Influence Of NUMB R630H On Uric Acid Metabolism

Background Patients with gout often suffer from multiple organ damage,manifesting as hyperuricemia with acute arthritic episodes,urate deposits,and joint deformities,often accompanied by kidney damage.In contrast,gout can trigger and exacerbate hypertension,cardiovascular disease,and diabetes,seriously improving people’s quality of life.Hyperuricemia,caused by disorders of purine metabolism and/or reduced uric acid excretion,is the basis for the development of gout.The incidence of gout and hyperuricemia increases with changes in lifestyle and diet.The study of the pathogenesis of hyperuricemia and gout is vital for the effective treatment and control of disease progression and for improving patients’ quality of life.The group has conducted a study on a large Han Chinese family with primary gout.A nucleotide G>A mutation in exon 11 of NUMB changed the highly conserved interspecies arginine at position 630 to histidine.No studies have reported the association of NUMB gene variants with the development of hyperuricemia and gout,and our study may have identified a gout-causing gene.PurposeTo investigate the effect of uric acid metabolism after the mutation of the NUMB R630 H gene.Methods1.To investigate the effect of the NUMB R630 H mutation on NUMB protein expression.Wild-type NUMB with a flag tag and the R630 H mutant overexpression plasmid were transfected separately into NUMB-GFP stably expressing cell lines,and total protein was extracted for Western blotting to The complete protein was extracted for Western blotting to determine whether the expression of the mutant affected the expression of wild-type NUMB.Based on the above experiments,NUMB protein expression was initially investigated by Western blotting after adding the autophagy pathway inhibitor 3-MA to investigate the mechanism by which the NUMB R630 H mutation causes changes in NUMB protein expression.2.The effect of the NUMB R630 H mutation on uric acid metabolism was studied at the cellular level.Construct a NUMB knockdown MDCK cell line,inoculate this cell line and the control MDCK cell line into transwell chambers with a pore size of 0.4 μ m,separately.Stable isotope-labeled urate was added to the medium in the chambers.The urate in the medium in the chambers was measured by mass spectrometry to calculate the rate of uric acid excretion.The Stable isotope-labeled urate was added to the intracellular medium,and urate in the intracellular medium was detected by mass spectrometry to calculate the uric acid reabsorption rate.The NUMB R630 H mutation overexpressing MDCK cell line,wild type NUMB gene overexpressing MDCK cell line,and MDCK cell line transfected with empty plasmid was inoculated into 0.4 μ m pore size transwell chambers,separately.Repeat the above experiments to compare uric acid excretion and reabsorption.3.Animal level study of the effect of the NUMB R630 H mutation on uric acid metabolism was performed.A custom-made NUMB R630 H knock-in mouse model(hereinafter referred to as KI mice)from the company was used to carry out the following studies.To measure blood uric acid and blood creatinine levels in KI mice,wild-type mice,and heterozygous mice.Take peripheral blood from the submandibular venous plexus of the mice.Serum was collected by centrifugation,and blood uric acid creatinine levels were measured.Urine was collected from wild-type and knock-in mice for 24 hours to measure urinary creatinine and urinary uric acid.The following parameters were calculated: 24-hour urinary uric acid/urinary creatinine ratio.Results1.According to Western blotting results,we can find that NUMB R630 H mutant overexpressing the HEK293 cell line showed significantly lower NUMB protein expression than NUMB wild type overexpressing HEK293 cell line.The addition of the autophagic pathway inhibitor 3-MA resulted in improved protein expression.The NUMB R630 H mutant overexpressing MDCK cell line showed a significant decrease in NUMB protein expression compared to the wild-type NUMB gene overexpressing MDCK cell line.2.The NUMB R630 H mutant overexpressing MDCK cell line showed a significant decrease in uric acid excretion compared to the wild-type NUMB overexpressing MDCK cell line.At the same time,the reabsorption process was not affected.3.At 48 weeks of age,both mutant male rats(R630H/R630H)and heterozygous male rats(+/R630H)showed a statistically significant decrease in urinary uric acid to urinary creatinine ratio compared to wild male rats(+/+)(P < 0.01).Serum uric acid was significantly higher in mutant male rats(R630H/R630H)than wild male rats(+/+).There was no significant difference in serum uric acid between heterozygous male rats(+/R630H)and wild male rats(+/+).ConclusionMutations in the NUMB R630 H gene can affect uric acid metabolism and may be a causative gene in the development of hyperuricemia and gout.