Urinary nephrin: A new predictive marker for pregnancies with preeclampsia and small-for-gestational age infants

양가영,이경아,박미혜,박혜숙,하은희,전선희,김영주 대한산부인과학회 2013 Obstetrics & Gynecology Science Vol.56 No.1

Objective The objective of this study was to determine the differences in urinary nephrin among controls, gravidas with preeclampsia (PE), and small-for-gestational age (SGA) infants. We also determined whether or not maternal urinary concentrations of nephrin are associated with the subsequent development of PE and SGA infants. Methods We analyzed maternal urinary levels of nephrin in women who were normal controls (n=50), women who were delivered SGA infants (n=40), and gravidas with PE (n=33) in the first, second and third trimesters. Urinary nephrin concentrations were measured with nephrin enzyme-linked immunosorbent assay kits. Results The levels of urinary nephrin were higher in gravida developing preeclampsia or SGA than in controls after adjusting serum creatinine (P<0.05 for both). Maternal urine concentrations of nephrin were higher in pregnancies complicated by SGA and PE in the third trimester (P<0.05), and also higher in pregnancies complicated by SGA in the first trimester (P<0.05). The sensitivity and specificity of nephrin in predicting SGA from normal pregnancies were 67% and 89% in the first trimester, 60% and 79% in the second trimester, and 80% and 84% in the third trimester, respectively. The sensitivity and specificity of nephrin in predicting PE from normal pregnancies were 67% and 83% in the first trimester and 73% and 79% in the third trimester, respectively. Conclusion We suggest that urinary nephrin can be used as an early marker in pregnancies at risk for developing PE and SGA infants.

생쥐에서 사구체내 Cyclooxygenase-2 과발현이 Puromycin에 의한 발세포 손상에 미치는 영향

조영일 ( Young Il Jo ) 대한신장학회 2007 Kidney Research and Clinical Practice Vol.26 No.2

목적: Cyclooxygenase-2 (COX-2) 억제제 치료가 단백뇨를 감소시키는 것으로 알려진 이후 COX-2가 발세포(podocyte) 손상에 관여할 가능성이 제기되었지만, COX-2가 발세포 손상에 어떤 역할을 하는지는 명확하지 않다. 한편, 생쥐는 발세포에 독성을 가진 puromycin (PAN)을 투여해도 발세포 손상과 단백뇨가 잘 유발되지 않는다. 저자는 COX-2가 발세포 손상에 관여한다면 사구체에 COX-2를 과발현시킨 생쥐는 PAN에 의해 단백뇨가 발생할 것이라고 가정하고, COX-2 유전자 도입 생쥐에 PAN 및 선택적 COX-2 억제제를 투여하여 COX-2가 발세포 손상에 어떤 영향을 미치는지를 알아보고자 하였다. 방법: Wild-type 생쥐 (B6/D2 strain)와 COX-2 유전자 도입 생쥐에 PAN을 투여하였고, 다른 한 군에는 PAN과 함께 선택적 COX-2 억제제 (SC58236)를 투여하였다. PAN은 실험 1일 (15 mg/100 g BW) 및 3일 (30 mg/100 g BW)의 시작 시점에 정맥 주사하였고, SC58236은 PAN을 주사한 직후부터 식용수에 용해시켜 실험기간 내내 경구 투여하였다. 생쥐는 실험 3일 및 10일 종료 시점에 희생시켰으며, 알부민뇨, 족돌기 소실 그리고 사구체내 COX-2와 nephrin의 발현 정도를 조사하였다. 결과: COX-2 유전자 도입 생쥐만 PAN을 투여한 후에 알부민뇨가 발생하였고, 알부민뇨는 선택적 COX-2 억제에 의해 유의하게 억제되었다. 알부민뇨는 3일째에 유의하게 증가하였고 이후에는 정상수준으로 점차 감소하였다. PAN 투여로 알부민뇨가 발생한 COX-2 유전자 도입 생쥐에서 사구 체내 COX-2 발현은 증가하였고 nephrin은 발현이 감소하는 양상을 보였으며, 이러한 변화는 COX-2 억제제 투여로 완화되었다. 이와는 달리, wild-type 생쥐는 PAN 투여 후에도 유의한 변화를 보이지 않았다. 결론: PAN에 반응을 보이지 않는 wild-type 생쥐와는 달리, COX-2 유전자 도입 생쥐는 PAN에 의해 알부민뇨, 발세포 손상 및 COX-2 발현 증가가 나타났고, 이는 선택적 COX-2 억제제에 의해 완화되었다. 이러한 결과는 COX-2가 생쥐에서 PAN에 의한 발세포 손상의 유발인자로 작용하며, COX-2 억제제가 발세포 손상을 완화시킬 수 있음을 시사한다. Purpose: The aim of this study is to investigate the effect of glomerular cyclooxygenase-2 (COX-2) overexpression on podocyte injury by puromycin aminonucleoside (PAN) in nephrin-driven COX-2 transgenic mice. Methods: We administrated PAN intravenously on day-1 (15 mg/100 g body weight) and day-3 (30 mg/100 g body weight) in wild type (male B6/D2 mice) and COX-2 transgenic mice. An additional group received a selective COX-2 inhibitor (SC58236) with PAN. The animals from each group were sacrificed at the end of day-3 and 10. We investigated albuminuria, foot process effacement and glomerular COX-2 and nephrin expression. Results: PAN induced albuminuria only in COX-2 transgenic mice, with the peak on day-3. Selective COX-2 inhibition significantly reduced albuminuria. EM examination demonstrated foot process effacement, which was improved partially by selective COX-2 inhibition, in COX-2 transgenic mice treated with PAN on day-3. Glomerular COX-2 expression increased significantly on day-3 in COX-2 transgenic mice treated with PAN, whereas expression of nephrin showed a tendency to decrease on day-3. These changes of expression of COX-2 and nephrin were partly attenuated by selective COX- 2 inhibition. Unlike COX-2 transgenic mice, wild-type mice did not show any changes even after PAN treatment. Conclusion: In COX-2 transgenic mice, PAN induced albuminuria, podocyte injury and up-regulation of glomerular COX-2, which were ameliorated by selective COX-2 inhibitor. These results suggest that COX-2 may play an important role in increasing susceptibility of podocytes to injury and selective COX-2 inhibition may ameliorate podocyte injury.

