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RATIONALE & OBJECTIVE: Preeclampsia, disproportionately affecting Black women, is a leading cause of preterm delivery and risk for future hypertension and chronic kidney disease (CKD). Apolipoprotein L1 (APOL1) kidney risk alleles, common among Blacks, contribute substantially to CKD disparities. Given the strong link between preeclampsia and CKD, we investigated whether maternal and fetal APOL1 risk alleles can jointly influence preeclampsia risk, and explored potential modifiers on the APOL1- preeclampsia association. STUDY DESIGN: Nested case-control study. SETTING & PARTICIPANTS: 426 Black mother-infant pairs (275 African-Americans; 151 Haitians) from the Boston Birth Cohort. EXPOSURE: Maternal and fetal APOL1 risk alleles OUTCOMES: Preeclampsia ANALYTICAL APPROACH: Logistic regression models with adjustment for demographic characteristics were applied to analyze associations between fetal and maternal APOL1 risk alleles and risk of preeclampsia, and to investigate effect modification by maternal country-of-origin. RESULTS: Fetal APOL1 risk alleles tended to be associated with an increased risk of preeclampsia, which was not statistically significant in the total genotyped population. However, this association was modified by maternal country-of-origin (P < 0.05 for interaction tests): fetal APOL1 risk alleles were significantly associated with an increased risk of preeclampsia among African-Americans under recessive (OR=3.6, 95% CI=1.3–9.7, P=0.01) and additive (OR=1.7, 95% CI=1.1–2.6, P=0.01) genetic models, but not in Haitian Americans. Also, maternal-fetal genotype discordance at the APOL1 locus was associated with a 2.6-fold higher risk of preeclampsia (P

Glomerular podocytes are highly specialized terminally differentiated cells that act as a filtration barrier in the kidney. Mutations in the actin-binding protein, α-actinin 4 (ACTN4), are linked to focal segmental glomerulosclerosis (FSGS), a chronic kidney disease characterized by proteinuria. Aberrant activation of NF-κB pathway in podocytes is implicated in glomerular diseases including proteinuria. We demonstrate here that stable knockdown of ACTN4 in podocytes significantly reduces TNFα-mediated induction of NF-κB target genes, including IL-1β and NPHS1, and activation of an NF-κB-driven reporter without interfering with p65 nuclear translocation. Overexpression of ACTN4 and an actin binding-defective variant increases the reporter activity. In contrast, an FSGS-linked ACTN4 mutant, K255E, which has increased actin binding activity and is predominantly cytoplasmic, fails to potentiate NF-κB activity. Mechanistically, IκBα blocks the association of ACTN4 and p65 in the cytosol. In response to TNFα, both NF-κB subunits p65 and p50 translocate to the nucleus, where they bind and recruit ACTN4 to their targeted promoters, IL-1β and IL-8. Taken together, our data identify ACTN4 as a novel coactivator for NF-κB transcription factors in podocytes. Importantly, this nuclear function of ACTN4 is independent of its actin binding activity in the cytoplasm.

Mutations in α-actinin 4 (ACTN4) are linked to familial forms of focal segmental glomerulosclerosis (FSGS), a kidney disease characterized by proteinuria due to podocyte injury. The mechanisms underlying ACTN4 mutant-associated FSGS are not completely understood. Although α-actinins are better known to cross-link actin filaments and modulate cytoskeletal organization, we have previously shown that ACTN4 interacts with transcription factors including estrogen receptor and MEF2s and potentiates their transcriptional activity. Nuclear receptors including retinoic acid receptor (RAR) have been proposed to play a protective role in podocytes. We show here that ACTN4 interacts with and enhances transcriptional activation by RARα. In addition, FSGS-linked ACTN4 mutants not only mislocalized to the cytoplasm, but also lost their ability to associate with nuclear receptors. Consequently, FSGS-linked ACTN4 mutants failed to potentiate transcriptional activation by nuclear hormone receptors in podocytes. In addition, overexpression of these mutants suppressed the transcriptional activity mediated by endogenous wild-type ACTN4 possibly by a cytoplasmic sequestration mechanism. Our data provide the first link between FSGS-linked ACTN4 mutants and transcriptional activation by nuclear receptor such as RARα and peroxisome proliferator-activated receptor γ.

