Segment-Dependent Gene Expression Profiling of the Cartilaginous Fish Nephron Using Laser Microdissection for Functional Characterization of Nephron at Segment Levels.

For adaptation to a high salinity marine environment, cartilaginous fishes have evolved a ureosmotic strategy. They have a highly elaborate "four-loop nephron" in the kidney, which is considered to be important for reabsorption of urea from the glomerular filtrate to maintain a high concentration of urea in the body. However, the function and regulation, generally, of the "four-loop nephron" are still largely unknown due to the complicated configuration of the nephron and its many subdivided segments. Laser microdissection (LMD) followed by RNA-sequencing (RNA-seq) analysis is a powerful technique to obtain segment-dependent gene expression profiles. In the present study, using the kidney of cloudy catshark, Scyliorhinus torazame, we tested several formaldehyde-free and formaldehyde-based fixatives to optimize the fixation methods. Fixation by 1% neutral buffered formalin for 15 min resulted in sufficient RNA and structural integrities, which allowed LMD clipping of specific nephron segments and subsequent RNA-seq analysis. RNA-seq from the LMD samples of the second-loop, the fourth-loop, and the five tubular segments in the bundle zone revealed a number of specific membrane transporter genes that can characterize each segment. Among them, we examined expressions of the Na + -coupled cotransporters abundantly expressed in the second loop samples. Although the proximal II segment of the second loop is known for the elimination of excess solutes, the present results imply that the PII segment is also crucial for reabsorption of valuable solutes. Looking ahead to future studies, the segment-dependent gene expression profiling will be a powerful technique for unraveling the renal mechanisms and regulation in euryhaline elasmobranchs.

Molecular and morphological investigations on the renal mechanisms enabling euryhalinity of red stingray Hemitrygon akajei.

Most cartilaginous fishes live in seawater (SW), but a few exceptional elasmobranchs (sharks and rays) are euryhaline and can acclimate to freshwater (FW) environments. The plasma of elasmobranchs is high in NaCl and urea concentrations, which constrains osmotic water loss. However, these euryhaline elasmobranchs maintain high levels of plasma NaCl and urea even when acclimating to low salinity, resulting in a strong osmotic gradient from external environment to body fluid. The kidney consequently produces a large volume of dilute urine to cope with the water influx. In the present study, we investigated the molecular mechanisms of dilute urine production in the kidney of Japanese red stingray, Hemitrygon akajei, transferred from SW to low-salinity environments. We showed that red stingray maintained high plasma NaCl and urea levels by reabsorbing more osmolytes in the kidney when transferred to low salinity. RNA-seq and qPCR analyses were conducted to identify genes involved in NaCl and urea reabsorption under the low-salinity conditions, and the upregulated gene expressions of Na+-K+-Cl- cotransporter 2 (nkcc2) and Na+/K+-ATPase (nka) were found in the FW-acclimated individuals. These upregulations occurred in the early distal tubule (EDT) in the bundle zone of the kidney, which coils around the proximal and collecting tubules to form the highly convoluted structure of batoid nephron. Considering the previously proposed model for urea reabsorption, the upregulation of nkcc2 and nka not only causes the reabsorption of NaCl in the EDT, but potentially also supports enhanced urea reabsorption and eventually the production of dilute urine in FW-acclimated individuals. We propose advantageous characteristics of the batoid-type nephron that facilitate acclimation to a wide range of salinities, which might have allowed the batoids to expand their habitats.

Facilitated NaCl Uptake in the Highly Developed Bundle of the Nephron in Japanese Red Stingray Hemitrygon akajei Revealed by Comparative Anatomy and Molecular Mapping.

Batoidea (rays and skates) is a monophyletic subgroup of elasmobranchs that diverged from the common ancestor with Selachii (sharks) about 270 Mya. A larger number of batoids can adapt to low-salinity environments, in contrast to sharks, which are mostly stenohaline marine species. Among osmoregulatory organs of elasmobranchs, the kidney is known to be dedicated to urea retention in ureosmotic cartilaginous fishes. However, we know little regarding urea reabsorbing mechanisms in the kidney of batoids. Here, we performed physiological and histological investigations on the nephrons in the red stingray (Hemitrygon akajei) and two shark species. We found that the urine/plasma ratios of salt and urea concentrations in the stingray are significantly lower than those in cloudy catshark (Scyliorhinus torazame) under natural seawater, indicating that the kidney of stingray more strongly reabsorbs these osmolytes. By comparing the three-dimensional images of nephrons between stingray and banded houndshark (Triakis scyllium), we showed that the tubular bundle of stingray has a more compact configuration. In the compact tubular bundle of stingray kidney, the distal diluting tubule was highly developed and frequently coiled around the proximal and collecting tubules. Furthermore, co-expression of NKAα1 (Na+/K +-ATPase) and NKCC2 (Na+- K+-2Cl- cotransporter 2) mRNAs was prominent in the coiled diluting segment. These findings imply that NaCl reabsorption is greatly facilitated in the stingray kidney, resulting in a higher reabsorption rate of urea. Lowering the loss of osmolytes in the glomerular filtrate is likely favorable to the adaptability of batoids to a wide range of environmental salinity.