Abstract
Growth hormone (GH) plays a significant role in normal renal function and overactive GH signaling has been implicated in proteinuria in diabetes and acromegaly. Previous results have shown that the glomerular podocytes, which play an essential role in renal filtration, express the GH receptor, suggesting the direct action of GH on these cells. However, the exact mechanism and the downstream pathways by which excess GH leads to diabetic nephropathy is not established. In the present article, using immortalized human podocytes in vitro and a mouse model in vivo, we show that excess GH activates Notch1 signaling in a γ-secretase-dependent manner. Pharmacological inhibition of Notch1 by γ-secretase inhibitor DAPT (N-[N-(3,5-Difluorophenacetyl)-l-alanyl]-S-phenyl glycine t-butylester) abrogates GH-induced epithelial to mesenchymal transition (EMT) and is associated with a reduction in podocyte loss. More importantly, our results show that DAPT treatment blocks cytokine release and prevents glomerular fibrosis, all of which are induced by excess GH. Furthermore, DAPT prevented glomerular basement membrane thickening and proteinuria induced by excess GH. Finally, using kidney biopsy sections from people with diabetic nephropathy, we show that Notch signaling is indeed up-regulated in such settings. All these results confirm that excess GH induces Notch1 signaling in podocytes, which contributes to proteinuria through EMT as well as renal fibrosis. Our studies highlight the potential application of γ-secretase inhibitors as a therapeutic target in people with diabetic nephropathy.
Highlights
Growth hormone (GH) plays a significant role in normal renal function and overactive GH signaling has been implicated in proteinuria in diabetes and acromegaly
To identify the biochemical pathways downstream of GH signaling that are hyperactivated in podocytes in response to excess GH, we previously performed microarray analysis (GEO accession number GSE21327) on immortalized human podocytes treated with GH and identified transcriptional activation of epithelial to mesenchymal transition (EMT) regulator “ZEB2” [24]
To reconfirm the up-regulation of Notch signaling pathway by GH in podocytes, we analyzed the expression of Notch1 and its targets Hes1 and Jag1 in nondiabetic mouse kidney (n ϭ 18) versus diabetic nephropathy mouse kidney (n ϭ 21) in the Hodgin diabetes mouse glomerulus data set available at Nephroseq,3 which shows that diabetic nephropathy kidneys overexpress Notch1 and its target genes, Hes1 and Jag1 (Fig. 1A)
Summary
To identify the biochemical pathways downstream of GH signaling that are hyperactivated in podocytes in response to excess GH, we previously performed microarray analysis (GEO accession number GSE21327) on immortalized human podocytes treated with GH and identified transcriptional activation of EMT regulator “ZEB2” [24]. Quantification of the Mas- with increased expression of cytokines (Fig. 5C) and fibrotic son’s trichrome-stained area revealed that GH enhanced glo- marker in GH-treated mice, as measured at both mRNA and merular fibrosis (50 Ϯ 10%, Fig. S3B). These data correlated protein (Fig. S3, C and D) levels. Our results show that whereas GH treatment in the mice leads to GBM thickening and podocyte foot process effacement, similar treatment of GH along with DAPT completely abrogated this effect (Fig. 5, F and G) All these results confirm that excess GH activates Notch signaling, which in turn leads to infiltration of circulating cells in the kidneys and leading to its fibrosis. Together the data suggest that excess GH impair the podocyte function and induces proteinuria through activated Notch signaling
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