HIV-1 induces renal epithelial dedifferentiation in a transgenic model of HIV-associated nephropathy. Background Human immunodeficiency virus-associated nephropathy (HIVAN) is the most common cause of renal failure in HIV-1–seropositive patients. Recent studies using an HIV-1 transgenic mouse model have demonstrated that expression of HIV-1 in the kidney is required for the development of HIVAN. What has remained unclear, however, is the renal cell type responsible for pathogenesis and the essential pathological process. Methods To address these issues, we used a transgenic murine model of HIVAN. We identified the cell types in kidney in which HIV transgene expression occurs using in situ hybridization. We evaluated evidence of proliferation by immunocytochemical analysis using an antibody to Ki-67 and cell type-specific markers, including WT-1, synaptopodin, Na + ,K + -ATPase, adducin, and desmin. TUNEL assay was used to evaluate apoptosis. Results We found that glomerular and tubular epithelial cells express the HIV-1 transgene early in the disease process when renal architecture is well preserved. Transgene expression is lost, however, in tubular epithelial cells when they lose their differentiated cuboidal phenotype. In glomerular epithelial cells, dedifferentiation occurs with reduced expression of WT-1 and synaptopodin, in association with activation of desmin expression. Tubular microcysts also form with mislocalization of Na + ,K + -ATPase expression to the lateral and apical cellular membranes. Conclusions These studies support the hypothesis that the glomerular and renal epithelial cells are the primary targets of HIV-1 pathogenesis in the kidney. The essential pathologic process is dysregulation of the epithelial cell cycle with increased proliferation, apoptosis, cellular dedifferentiation, and altered cellular polarity.

Transport of phosphorothioate oligonucleotides in the kidney: Implications for molecular therapy. The systemic administration of phosphorothioated antisense oligonucleotides has been demonstrated to be an effective strategy for the control of gene expression. Because previous studies have suggested both hepatic and renal accumulation of systemically administered oligonucleotides, we explored whether the kidney might be a site of free DNA transport. [ 32 P]-phosphorothioate oligonucleotides (20 mers) were excreted in urine but cleared at only 30% of glomerular filtration rate. Plasma clearance of the label was very rapid (t 1/2 ˜5 min) but the half life of labeled S-deoxynucleotide excreted in urine was much slower (28 min). Infused oligonucleotide appeared in urine with little degradation. By autoradiography of renal tissue, labeled antisense oligonucleotides appeared within Bowman's capsule and the proximal tubule lumen. DNA was detected in association with brush border membrane and within tubular epithelial cells. Brush border membrane preparations from rat kidney contained oligonucleotide binding proteins as determined by gel mobility shift and UV cross linking assays. Because renal epithelial cells efficiently take up phosphorothioate oligonucleotides without apparent degradation, the kidney appears to be an excellent target for site-directed antisense therapy, but may be a site of anti-sense toxicity as well.

Thromboxane and prostacyclin differentially regulate murine extracellular matrix gene expression. Alterations in the arachidonic acid metabolites thromboxane and prostacyclin are known to contribute to hemodynamic changes observed in certain models of acute and chronic renal failure. We have previously shown that thromboxane may have an important role in mediating glomerulosclerosis by stimulating the expression of certain extracellular matrix proteins. In the present study, we compared the effects of thromboxane and prostacyclin on the expression of genes encoding basement membrane proteins using a murine teratocarcinoma cell line, that when differentiated to an endodermal phenotype synthesizes abundant extracellular matrix. Incubation of these cells with stable analogs of thromboxane and prostacyclin for four hours resulted in changes in basement membrane gene expression. Thromboxane increased steady-state mRNA levels for all three laminin chains, type IV collagen, and fibronectin, but decreased the level of mRNA for heparan sulfate proteoglycan. In contrast, incubation with carbo-prostacyclin, a stable analog of prostacyclin, decreased the steady-state mRNA level for the laminin A and B1 chains, type IV collagen and fibronectin, and increased the mRNA level for heparan sulfate proteoglycan and laminin B2. Carbo-prostacyclin did not affect cellular proliferation or thymidine incorporation. These results indicate that eicosanoids directly modulate matrix gene expression independently of hemodynamic influence, and independently of effects mediated by platelets, or mitogenesis. Furthermore, these findings suggest that the alterations in renal eicosanoid metabolism may directly participate in the pathogenesis of glomerulosclerosis and thus provide a rationale for therapy directed toward the specific inhibition of thromboxane in the treatment of progressive glomerular sclerosis.

