Objective To establish heterologous expression system of Na+-glucose cotransporter 2 (SGLT2) gene.Methods Human SGLT2 cDNA from normal kidney, generated by RT-PCR,was subclonedintoPEXL-GFP vector andtransfectedinto HEK293cells. After 24hours of incubation, the expression of SGLT2-GFP fusion protein was detected by Western blotting and laser confocal microscopy.Transport activity of SGLT2-GFP fusion proteins in cultured human HEK293 cells was evaluated with the uptake test of glucose analogue.ResultsSGLT2-GFP fusion protein was expressed in cultured human HEK293 cells.Furthermore, confocal microscopy using green fluorescent protein(GFP) revealed a punctate membrane pattern of SGLT2.Glucose analogue uptake increased in HEK293 cells transfected with SGLT2-GFP at least by 3.5 folds compared with HEK293 cells transfected with GFP vector only(P＜0.01).Conclusion Heterologous expression of SGLT2 gene in HEK293 cells is successfully established, which provides valuable approach for the functional and pathological study of SGLT2 gene.

Objective:This study aimed to compare rectal cancer tumor volume parameters measured by MRI sequences (T1WI, T2WI, and DWI) and/or CT with those by pathological specimen. Methods:Twenty-two patients with rectal cancer were prospectively enrolled. MRI sequences including T1WI, T2WI, and DWI, and/or CT of the pelvis were performed before operation. Volume parameters, such as tumor length along the rectal axis, maximum tumor width perpendicular to rectal axis, and tumor actual area in that perpendicular plane, were measured on T1WI, T2WI, DWI, and CT, respectively, for each patient. The respective pathological parameters were further measured in surgical specimen after total mesorectal excision. The two kinds of parameter values measured in imaging and pathology were statistically compared and accuracy appraisal was performed. Results:The mean Lpath-L was 4.06±1.14 cm. The mean LT1-L, LT2-L, LDWI-L, and LCT-L were 3.91± 1.51, 4.62±1.41, 3.39±1.05, and 3.94±1.23 cm, respectively. Correlation coefficients were 0.688, 0.635, 0.688, and 0.720 (P<0.05). An average 6 mm overestimation was found in T2WI, and 1 to 6 mm underestimation in T1WI, DWI, and CT in length values compared with those measured in surgical specimen. The mean Lpath-W was 2.56 ±0.94 cm. The mean LT1-W, LT2-W, LDWI-W, and LCT-W were 3.62±0.99, 3.66±0.76, 3.23±0.58, and 3.64±1.04 cm, respectively. The magnitude of mean overestimation ranged from 5.1 to 11.1 mm. The Apath was 4.30 ±2.83 cm2. AT1, AT2, ADWI, and ACT were 8.98±3.90, 8.99±3.43, 8.41±3.09, and 9.63±4.40 cm2, respectively, which double overestimated the tumor area in the perpendicular rectal plane. Conclusion:The difference in longitudinal length between MRI sequences/CT and pathological specimen was in the range of?6 mm to 6 mm. The mean maximum tumor width and areas in the maximum tumor perpendicular plane were overestimated. This study indicated that gross tumor volume delineation based on CT or MRI for rectal cancer irradiation should be conservative in the axial images of rectum, and meticulous consideration is required along the rectum.

Objective:To define more information for familial hematuria by genetic screening in a pedigree with familial hematuria.Methods:This was a 4 generation pedigree included 20 family members. The clinical data and laboratory manifestations of the family members were reviewed and collected from medical records. Meanwhile, the peripheral blood samples of 11 family members of the pedigree were collected, and then DNA samples were extracted by salting out method for genetic analysis. For genetic analysis, firstly, three family members including the proband were selected for whole exome sequencing, and the genetic variations were screened according to the sequence variation interpretation guidelines from the American College of Medical Genetics and Genomics (ACMG) for diagnostic sequence interpretation. Then PCR and Sanger sequencing were used to verify the identified pathogenic variants in all family members in the pedigree.Results:In the pedigree, 6 female members had persistent hematuria. Among them, 2 died due to end-stage renal disease, 2 died due to non-renal diseases, and 2 maintained stable renal function. One of the two members with stable renal function was diagnosed as IgA nephropathy by renal biopsy. Moreover, diffuse basement membrane lesions were identified in her renal biopsy sample after the electron microscope examination, which resulted in the suspected diagnosis of Alport syndrome. Genetic testing in this pedigree revealed two novel mutations in COL4A4 gene (NM_000092), c.G446T:p.G149V in exon 7 and c.G1249A:p.G417R in exon 20. Conclusion:Two novel mutations of COL4A4 gene (c.G446T:p.G149V in exon 7 and c.G1249A:p.G417R in exon 20) in a hematuria pedigree are related with phenotype of familial hematuria.