A novel NPHS2 mutation (c.865A > G) identified in a Chinese family with steroid-resistant nephrotic syndrome alters subcellular localization of nephrin

Wu Na,Zhu Yingchuan,Jiang Wenhao,Song Yue,Yin Lan,Lu Yilu,Tao Dachang,Liu Yunqiang,Ma Yongxin 한국유전학회 2022 Genes & Genomics Vol.44 No.5

Background: NPHS2 is the causative gene of nephrotic syndrome type 2 (MIM 600995) which often clinically manifests as steroid-resistant nephrotic syndrome (SRNS). The NPHS2 gene encodes a slit diaphragm (SD) associated protein podocin. Objective: This study reported a novel disease-causing mutation of NPHS2 in a Chinese family with SRNS. We also investigated the pathogenic mechanism of the variants in this family. Method: A Chinese family with SRNS was recruited. Whole exome sequencing was performed to screen for disease-causing mutation. Sanger sequencing was used to confirm the results. In vitro functional experiments including immunoblotting, co-immunoprecipitation and double immunofluorescence staining were performed to explore the pathogenic mechanisms of mutations. Results: In this family, compound heterozygous mutations of NPHS2 (c.467dupT and c.865A > G) were identified and segregated with the disease. The maternal c.865A > G was a novel variant, leading to amino acid substitution (p.K289E). In vitro functional assays indicated that c.467dupT (p.L156FfsX11) mutant lost interaction with nephrin. Both K289E and L156FfsX11 mutants showed sharply diminished plasma membrane localization. Furthermore, abnormal distribution of podocin mutants also altered the cell membrane localization of nephrin. Conclusion: We reported a family with SRNS caused by compound heterozygous mutations of NPHS2 (c.467dupT and c.865A > G). c.865A > G (p.K289E) in NPHS2 was a novel causative variant associated with SRNS. Both variants in this family not only affected the normal cell membrane localization of podocin, but also altered the cell membrane localization of nephrin which is the major architectural protein of SD.

