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. 2022 Jun 15;9(6):296.
doi: 10.3390/vetsci9060296.

Analysis of the Expression of Neurotrophins and Their Receptors in Adult Zebrafish Kidney

Pietro Cacialli  1 , Carla Lucini  2
Affiliations

Analysis of the Expression of Neurotrophins and Their Receptors in Adult Zebrafish Kidney

Pietro Cacialli et al. Vet Sci. .

Abstract

Neurotrophins and their receptors are involved in the development and maintenance of neuronal populations. Different reports have shown that all neurotrophin/receptor pathways can also play a role in several non-neuronal tissues in vertebrates, including the kidney. These signaling pathways are involved in different events to ensure the correct functioning of the kidney, such as growth, differentiation, and regulation of renal tubule transport. Previous studies in some fish species have identified the neurotrophins and receptors in the kidney. In this study, for the first time, we compare the expression profiles (mRNA and protein) of all neurotrophin/receptor pathways in the kidney of the adult zebrafish. We quantify the levels of mRNA by using qPCR and identify the expression pattern of each neurotrophin/receptor pathway by in situ hybridization. Next, we detect the proteins using Western blotting and immunohistochemistry. Our results show that among all neurotrophins analyzed, NT-3/TrkC is the most expressed in the glomerule and tubule and in the hematopoietic cells, similar to what has been reported in the mammalian kidney.

Keywords: Trk receptor; kidney; neurotrophin; zebrafish.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Analysis of neurotrophin and receptor expression by qPCR. (a) qPCR analysis for neurotrophic factors: ngf; bdnf; nt3; nt4. ngf is more expressed than nt4 (not significatively expressed), but less than bdnf. nt3 is highly expressed compared with previous neurotrophins (* p < 0.018; ** p < 0.006; **** p < 0.0001). (b) qPCR analysis for receptors of tyrosine kinase: trkA; trkB; trkC. trkA is more expressed than trkB. However, trkC is highly expressed in the kidney compared with the expression of other receptors (* p < 0.020; ** p < 0.001; *** p < 0.0001).
Figure 2
Figure 2
Chromogenic and fluorescence in situ hybridization for ngf and bdnf on cryostat and paraffin sections of adult zebrafish kidney. (a) Chromogenic and (b) fluorescence ISH for ngf-mRNA in adult zebrafish kidney; it is expressed in proximal tubules (indicated with abbreviation PT). (c) Chromogenic and (d) fluorescence ISH for Bdnf-mRNA in adult zebrafish kidney; it is expressed in distal tubules (DT). Scale bars: 100 μm (a,c); 50 μm (b,d).
Figure 3
Figure 3
Chromogenic and fluorescence in situ hybridization for neurotrophins nt3 and nt4 on cryostat and paraffin sections of adult zebrafish kidney. (a) Chromogenic and (b) fluorescence ISH for nt3 in adult zebrafish kidney; it is highly expressed in different regions: proximal and distal tubules, interstitial hematopoietic cells (respective abbreviations: PT, DT, and Hc). (c) Chromogenic and (d) fluorescence ISH for nt4; it is not detected in adult zebrafish kidney. Scale bars: 100 μm (a–d).
Figure 4
Figure 4
Chromogenic and fluorescence in situ hybridization for tyrosine kinase receptors trka, trkb, and trkc on cryostat and paraffin sections of adult zebrafish kidney. (a) Chromogenic and (b) fluorescence in situ hybridization of trkA-mRNA in adult zebrafish kidney; it is expressed in proximal tubules (PT). (c) Chromogenic and (d) fluorescence in situ hybridization of trKB-mRNA and cell nuclei marked with DAPI staining in adult zebrafish kidney; trkb is expressed in hematopoietic cells (Hc). (e) Chromogenic and (f) fluorescence in situ hybridization of trkC-mRNA; it is expressed in hematopoietic cells, and proximal and distal tubules (respectively, Hc, PT, and DT). Scale bars: 100 μm (a–f).
Figure 5
Figure 5
Western blotting analysis for neurotrophins and receptors in adult zebrafish kidney. (a) Western blot to detect BDNF, NGF, NT-3, and NT-4 in adult zebrafish kidney. Two bands for BDNF of 14 kD and 37 kD; 3 bands for NGF of 30 kD, 34 kD, and 37 kD; 3 bands for NT-3 of 30 kD, 34 kD, and 37 kD. NT4 is not detected. (b) Western blot to detect TrkA, TrkB, and TrkC in adult zebrafish kidney. Three bands for TrKA at 75 kD, 115 kD, and 140 kD. One band of 140 kD for TrkB and 2 bands of 115 kD and 140 kD for TrkC.
Figure 6
Figure 6
Immunohistochemistry to detect NGF and BDNF on paraffin sections of adult zebrafish kidney. (a) NGF protein in adult zebrafish kidney is detected in distal and proximal tubules (respectively, DT and PT). (b) BDNF protein is distributed in distal tubules and a few hematopoietic cells (DT and Hc). Scale bars: 200 μm and 50 μm (a,b).
Figure 7
Figure 7
Immunohistochemistry to detect NT-3 and NT-4 on paraffin sections of adult zebrafish kidney. (a) NT-3 protein in adult zebrafish kidney is detected in distal and proximal tubules, and hematopoietic cells (DT, PT, and Hc). (b) NT-4 protein is not detected. Scale bars: 200 μm and 50 μm (a,b).
Figure 8
Figure 8
Immunohistochemistry to detect TrkA, TrkB, and TrkC on paraffin sections of adult zebrafish kidney. (a) All receptors are expressed in adult zebrafish kidney in different regions. TrkA is expressed in the collecting duct and proximal tubule. (b) TrkB is detected in hematopoietic cells. (c) TrkC protein is abundantly distributed in different regions: hematopoietic cells; distal and proximal tubules (Hc, DT, and PT). Scale bars: 200 μm and 50 μm (a–c).
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