Objective To investigate the expression of regulators of G protein signaling(RGS), including RGS2, RGS3 and RGS4 in OLETF rats, as well as the effects of metformin on these expressions. Methods LETO rats were used as control group. Eight-week-old male OLETF rats were assigned to two guoups randomly:model and trial(metfomin dose during 8~(th) to 22~(nd) weeks:300mg kg~(-1)·d~(-1);during 23rd to 28th weeks:400 mg·kg~(-1) ·d~(-1))groups. Expressions of RGS mRNA in aorta and heart werequantified by real-time PCR. Results RGS2, RGS3 and RGS4 mRNA of the thoracic aorta and left ventricle were significantly higher in model group than in control group (P<0.01). Compared with model group, metformin significantly reduced their mRNA in trial group (P<0.01). Conclusions Upregulation of RGS2, RGS3 and RGS4 mRNA expression in the thoracic aorta and left ventricle of OLETF rats is in correlation with cardiovascular lesions; while downregulation of their expression is in correlation with the action of metformin.

Objective: To measure the distance of the lateral, inferior, and superior microfoci from a gross tumor in a pathological speci-men and to provide scientific evidence for margin extension to form the clinical target volume (CTV) in high-dose radiotherapy for rec-tal cancer. Methods: Twenty-eight surgical specimens were collected from patients with rectal cancer who underwent total mesorectal excision (TME) in Hunan Cancer Hospital between October 2016 and April 2017. The nearest distance of the farthest peripheral micro-foci from the gross tumor was measured. The in vivo-in vitro tumor retraction factor (R1) was calculated by measuring the ratio of the tumor's perpendicular depth based on magnetic resonance imaging and immediate surgical specimens. The retraction factor (R2) in the process of pathological specimen makeup was calculated by knot labeling. The distance of microfoci extension was calculated based on that measured in pathological specimens including corrections with R1 and R2 and record as microcarcinoma extension mea-sured in vivo,MEin vivo. Results: Among the 28 pathological specimens, lateral, inferior, and superior microfoci were found in 17 (60.7%), 3 (10.7%), and 0 cases, respectively. The mean R1 was 0.913 and mean R2 was 0.803. The farthest distance measured inferiorly was 28 mm in vivo after correction. The maximum, minimum, and mean measured lateral distances were 12.03 mm, 3.03 mm, and 7.50 mm after correction, respectively. The 95% frequency value was within 10 mm. Conclusions: The lateral microfoci extension was within 10 mm for 95% of the rectal cancer patients. The margin expansion to form the CTV was suggested to be 10 mm for a late-course boost of high-dose radiotherapy for rectal cancer.

Objective:Through the investigation of the pathogenicity of COL4A4 heterozygous splicing mutations and the genotype-phenotype correlation in autosomal dominant Alport syndrome (ADAS), to better understand the impact of COL4A4 heterozygous splicing mutations on ADAS. Methods:The study was a case series analysis. Patients from 5 ADAS families with COL4A4 heterozygous splicing mutations detected by whole exome sequencing were recruited by three hospitals. In vivo transcriptional analysis and/or in vitro minigene splicing assay were conducted to determine the splicing patterns and assess the pathogenicity of COL4A4 heterozygous splicing mutations. Results:In the five ADAS pedigrees carrying COL4A4 heterozygous splicing mutations, four novel ADAS splicing patterns were described. In pedigree 1-4, most patients presented with continuous hematuria or/and microalbuminuria. Otherwise,the proband in pedigree 4 presented with macroalbuminuria and the proband in pedigree 1 had progressed to chronic kidney disease stage 2 at the age of 70 years old. In pedigree 5, all patients developed end-stage renal disease between 28 and 41 years old. c.735+3A>G detected in pedigree 1 and pedigree 2 and c.694-1G>C detected in pedigree 3 both led to exon 12 skipping in COL4A4, resulting in 42 nucleotides in-frame deletion (c.694_735del). c.2056+3A>G detected in pedigree 4 led to COL4A4 exon 26 skipping, which caused in-frame deletion of 69 nucleotides (c.1988_2056del). c.2716+5G>T detected in pedigree 5 led to a 360 nucleotides large in-frame deletion, including 100 bp sequence at the 3'end of exon 29,the whole sequence of exon 30 and 89 bp sequence at the 5'end of exon 31 (c.2446_2805del). Conclusions:Renal prognosis differs significantly for patients with small in-frame deletions versus large in-frame deletion splicing abnormalities. Determination of the pathogenicity and the splicing patterns of COL4A4 heterozygous splicing mutations using in vivo and in vitro transcriptional analysis may provide renal prognostic information.