Difference In Serum Nephrin Expression Between Normal And Preeclamptic Pregnancies; A Preliminary Study

( Bit Na Rae Kim ),( Ja Young Kwon ),( Ye Jin Park ),( Myung Hwa Kang ),( Jong Rak Choi ),( Young Geun Kwon ),( Young Han Kim ),( Yong Won Park ) 대한산부인과학회 2012 Obstetrics & Gynecology Science Vol.55 No.8

Objective Nephrin is one of the slit membrane proteins of podocytes in the kidney. It is known that the nephrin is shed in the urine in nephropathy accompanying proteinuira. So the aim of this study was to evaluate the difference in the serum nephrin expression between normal and preeclamptic pregnancies. Methods A total of 20 pregnant women from May to September 2008 who received prenatal care and underwent delivery at our institute participated in the study. The preeclamptic group includes 13 women diagnosed as preeclampsia and a normal group of 7. Their serum were collected before delivery and analyzed by Western blotting for comparing serum nephrin expression. Results There was no difference in age, body weight of pregnant women, blood urea nitrogen, serum creatinine, urine creatinine level and gestational age between two groups. However, preeclampsia group had significantly higher systolic and diastolic blood pressure ( P<0.001), serum soluble fms-like tyrosine kinase-1 level ( P= 0.002), and lower birth weight ( P=0.011). In serum Western blot analysis, serum nephrin was detected in 10 of 13 in preeclampsia women (76.9%) but only in 2 of 7 (28.6%) in normal pregnancy women showing statistically significant difference ( P=0.032). Conclusion A higher prevalence of nephrin expression in the maternal serum was found in the preeclampsia when compared to the normal pregnancy.

Sirolimus 사용 후 사구체 기저막 세극막 관련 분자의 변화

최정연,한기동,김용진,박용훈,Choi, Jung-Youn,Han, Gi-Dong,Kim, Yong-Jin,Park, Yong-Hoon 대한소아신장학회 2010 Childhood kidney diseases Vol.14 No.2

목 적 : 최근 신이식 환자들에서 cyclosporine A (CsA)의 대체로 sirolimus를 투여 받은 후 단백뇨가 발생한다는 임상보고가 있으나 단백뇨 발생기전의 정확한 메커니즘에 대한 연구가 없다. 이에 sirolimus에 의한 여러 단백뇨 발생기전 중 sirolimus와 CsA 투여 후 족세포의 세극막 관련 분자 변화를 조사하여 족세포와의 직접적인 영향에 대해 알아보고자 하였다. 방 법 : 생체 외 실험- 마우스 족세포를 완충액, CsA ($10\;{\mu}g/mL$) 처리 후 sirolimus ($10\;{\mu}g/mL$) 처리군, sirolimus 단독군, CsA와 sirolimus 동시 처리한 군으로 나누어 RT-PCR을 이용하여 12, 24, 48시간에 족세포의 세극막 관련 분자 변화를 측정하였다. 생체 내 실험- SPF Wistar 쥐 24마리를 각각 4군(완충액, CsA 2주 투여 후 sirolimus 2주간 투여, sirolimus 4주간 투여, CsA와 sirolimus를 4주간 동시투여)으로 분류하여 하루 걸러서 한번 복강 내 약물을 주입하였다(CsA: 25 mg/kg, sirolimus: 0.5 mg/kg). 모든 쥐는 약물주입 후 4주에 희생되어 병리조직은 오른쪽 신장의 일부분을 이용하고, 나머지 신장은 RT-PCR을 이용하여 세극막 관련 분자의 mRNA 발현 변화를 측정하였다. 결 과 : 생체 외 실험에서 CsA와 sirolimus 동시투여 또는 CsA 처리 후 sirolimus 처리군에서 nephrin 발현이 의미 있게 감소하였다. 생체 내 실험에서 nephrin 발현은 sirolimus를 사용한 모든 군에서, podocin 발현은 CsA와 sirolimus 동시투여 또는 CsA 처리 후 sirolimus 처리군에서 의미 있게 감소하였다. 광학현미경에서 CsA 투여군은 세뇨관 상피세포에서 공포형성 및 석회화가 관찰되었으며, 면역 조직화학검사에서 사구체 모세혈관의 nephrin, podocin항체 침착은 감소되지 않았다. CsA 투여군의 전자 현미경소견에서 사구체 족돌기의 국소적 융합이 있었으며, sirolimus 단독군에서는 특이소견이 없었다. 결 론 : 본 연구를 통해 sirolimus로 발생한 단백뇨의 많은 기전 중 하나로 sirolimus가 세극막 관련 분자 중 nephrin 및 podocin의 mRNA 발현을 감소시킬 수 있으며 sirolimus 단독보다는 CsA와 함께 작용했을 때 효과가 더 커질 수 있음을 제시하는 바이다. Purpose: Recently, massive proteinuria has been observed in some transplant patients after switching cyclosporine A (CsA) to sirolimus. To evaluate the pathogenesis of sirolimus-associated proteinuria, we investigated the early changes in slit diaphragm molecules by various administrative conditions of sirolimus and CsA. Methods: In vitro-Mouse podocytes were incubated with buffer (C), sirolimus ($10\;{\mu}g/mL$) after CsA ($10\;{\mu}g/mL$) (C-S), sirolimus only (S) and CsA and sirolimus simultaneously (C+S) for 12, 24, and 48 hours. In vivo- twenty four SPF female Wistar rats were divided into 4 groups buffer (C), sirolimus after 2 weeks of CsA (C-S), sirolimus only (S) and CsA and sirolimus simultaneously (C+S). All groups were treated by intraperitoneal injection every other day for 4 weeks (CsA: 25 mg/kg, sirolimus: 0.5 mg/kg). The changes in mRNA of slit diaphragm molecules were examined by RT-PCR. Results: The mRNA of nephrin was significantly decreased in group C-S and C+S in vitro. In vivo, the mRNA of nephrin in all groups using sirolimus and the mRNA of podocin in group C-S and C+S were decreased. Microscopically, group C-S and C+S showed small vacuolization and calcification in proximal tubular epithelial cells. Immunohistochemistry using nephrin and podocin antibodies did not show remarkable decrease of staining along the glomerular capillaries. Electron-microscopically, focal fusion of foot processes was seen in group C-S and C+S. Conclusion: This study suggests the decrease of slit diaphragm molecules (nephrin and podocin) in podocyte may be one of the causes of sirolimus associated proteinuria, and podocyte injury by sirolimus may need a primary hit by CsA to develop the proteinuria.

