MCP is a derivative of pectin; a soluble dietary fibre found in the peel and pulp of citrus fruits [20], and has inhibitory effects on the progression of several animal models of cancer [24], [30]. MCP is rich in β-galactoside residues and binds to the carbohydrate recognition domain of galectin-3 [22], [23] thereby impairing the lectin’s carbohydrate binding-related functions. Therefore, it could be postulated that if MCP acted only through galectin-3 in FA nephropathy it would not have any effect on intracellular actions including proliferation and apoptosis, while modulating extracellular functions such as inflammation. This proved not the case; MCP treatment reduced tubular proliferation two days following FA administration with no differences in galectin-3 expression.

Most studies have only considered the effects of MCP in relation to galectin-3, but many other pathways could modulate proliferation here, such as MAP kinase activation [31]. One could speculate that an alternative explanation is that it is not just galectin-3 levels but also bioavailability that should be considered. It is possible that similar levels of galectin-3 have less biological effects when MCP is present because its carbohydrate binding roles will be abrogated. This cannot be measured in-vivo at present, but in-vitro studies have shown that both cell migration [24] and agglutination [32] are diminished in the presence of MCP when induced with similar concentrations of galectin-3. The reduced proliferation could also suggest renal recovery is slower in MCP-treated mice or alternatively that MCP may protect the kidney against structural injury. This second explanation is supported by the fact MCP mice have preserved body weight and their kidneys did not exhibit such acute gross swelling as those exposed to FA but maintained on water alone.

MCP decreased renal mRNA and protein levels of galectin-3 at 14 days after FA injection, in concert with significantly improved renal fibrosis as assessed by reduced expression of multiple fibrotic genes. MCP had no effect on galectin-1 and galectin-9, which are also expressed in the kidney [33], [34], suggesting these effects do not result from MCP interactions with other galectins. The direct correlation of less galectin-3 with less renal injury is consistent with studies by Henderson and colleagues [35] wherein mice lacking galectin-3 have less fibrosis and decreased collagen and α-SMA expression seven days after UUO. In contrast, a recent paper reported that fibrosis severity was increased by day 14 post UUO in galectin-3 deficient mice [11]. These observations may not relate directly to our model, however, because the pathology of chronic UUO is very different to FA-induced injury and both of these studies focussed on knock-out mice where both carbohydrate binding-dependent and -independent functions are abrogated.

The growth factor TGF-β plays a key role in the progression of renal fibrosis by promoting myofibroblastic differentiation [36] and galectin-3 has been implicated in this type of differentiation and extracellular matrix production in hepatic stellate cells [37]. MCP reduced TGF-β mRNA here, which may have contributed to reduced myofibroblast formation as evidenced by decreased α-SMA levels. An unexpected finding was the lack of significant differences in collagen III expression with MCP, which contrasts with the other profibrotic factors examined.

A correlation between galectin-3 and collagen I, but not with collagen III, was recently reported by Okamura and colleagues [11]: they noted that lack of galectin-3 led to a relative fall in collagen I but not III over time in UUO. Sharma et al. also observed differential effects of galectin-3 on different collagens [38]: they initially found that galectin-3 was strongly upregulated in an animal model of heart failure, and then infused galectin-3 into the pericardial sac of healthy rats to induce left ventricular dysfunction; this led to a 3-fold increase of collagen I over collagen III. A potential mechanism may be that the collagen I promoter, contains a high number of Sp-1 binding sites [39] through which galectin-3 might act [3] but these are not present for collagen III.

MCP also reduced inflammatory responses at day 14 of FA nephropathy which was accompanied by a loss of galectin-3 positive interstitial cells in the kidney. Galectin-3 is upregulated in several other renal diseases with prominent inflammatory components including systemic lupus erythematosus [40], experimental glomerulonephritis [41] and unilateral ureteral obstruction [11], [35]. An alternative name for galectin-3 is Mac-2 as it is abundantly expressed by subsets of macrophages [42]. Galectin-3 can act in an autocrine or parcrine fashion to induce monocyte migration [43] or alternative macrophage activation [7] and regulate cytokine expression [44]. Therefore, as part of this process is mediated by extracellular actions of galectin-3 [9], we propose that MCP effects on inflammation in FA-nephropathy may in part be mediated by galectin-3.

We also observed a small but highly significant reduction in apoptosis with MCP in the later disease phase. This difference is likely to be biologically important because Coles and colleagues [45] previously demonstrated that small changes in measured apoptosis in the kidney actually reflect larger overall changes in cell death due to apoptotic cells being cleared so quickly. Galectin-3 and MCP have different effects on apoptosis: galectin-3 has a BH1 domain of BCL2 that can protect cells from apoptosis [46], [47] and transfection with the lectin makes T-cells resistant to this type of cell death [48]; MCP, in contrast, promotes apoptosis in angiosarcoma [49] and prostate cancer cell lines [31]. This pro-apoptotic effect of MCP is formulation-dependent, however, because alterations in pH and heat-treatment (as we used here to prepare the MCP) can abrogate these pro-apoptotic effects [50]. We suspect that our observed reduction in apoptosis is not directly related to MCP actions on galectin-3 but simply reflects the reduction in disease severity with the modified pectin leading to less remodelling being required; in this case, decreased early proliferation might generate less ‘unwanted’ cells that need to be deleted by apoptosis later.

In conclusion, our data indicates that MCP is protective in experimental nephropathy through modulation of proliferation, apoptosis, fibrosis and inflammation. However, there are two caveats that need to be considered when interpreting this study. Firstly, it is possible that MCP affects other molecular pathways other than galectin-3 in FA nephropathy which need to be investigated further. Secondly, we need to exert caution when interpreting the effect of MCP on galectin-3 levels as these measurements do not account for the bioavailability of the lectin which may be altered. Nevertheless, this study does identify a new potential therapy for acute kidney injury and further experiments are warranted to examine effects of different doses of MCP, timing of treatment and roles in other renal diseases.