Transforming growth factor TGF-beta 2 is encoded by multiple mRNA transcripts of 5.8, 5.1, 4.0, 3.8, and 2.8 kilobase pairs (kb) that are expressed in various human and monkey cells. Northern blot analysis using genomic fragments of DNA was used to demonstrate that some of this size heterogeneity is due to differences in the length of the 5'-untranslated region. Probes that were colinear with the first 600 nucleotides of the 5'-untranslated region detected only the 5.8-, 4.0-, and 3.8-kb transcripts. In order to identify DNA elements that regulate the transcription of these mRNA transcripts, deletion constructs of 5'-flanking DNA were ligated to the coding region for chloramphenicol acetyltransferase (CAT) and analyzed for promoter activity in several cell lines. Sequences responsible for putative enhancer and silencer regions were identified between -778 and -40 relative to the transcription initiation site. Addition of a cyclic AMP-responsive element/activating transcription factor-like element at -74 resulted in a 5-10-fold increase in CAT activity over that expressed with a construct that contained only the TATA box. This increase in CAT activity was suppressed by the addition of DNA sequences between -257 and -187, whereas sequences between -778 and -257 stimulated CAT activity. Point mutations within the ATF binding site at -74 resulted in a marked decrease in CAT expression. Cotransfection with ATF-1 or ATF-2 expression plasmids resulted in both dose-dependent stimulatory and inhibitory activities that were cell type-dependent. These studies identify multiple transcription initiation sites for TGF-beta 2 and demonstrate that transcription from one of these promoters is dependent upon an ATF binding site located 5' of the TATA box.

A PCR method for the quantitative assessment of mRNA for laminin A, B1, and B2 chains. Laminin, a basement membrane glycoprotein, is involved in the development of normal kidney and its dysregulation contributes to glomerulosclerosis in renal disease. Studies designed to assess the regulation of this molecule at the level of transcription have been hindered by the relatively low abundance of the mRNA, making standard techniques such as Northern hybridization and RNase protection difficult and inaccurate. In this report, we have utilized the polymerase chain reaction (PCR) to quantitate differences in laminin mRNA expression during normal development of the mouse kidney. We have constructed a synthetic template to be used as an internal standard for mRNA quantitation of laminin chains A, B1 and B2, and β-actin. This DNA template can be used to generate complementary RNA which can be reverse transcribed and amplified simultaneously with 0.5 µg of total cellular mRNA allowing for accurate and absolute quantitation of laminin mRNA by PCR.

Two regulatory regions in the murine collagen IV enhancer were identified. Transient transfection assays delimited a 210-base pair fragment within the first intron of the alpha 1(IV) collagen gene that had significant transcriptional enhancer activity. DNase I protection and gel mobility shift confirmed that two regions, designated footprints A and B, within this fragment bound nuclear factors. Gel shift studies suggested that the CCTTATCTCTGATGG motif (A-34) in the footprint A region was important for specific nuclear factor binding. Mutations in the A-34 motif abolished factor binding as detected by gel shift and resulted in a significant decrease in enhancer activity in transient transfection assays of F9 teratocarcinoma cells. Two putative transcription factors of Mr = 37,000 and Mr = 94,000, which interact with the A-34 motif, were purified from Engelbreth-Holm-Swarm tumor tissue using DEAE-Sephacel, heparin-Sepharose, salmon sperm DNA-Sepharose, and specific A-34 oligonucleotide affinity chromatography. Southwestern analysis revealed that both of these factors were capable of binding the A-34 oligonucleotide directly and did not require additional subunits for binding. These data suggest that positively acting transcription factor(s) interact with the A-34 site in the enhancer and are required for efficient transcription of the alpha 1 and alpha 2(IV) collagen chain genes.