The Level of Serum and Urinary Nephrin in Normal Pregnancy and Pregnancy with Subsequent Preeclampsia

정윤지,권자영,조희영,조시현,김영한,전진동,김영진,이상후,박지명,김하얀,박용원 연세대학교의과대학 2017 Yonsei medical journal Vol.58 No.2

Purpose: The aim of this study was to evaluate serum and urinary nephrin levels of normal pregnancy to establish a standard referencevalue and to compare them with patients who subsequently developed preeclampsia (PE). Materials and Methods: In this prospective study, 117 healthy singleton pregnancies were enrolled between 6 to 20 weeks of gestationat 2 participating medical centers during October 2010 to March 2012. Urine and serum samples were collected at the time of enrollment, each trimester, and at 4 to 6 weeks postpartum. Enzyme-linked immunosorbent assay for nephrin was performed and samples from patients who subsequently developed PE were compared to the normal patients. Results: Of 117 patients initially enrolled, 99 patients delivered at the study centers and of those patients, 12 (12.1%) developed PE at a median gestational age of 34+4 weeks (range 29+5–36+6). In the normal patients (n=68), serum nephrin level decreased and urinarynephrin level increased during the latter of pregnancy. In 12 patients who subsequently developed PE, a significant rise in the 3rd trimester serum and urinary nephrin levels, compared to the controls, was observed (p<0.001), and this increase occurred 9 days prior to the onset of clinical disease. Conclusion: As the onset of PE was preceded by the rise in the serum and urinary nephrin in comparison to normal pregnancy, serum and urinary nephrin may be a useful predictive marker of PE.

The reno-protective effect of a phosphoinositide 3-kinase inhibitor wortmannin on streptozotocin-induced proteinuric renal disease rats

Sang Hoon Kim,Young-Woo Jang,Patrick Hwang,Hyun-Jung Kim,Gi-Yeon Han,김찬화 생화학분자생물학회 2012 Experimental and molecular medicine Vol.44 No.1