Objective:To evaluate the clinicopathological characteristics and prognosis of IgA nephropathy (IgAN) patients with microangiopathy lesions and with no hypertension.Methods:Adult IgAN patients without hypertension were selected from Peking University First Hospital. All kidney biopsies were independently reviewed by 2 investigators. Patients were divided into three groups (microangiopathy group, simple arterio/arteriolosclerosis group and normal vascular group) by renal arteriolar lesions. Composite kidney end point event defined as a ≥30% reduction in estimated glomerular filtration rate (eGFR) and end-stage kidney disease. Cox regression analysis was used to test the association between microangiopathy lesions and the outcomes.Results:A total of 420 patients were included in this study, of which 37(8.8%) patients had renal arteriolar microangiopathy lesions, 134 (31.9%) patients had simple arterio/ arteriolosclerosis, and the others had no vascular lesion. Compared with simple arterio/arteriolosclerosis group or non-vascular lesion group, patients with renal arteriolar microangiopathy lesions had more severe urine protein ( P=0.002), worse renal function ( P<0.001), higher proportion of segmental glomerulosclerosis and/or balloon adhesion (S1), tubular atrophy/interstitial fibrosis (T1/2), cellular/fibrocellular crescents (C1/2) (all P<0.05). During the follow-up, 20(54.1%) patients with microangiopathy lesions, 45(33.6%) patients with simple arterio/arteriolosclerosis and 82(32.9%) patients without vascular lesion reached the composite kidney end points ( χ2=6.491, P=0.039). In a multivariable Cox regression model, the presence of microangiopathy lesions was an independent risk factor for kidney disease progression in IgAN patients ( HR=1.872, 95% CI 1.044-3.357, P=0.035), and simple arterio/arteriolosclerosis was not a risk factor for kidney disease progression. Conclusion:It is not uncommon for non-hypertensive patients with IgAN having microangiopathy lesions, which suggests that hypertension is not the sole risk factor for microangiopathy lesions.

The paper reports a case of coenzyme Q10 deficiency nephrotic syndrome associated with coenzyme Q2 gene mutation and reviews the literature on this topic. The patient presented with hematuria, proteinuria, and a diminution of vision as clinical manifestations. But the proteinuria was not relieved after sufficient doses of glucocorticoids for over 2 months. The patient′s birth history was unremarkable, and his parents were both healthy and not consanguineous. Whole exome sequencing revealed that the patient had a mutation of coenzyme Q2 gene at c.973A>G(p.T325A) and c.517C>T(p.R173C). Combined with renal biopsy pathology, the patient was diagnosed with hereditary nephropathy and started the supplements of coenzyme Q10 after stopping glucocorticoid treatments immediately. After 5 weeks of therapy, the patient′s 24-h urine protein quantification decreased from 6.01 g to 1.53 g.

OBJECTIVE To systematically review the efficacy and safety of kidney -tonifying Chinese patent medicine combined with phosphodiesterase type 5（PDE5）inhibitors in the treatment of erectile dysfunction （ED），and to provide evidence - based reference for clinical medication . METHODS Retrieved from PubMed ，Embase，Cochrane Library ，Web of Science ，VIP， China Biomedical Literature Database ，CNKI and Wanfang database ，randomized controlled trials （RCTs）of kidney -tonifying Chinese patent medicine combined with PDE 5 inhibitors（trial group ）versus PDE 5 inhibitors or kidney -tonifying Chinese patent medicine alone （control group ）were collected . The search period was from the establishment of the database to February 15，2022. After literature screening and data extraction ，the risk of bias assessment tool recommended in Cochrane System Reviewer ’s Handbook 6.1.0 was used to evaluate the quality of the included literature ；Stata 16.0 software was used for network meta -analysis； and the funnel plot was used for publication bias analysis . RESULTS A total of 23 RCTs were included ，with a total of 2 417 patients， involving 8 kinds of Chinese patent medicines ， including Congrong yishen granule ，Canrong zhutian capsule ， Compound xuanju capsule ， Huanshao capsule ， Shanhaidan granule， Shengjing capsule ， Shisanwei ziyin zhuangyang capsule，Shugan yiyang capsule . The results of network meta analysis showed that in terms of total effective rate ，Compound xuanju capsule combined with PDE 5 inhibitor，Shengjing capsule combined with PDE 5 inhibitor，Shugan yiyang capsule combined with PDE 5 inhibitor had higher total effective rate ；in terms of international index of erectile function 5，the scores of Compound xuanju capsule combined with PDE 5 inhibitor，Congrong yishen granule combined with PDE 5 inhibitor，and Canrong zhutian capsule combined with PDE 5 inhibitor were higher ；in terms of safety ，the incidence of adverse drug reactions caused by Huanshao capsule ，Shanhaidan granule and Shugan yiyang capsule were lower . CONCLUSIONS Kidney-tonifying Chinese patent medicine combined with PDE 5 inhibitor can improve the erectile function of ED patients and reduce the occurrence of adverse drug reactions . In terms of efficacy and safety ，Compound xuanju capsule combined with PDE 5 inhibitor is the best .