Proximal tubule overexpression of a locally acting IGF isoform, Igf-1Ea, increases inflammation after ischemic injury

https://doi.org/10.1016/j.ghir.2011.11.002Get rights and content

Abstract

Objective

IGF-1 is an important regulator of postnatal growth in mammals. In mice, a non-circulating, locally acting isoform of IGF-1, IGF-1Ea, has been documented as a central regulator of muscle regeneration and has been shown to improve repair in the heart and skin. In this study, we examine whether local production of IGF1-Ea protein improves tubular repair after renal ischemia reperfusion injury.

Design

Transgenic mice in which the proximal-tubule specific promoter Sglt2 was driving the expression of an Igf-1Ea transgene. These animals were treated with an ischemic-reperfusion injury and the response at 24 h and 5 days compared with wildtype littermates.

Results

Transgenic mice demonstrated rapid and enhanced renal injury in comparison to wild type mice. Five days after injury the wild type and low expressing Igf-1Ea transgenic mice showed significant tubular recovery, while high expressing Igf-1Ea transgenic mice displayed significant tubular damage. This marked injury was accompanied by a two-fold increase in the number of F4/80 positive macrophages and a three-fold increase in the number of Gr1-positive neutrophils in the kidney. At the molecular level, Igf-1Ea expression resulted in significant up-regulation of proinflammatory cytokines such as TNF-α and Ccl2. Expression of Nfatc1 was also delayed, suggesting reduced tubular proliferation after kidney injury.

Conclusions

These data indicate that, unlike the muscle, heart and skin, elevated levels of IGF-1Ea in the proximal tubules exacerbates ischemia reperfusion injury resulting in increased recruitment of macrophages and neutrophils and delays repair in a renal setting.

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.

References (41)

  • C.M. Prele et al.

    Insulin-like growth factor-1 overexpression in cardiomyocytes diminishes ex vivo heart functional recovery after acute ischemia

    Cardiovasc. Pathol.

    (Jan 2012)
  • J. Zhao et al.

    Insulin-like growth factor-I reduces stress-induced gastric mucosal injury by inhibiting neutrophil activation in mice

    Growth Horm. IGF Res.

    (Apr 2009)
  • K. Furuichi et al.

    Chemokine/chemokine receptor-mediated inflammation regulates pathologic changes from acute kidney injury to chronic kidney disease

    Clin. Exp. Nephrol.

    (Feb 2009)
  • A.M. Versteilen et al.

    Molecular mechanisms of acute renal failure following ischemia/reperfusion

    Int. J. Artif. Organs

    (Dec 2004)
  • T. Doi et al.

    Progressive glomerulosclerosis develops in transgenic mice chronically expressing growth hormone and growth hormone releasing factor but not in those expressing insulinlike growth factor-1

    Am. J. Pathol.

    (Jun 1988)
  • S.A. Rogers et al.

    Insulin-like growth factor I regulates renal development in rodents

    Dev. Genet.

    (1999)
  • R. Hirschberg et al.

    Effects of growth hormone and IGF-I on renal function

    Kidney Int. Suppl.

    (Nov 1989)
  • M. Fernandez et al.

    Exacerbated inflammatory response induced by insulin-like growth factor I treatment in rats with ischemic acute renal failure

    J. Am. Soc. Nephrol.

    (Sep 2001)
  • A.A. Martin et al.

    Effects of insulin-like growth factor-I peptides in rats with acute renal failure

    J. Endocrinol.

    (Jan 1994)
  • R. Clark et al.

    Recovery from acute ischaemic renal failure is accelerated by des-(1–3)-insulin-like growth factor-1

    Clin. Sci. (Lond.)

    (Jun 1994)
  • View full text