results in hyperplasia and increased glomerular size [3]. Igf-1 deficient animals are smaller than wild type littermates and their kidneys have proportionally lower weight, along with reduced glomerular size and number of nephrons [4]. This may indicate a role for IGF-1 in determining nephron number [4] or this may simply be the consequence of reduced size. It has been proposed that the therapeutic use of growth factors, such as IGF-1, may exert a beneficial effect on the ischemia-induced chain of events because such growth factors are mitogenic and anti-apoptotic. In addition, circulating IGF-1 increases renal blood flow and glomerular filtration rate [5]. Several investigators have studied the use of circulating IGF-1 for the treatment of acute renal failure. The majority, although not all [6], [7], studies have found that administration of exogenous IGF-1 is of some benefit in rats with acute kidney failure [8], [9], [10], [11]. Despite this, two double-blind studies in human acute kidney failure yielded negative results, even when IGF-1 was given early in the course of disease [12], [13].
IGF-1 protein is produced in multiple isoforms that differ in their amino-terminal signal peptides and carboxy-terminal extension peptide [14], [15], [16], [17]. The different isoforms vary in structure and function and were initially referred to as circulating (Class 2) and local (Class 1) IGF-1 [14], [15], although recent evidence suggests that Class 1 IGF-1 can completely compensate for a lack of Class 2 transcripts and is secreted into the circulation by the liver [16]. In this study, we have investigated the function of the IGF-1Ea isoform, comprising a Class 1 signal peptide and a C-terminal Ea extension peptide [14], [17]. This isoform is expressed at high levels in neonatal tissues and adult liver, but decreases with age in extrahepatic tissues, where its expression is activated transiently in response to local damage [18]. The Ea C-terminal extension is believed to cause retention of IGF-1Ea in the tissue of synthesis so it does not enter the circulation (M. Hede and N. Rosenthal, personal communication), thereby avoiding hypertrophic effects on distant organs such as the heart, and eliminating the risk of neoplasms induced by inappropriately high levels of circulating IGF-1. How local activity is achieved is not clear as other in vitro studies have detected IGF1 with C-terminal extensions in conditioned media, suggesting a capacity for these to be freely secreted [19].
This locally synthesized IGF-1 isoform has been well characterised in muscle regeneration and, when rat Igf-1Ea gene is overexpressed in the skeletal muscle of transgenic mice, it appears to safely enhance and preserve muscle fibre integrity suggesting that IGF-1Ea acts as a survival factor by prolonging the regenerative potential of skeletal muscle through increases in satellite cell activity [20]. Furthermore, overexpression of Igf-1Ea in both muscle and heart has been shown to modulate the inflammatory response after injury by down-regulating pro-inflammatory cytokines and increasing anti-apoptotic signalling [21], [22]. For this reason, we generated transgenic mice expressing rat Igf-1Ea under the control of a previously defined proximal tubule specific promoter [23] to determine whether elevation of IGF-1Ea protein could also improve repair in a model of acute renal injury without the negative effects associated with circulating IGF-1. In contrast to what has been reported in heart, chronic expression of Igf-1Ea from the proximal tubules resulted in a more severe inflammatory response to ischemic injury.
Section snippets
Animal experimentation
Sglt2-Igf1-Ea transgenic mice were generated on the inbred FVB mouse strain at the European Molecular Biology Laboratory, Rome, Italy. All animals used for experimentation were housed within the University of Queensland Biological Resources in a clean, temperature-controlled mouse facility on a 12-hour light/dark cycle and standard diet. Animal experiments were approved in advance by the University of Queensland Animal Ethics Committee (Molecular Biosciences) and adhered to the ‘Australian Code
Characterisation of the Sglt2 Igf-1Ea transgenic mice
A locally acting isoform of IGF-1 (IGF-1Ea), which comprises a class 1 signal peptide derived from exon 1 and an Ea extension peptide derived from exons 4 and 6 (Fig. 1A), has been shown to improve muscle, heart and skin regeneration in mice [20], [22], [27]. To test whether this IGF-1 isoform could also improve kidney regeneration, transgenic mice were generated with a rat Igf-1Ea cDNA driven by a mouse Sglt2 promoter (Fig. 1A). Murine Sglt2 is expressed specifically in the renal proximal
Discussion
In this study we examined whether the overexpression of the locally acting IGF-1Ea isoform in the proximal tubules of the kidney led to improved repair in a model of acute renal injury. Unexpectedly we found that Igf-1Ea expression resulted in greater initial damage (both necrosis and apoptosis) 24 h after injury, particularly in the Tglow transgenic mice, and this damage was unresolved after 5 days in the Tghigh mice. These results are in contrast to those reported in the heart and muscle where
Acknowledgements
We thank Bree A. Rumballe for her technical input and the EMBL Monterotondo Mouse Core for generation of the transgenic mouse lines. MHL is a Principal Research Fellow and FR is an Industry Fellow supported by the National Health and Medical Council, Australia. TM was supported by a sponsored research agreement from Novartis Pharma AG.
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