Diabetic nephropathy (DN) is a progressive kidney disease that is caused by injury to kidney glomeruli. Podocytes are glomerular epithelial cells and play critical roles in the glomerular filtration barrier. Recent studies have shown the importance of regulating the podocyte actin cytoskeleton in early DN. The phosphoinositide 3-kinase (PI3K) inhibitor, wortmannin, simultaneously regulates Rac1 and Cdc42, which destabilize the podocyte actin cytoskeleton during early DN. In this study, in order to evaluate the reno-protective effects of wortmannin in early DN by regulating Rac1and Cdc42, streptozotocin (STZ)-induced proteinuric renal disease (SPRD) rats were treated with wortmannin. The albuminuria value of the SPRD group was 3.55± 0.56 mg/day, whereas wortmannin group was 1.77± 0.48 mg/day. Also, the albumin to creatinine ratio (ACR) value of the SPRD group was 53.08 ± 10.82mg/g, whereas wortmannin group was 20.27 ± 6.41mg/g. Changes in the expression level of nephrin, podocin and Rac1/Cdc42, which is related to actin cytoskeleton in podocytes, by wortmannin administration were confirmed by Western blotting. The expression levels of nephrin (79.66 ± 0.02), podocin (87.81 ± 0.03) and Rac1/Cdc42 (86.12 ± 0.02) in the wortmannin group were higher than the expression levels of nephrin (55.32 ± 0.03), podocin (53.40 ±0.06) and Rac1/Cdc42 (54.05 ± 0.04) in the SPRD group. In addition, expression and localization of nephrin, podocin and desmin were confirmed by immunofluorescence. In summary, we found for the first time that wortmannin has a reno-protective effect on SPRD rats during the early DN. The beneficial effects of wortmannin in SPRD rats indicate that this compound could be used to delay the progression of the disease during the early DN stage.

The reno-protective effect of a phosphoinositide 3-kinase inhibitor wortmannin on streptozotocin-induced proteinuric renal disease rats

Kim, Sang-Hoon,Jang, Young-Woo,Hwang, Patrick,Kim, Hyun-Jung,Han, Gi-Yeon,Kim, Chan-Wha Korean Society for Biochemistry and Molecular Bion 2012 Experimental and molecular medicine Vol.44 No.1

Diabetic nephropathy (DN) is a progressive kidney disease that is caused by injury to kidney glomeruli. Podocytes are glomerular epithelial cells and play critical roles in the glomerular filtration barrier. Recent studies have shown the importance of regulating the podocyte actin cytoskeleton in early DN. The phosphoinositide 3-kinase (PI3K) inhibitor, wortmannin, simultaneously regulates Rac1 and Cdc42, which destabilize the podocyte actin cytoskeleton during early DN. In this study, in order to evaluate the reno-protective effects of wortmannin in early DN by regulating Rac1 and Cdc42, streptozotocin (STZ)-induced proteinuric renal disease (SPRD) rats were treated with wortmannin. The albuminuria value of the SPRD group was $3.55{\pm}0.56$ mg/day, whereas wortmannin group was $1.77{\pm}0.48$ mg/day. Also, the albumin to creatinine ratio (ACR) value of the SPRD group was $53.08{\pm}10.82$ mg/g, whereas wortmannin group was $20.27{\pm}6.41$ mg/g. Changes in the expression level of nephrin, podocin and Rac1/Cdc42, which is related to actin cytoskeleton in podocytes, by wortmannin administration were confirmed by Western blotting. The exexpression levels of nephrin ($79.66{\pm}0.02$), podocin ($87.81{\pm}0.03$) and Rac1/Cdc42 ($86.12{\pm}0.02$) in the wortmannin group were higher than the expression levels of nephrin ($55.32{\pm}0.03$), podocin ($53.40{\pm}0.06$) and Rac1/Cdc42 ($54.05{\pm}0.04$) in the SPRD group. In addition, expression and localization of nephrin, podocin and desmin were confirmed by immunofluorescence. In summary, we found for the first time that wortmannin has a reno-protective effect on SPRD rats during the early DN. The beneficial effects of wortmannin in SPRD rats indicate that this compound could be used to delay the progression of the disease during the early DN stage.

Nephrin 유전자(NPHS1)의 변이가 증명된 선천성 신증후군 1례

안요한 ( Yo Han Ahn ),이범희 ( Boem Hee Lee ),최현진 ( Hyun Jin Choi ),문경철 ( Kyung Chul Moon ),하일수 ( Il Soo Ha ),정해일 ( Hae Il Cheong ),최용 ( Yong Choi ) 대한신장학회 2008 춘계학술대회 초록집 Vol.28 No.1

Nephrin expression in human epidermal keratinocytes and its implication in poor wound closure

( Ji Young Kim ),( Eun Jung Lee ),( Jimyung Seo ),( Yangsin Lee ),( Yuri Ahn ),( Sujin Park ),( Yu Jeong Bae ),( Jinu Lee ),( Beom Jin Lim ),( Doyoung Kim ),( Jin Won Cho ),( Sang Ho Oh ) 대한피부과학회 2022 대한피부과학회 학술발표대회집 Vol.74 No.1