Effects of Hyperosmolality on Expression of Urea Transporter A2 and Aquaporin 2 in Mouse Medullary Collecting Duct Cells

In this study,the effects of hyperosmolality on the expression of urea transporter A2 (UTA2) and aquaporin 2 (AQP2) were investigated in transfected immortalized mouse medullary collecting duct (mIMCD3) cell line.AQP2-GFP-pCMV6 and UTA2-GFP-pCMV6 plasmids were stably transfected into mIMCD3 cells respectively.Transfected mIMCD3 and control cells were cultured in different hy-pertonic media,which were made by NaCl alone,urea alone,or an equiosmolar mixture of NaCl and urea.The mRNA and protein expression of AQP2 was elevated by the stimulation of NaCl alone,urea alone and NaCl plus urea in AQP2-mIMCD3 cells;whereas NaCl alone and NaCl plus urea rather than urea alone increased the mRNA and protein expression of UTA2 in UTA2-mIMCD3 cells,and all the expression presented an osmolality-dependent manner.Moreover,the mRNA and protein expression of UTA2 rather than AQP2 was found to be synergistically up-regulated by a combination of NaCl and urea in mIMCD3 cells.It is concluded that NaCl and urea synergistically induce the expression of UTA2 rather than AQP2 in mIMCD3 cells,and hyperosmolality probably mediates the expression of AQP2 and UTA2 through different mechanisms.

Discovery of novel diarylamides as orally active diuretics targeting urea transporters

Urea transporters(UT)play a vital role in the mechanism of urine concentration and are recognized as novel targets for the development of salt-sparing diuretics.Thus,UT inhibitors are promising for development as novel diuretics.In the present study,a novel UT inhibitor with a diarylamide scaffold was discovered by high-throughput screening.Optimization of the inhibitor led to the identifi-cation of a promising preclinical candidate,N-[4-(acetylamino)phenyl]-5-nitrofuran-2-carboxamide(1 H),with excellent in vitro UT inhibitory activity at the submicromolar level.The half maximal inhibitory concentrations of 1 H against UT-B in mouse,rat,and human erythrocyte were 1.60,0.64,and0.13 mmol/L,respectively.Further investigation suggested that 8 mmol/L 1 H more powerfully inhibited UT-A1 at a rate of 86.8%than UT-B at a rate of 73.9%in MDCK cell models.Most interestingly,we found for the first time that oral administration of 1 H at a dose of 100 mg/kg showed superior diuretic effect in vivo without causing electrolyte imbalance in rats.Additionally,1 H did not exhibit apparent toxicity in vivo and in vitro,and possessed favorable pharmacokinetic characteristics.1 H shows promise as a novel diuretic to treat hyponatremia accompanied with volume expansion and may cause few side effects.

Urea transport and hydrolysis in the rumen: A review

Inefficient dietary nitrogen(N)conversion to microbial proteins,and the subsequent use by ruminants,is a major research focus across different fields.Excess bacterial ammonia(NH3)produced due to degradation or hydrolyses of N containing compounds,such as urea,leads to an inefficiency in a host’s ability to utilize nitrogen.Urea is a non-protein N containing compound used by ruminants as an ammonia source,obtained from feed and endogenous sources.It is hydrolyzed by ureases from rumen bacteria to produce NH_(3) which is used for microbial protein synthesis.However,lack of information exists regarding urea hydrolysis in ruminal bacteria,and how urea gets to hydrolysis sites.Therefore,this review describes research on sites of urea hydrolysis,urea transport routes towards these sites,the role and structure of urea transporters in rumen epithelium and bacteria,the composition of ruminal ureolytic bacteria,mechanisms behind urea hydrolysis by bacterial ureases,and factors influencing urea hydrolysis.This review explores the current knowledge on the structure and physiological role of urea transport and ureolytic bacteria,for the regulation of urea hydrolysis and recycling in ruminants.Lastly,underlying mechanisms of urea transportation in rumen bacteria and their physiological importance are currently unknown,and therefore future research should be directed to this subject.

The role of rumen epithelial urea transport proteins in urea nitrogen salvage:A review

The symbiotic relationship between the host and the rumen microbiome plays a crucial role in ruminant physiology.One of the most important processes enabling this relationship is urea nitrogen salvaging(UNS).This process is important for both maintaining ruminant nitrogen balance and supporting production of their major energy supply,bacterially-derived short chain fatty acids(SCFA).The key step in UNS is the trans-epithelial movement of urea across the ruminal wall and this is a highly regulated process.At the molecular level,the key transport route is via the facilitative urea transporter-B2,localized to ruminal papillae epithelial layers.Additional urea transport through aquaporins(AQP),such as AQP3,is now also viewed as important.Long-term regulation of these ruminal urea transport proteins appears to mainly involve dietary fermentable carbohydrates;whereas,transepithelial urea transport is finely regulated by local conditions,such as CO_(2) levels,pH and SCFA concentration.Although the key principles of ruminal urea transport physiology are now understood,there remains much that is unknown regarding the regulatory pathways.One reason for this is the limited number of techniques currently used in many studies in the field.Therefore,future research in this area that combines a greater range of techniques could facilitate improvements to livestock efficiency,and potentially,reductions in the levels of waste nitrogen entering the environment.

A novel mutation at the JK locus causing Jknull phenotype in a Chinese family

Urea transporters are a group of proteins that facilitate urea movement across biological membranes. Kidd blood group (JK antigen) and urea transporter of human erythrocytes are carried by the same protein UT-B. To investigate the molecular basis of the Jknull phenotype in the Chinese population, blood samples from Chinese individuals were screened using the 2 mol/L urea solution hemolysis test. Urea and water permeability of erythrocytes membrane was measured by stopped-flow light scattering. Genomic DNA was extracted from lymphocytes. UT-B gene of Jknull's family was analyzed using genomic PCR by primers designed to cover sequences of all exons and exon-intron boundaries in human UT-B gene. One Jknull subject was found from twenty thousand screened Chinese individuals, and it was confirmed that this individual did not express the erythrocyte urea transporter. Genomic sequence analysis of the Jknull individual showed that there were two point mutations, G→C, which is novel, and G→A, at the 3(-acceptor splice site (AG) of intron 5 of UT-B gene. Exon 6 is spliced out in the UT-B transcript due to either of these mutations. Water permeability in Jknull erythrocytes (Pf, ~0.00037 cm/s) was significantly lower than that in normal erythrocytes (Pf, ~0.00062 cm/s) after HgCl2 incubation, providing evidence for UT-B facilitated water transport in human erythrocytes.