PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-7 (7)
 

Clipboard (0)
None
Journals
Year of Publication
Document Types
1.  Tetracycline-Inducible Gene Expression in Conditionally Immortalized Mouse Podocytes 
American journal of nephrology  2008;29(3):153-163.
Background
Conditionally immortalized podocytes are valuable research tools but are difficult to efficiently transfect and do not provide graded transgene expression.
Methods
Conditionally immortalized mouse podocyte cell lines were established employing a tetracycline-inducible system. Glomerular cells, isolated from transgenic mice bearing two transgenes, NPHS2-reverse tetracycline-controlled transactivator, rtTA (A transgene) and H2-Kb-thermosensitive SV40 T, ts58A (I transgene), were cloned. One clone (AI podocytes) expressing WT1 and synaptopodin was transfected with pBI-EGFP (enhanced green fluorescent protein, G transgene) and separately with ptTS-Neo (transcriptional suppressor, T transgene) to produce stable transformants, AIG podocytes and AIT podocytes.
Results
AIG podocytes expressed EGFP at 33 and 37°C after doxycycline treatment, and retained podocin and rtTA mRNA expression and temperature-sensitive growth regulation. AIT podocytes, transiently transfected with luciferase-BI-EGFP (LG transgene), showed reduced background expression of EGFP and luciferase in the absence of doxycycline. In AITLG podocytes, generated by stable transfection of AIT podocytes with the LG transgene, luciferase expression was tightly regulated by doxycycline in a time- and concentration-dependent manner both at 33 and 37°C, although background expression was not entirely eliminated. These podocytes retained temperature-sensitive growth regulation and expression of podocyte differentiation markers.
Conclusion
Mouse podocytes expressed tetracycline-induced transgenes efficiently while retaining differentiation markers.
doi:10.1159/000151770
PMCID: PMC2698022  PMID: 18753740
Tetracycline-inducible system; Conditional immortalization; Transcription; Gene of interest
2.  Tetracycline-Inducible Gene Expression in Conditionally Immortalized Mouse Podocytes 
American Journal of Nephrology  2008;29(3):153-163.
Background
Conditionally immortalized podocytes are valuable research tools but are difficult to efficiently transfect and do not provide graded transgene expression.
Methods
Conditionally immortalized mouse podocyte cell lines were established employing a tetracycline-inducible system. Glomerular cells, isolated from transgenic mice bearing two transgenes, NPHS2-reverse tetracycline-controlled transactivator, rtTA (A transgene) and H2-Kb-thermosensitive SV40 T, ts58A (I transgene), were cloned. One clone (AI podocytes) expressing WT1 and synaptopodin was transfected with pBI-EGFP (enhanced green fluorescent protein, G transgene) and separately with ptTS-Neo (transcriptional suppressor, T transgene) to produce stable transformants, AIG podocytes and AIT podocytes.
Results
AIG podocytes expressed EGFP at 33 and 37°C after doxycycline treatment, and retained podocin and rtTA mRNA expression and temperature-sensitive growth regulation. AIT podocytes, transiently transfected with luciferase-BI-EGFP (LG transgene), showed reduced background expression of EGFP and luciferase in the absence of doxycycline. In AITLG podocytes, generated by stable transfection of AIT podocytes with the LG transgene, luciferase expression was tightly regulated by doxycycline in a time- and concentration-dependent manner both at 33 and 37°C, although background expression was not entirely eliminated. These podocytes retained temperature-sensitive growth regulation and expression of podocyte differentiation markers.
Conclusion
Mouse podocytes expressed tetracycline-induced transgenes efficiently while retaining differentiation markers.
doi:10.1159/000151770
PMCID: PMC2698022  PMID: 18753740
Tetracycline-inducible system; Conditional immortalization; Transcription; Gene of interest
3.  Microalbuminuria in HIV Disease 
American journal of nephrology  2013;37(5):10.1159/000350384.
Background/Aims
Microalbuminuria is a marker for early kidney disease and cardiovascular risk. The purposes of this study were to determine the prevalence of microalbuminuria in an HIV-infected clinic population, to test the predictive value of a single urine albumin-creatinine ratio (ACR) to identify persistent microalbuminuria and to examine covariates of microalbuminuria.
Methods
We conducted a prospective cohort study of HIV-infected subjects (n=182) without proteinuria (P/C ratio ≥0.5 g/g), elevated serum creatinine, diabetes, or chronic inflammatory conditions. Subjects completed three research visits within nine months. Microalbuminuria was defined as the geometric mean ACR of 25–355 mg/g for women and 17–250 mg/g for men.
