Fish kidney cells show higher tolerance to hyperosmolality than amphibian

Lang Gui ,Tao Li ,Qiya Zhang

a College of Fisheries and Life Science,Shanghai Ocean University,Shanghai 201306,China

b State Key Laboratory of Freshwater Ecology and Biotechnology,Institute of Hydrobiology,Chinese Academy of Sciences,Wuhan 430072,China

1.Introduction

The Chinese giant salamander(Andrias davidianus)belongs to the family Cryptobanchidae and is the world's largest amphibian only found in China(National environmental protection agency and Endangered species scientific commission.P.R.C.,1998).As a living fossil with over 350 million years of existence,the evolutionary history of this species involves adaptations from aquatic to terrestrial environments(Gao & Shubin,2003;Heiss,Natchev,Gumpenberger,Weissenbacher,& Van Wassenbergh,2013).Terrestrial amphibians are more tolerant to dehydration than other vertebrates(Hillyard,1999).Specifically,the Chinese giant salamander seems to endure what appears to be osmotic stress under normal conditions(dry environment),making this species a valuable model for research on osmotic stress.However,the osmoregulatory capacity of the Chinese giant salamander is not yet well understood.

The grass carp(Ctenopharyngodon idella)is one of the most important teleost species in the Chinese fishery industry,accounting for 18%of the freshwater aquaculture production,with an output of 4.22 million tons in 2010.Previously,we have used the grass carp C.idella kidney cell line(CIK),established in 1986(Zuo,Qian,Xu,Du,&Yang,1986),to examine the osmoregulatory response(Gui et al.,2016).The kidney plays a key role in maintaining osmoregulation in fishes(Varsamos,Nebel,&Charmantier,2005)as in amphibians(Hillyard,1999).For thisreason,CIK cell line and Chinese giant salamander kidney cell line(GSK)(Yuan,Chen,Huang,Gao,&Zhang,2015)were used to assess the osmoregulatory capacity of the kidney in freshwater fish and amphibian in vitro.

Anisosmotic environments cause an imbalance between extracellular and intracellular compartments and cell morphology can be affected.In a previous study,we have described the effect of osmotic stressin kidney cell morphology(Gui et al.,2016).Here,we describe kidney cell morphology changes in the freshwater grass carp and the Chinese giant salamander after anisosmotic treatments detected by optical and transmission electron microscopy.The results show that freshwater fishes and amphibians have different osmoregulatory capacities.

2.Materials and methods

2.1.Cells and culture conditions

CIK and GSK cells were maintained at the state key laboratory of freshwater ecology and biotechnology,institute of hydrobiology,Chinese Academy of Sciences.CIK was established in 1986(Zuo et al.,1986)and CSK was initially established in the lab in 2015(Yuan et al.,2015).

CIK and GSK cells were grown in TC199 medium supplemented with 10%foetal bovine serum(FBS;Gibco BRL,Grand Island,NY,USA)at 25°C.The osmolarity of the cell culture medium was modified(increased or decreased)by adding double-distilled H2O or NaCl.The osmolarity of the prepared medium was measured by a Vapro 5520 pressure osmometer(Wescor;Logan,UT).The osmolality of the standard medium was 300 m Osm,whereas that of hyposmolar and hyperosmolar media were 100 and 700 m Osm,respectively.Cells were seeded in plastic flasks and grown to approximately 90%con fluency and the standard cell medium was replaced with media of varied osmolarities(100,300 and 700 m Osm)and cells were cultured for 16 h under these conditions.

2.2.Optical and electron microscopy observations

For optical microscopy,cells were observed by phase contrast microscopy or stained with crystal violet solution and observed under a Leica DM IRB light microscope(Leica,Wetzlar,Germany).

For transmission electron microscopy(TEM),cells were harvested by scraping and were subsequently centrifuged at 2000×g for 5 min.Cell pellets were pre fixed with 2.5%glutaraldehyde,post fixed with 1%osmium tetroxide,dehydrated using a graded series of ethanol and then embedded in Epon-812.Blocks were sectioned using a Leica Ultracut Rmicrotome and ultrathin sections(60 nm)were double stained with 1%uranyl acetate and lead citrate,and examined under a JEM-1230 electron microscope(JEOL,Tokyo,Japan)at 80 k V.

Fig.1.Images obtained by phase contrast microscopy showing the effect of the different osmotic pressures on the morphology of CIK and GSK cells after 16 hours:(A,B)hyposmotic medium(100 mOsm);(C,D)isosmotic medium(300 m Osm,control);and(E,F)hyperosmotic medium(700 mOsm).Bars=40μm.

Fig.2.Optical microscopy images showing the effects of the different osmotic pressures on CIK and GSK cells after 16 h of exposure:(A,B)hyposmotic medium(100 m Osm);(C,D)isosmotic medium,(300 m Osm,control)and(E,F)hyperosmotic medium(700 m Osm).Arrows point to cytoplasmic vacuoles in unhealthy cells.Cells were stained with crystal violet solution.Bars=20μm.

