Clinical characterization and diagnosis of cystic fibrosis through exome sequencing in Chinese infants with Bartter-syndrome-like hypokalemia alkalosis / 医学前沿

Cystic fibrosis (CF) is a fatal autosomal-recessive disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. CF is characterized by recurrent pulmonary infection with obstructive pulmonary disease. CF is common in the Caucasian population but is rare in the Chinese population. The symptoms of early-stage CF are often untypical and may sometimes manifest as Bartter syndrome (BS)-like hypokalemic alkalosis. Therefore, the ability of doctors to differentiate CF from BS-like hypokalemic alkalosis in Chinese infants is a great challenge in the timely and accurate diagnosis of CF. In China, sporadic CF has not been diagnosed in children younger than three years of age to date. Three infants, who were initially admitted to our hospital over the period of June 2013 to September 2014 with BS-like hypokalemic alkalosis, were diagnosed with CF through exome sequencing and sweat chloride measurement. The compound heterozygous mutations of the CFTR gene were detected in two infants, and a homozygous missense mutation was found in one infant. Among the six identified mutations, two are novel point mutations (c.1526G > C and c.3062C > T) that are possibly pathogenic. The three infants are the youngest Chinese patients to have been diagnosed with sporadic CF at a very early stage. Follow-up examination showed that all of the cases remained symptom-free after early intervention, indicating the potential benefit of very early diagnosis and timely intervention in children with CF. Our results demonstrate the necessity of distinguishing CF from BS in Chinese infants with hypokalemic alkalosis and the significant diagnostic value of powerful exome sequencing for rare genetic diseases. Furthermore, our findings expand the CFTR mutation spectrum associated with CF.

Sublytic C5b-9 induces protective autophagy in cultured podocytes / 中国病理生理杂志

AIM:In podocytes , autophagy occurs at a high basal level and dysregulated autophagy is associa -ted with a variety of podocytopathies .This paper is to investigate the role of autophagy in sublytic C 5b-9-induced podocyte injury.METHODS: Sublytic complement C5b-9 stimulation was used as an in vitro model.Autophagosomes were con-firmed using monodansylcadaverine (MDC) staining.Immunoblotting was used to measure the change of autophagy-related markers.Cellular morphological changes were observed by Wright-Giemsa staining.Immunofluorescence staining and con-focal microscopy were used to detect the expression and distribution of nephrin .The cell viability was assessed by methylth-iazol tetrazolium (MTT) assay.The cell apoptosis was assessed by Annexin V-fluorescein isothiocyanate/PI staining.RE-SULTS:For ensuring sublytic complement injury , the maximal amounts of anti-podocyte antiserum and 160 ×-diluted nor-mal human serum were used without inducing cell lysis (defined as >5%LDH release).Sublytic C5b-9 promoted autoph-agy of podocytes in vitro.The proautophagic effect of sublytic C 5b-9 manifested in the form of accumulated MDC-labeled vesicles and enhanced the expression of LC 3-Ⅱ.Autophagy inhibitor 3-methyladenosine (3-MA) promoted sublytic C5b-9-induced podocyte morphological abnormalities .Compared with the sublytic C5b-9-injured podocytes, 3-MA exposure further decreased the expression of nephrin .3-MA enhanced sublytic C5b-9-induced podocyte apoptosis .CONCLUSION: Sub-lytic C5b-9 attack induces autophagy , which may play a protective role against complement-mediated podocyte injury .

