Understanding the cellular and molecular basis for changes in podocyte structure and function in diabetes mellitus may lead to novel diagnostic tools and treatment strategies for diabetic nephropathy.

Growth hormone (GH) excess results in structural and functional changes in the kidney and is implicated as a causative factor in the development of diabetic nephropathy (DN). Glomerular podocytes are the major barrier to the filtration of serum proteins, and altered podocyte function and/or reduced podocyte number is a key event in the pathogenesis of DN. We have previously shown that podocytes are a target for GH action. To elucidate the molecular basis for the effects of GH on the podocyte, we conducted microarray and RT-quantitative PCR analyses of immortalized human podocytes and identified zinc finger E-box-binding homeobox 2 (ZEB2) to be up-regulated in a GH dose-and time-dependent manner. We established that the GH-dependent increase in ZEB2 levels is associated with increased transcription of a ZEB2 natural antisense transcript required for efficient translation of the ZEB2 transcript. GH down-regulated expression of E-and P-cadherins, targets of ZEB2, and inhibited E-cadherin promoter activity. Mutation of ZEB2 binding sites on the E-cadherin promoter abolished this effect of GH on the E-cadherin promoter. Whereas GH increased podocyte permeability to albumin in a paracellular albumin influx assay, shRNA-mediated knockdown of ZEB2 expression abrogated this effect. We conclude that GH increases expression of ZEB2 in part by increasing expression of a ZEB2 natural antisense transcript. GH-dependent increase in ZEB2 expression results in loss of P-and E-cadherins in podocytes and increased podocyte permeability to albumin. Decreased expression of P-and E-cadherins is implicated in podocyte dysfunction and epithelial-mesenchymal transition observed in DN. We speculate that the actions of GH on ZEB2 and P-and E-cadherin expression play a role in the pathogenesis of microalbuminuria of DN. Pituitary growth hormone (GH)5 is essential for postnatal growth in mammals. In addition to growth, GH affects the metabolism of fat, protein, and carbohydrate (1). GH exerts these actions both by its direct effect on target organs and by stimulating the production of insulin-like growth factor-1 (IGF-1). At the tissue level, these pleiotropic actions of GH result from the interaction of GH with a specific cell surface receptor, the GH receptor (GHR). Whereas the GHR is ubiquitously expressed, the role and effects of GH have been most intensely investigated in organs and tissues such as liver, bone, muscle, and adipocytes in which GHR expression is substantial and are thus considered canonical targets of GH action. However, recent reports have highlighted the biological effects and physiological relevance of GH action in non-canonical targets such as the blastocyst (2), colonic epithelial cells (3), cardiomyocytes (4), and neurons (5).GH excess in both the human (acromegaly) and in transgenic animal models is characterized by significant structural and functional changes in the kidney (6 -8). An overactive GH/ GHR axis is implicated as a causative factor in the development of diabetic nephropathy (9 -11). Our previous study...

GH excess in both the human and transgenic animal models is characterized by significant changes in blood pressure and renal function. The GH/GH receptor (GHR) axis is also implicated in the development of diabetic nephropathy. However, it is not clear whether GH's actions on renal function are due to indirect actions mediated via changes in blood pressure and vascular tone or due to direct action of GH on the kidney. We hypothesized that functional GHRs are expressed on the glomerular podocyte enabling direct actions of GH on glomerular function. Real-time PCR, immunohistochemistry, and Western blot analysis of murine podocyte cells (MPC-5) and kidney glomeruli demonstrated expression of GHR mRNA and protein. Exposure of both murine and human podocytes to GH (50-500 ng/ml) resulted in an increase in abundance of phosphorylated signal transducer and activator of transcription-5, Janus kinase-2, and ERK1/2 proteins. Exposure of podocytes to GH also caused changes in the intracellular distribution of the Janus kinase-2 adapter protein Src homology 2-Bbeta, stimulation of focal adhesion kinase, increase in reactive oxygen species, and GH-dependent changes in the actin cytoskeleton. We conclude that glomerular podocytes express functional GHRs and that GH increases levels of reactive oxygen species and induces reorganization of the actin cytoskeleton in these cells. These results provide a novel mechanistic link between GH's actions and glomerular dysfunction in disorders such as acromegaly and diabetic glomerulosclerosis.

GnRH is a potent hypothalamic regulator of gonadotropin hormones, LH and FSH, which are both expressed within the pituitary gonadotrope and are necessary for the stimulation of gametogenesis and steroidogenesis in the gonads. Differential regulation of LH and FSH, which is essential for reproductive fitness, is achieved, in part, through the varying of GnRH pulse frequency. However, the mechanism controlling the increase in FSH during the periods of low GnRH has not been elucidated. Here, we uncover another level of regulation by GnRH that contributes to differential expression of the gonadotropins and may play an important role for the generation of the secondary rise of FSH that stimulates folliculogenesis. GnRH stimulates LHβ and FSHβ subunit transcription via induction of the immediate early genes, Egr1 and c-Fos, respectively. Here, we determined that GnRH induces rapidly both Egr1 and c-Fos, but specifically decreases the rate of c-Fos degradation. In particular, GnRH modulates the rate of c-Fos protein turnover by inducing c-Fos phosphorylation through the ERK1/2 pathway. This extends the half-life of c-Fos, which is normally rapidly degraded. Confirming the role of phosphorylation in promoting increased protein activity, we show that a c-Fos mutant that cannot be phosphorylated by GnRH induces lower expression of the FHSβ promoter than wild-type c-Fos. Our studies expand upon the role of GnRH in the regulation of gonadotropin gene expression by highlighting the role of c-Fos posttranslational modification that may cause higher levels of FSH during the time of low GnRH pulse frequency to stimulate follicular growth.