CCN2 Binds to Tubular Epithelial Cells in the Kidney

Abstract

Cellular communication network-2 (CCN2), also called connective tissue growth factor (CTGF), is considered a fibrotic biomarker and has been suggested as a potential therapeutic target for kidney pathologies. CCN2 is a matricellular protein with four distinct structural modules that can exert a dual function as a matricellular protein and as a growth factor. Previous experiments using surface plasmon resonance and cultured renal cells have demonstrated that the C-terminal module of CCN2 (CCN2(IV)) interacts with the epidermal growth factor receptor (EGFR). Moreover, CCN2(IV) activates proinflammatory and profibrotic responses in the mouse kidney. The aim of this paper was to locate the in vivo cellular CCN2/EGFR binding sites in the kidney. To this aim, the C-terminal module CCN2(IV) was labeled with a fluorophore (Cy5), and two different administration routes were employed. Both intraperitoneal and direct intra-renal injection of Cy5-CCN2(IV) in mice demonstrated that CCN2(IV) preferentially binds to the tubular epithelial cells, while no signal was detected in glomeruli. Moreover, co-localization of Cy5-CCN2(IV) binding and activated EGFR was found in tubules. In cultured tubular epithelial cells, live-cell confocal microscopy experiments showed that EGFR gene silencing blocked Cy5-CCN2(IV) binding to tubuloepithelial cells. These data clearly show the existence of CCN2/EGFR binding sites in the kidney, mainly in tubular epithelial cells. In conclusion, these studies show that circulating CCN2(IV) can directly bind and activate tubular cells, supporting the role of CCN2 as a growth factor involved in kidney damage progression.

Trial registration: ClinicalTrials.gov NCT02606136 NCT04229004 NCT03955146.

Keywords: CCN2; CTGF; EGFR; kidney damage.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1

Evaluation of CCN2(IV) binding to renal tissue in the model of intra-renal injection. C57BL/6 mice were injected with Cy5-CCN2(IV) (dose of 2.5 ng/g of body weight, n = 3 mice per group) in the right kidney and sacrificed at different times (from 10 to 30 min). Frozen OCT-embedded renal samples were used for confocal microscopy. Red immunostaining (corresponding to CCN2(IV) labeled with Cy5 fluorophore) was found in tubular epithelial cells, showing CCN2(IV) binding to renal cells. Nuclei were stained with DAPI to distinguish renal structures. The overlaid images in red and blue (merge) indicated CCN2(IV) localization mainly in the membrane of tubuloepithelial cells. Figures show one representative mouse per group, and scale bar represents 5 μM.

Figure 2

CCN2 (IV) binding was located in the tubuli but not in other renal structures in the kidney. Mice were injected with Cy5-CCN2(IV) in the right kidney and studied 30 min later. Red immunostaining was only found in the tubuli but was not present in the glomeruli. Renal tissue autofluorescence (green) and DAPI staining (blue) show renal structures. The overlaid images indicate CCN2(IV) localization only in the tubuli. Figures show a representative mouse per group, and scale bar represents 20 μM.

Figure 3

Evaluation of CCN2(IV) binding to the kidney in the model of systemic administration in mice. C57BL/6 mice were i.p. injected with 2.5 ng/g of body weight of recombinant Cy5- CCN2(IV) and sacrificed 30, 45, and 60 min later. In the kidneys from Cy5-CCN2(IV)-injected mice, red immunostaining corresponding to CCN2(IV) labeled with Cy5 fluorophore (red staining) was observed by confocal microscopy. Nuclei were stained with DAPI to locate renal structures. The overlaid images in red and blue (merge) indicated CCN2 (IV) localization mainly in the membrane of tubular epithelial cells. Figures show a representative mouse per group of 3 done, and scale bar represents 5 μM.

Figure 4

Determination of CCN2(IV) binding to proximal or distal tubuli in the kidney in the model of systemic administration of CCN2(IV). Co-localization of Cy5-CCN2(VI) (red) staining with markers of proximal (LotusTetragonolobus Lectin, green) or distal (Dolichos Biflorus Lectin, green) tubuli in CCN2(IV)-injected mice. Proximal tubuli are the main sites of Cy5-CCN2 binding sites. The scale bar represents 5 μM.

Figure 5

Localization of CCN2(IV) binding and EGFR activation in tubuloepithelial cells in vivo. C57BL/6 mice were injected with CCN2(IV)-Cy5 (2.5 ng/g of body weight, n = 3 mice per group) in the right kidney, using its corresponding contralateral kidney as control and sacrificed at 30 min. Figure shows Cy5-CCN2(IV) binding in red, activated EGFR in green (corresponding to phosphorylated EGFR (Alexa fluor® 488)), and nuclei in blue (DAPI). The overlaid images show the colocalization of CCN2(IV) with EGFR activated in several tubule epithelial cells. A magnification is shown on the right. Figures show one representative mouse per group, and scale bar represents 5 μM.

Figure 6

In vitro binding of Cy5-CCN2(IV) to tubular epithelial cells via EGFR. Serum-starved human tubular epithelial cells were transfected with a siRNA against EGFR or its corresponding scramble control siRNA, both FAM-labeled (green) and then stimulated with Cy5-CCN2(IV) for 10 min. Only cells expressing EGFR can bind to Cy5-CCN2(IV) (red signal), mainly located in the cellular membrane, whereas this signal is not found in EGFR-silenced cells. The scale bar represents 20 μM.