Role of podocyte autophagy in passive Heymann nephritis / 中华肾脏病杂志

Objective To investigate the role of autophagy in podocyte damage,and the intracellular mechanism of autophagy activation through passive Heymann nephritis (PHN) animal model.Methods Male Sprague-Dawley rats (n=40) were studied on day 0,2,4,7,and 21 after induction of PHN by injection of anti-Fx1A.Podocyte morphology and autophagosomes were observed by transmission electron microscopy.Podocyte numerical density was estimated by Weibel-Gomez =method.Cell apoptosis was detected by TUNEL assay and caspase-3 immunohistochemical staining.Expressions of autophagy markers and endoplasmic reticulum stress (ERS)-associated proteins were analyzed by Western blotting.Results (1) In PHN rats,immunohistochemical staining showed that C5b-9 deposited along glomerular basement membrane on day 4 to day 21.Small subepithelial electron -dense deposits and a part of foot process fusion were detected in the glomerulus of PHN rats on day 4 by transmission electron microscope,and podocyte damage was aggravated on day 21.Furthermore,compared with control,the urinary protein levels of PHN rats began to increase on day 3,and reached the top on day 21 [(50.6±6.0) mg/24 h].(2) The number of podocytes significantly decreased in PHN rats compared with control group on day 21(P ＜ 0.05).(3) In PHN rats,apoptotic podocytes were found by caspase-3 immunohistochemical staining and TUNEL assay on day 21.(4) The expression of autophagy marker LC3 Ⅱ was markedly increased on day 7 and 21,but down-regulated on day 21 compared with day 7.Moreover,accumulated autophagosomes in podocytes were detected on day 7 and 21 by transmission electron microscope.(5) The level of GRP78 was significantly increased on day 2 and 7 but reduced to baseline on day 21.At the same time,the downstream pathways (ATF6α,p-PERK and p-JNK) of unfolded protein response were also up-regulated in the early process of PHN and down-regulated later.Conclusions Autophagy is an important way to protect against immunemediated podocyte injury in membranous nephropathy.Autophagy activation is mainly related to endoplasmic reticulum stress induced by complement attack.This provides an important basis for a thorough understanding of the role of autophagy in the process of podocyte damage and the pathogenesis of membranous nephropathy.

Rapamycin markedly slows disease progression in a rat model of passive Heymann nephritis / 中国病理生理杂志

AIM: To determine the effect of rapamycin on the progression of passive Heymann nephritis (PHN), and whether autophagy is involved in this process .METHODS:Male Sprague-Dawley rats (n=24) were ran-domly divided into 3 groups:control group , PHN group and rapamycin treatment group .The rat PHN model was induced by injection of anti-Fx1A serum through penile vein , and all rats were sacrificed on day 21.Automatic biochemical analyzer was used to detect 24 h urine protein , blood urea nitrogen and serum creatinine .Renal damage was observed through per-iodic acid-silver methenamine staining .The number of podocyte was estimated by Weibel-Gomez method .The glomerular deposition of C5b-9, the expression of caspase-3 and expression of autophagy marker LC 3 in glomeruli were examined by immunofluorescence staining , immunohistochemical staining and Western blotting , respectively.RESULTS: Rapamycin significantly reduced proteinuria in the PHN rats (P<0.05), while the renal functions in 3 groups were normal, without significant difference .Although rapamycin limited weight gain in the rats , the health of the rats during drug treatment was not affected .Rapamycin retarded glomerular basement membrane thickening in the PHN rats .Rapamycin significantly re-duced the podocyte deletion by preventing podocyte apoptosis .Rapamycin enhanced the level of autophagy of glomerular in-herent cells .CONCLUSION:In the disease process of PHN , appropriate strength of autophagy plays a protective role . Rapamycin appropriately enhances autophagy and prevents podocyte apoptosis , thus reducing nephropathy and proteinuria . This may be one of the important mechanisms of rapamycin to slow down the progress of PHN .

Rapamycin reduces podocyte adhesion damage caused by sublytic C5b-9 via autophagy activation / 中华肾脏病杂志

Objective To determine the effect of rapamycin on sublytic C5b-9-induced podocyte adhesion damage,and whether autophagy is involved in this progression.Methods Sublytic complement C5b-9 stimulation was used in vitro.Autophagosomes were viewed using electron microscopy.Western blotting was used to measure the change of autophagy-related markers.Attachment assay was used to assess the adhesion of podocyte.Confocal microscopy was used to explore the expression patterns of cytoskeletal protein F-actin.Flow cytometry was used to measure the level of adhesion-associated protein integrin α3.Results (1) For ensuring sublytic complement injury,the maximal amounts of anti-podocyte antiserum and 160×-diluted normal human serum were used without inducing cell lysis (defined as ＞ 5％ LDH release).(2) Sublytic C5b-9 promoted autophagy in podocyte in vitro.The proautophagic effect of sublytic C5b-9 manifested in the form of accumulated autophagosomes and enhanced expression of LC3-lⅡ.(3) Inhibition of autophagy by 3-methyadenine enhanced the effect of sublytic C5b-9-induced podocyte injury,including serious cytoskeleton damage and markedly reduced adhesion of podocyte.(4) Rapamycin treatment significantly improved the above lesions.(5) Rapamycin enhanced autophagy induced by sublytic C5b-9 in podocyte.Conclusions In summary,rapamycin can improve sublytic CSb-9-induced podocyte adhesion damage by appropriate autophagy activation.These findings provide important information for the development of appropriate protocols for the application of mTOR (mammalian target of rapamycin) inhibitors in podocytopathy.