Chronic kidney disease (CKD) remains a major public health problem and is one of the leading causes of mortality and morbidity worldwide (1–3). CKD patients are generally asymptomatic, so it is difficult to recognize the early clinical signs or symptoms of CKD (4). Current biomarkers, such as serum blood urea nitrogen, serum creatinine and proteinuria, lack sensitivity for the early detection of the development of CKD (5, 6). Interstitial fibrosis is characterized by the accumulation of collagen-rich extracellular matrix in the interstitium (7, 8). Tubular atrophy is defined as the presence of tubular epithelial thinning with pyknotic nuclei or dilated and thin tubules (9). Interstitial fibrosis and tubular atrophy are considered as the common pathway for CKD progression (9–11). If we can identify these patients in the early disease stage, their renal function decline may be slowed or even reversed. Therefore, it is important to find useful predictive biomarkers to identify individuals at risk for renal fibrosis at the earliest possible stage (12).

Human galectin-3 (Gal-3) is a β-galactoside-binding lectin protein encoded by a single gene (LGALS3) located on chromosome 14 (13). Gal-3 plays a role in signaling in angiogenesis, oncogenic transformation, inflammatory responses and fibrogenic processes (14, 15). Upregulation of Gal-3 expression has been considered a key regulator of inflammation and fibrosis and may contribute to multiple organ fibrosis (16–18). In an in vitro model, Gal-3^−/− fibroblasts showed reduced collagen production in response to transforming growth factor-beta 1 (TGF-β1). Moreover, in an in vivo model, Gal-3 knockout mice exhibited a decrease in collagen accumulation and resistance to the development of fibrosis (19, 20). Because there is increasing evidence that Gal-3 is associated with the development of organ fibrosis (21), Gal-3 may serve as a potential biomarker for renal fibrosis through its possible pathogenic mechanisms of action.

In the general population, data from the Framingham Offspring Study, which involved 2,450 subjects, showed that higher plasma levels of Gal-3 were associated with a higher risk of CKD but not with the risk of albuminuria (21). Other studies found that higher plasma Gal-3 levels were associated with poorer survival, renal insufficiency and late allograft failure (22, 23). However, another study consisting of 7,968 subjects from the Prevention of REnal and Vascular ENd-stage Disease (PREVEND) cohort found that the correlation between Gal-3 and renal function was weak (24). Because some inconsistent results exist, this topic deserves further investigation of the links between plasma Gal-3 levels, Gal-3 gene expression and detailed renal pathologic findings.

To address this problem, we performed a prospective study on patients who underwent kidney biopsy to examine the association between their plasma Gal-3 levels and renal histopathology findings. In addition, we examined the gene expression of Gal-3 using RNA-sequencing analysis in kidney specimens. The present study aimed to (1) characterize the associations between plasma Gal-3 and clinical variables and laboratory results, (2) investigate whether plasma Gal-3 was associated with the pathological findings of CKD, such as renal fibrosis and/or tubular atrophy, and (3) identify the gene expression of Gal-3 using RNA-sequencing analysis in kidney biopsy specimens. We also attempted to perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses to explore the possible pathogenesis and potential pathways of Gal-3 in the development of renal fibrosis.

Our study aims to explore the association between the expression of Gal-3, pathological findings of kidney biopsy and RNA-sequencing analysis. First, we found that higher Gal-3 levels were associated with an increased risk of renal fibrosis, and Gal-3 was inversely associated with eGFR. Second, we further demonstrated that Gal-3 was associated with interstitial fibrosis, tubular atrophy and vascular intimal fibrosis. Finally, the expression of Gal-3 was upregulated in fibrotic kidney biopsy samples in RNA-sequence analysis. GO enrichment analysis found that the DEGs were mainly enhanced in T cell activation, monocyte cell differentiation, and the regulation of cell-cell adhesion. KEGG analyses also found an association between cytokine-cytokine receptor interactions and chemokine signaling pathways. Taken together, our data suggest that Gal-3 may be associated with a risk of lower eGFR and the development of renal fibrosis through the involved immune response and pathway.

The Framingham Offspring Study, which investigated 2,450 participants from the general population with 10 years of follow-up, found that higher levels of Gal-3 were associated with CKD despite adjustment for other confounding factors (21). The Atherosclerosis Risk in Communities Study consisted of 9,148 African American and white men and women without pre-existing heart failure. Higher plasma Gal-3 levels were also associated with an increased risk of incident CKD, and the association became stronger in patients with hypertension (35). In contrast, however, in the Cardiovascular Heart Study including 2,763 subjects, the association between baseline Gal-3 levels and CKD was not statistically significant in the final analyses (36). Our study provided support that Gal-3 was inversely associated with eGFR in our study participants.

A hospital-based study including 205 outpatients found that Gal-3 levels were considered an early marker for renal dysfunction and the progression of cardiorenal syndrome in heart failure. Stepwise regression analysis found that Gal-3 was not associated with microalbuminuria (37). In contrast, the Framingham Offspring Study including 2,450 participants from 1995 to 2008 found that higher Gal-3 levels were associated with higher risks of incident CKD but were not associated with increased risks of albuminuria. Similar to the findings of the Framingham Offspring Study, our study found that Gal-3 was not associated with either the UPCR or UACR.