3.Results

3.1.Morphological changes of kidney cells

3.1.1.Optical microscopy

Both CIK and GSK cells had an epithelial-like morphology in an isosmotic medium(300 m Osm)(Fig.1Cand D,Fig.2Cand D).After the hyposmotic treatment(100 m Osm),CIK and GSK cells swelled(Fig.1A and B)and numerous cytoplasmic vacuoles were observed(Fig.2A and B).Subsequent to the hyperosmotic treatment(700 m Osm),most of the fish and amphibian cells detached(Fig.1Eand F),and the attached CIK cells possessed a multipolar shape(Fig.2E)and GSK cells possessed a spherical shape(Fig.2F).

Fig.3.Transmission electron microscopy image showing the effects of different osmotic pressures on CIK and GSK cells after 16 h:(A-D)hyposmotic medium(100 m Osm);(E,F)isosmotic medium(300 m Osm,control);and(G-I)hyperosmotic medium(700 m Osm).N:Nucleus.Stars point to condensed chromatin.Arrows point to swollen mitochondria.Ultrathin sections.Bars=2μm.

Table 1 Effects of the osmotic stress on CIK and GSK morphology and intracellular content.

3.1.2.Electron microscopy

The images of electron microscopy revealed that CIK(Fig.3E)and GSK cells(Fig.3F)cultured in isosmotic medium(300 m Osm)had an epithelial-like morphology.In hyposmotic medium(100 m Osm),condensed chromatin(Fig.3A and B)and swollen mitochondria(Fig.3C and D)were detected in both CIK and GSK cells,and for CIK,cell debris(Fig.3A)and numerous cytoplasmic vacuoles(Fig.3C)were also observed.By increasing the osmoticity(700 m Osm),CIK cells exhibited pale or lightly stained cytoplasm,condensed chromatin (Fig.3G),swollen mitochondria and numerous cytoplasmic vacuoles(Fig.3H).In contrast,GSK cells were seriously damaged after cell lysis and extensive cell debris and defective organelle was detected(Fig.3I).

3.2.Comparison of CIK and GSK cells after anisosmotic treatment

The effects of different osmotic pressure on CIK and GSK cells was observed by optical and electron microscopy and changes in cell morphology and intracellular content were compared and shown in Table 1.In isosmotic medium,both CIK and GSK cells presented a similar epithelial-like morphology and became unhealthy after osmotic stress.Both CIK and GSK cells presented numerous damaged cell structures when cultured in hyposmotic medium,whereas hyperosmotic medium only affected few CIK cells but completely lysed most of GSK cells.

4.Discussion

The kidneys are one of the major osmoregulatory organs in fishes(Engelund&Madsen,2011)as well as in amphibians(Hillyard,1999).Amphibians represent the crucial evolutionary link between water-dwelling fish and land-dwelling animals.Thus,it is important to compare the biological role and adaptability of kidney cells during osmotic stress between fishes and amphibians.

To mimic the natural setting of varying osmolarity in vitro,we measured a range of different osmotic stimulations from 50 to 700 m Osm and different periods of stimulation from 6 to 24 h on fish CIK cells(unpublished data).We found that CIK cells were able to tolerate to some extend lower salinities(100 m Osm)and higher salinity(700 m Osm)during short periods,but present visible morphological changes after 16 h of exposure to the two environments.In contrast,amphibian GSK cells became extremely unhealthy suggesting that CIK rather than GSK cells have a higher capacity to tolerate hyperosmolality.

Research on amphibian osmoregulation has mainly focused on frogs.It was found that the osmotic pressure of the excreted urine was always slightly lower than that of the body fluids,which explains why frogs in hyperosmotic solutions cannot establish normal water balance(Jorgensen,1997).This may explain why kidney cells of the Chinese giant salamander were more sensitive to hyperosmotic challenge when compared to fish.In adult frogs,gut,kidney,urinary bladder and skin are important in osmoregulation organs.For example,skin can adapt to changes in water permeability,and it is important for the maintenance of a constant osmotic pressure(Mori et al.,2012).Due to changes between dehydration on land and rehydration in the water,the osmoregulatory capacity of the Chinese giant salamander is mainly regulated by skin and kidney.However,little was previously known about the osmoregulation of the Chinese giant salamander and the nature of the integrated mechanisms are still not well understood.

Further research on other cell types of the Chinese giant salamander will provide new insights on the relationships between osmotic stress and cell responses in amphibians.The Chinese giant salamander is an expensive delicacy and farming is rapidly growing in China in recent years(Cunningham et al.,2016).A better understanding of the mechanism of osmotic regulation may provide essential data for artificial breeding.

Acknowledgements

This work was supported by the National Natural Science Foundation of China(31302214).The authors gratefully acknowledge Junbin Zhang for excellent suggestions and comments.

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