Functional transposition of renal functions to the posterior intestine during maturation in male three-spined stickleback

Abstract

During the breeding season, the male stickleback proximal tubule of the kidney undergoes hypertrophy. This is due to the synthesis of the nest building protein spiggin, in response to increased levels of 11-ketotestosterone. The increased protein synthesis that is initiated during breeding alters the kidney function and the ability to secrete excess water, to osmoregulate, in fresh water. It has earlier been shown that there exist organ specific differences in transport proteins between mature and non-mature three-spined stickleback. To understand the molecular mechanisms compensating for kidney functions, this study examined transport genes responsible for functional changes between the kidney and intestine. RNA sequencing was performed on castrated and 11-ketoandrostenedione (11KA)-treated male stickleback. Results showed organ-specific responses: 2,549 differentially expressed genes (DEGs) in the kidney and 885 in the posterior intestine, with 210 shared between the organs. Solute transporters, aquaporin 10a and cadherin-17, were upregulated in the posterior intestine but downregulated in the kidney in 11KA treated males. Enrichment analysis revealed distinct biological processes, primarily involving solute transporters, indicating functional adaptation. While amino acid and ion transport were downregulated in the kidney, compensatory transport was observed in the posterior intestine. However, cellular hexose transporters were downregulated in both organs, suggesting a reduction in glucose absorption and passive water diffusion. The present study shows that androgens alter the expression of cellular transporters and redirect functions of the kidney to the posterior intestine. The results also indicate reduced glucose absorption in breeding, male three-spined stickleback.

Keywords: 11-ketoandrostenedione; Androgen; Glucose; Kidney; Solute transporters.

Fig. 1

(A) Principal component analysis was conducted using normalized transcripts with equal feature contribution. (B) Venn diagram shows DEGs in the kidney and posterior intestine of the male three-spined stickleback. Genes showing significant fold changes between 11 KA-treated and castrated males in each organ are shown (p < 0.05, FDR ≤ 0.1, log2 FC ≤ −1.2 or ≥ 1.2). CAS_K (Castrated kidney); 11 KA_K (11 KA-treated kidney); CAS_PI (Castrated posterior intestine), 11 KA_PI (11 KA-treated posterior intestine); CAS_AI (Castrated anterior intestine); 11 KA_AI (11 KA-treated anterior intestine); K_up (upregulated in the kidney), K_down (Downregulated in the kidney), PI_up (upregulated in the posterior intestine), PI_down (Downregulated in the posterior intestine).

Fig. 2

qPCR was conducted to compare the expression levels of the observed DEGs. Genes showing significant fold changes between 11 KA-treated and castrated males in each organ are indicated by red (upregulated) and green (downregulated) (*= p < 0.05 indicate difference between castrated and 11 KA implanted fish), nd; not detected, n = 4.

Fig. 3

KEGG and Reactome analyses were performed to identify affected transport pathways in the kidney and posterior intestine. The number of upregulated and downregulated DEGs in each tissue are represented by red and green, respectively. Genes showing significant fold changes between 11 KA-treated and castrated males in each organ are indicated by red (upregulated) and green (downregulated) gradients (*= p < 0.05, FDR ≤ 0.1, FC ≤ −1.2 or ≥ 1.2).

Fig. 4

Biological gene ontology was constructed in the STRING database using DEGs for each tissue. (A) Kidney, (B) posterior intestine, (C) anterior intestine. False discovery rate (FDR)-based significance level was used to rank enriched terms. The top ten enriched terms in biological process (BP), molecular function (MF), and cellular component (CC) ontologies are shown. Enrichment is -log10 (FDR). The color and size of each dot represents the significance level of each term and the number of genes that enriched them. Genes showing significant fold changes between 11 KA-treated and castrated males in each organ are indicated by red (highly significant) and green (low significant) gradients.

Fig. 5

The expression levels of DEGs in enriched transport ontology terms common in the kidney and posterior intestine are shown. (A) Transport biological process, (B) transport molecular function activity, and (C) membrane cellular component terms were highly enriched in tissues with genes that showed distinct differences in expression levels between the kidney and posterior intestine. (D) Genes upregulated in the kidney or downregulated in the posterior intestine are shown. Genes showing significant fold changes between 11 KA-treated and castrated males in each organ are shown.

Fig. 6

Gene counts of solute transporters in the kidney and posterior intestine. (A) Inorganic ion transporters, (B) cellular hexose transporters, (C) vitamins, nucleosides, and related molecule transporters, (D) amino acid transporters, and (E) bile salts, organic acids, metal ions, and amine transporters. Genes showing significant fold changes between 11 KA-treated and castrated males in each organ are shown (*= p < 0.05, FDR ≤ 0.1, FC ≤ −1.2 or ≥ 1.2).

Fig. 7

Scatter plots comparing the regulation of transporters between the kidney and posterior intestine. The ratio of gene counts from the 11 KA-treated group normalized against the castrated group. (A) Inorganic cation/anion transporters, (B) cellular hexose transporters, (C) vitamins, nucleosides, and related transporters, (D) amino acid transporters, and (E) bile salts, organic acids, metal ions, and amine transporters. Gene regulation in the posterior intestine is shown in the color gradient, with upregulated genes represented by red and downregulated or unexpressed genes represented by green.

Fig. 8

Heatmap showing the differential regulation of analogs and paralogs of (A) aquaporins and (B) cell junction and vesicle transport proteins. Genes showing significant fold changes between 11 KA-treated and castrated males in each organ are indicated by red (upregulated) and green (downregulated) gradients (*= p < 0.05, FDR ≤ 0.1, FC ≤ −1.2 or ≥ 1.2). In each organ, non-expressed genes were shown by white shading. K-Kidney; PI-Posterior intestine.