Although Gal-3 might play a causal role in tissue remodeling and the development of organ fibrosis (38), a previous study focused on kidney biopsy findings in humans remains scarce (39). In our study, we found that Gal-3 was associated with interstitial fibrosis, tubular atrophy and vascular intimal fibrosis, irrespective of the etiology of renal diseases. Gal-3 is widely expressed in immune cells (40–42), especially in monocytes/macrophages, which profoundly affect critical macrophage functions, such as phagocytosis and phenotype transition (43–45). A mouse model of unilateral ureteral obstruction showed that Gal-3 expression was upregulated, and depletion of Gal-3 protected against the accumulation of renal myofibroblasts and further fibrosis (45). Profibrotic microphages also demonstrated upregulation of several profibrotic genes to stimulate the production of fibronectin and extracellular matrix proteins (46). In addition, extracellular Gal-3 also has the ability to regulate T cell function to regulate further immune responses during fibrotic processes (47, 48). In our RNA sequence analysis, we found that LGALS3 was significantly upregulated in fibrotic groups compared to controls. For the GO enrichment analyses, the DEGs were mainly enhanced for monocyte cell differentiation, T cell activation and cell-cell adhesion. The KEGG pathways also showed that DEGs were enhanced in cytokine-cytokine receptor interactions and chemokine signaling pathways. Laminin, fibronectin and integrins are known ligands for Gal-3, and therefore, Gal-3 may promote pathological fibrotic processes by regulating cell-cell adhesion and cell proliferation (49, 50). Therefore, our study may provide some evidence that Gal-3 plays a multifaceted role in the regulation of inflammatory responses to promote cell-cell interactions and adhesions, which contributes to renal fibrosis that is associated with renal failure.

The strengths of our study are the well-characterized sample, completeness of the data collection and verification of participating patients. Our study showed a positive correlation between Gal-3 and the risk of renal fibrosis. We provided new data indicating that patients with higher levels of Gal-3 have a significantly higher risk of interstitial fibrosis, vascular intimal fibrosis and tubular atrophy than those with lower levels of Gal-3. Another major strength of this study is that it provides RNA-sequencing evidence of the gene expression of Gal-3 and renal pathological findings, which lends further credence to the study findings.

Our study has some limitations that should be considered. All eGFRs were measured once at the time of kidney biopsy. Therefore, it is possible that acute kidney injury or variability in renal function may not have been detected in our analyses. However, all participants in our study had undergone kidney biopsy, and therefore, the definitive etiology of CKD may be assured. Detailed biopsy information, such as the degree of tubular atrophy and interstitial fibrosis, was also identified in our study. Second, the data of our participants were based on a tertiary medical center, which may include more complicated cases with more underlying diseases, and thus, this may limit the generalizability of our results. Finally, we did not perform serial measurements of plasma Gal-3 over time, so whether the change in Gal-3 was associated with renal function progression was unknown. Further prospective studies may be needed to confirm our results and the potential ability of Gal-3 as a biomarker of CKD progression.

In conclusion, we found an association between plasma Gal-3 and the risk of renal fibrosis in kidney biopsy specimens. Gal-3 may be considered a potential biomarker for detecting renal fibrosis, potentially affording an opportunity for early intervention to prevent disease progression. However, the precise mechanisms of Gal-3 in the development of CKD warrant further investigation.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/bioproject/770300. Accession number(s):PRJNA770300.

The studies involving human participants were reviewed and approved by Taipei Veteran General Hospitals (2018-06-008B). The patients/participants provided their written informed consent to participate in this study.

S-MO, M-TT, H-YC, F-AL, W-CT, K-HL, F-PC, Y-PL, R-BY, and D-CT: conception and study design. S-MO, M-TT, H-YC, F-AL, W-CT, K-HL, F-PC, and D-CT: data acquisition. S-MO, M-TT, R-BY, and D-CT: data analysis/interpretation and statistical analysis. S-MO, M-TT, H-YC, F-AL, W-CT, K-HL, F-PC, Y-PL, R-BY, and D-CT: drafting of the manuscript. All authors contributed to the article and approved the submitted version.

This work was supported in part by the Ministry of Science and Technology, Taiwan (MOST 106-2314-B-010-039-MY3, MOST 107-2314-B-075-052, MOST 108-2314-B-075-008, MOST 109-2314-B-075−067-MY3, MOST 109-2320-B-075-006, MOST 110-2321-B-A49-003); Taipei Veterans General Hospital (V107B-027, V108B-023, V108C-103, V108D42-004-MY3-1, V108D42-004-MY3-2, V108D42-004-MY3-3, V109B-022, V109C-114, V109D50-001-MY3-1, V109D50-001-MY3-2, V110C-152, V110E-003-2); Taipei, Taichung, Kaohsiung Veterans General Hospital, Tri-Service General Hospital, Academia Sinica Joint Research Program (VTA110-V1-3-1); Taipei Veterans General Hospital-National Yang-Ming University Excellent Physician Scientists Cultivation Program (No. 104-V-B-044); Institute of Biomedical Sciences, Academia Sinica (IBMS-CRC110-P04 and AS-VTA-110-03) and Foundation for Poison Control (FPC-109-002).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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