Modified Citrus Pectin Reduces Galectin-3 Expression and Disease Severity in Experimental Acute Kidney Injury

Long DA (2011) Modified Citrus Pectin Reduces Galectin-3 Expression and Disease Severity in Experimental Acute Kidney Injury. PLoS ONE 6(4): e18683. doi:10.1371/journal.pone.0018683 Modified Citrus Pectin Reduces Galectin-3 Expression and Disease Severity in Experimental Acute Kidney Injury Maria Kolatsi-Joannou 0 Karen L. Price 0 Paul J. Winyard 0 David A. Long 0 Niels Olsen Saraiva Camara, Universidade de Sao Paulo, Brazil 0 Nephro-Urology Unit, UCL Institute of Child Health , London , United Kingdom Galectin-3 is a b-galactoside binding lectin with roles in diverse processes including proliferation, apoptosis, inflammation and fibrosis which are dependent on different domains of the molecule and subcellular distribution. Although galectin-3 is known to be upregulated in acute kidney injury, the relative importance of its different domains and functions are poorly understood in the underlying pathogenesis. Therefore we experimentally modulated galectin-3 in folic acid (FA)-induced acute kidney injury utilising modified citrus pectin (MCP), a derivative of pectin which can bind to the galectin-3 carbohydrate recognition domain thereby predominantly antagonising functions linked to this role. Mice were pre-treated with normal or 1% MCP-supplemented drinking water one week before FA injection. During the initial injury phase, all FAtreated mice lost weight whilst their kidneys enlarged secondary to the renal insult; these gross changes were significantly lessened in the MCP group but this was not associated with significant changes in galectin-3 expression. At a histological level, MCP clearly reduced renal cell proliferation but did not affect apoptosis. Later, during the recovery phase at two weeks, MCP-treated mice demonstrated reduced galectin-3 in association with decreased renal fibrosis, macrophages, proinflammatory cytokine expression and apoptosis. Other renal galectins, galectin-1 and -9, were unchanged. Our data indicates that MCP is protective in experimental nephropathy with modulation of early proliferation and later galectin-3 expression, apoptosis and fibrosis. This raises the possibility that MCP may be a novel strategy to reduce renal injury in the long term, perhaps via carbohydrate binding-related functions of galectin-3. - Galectins are low molecular weight, calcium-independent, bgalactoside-binding lectins [1]. Galectin-3 is a multi-domain molecule which includes an N-terminal proline-rich domain and a C-terminal carbohydrate recognition domain essential for binding simple b-galactosides such as lactosamine and Galb14GlcNAc; and for higher affinity binding to polylactosamine chains [2]. Galectin-3 plays a key role in several intracellular physiological and pathological processes including proliferation and apoptosis, via carbohydrate-independent mechanisms [35]. In addition, galectin-3 is involved in modulation of cell-cell interactions and inflammation, predominately through extracellular carbohydrate binding functions [69]. In the kidney, galectin-3 is strongly expressed in the ureteric bud and its derivatives, the collecting ducts, in normal development and the mature organ [8,10]. Lower levels are also sometimes observed in mature tubules [11] but the lectin is expressed in a more widespread distribution in models of acute kidney damage such as ischemia-reperfusion injury or high-dose folic acid (FA) treatment [12]. In this latter model, FA initially undergoes glomerular filtration following systemic injection, and precipitates in the tubules which become damaged with a loss of epithelial cell integrity due to necrosis and apoptosis [13,14]. After two days, the majority of the tubules show regenerative changes as new cells proliferate and migrate to repair the denuded areas of the tubule [15]. However, over the next two weeks there is incomplete healing in some areas of the kidney, as evidenced by patchy interstitial fibrosis, loss of peritubular capillaries and inflammation with macrophage infiltration [16,17]. In the FA model, galectin-3 expression is initially observed in both proximal and distal tubules, and thick ascending limbs as well as collecting ducts; later, it is detectable in macrophages, particularly in areas of inflammation [12]. Several lines of evidence suggest that galectin-3 is beneficial in experimental kidney diseases such as polycystic kidney disease [18], nephrotoxic nephritis [19] and unilateral ureteric obstruction (UUO) [11], but its functional importance in FA-induced acute kidney injury is unknown. Therefore, we utilised this model and modulated galectin-3 levels using modified citrus pectin (MCP), a derivative of pectin; which is a soluble dietary fibre found in the peel and pulp of citrus fruits [20]. MCP contains fragments of the original pectin molecule, including rhamnogalacturonan 1 regions which contain galactan side-chains [21] which bind to the carbohydrate recognition domain of galectin-3 [22,23], hence modulating galectin-3 bioactivity by altering extracellular functions such as cell-cell interactions and inflammation. Experimental Strategy Reagents were obtained from Sigma Chemical Company (Poole, UK) unless otherwise stated. Eight week-old male C57Bl/ 6J mice (Charles River Laboratories, Margate, UK), of average weight 25 g were used in procedures approved by the UCL local ethics committee and the UK Home Office (project licence PPL 70/6627). The three main experimental groups are depicted in Figure 1. Group I (n = 8), the sham controls, were provided with normal drinking water ad libitium throughout, injected with intraperitoneal (IP) sodium bicarbonate (NaHCO3, 0.2 ml, 0.3 M; the vehicle used for FA administration) at Day 0; and were killed either at Day 2 (n = 4) or Day 14 (n = 4). Groups II and III were used to induce FA-nephropathy. Group II (n = 14), were also provided with normal drinking water throughout but were injected on Day 0 with 240 mg/g body weight FA, a dose which reliably caused acute tubular necrosis in all mice, with a low mortality rate (less than 5%) over 14 days observation [16,17]. Group III (n = 14), were administered 1% MCP (Pectasol, EcoNugenics, Santa Rosa, CA) in the drinking water for seven days before the IP FA and then continued on MCP throughout the protocol. This dose of MCP previously led to effective galectin-3 blockade and was non-toxic in murine cancer studies examining parameters such as tumour growth, angiogenesis and spontaneous metastasis [24]. Lower doses of MCP also prevent galectin-3 mediated functions in-vitro including chemotaxis and cell adhesion [24,25], but there is no data showing this would be replicated in-vivo. Experiments were also performed to ensure that pectin itself did not have adverse effects. In both Groups II and III, one mouse died spontaneously at Day 1 and the remainder were killed at either Day 2 or Day 14 of the protocol. All of the assessments were made blinded to experimental groups. Histology and Blood Analyses Body and kidn (...truncated)