Intrauterine growth restriction (IUGR) increases the risk of serious adult morbidities such as hypertension. In an IUGR rat model of hypertension, we reported a persistent decrease in kidney 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) mRNA and protein levels from birth through postnatal (P) day 21. This enzyme deficiency can lead to hypertension by limiting renal glucocorticoid deactivation. In the present study, we hypothesized that IUGR affects renal 11beta-HSD2 epigenetic determinants of chromatin structure and alters key transcription factor binding to the 11beta-HSD2 promoter in association with persistent downregulation of its mRNA expression. To test this hypothesis, we performed bilateral uterine artery ligation on embryonic day 19.5 pregnant rats and harvested kidneys at day 0 (P0) and P21. Key transcription factors that can affect 11beta-HSD2 expression include transcriptional enhancers specificity protein 1 (SP1) and NF-kappaB p65 and transcriptional repressors early growth response factor (Egr-1) and NF-kappaB p50. Our most important findings were as follows: 1) IUGR significantly decreased SP1 and NF-kappaB (p65) binding to the 11beta-HSD2 promoter in males, while it increased Egr-1 binding in females and NF-kappaB (p50) binding in males; 2) IUGR increased CpG methylation status, as well as modified the pattern of methylation in several CpG sites of 11beta-HSD2 promoter at P0 also in a sex-specific manner; and 3) IUGR decreased trimethylation of H3K36 in exon 5 of 11beta-HSD2 at P0 and P21 in both genders. We conclude that IUGR is associated with altered transcriptional repressor/activator binding in connection with increased methylation in the 11beta-HSD2 promoter region in a sex-specific manner, possibly leading to decreased transcriptional activity. Furthermore, IUGR decreased trimethylation of H3K36 of the 11beta-HSD2 gene in both genders, which is associated with decreased transcriptional elongation. We speculate that alterations in transcription factor binding and chromatin structure play a role in in utero reprogramming.

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Juxtaglomerular cells are crucial for blood pressure and fluid-electrolyte homeostasis. The factors that maintain the life of renin cells are unknown. In vivo, renin cells receive constant cell-to-cell, mechanical, and neurohumoral stimulation that maintain their identity and function. Whether the presence of this niche is crucial for the vitality of the juxtaglomerular cells is unknown. Integrins are the largest family of cell adhesion molecules that mediate cell-to-cell and cell-to-matrix interactions. Of those, β1-integrin is the most abundant in juxtaglomerular cells. However, its role in renin cell identity and function has not been ascertained. To test the hypothesis that cell-matrix interactions are fundamental not only to maintain the identity and function of juxtaglomerular cells but also to keep them alive, we deleted β1-integrin in vivo in cells of the renin lineage. In mutant mice, renin cells died by apoptosis, resulting in decreased circulating renin, hypotension, severe renal-vascular abnormalities, and renal failure. Results indicate that cell-to-cell and cell-to-matrix interactions via β1-integrin is essential for juxtaglomerular cells survival, suggesting that the juxtaglomerular niche is crucial not only for the tight regulation of renin release but also for juxtaglomerular cell survival—a sine qua non condition to maintain homeostasis.

1. Four types of yolk spheres with variable structure, chemical composition and frequency of occurrence in yolk plasma of hierarchical follicles (F(4), F(3), F(2) and F(1) with diameters of 10.0, 15.5, 20.0 and 35.0 mm, respectively) of the hen ovary were identified using histochemical methods for localising lipids, carbohydrates and proteins. 2. Yolk spheres of the first type (YS(1)) had a phospholipoprotein membrane surrounding fluid matrix which stained lightly for phospholipids, proteins and acidic mucopolysaccharides. Two types of droplets were observed in the matrix of YS(1). Spheres of the second type (YS(2)) had a lipoprotein- and acidic mucopolysaccharide-rich peripheral region and a single large droplet in its fluid matrix. Droplets of YS(2), unlike YS(1), showed three regions and metachromatic staining with ninhydrin-Schiff reagent. The third type of sphere (YS(3)) had a homogeneous matrix staining for proteins, neutral lipids and florescent yellow with alcian blue and differentially with ninhydrin-Schiff reagent; it was bounded by a phospholipids- and acidic mucopolysaccharide-containing thick peripheral region. Its fluid matrix also showed toluidine-blue-positive, densely packed granules and small droplets. The fourth type (YS(4)) was seen only in bromophenol blue and Nile blue preparations, revealing the presence of proteins and neural lipids in their matrix and peripheral regions. 3. Quantitative data on the relative abundance of yolk spheres in F(4) to F(1) follicles revealed more YS(3) (51.1 to 64.7%) than YS(1) (16.2 to 28.3%) and YS(2) (19.1 to 23.2%). The percentage of YS(1) increased and that of YS(3) decreased as follicle size increased.

Integrins are the largest family of cell adhesion molecules. β1-Integrin (Itgb1) is the most abundantly expressed β subunit and is present in almost every cell type. Previous studies showed that Itgb1 is required for normal development of the ureteric bud and podocytes and for the function of the proximal tubule. However, its role in the kidney vasculature has not been explored. Renin cells are crucial for blood pressure homeostasis and for normal nephrovascular development. The mechanisms involved in their morphogenetic functions are not well understood. We found that Itgb1 is highly expressed in renin expressing cells throughout development. Therefore, to study the role of Itgb1 in renin cells we generated a conditional deletion (cKO) of Itgb1 in cells of the renin lineage by crossing floxed Itgb1 mice with mice expressing cre recombinase driven by the renin locus. Itgb1 cKO mice were smaller in size (20.32 ± 4.35 g vs 29.55 ± 7.39 g, p=0.016 ), had smaller kidney to body weight ratio (0.92 ± 0.30 vs 1.31 ± 0.30, p=0.017 ), hypotension (MABP 82.33 ± 4.00 mmHg vs 92.55 ± 7.72 mmHg, p=0.02 ), anemia (hemoglobin 11.26 ± 1.48 g/dL vs 15.07 ± 1.31 d/dL, p=0.003 ; hematocrit 40.39 ± 5.48% vs 53.8 ± 4.21%, p=0.002 ), renal failure (BUN 71.44 ± 32.81 mg/dL vs 29.4 ± 7.67mg/dL, p=0.004), and lower plasma renin levels (6664.58 ± 3251.92 vs 43357.09 ± 17032.63 pg/ml, p =0.001 ). Mutants also developed hyposthenuria (urine osmolality 452 ± 138.98 mOsm/kg vs 1460.5 ± 482.09 mOsm/kg, p<0.02 ). Histological analysis revealed excessive collagen deposition in the interstitium and peri-glomerular areas; fibrocystic glomeruli; tubular dilatation and protein casts in the tubules. Immunostaining for renin and α-smooth muscle actin showed a marked decrease in renin protein expression and abundant α-smooth muscle actin expression in the interstitium. Microdissection of the renal arterial tree combined with renin immunostaining confirmed the marked decrease in renin and evidenced the overall vascular abnormalities including fewer and shorter arterial and arteriolar branches. Overall, this study shows that β1-Integrin in cells of the renin lineage is crucial for renin expression, morphogenesis of the renal vasculature and maintenance of the normal kidney architecture and function.