Results
The prevalence of microalbuminuria was 14%. The negative predictive value of a single urine ACR determination was 98%, whereas the positive predictive value was only 74%. Microalbuminuria was similar among Black (15%) and non-Black (14%) subjects (p=0.8). Subjects with microalbuminuria were more likely to have hypertension (p=0.02) and metabolic syndrome (p=0.03). While duration of HIV infection and the level of HIV viremia were similar between groups, those with microalbuminuria were more likely to have a CD4 count <200 cells/μL (p=0.0003). In a multivariate logistic regression analysis, the only significant independent predictors of microalbuminuria were low CD4 count (p=0.018) and current ritonavir exposure (p=0.04).
Conclusion
The prevalence of microalbuminuria in an HIV-infected clinic population was similar to earlier reports, and was associated with hypertension and impaired immune function. A single normal ACR determination effectively excludes microalbuminuria, whereas an elevated ACR requires confirmation.
doi:10.1159/000350384
PMCID: PMC3809894  PMID: 23615312
HIV infection; microalbuminuria; urinary albumin-creatinine ratio
4.  Trophoblast Glycoprotein: Possible Candidate Mediating Podocyte Injuries in Glomerulonephritis 
American Journal of Nephrology  2010;32(6):505-521.
Background
Trophoblast glycoprotein (Tpbg), a 72-kDa transmembrane glycoprotein, is known to regulate the phenotypes of epithelial cells by modifying actin organization and cell motility. Recently, a microarray study showed that Tpbg is upregulated in Thy1 glomerulonephritis (Thy1 GN). We hypothesized that Tpbg regulates cytoskeletal rearrangement and modulates phenotypic alteration in podocytes under pathological conditions.
Methods
We examined Tpbg expression in Thy1 GN and Tpbg function in mouse podocytes.
Results
We demonstrated that Tpbg is upregulated in the injured podocytes of Thy1 GN. In vitro, immunofluorescence studies revealed that Tpbg colocalized with the focal adhesion protein, vinculin, in parallel with stress fiber formation. This colocalization was observed even when actin filaments were depolymerized with cytochalasin D. Tpbg localization at focal adhesions was induced by dominant-active RhoA and suppressed by the ROCK1 inhibitor Y-26732. In addition, transforming growth factor-β increased Tpbg expression at focal adhesions concurrently with rearrangement of stress fibers. Stress fiber formation was suppressed in differentiated podocytes transfected with full-length Tpbg. Furthermore, knockdown of Tpbg using small interfering RNA decreased podocyte motility.
Conclusion
Our findings suggest a novel role of Tpbg in the phenotypic alteration of injured podocytes, and we accordingly propose a new mechanism of glomerular injury in glomerulonephritis.
doi:10.1159/000321366
PMCID: PMC2992649  PMID: 20980737
Trophoblast glycoprotein; Podocyte; Thy1 glomerulonephritis; Transforming growth factor-β; Cell motility; Stress fiber
5.  The Non-Muscle Myosin Heavy Chain 9 Gene (MYH9) Is Not Associated with Lupus Nephritis in African Americans 
American Journal of Nephrology  2010;32(1):66-72.
Background
African Americans (AA) disproportionately develop lupus nephritis (LN) relative to European Americans and familial clustering supports causative genes. Since MYH9 underlies approximately 40% of end-stage renal disease (ESRD) in AA, we tested for genetic association with LN.
Methods
Seven MYH9 single nucleotide polymorphisms (SNPs) and the E1 risk haplotype were tested for association with LN in three cohorts of AA.
Results
A preliminary analysis revealed that the MYH9 E1 risk haplotype was associated with ESRD in 25 cases with presumed systemic lupus erythematosus (SLE)-associated ESRD, compared to 735 non-SLE controls (odds ratio 3.1; p = 0.010 recessive). Replication analyses were performed in 583 AA with SLE in the PROFILE cohort (318 with LN; 265 with SLE but without nephropathy) and 60 AA from the NIH (39 with LN; 21 with SLE but without nephropathy). Analysis of the NIH and larger PROFILE cohorts, as well as a combined analysis, did not support this association.
Conclusions
These results suggest that AA with ESRD and coincident SLE who were recruited from dialysis clinics more likely have kidney diseases in the MYH9-associated spectrum of focal segmental glomerulosclerosis. PROFILE and NIH participants, recruited from rheumatology practices, demonstrate that MYH9 does not contribute substantially to the development of LN in AA.
doi:10.1159/000314688
PMCID: PMC2914393  PMID: 20523037
African Americans; Genetics; Lupus nephritis; Kidney; MYH9; Systemic lupus erythematosus
6.  Polymorphisms in the Nonmuscle Myosin Heavy Chain 9 Gene (MYH9) Are Associated with Albuminuria in Hypertensive African Americans: The HyperGEN Study 
American Journal of Nephrology  2009;29(6):626-632.
