Modified citrus pectin reduces galectin-3 expression and disease severity in experimental acute kidney injury

Abstract

Galectin-3 is a β-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 FA-treated 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, pro-inflammatory 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.

Figure 1. Experimental groups I-III.

Figure 2. Galectin-3 expression in FA nephropathy.

A. Sham control kidneys contained positive galectin-3 expression primarily in collecting ducts. B. Two days after FA administration, prominent galectin-3 expression was observed in dilated tubules (indicated by *, g  =  glomerulus). This was maintained in the recovery phase of FA nephropathy after 14 days, but in addition positive galectin-3 was observed in infiltrating cells in fibrotic areas (arrows, C). Bars are 50 µm.

Figure 3. Effect of MCP on galectin expression in FA nephropathy.

Quantitative RT-PCR for renal galectin-3 (A), galectin-1 (B) and galectin-9 (C) on kidney RNA from animals in Group I (water throughout, NaHCO₃ injection), Group II (water throughout, FA injection) and Group III (MCP throughout and FA injection), two and fourteen days after FA administration (n = 4 in Group I, 6–7 at each time-point in Group II and Group III). All galectins were significantly upregulated in FA-treated animals at both time-points, but MCP only diminished galectin-3 levels fourteen days after induction of FA nephropathy. (D) Western blotting for galectin-3 in kidney samples from Groups II and III collected 14 days after induction of FA nephropathy. Following densitometry analysis (n = 6–7 in each group), galectin-3 protein levels were shown to be significantly decreased in FA animals administered MCP. ** indicates p<0.01 between Groups while * indicates p<0.05 between Groups.

Figure 4. Assessment of kidney and body weights.

Body (A), kidney (B) and kidney/body weights (C) were assessed in Group I (normal water throughout, injected with vehicle NaHCO₃ as sham control), Group II (normal water throughout, injected with FA) and Group III (normal water supplemented with 1% MCP for 7 days prior to injection with FA, and continued thereafter). Two days after FA, animals drinking normal water had elevated kidney weights, increased kidney/body weights and decreased body weights compared to sham controls. MCP administration prevented the loss in body weight at this time-point with levels similar to sham controls. Kidney weights and kidney/body weights in MCP FA-exposed animals two days after FA exposure remained higher than sham controls but were significantly reduced compared to FA animals drinking normal water. By Day 14 of the protocol, the initial swelling in FA-exposed kidneys subsided, with kidney, body and kidney/body weight ratios returning to those observed in sham controls; administration of MCP did not alter kidney/body weight ratios at this time-point. ** indicates p<0.01 between Groups while * indicates p<0.05 between Groups.

Figure 5. Kidney histology two days after FA exposure.

A–C. PAS staining on a representative sham control and FA kidneys after 2 days. Marked kidney damage was noted in all animals administered FA, with flattened proximal and distal tubule epithelia with casts in tubule lumens (indicated by *, g  =  glomerulus). There was no observed difference between FA animals administered normal drinking water (B) or those supplemented with 1% modified citrus pectin (C). D–F. Assessment of proliferation by PCNA immunostaining. D. Occasional positive cells (brown signal, arrows indicate some of these positive cells) were observed in sham controls. E. Kidneys of animals injected with FA showed markedly increased numbers of PCNA positive cells predominately in tubular epithelium. Kidneys from FA animals provided with MCP in the drinking water had significantly decreased numbers of PCNA positive cells (F, p<0.05). Bars are 50 µm.

Figure 6. Histological observations fourteen days after FA nephropathy.

Immunohistochemistry with collagen I (A–C) and III (D–F) demonstrated patchy fibrotic areas 14 days following FA administration (brown signals, g =  glomerulus) and quantification revealed a marked increase compared to sham controls. Collagen I staining, but not collagen III was reduced in FA-animals administered MCP in their drinking water compared to those drinking normal water. The area of the kidney containing macrophages was increased in animals exposed to FA versus sham controls as assessed by F4/80 staining (G–I) and this increase was attenuated in MCP FA mice. Bars are 50 µm in A–F and 100 µm in G–I.

Figure 7. Renal gene expression of fibrotic genes and cytokines.

Quantitative RT-PCR for renal α-SMA (A), collagen I (B), collagen III (C), fibronectin (D), TGFβ-1 (E), MCP-1 (F), IL-1β (G), IL-6 (H) and TNF-α (I) on kidney RNA from animals in Group I (water throughout, NaHCO₃ injection), Group II (water throughout, FA injection) and Group III (MCP throughout and FA injection), two weeks after FA administration (n = 4 in Group I, 6 in Group II and 7 in Group III). All genes were significantly upregulated in FA nephropathy compared to sham controls, but this was attenuated when MCP was provided in the drinking water, except for collagen III, MCP-1 and IL-6. ** indicates p<0.01 while * indicates p<0.05.