Chronic Activation of the MAP Kinase Pathway in Isolated Proximal Tubules of Adult Male Sheep with Antenatal Glucocorticoid Exposure

The administration of antenatal glucocorticoids is a well‐accepted therapeutic approach to improve the survivability of infants; an estimated 100,000 women annually who are in jeopardy of premature delivery are treated with steroids. Although antenatal steroid treatment has clear benefit for pulmonary development and improved mortality of the newborn, the long‐term consequences of glucocorticoid exposure during a critical period in fetal development for the adult offspring are not clear. We established a model of fetal programming in the sheep whereby pregnant ewes are exposed to the same dose of glucocorticoids and at the same period of gestation as that applied clinically. The betamethasone‐exposed (BMX) offspring exhibit indices of cardiovascular dysregulation including elevated blood pressure, a reduced baroreflex response and an imbalance in the kidney and brain renin‐angiotensin systems (RAS). The adult exposed males also exhibit a reduced capacity to excrete a sodium (Na+) load that may contribute to the sustained increase in blood pressure. In this regard, isolated proximal tubule cells from male BMX sheep show an enhanced basal increase in Na+ uptake and an exaggerated Na+ response to angiotensin II (Ang II); however, the natriuretic response to Ang‐(1‐7) is blunted suggesting that antenatal steroid exposure impairs the regulation of Na+ in the proximal tubules of adult males. The present study investigated the downstream signaling events in isolated proximal tubules that may reflect the altered expression of the Ang II‐AT 1 receptor and Ang‐(1‐7)‐AT 7 /Mas receptor pathways by antenatal steroid exposure. Proximal tubules (PTs) were isolated from kidneys of control and BMX adult male sheep (~1.2 years of age) and the soluble PT extracts were initially analyzed by a multiplex intracellular signaling array (Cell Signaling, Danvers MA). The array data revealed that components of the MAP kinase pathway [ERK1/2, p38, JNK] were elevated in the PT extracts from the BMX sheep. We then sought to confirm that the MAP kinase pathway was activated in the PTs of exposed sheep using individual antibodies for the phosphorylated (phospho) and total isoforms for each kinase. The MAP kinase phospho‐ERK1/2 was increased 3.7‐fold in the BMX PTs as compared to the non‐exposed controls [2.28±0.57 vs. 0.62±0.07; n = 4 per group, p<0.05]. The phospho‐p38 isoform was increased 2.6‐fold in the PTs from the exposed BMX sheep as compared to the non‐exposed group [1.46±0.24 vs. 0.57±0.22; n = 4 per group, p<0.05]. The chronic activation of the MAP kinase pathway in the BMX PTs may reflect an attenuated expression of cellular phosphatases. One potential target involved in the regulation of both ERK1/2 and p38 is the dual specificity phosphatase DUSP‐1; however, immunoblot analysis did not reveal a difference in the expression of DUSP‐1 between the BMX and control sheep. In summary, the present data demonstrate that the MAP kinase pathway is chronically stimulated in the tubules of BMX male sheep. We suggest that both an activated Ang II‐AT 1 receptor axis and an attenuated Ang‐(1‐7)‐AT 7 /Mas receptor pathway contribute to the long‐term alteration of the MAP kinase system within the renal tubules that potentially accounts for the altered Na+ handling in this model of glucocorticoid‐dependent fetal programming. Support or Funding Information NICHD HD084227, HD‐047584 and HD‐017644