Background
MYH9 is a podocyte-expressed gene encoding nonmuscle myosin IIA that is associated with idiopathic and human immunodeficiency virus-associated focal segmental glomerulosclerosis (FSGS) and hypertensive end-stage renal disease in African Americans.
Methods
Four single nucleotide polymorphisms comprising the major MYH9 E1 risk haplotype were tested for association with estimated glomerular filtration rate (eGFR) and urine albumin:creatinine ratio (ACR) in 2,903 HyperGEN participants (1,458 African Americans (AA) in 895 families and 1,445 European Americans (EA) in 859 families) to determine the role of MYH9 in subclinical nephropathy. Association analyses employed general linear models in unrelated probands and generalized estimating equations in families. Adjustment was performed for age, sex, diabetes, BMI, medications, and mean arterial pressure separately in each race.
Results
Mean (SD) eGFR and ACR were 74.3 (16.0) ml/min/1.73 m2 and 20.3 (119.9) mg/g in EA, and 88.6 (20.9) ml/min/1.73 m2 and 76.8 (394.5) mg/g in AA (both p < 0.0001 across ethnicities). Urine ACR was associated with rs3752462 (p = 0.01) and rs4821481 (p = 0.05) in unrelated AA and with rs4821481 (p = 0.03), rs2032487 (p = 0.04) and the E1 3224 haplotype (p = 0.013) in AA families. Single nucleotide polymorphisms and the haplotype were not associated with ACR in EA or with eGFR in either ethnic group.
Conclusions
MYH9 variants are associated with albuminuria in hypertensive AA. The strength of the association was weaker than that in FSGS and hypertensive end-stage renal disease. MYH9 risk variants appear to be associated with primary FSGS with secondary hypertension, although nephrosclerosis may develop in response to hypertension in subjects homozygous for the MYH9 E1 risk haplotype.
doi:10.1159/000194791
PMCID: PMC2749685  PMID: 19153477
African Americans; Albuminuria; Chronic kidney disease; Essential hypertension; HyperGEN study; MYH9 gene
7.  Polymorphisms in the Nonmuscle Myosin Heavy Chain 9 Gene (MYH9) Are Associated with Albuminuria in Hypertensive African Americans: The HyperGEN Study 
American journal of nephrology  2009;29(6):626-632.
Background
MYH9 is a podocyte-expressed gene encoding nonmuscle myosin IIA that is associated with idiopathic and human immunodeficiency virus-associated focal segmental glomerulosclerosis (FSGS) and hypertensive end-stage renal disease in African Americans.
Methods
Four single nucleotide polymorphisms comprising the major MYH9 E1 risk haplotype were tested for association with estimated glomerular filtration rate (eGFR) and urine albumin:creatinine ratio (ACR) in 2,903 HyperGEN participants (1,458 African Americans (AA) in 895 families and 1,445 European Americans (EA) in 859 families) to determine the role of MYH9 in subclinical nephropathy. Association analyses employed general linear models in unrelated probands and generalized estimating equations in families. Adjustment was performed for age, sex, diabetes, BMI, medications, and mean arterial pressure separately in each race.
Results
Mean (SD) eGFR and ACR were 74.3 (16.0) ml/min/1.73 m2 and 20.3 (119.9) mg/g in EA, and 88.6 (20.9) ml/min/1.73 m2 and 76.8 (394.5) mg/g in AA (both p < 0.0001 across ethnicities). Urine ACR was associated with rs3752462 (p = 0.01) and rs4821481 (p = 0.05) in unrelated AA and with rs4821481 (p = 0.03), rs2032487 (p = 0.04) and the E1 3224 haplotype (p = 0.013) in AA families. Single nucleotide polymorphisms and the haplotype were not associated with ACR in EA or with eGFR in either ethnic group.
Conclusions
MYH9 variants are associated with albuminuria in hypertensive AA. The strength of the association was weaker than that in FSGS and hypertensive end-stage renal disease. MYH9 risk variants appear to be associated with primary FSGS with secondary hypertension, although nephrosclerosis may develop in response to hypertension in subjects homozygous for the MYH9 E1 risk haplotype.
doi:10.1159/000194791
PMCID: PMC2749685  PMID: 19153477
African Americans; Albuminuria; Chronic kidney disease; Essential hypertension; HyperGEN study; MYH9 gene

Results 1-7 (7)