Studies On Expression Of Renal V2R And AQPs In Bactrian Camel(Camelus Bactrianus)

【Objective】The Bactrian camel(Camelus bactrianus), as a typical desert mammal, has extremely strong tolerance for arid and hemi-arid environments in China. It can good adapt to a lack of water essential and live as well in survival, reproduction and production, which is closely related to its special ability of water metabolism. In mammals, V2 vasopressin receptor(V2R) and aquaporins(AQPs) are both mainly expressed in kidney, The former regulates AQPs-mediated water reabsorption in glomerular filtrate and urine concentration abilities, thereby provides an advantage to maintain the water balance and electrolyte homeostasis. It is widely concerned on mechanism of camel in adaptation to arid environments. Therefore, the camels, as research objects [cattles(Bos Taurus) as a control] in this paper, of which expression and mRNA of their kidney V2 R, AQP2, AQP3 and AQP4 and bioinformatics characteristics(part) would be as a breakthrough point, combining with renal structural indices and ultrastructure, to explore structural and molecular bases and regulation of molecular mechanisms in adaptation to arid and hemi-arid environments for camels systematically. 【Methods】1. Histological methods, both cattle and camel kidneys fixed in the PFA were observed as a whole under the fume hood, and dissected the characteristics of renal structure. For measurement of renal structure indices, here including percentage of medulla thickness(PMT), percentage of medulla area(PMA), relative medulla thickness(RMT), relative medulla area(RMA), percentage of papilla(medulla internal) thickness(PPT) and percentage of papilla area(PPA), respectively. Histological sections and HE staining,scanning and transmission electron microscopy.2. Paraffin section-immunohistochemical reaction, sections were carried out using light microscopy. Immunopositive production(AQP2, AQP3, AQP4, V2 R and AVP) were carried out using an image analysis software(Image-Pro Plus 6.0). The results are expressed as total optical density of the positive cells(IOD) value of the stained area.3. QRT-PCR reaction, oligonucleotide primer sets(AQP2, AQP3, AQP4, V2 R and β-actin) were designed from cattle and camel by Oligo 6.0 and were synthesized by Sangon Biotech, China. Reverse transcription products cDNA as templates were using for PCR reaction.4. Bioinformatics methods, bioinformatics softwares were used to analyze the differences of V2 R and AQP2 between cattle and camel, e.g. amino acid sequence, putative tertiary structure, hydrophobicity/hydrophily, transmembrane structure and phosphorylation sites. 【Results】1. The average thickness ratio of renal medulla and cortex of camel and cattle were about 4/1 and 1.5/1, respectively. Medulla can be further divided into the outer and inner medulla.The thickness of the outer medulla was twice as big as that of the inner medulla for camel. But the thickness of the outer and inner medulla was considerable for cattle. The results showed that renal structure indices of camel(PMT, PMA, RMA, PPA, RMT and PPT) were higher than those of cattle(P<0.001), especially RMA and PPA. It was found that the number of the renal corpuscle in the shallow-middle cortex of cattle(9.0±1.58) was higher than that of camel(4.6±1.52) under SEM, and there was a extremely significant difference(P<0.01). Compared with the cattle, the volume of mitochondria in the epithelial cells of cortical and outer medullary uriniferous tubule in camel was smaller, but the number was more than those of cattle. In addition, for inner medullary uriniferous tubule, even though the volume of mitochondria in the epithelial cells in camel were nearly consistent with those of cattle, the number of former was more.2. In the outer medullary collecting duct(OMCD) and inner medullary collecting duct(IMCD), expression of AQP2 showed more strong in the apical plasma membrane and intracellular vesicles of principal cells in camel respectively than the control under immunolight microscopy(P<0.01). AQP3 expression was more strong in the basolateral plasma membrane of the OMCD and IMCD of camel respectively than in control(P<0.01). Expression of AQP4 showed more strong in the basolateral plasma membrane of principal cells in the cortical collecting duct(CCD) and IMCD of cattle respectively than camel(P<0.01). In addition, in the outer medulla(OM) and inner medulla(IM) of camel, V2 R was expressed in the cytoplasm of epithelial cells except the apical and basolateral plasma membrane, and the expression revealed stronger than cattle(P<0.01). However, V2 R expression abundance in the cortex(CT) of cattle was higher than that of camel(P<0.01). Expression of AVP in the magnocellular neurons of supraoptic nucleus(SON) of camel was consistent with that of cattle. But in the paraventricular nucleus(PVN), AVP expression of camel was more strong than cattle.3. Real time quantitative RT-PCR analysis showed that expression trend of AQP2 and V2 R mRNA in the CT-OM-IM in camel were homogeneous basically, but their expression levels were higher in the IM of camel than cattle(P<0.001) significantly. In addition, AQP3 mRNA expression level in the kidney of camel were higher than that of cattle(P<0.001). However, expression level of AQP4 mRNA in camel was very weak/absent in the collecting ducts, and lower than that of cattle(P<0.001) significantly.4. The overall structural conservation of V2 R in camel was not significantly different when compared with cattle, and the core sequences V2 R genes were highly conserved. It was found that V2 R genes of camel and cattle were respectively 17 and 12 potential phosphorylation sites. In addition, AQP2 phosphorylation sites between camel and cattle were also different, 10 and 8 phosphorylation sites of tryptophan(Ser) or threonine(Thr), respectively. 【Conclusion】1. The changes in kidney morphology, e.g. the wider medulla/longer the Henle’s loop and lots of mitochondria in the renal tubular epithelial cells of camels might have provided structural foundation for high osmotic pressure gradient in the medulla.2. Camels, due to long-term evolution and adaption to arid environments, may have the increased V2 R in basal activity, the latter induces a series of signal cascades, which mediates upregulation of AQP2 and AQP3 protein and mRNA in collecting duct epithelial cells of renal cortex, outer and inner medulla, eventually thus to strengthen the ability to reduce water loss by water reabsorption and excretion of high concentrated urine.