The glomerular podocyte as a target of growth hormone action: implications for the pathogenesis of diabetic nephropathy

Involvement of the growth hormone (GH) / insulin-like growth factor 1 (IGF-I) axis in the pathogenesis of diabetic nephropathy (DN) is strongly suggested by studies investigating the impact of GH excess and deficiency on renal structure and function. GH excess in both the human (acromegaly) and in transgenic animal models is characterized by significant structural and functional changes in the kidney. In the human a direct relationship has been noted between the activity of the GH/IGF-1 axis and renal hypertrophy, microalbuminuria, and glomerulosclerosis. Conversely, states of GH deficiency or deficiency or inhibition of GH receptor (GHR) activity confer a protective effect against DN. The glomerular podocyte plays a central and critical role in the structural and functional integrity of the glomerular filtration barrier and maintenance of normal renal function. Recent studies have revealed that the glomerular podocyte is a target of GH action and that GH's actions on the podocyte could be detrimental to the structure and function of the podocyte. These results provide a novel mechanism for GH's role in the pathogenesis of DN and offer the possibility of targeting the GH/IGF-1 axis for the prevention and treatment of DN.     

The podocyte as a target for therapies--new and old

Injury to the podocyte results in proteinuria and often leads to progressive kidney disease. As podocytes have limited ability to repair and/or regenerate, the extent of podocyte injury is a major prognostic determinant in diabetic nephropathy and other common causes of end-stage renal disease. Therapies aimed at preventing or limiting podocyte injury and/or at promoting podocyte repair or regeneration therefore have major potential clinical and economic benefits. Many current therapies--including glucocorticosteroids and calcineurin antagonists--have potent effects on podocytes. The nonspecific natures of these agents lead to undesirable systemic adverse effects: an agent with a more specific focus on podocytes would cause less treatment-associated morbidity. Recent years have seen dramatic advances in our understanding of podocyte biology and in particular regulation of its actin cytoskeleton, the major determinant of the complex architecture on which these cells depend for their function. This advance has allowed the identification of potential therapeutic targets and the next few years should see the development and testing of specific therapies aimed at the podocyte. Thus we are about to move from a situation where some of our 'blunderbuss' older therapies fortuitously happened to have beneficial effects on podocytes to a new era where advances in biological knowledge about a key cell type in the kidney will allow targeted drug design. As well as being intellectually more satisfying, every reason exists to believe that patients of the future will benefit and that the scourge of progressive kidney disease will be more effectively tackled.     

Myostatin is a skeletal muscle target of growth hormone anabolic action

Myostatin is a cytokine that has recently been shown to selectively and potently inhibit myogenesis. To investigate the mechanisms of anabolic actions of GH on skeletal muscle growth, we examined the in vitro and in vivo effects of GH on myostatin regulation. Twelve GH-deficient hypopituitary adult subjects were treated with recombinant GH (5 micro g/kg.d) in a double-blind, placebo-controlled fashion. Body composition and physical function were assessed and skeletal muscle biopsies from the vastus lateralis performed at 6-monthly intervals during 18 months of treatment. Myostatin mRNA expression was significantly inhibited to 31 +/- 9% (P < 0.001) of control by GH but not by placebo administration (79 +/- 11%) as determined by quantitative real-time PCR normalized for the housekeeping glyceraldehyde-3-phosphate dehydrogenase gene. The inhibitory effect of GH on myostatin was sustained after 12 and 18 months of GH treatment. These effects were associated with increases in lean body mass and translated into enhanced aerobic performance as determined by maximal oxygen uptake and ventilation threshold. Parallel in vitro studies of skeletal muscle cells demonstrated significant reduction of myostatin expression by myotubes in response to GH, compared with vehicle treatment. Conversely, GH receptor antagonism resulted in up-regulation of myostatin in myoblasts. Given the potent catabolic actions of myostatin, our data suggest that myostatin represents a potential key target for GH-induced anabolism.     

Identification of placental human growth hormone as the growth hormone-V gene expression product

A GH variant of placental origin, placental GH, has recently been shown to replace pituitary GH in maternal serum during pregnancy. Besides, the GH variant (GH-V) gene has been demonstrated to be expressed in the placenta. The similarities between their known properties strongly suggest that the placental GH and the GH-V protein are the same molecular species. Here we provide final evidence that this is indeed the case by sequence analysis of both the 22K and 25K forms. Furthermore, the 25K form is shown to be glycosylated, while the 22K form is not. Both size variants of placental GH are, thus, likely to reflect the partial glycosylation of a unique peptidic chain.     

Morphoangiogenesis: a unique action of growth hormone

A new form of angiogenesis, morphoangiogenesis (M-A), produces stem cells in vivo in adults. M-A was first identified in the knees of adult rabbits used to test intraarticular growth hormone (IAGH) injections as a method of regenerating articulophyseal cartilage (APC). M-A produces two structures capable of generating the stem cells. The first structure, which resembles cartilage canals normally observed only in fetuses, starts to develop 24 h after an IAGH injection. The second structure, which resembles renal glomeruli, develops several weeks later. Both structures contained thin-walled fenestrated capillaries and produced stem cells which contributed to the cascade generating articular cartilage. It was thought that other uses for morphoangiogenesis could be developed. These include tissue and organ formation using stem cells produced by M-A and the obstruction of neovascularization of cancers by M-A's ability to form structures which would obstruct nutritive vessels and thereby starve tumors.     

Bone marrow adipocytes: a neglected target tissue for growth hormone

Bone marrow (BM) contains numerous adipocytes. These share a common precursor with osteoblasts and chondrocytes, but their function is unknown. It is unclear what regulates the differentiation of these three different cell types, though their subsequent metabolic activity is under hormonal regulation. GH and estrogen stimulate bone growth and mineralization, by direct effects on chondrocytes and osteoblasts. GH also stimulates lipolysis in subcutaneous and visceral adipocytes. However, adipocytes in BM have largely been ignored as potential targets for GH or estrogen action. We have addressed this by measuring BM adipocyte number, perimeter and area as well as bone area and osteoblast activity in GH-deficient dwarf (dw/dw), normal, or ovariectomized (Ovx) rats, with or without GH, IGF-1, PTH, or estrogen treatment or high fat feeding. Marrow adipocyte numbers were increased 5-fold (P < 0.001) in dw/dw rats, and cell size was also increased by 20%. These values returned toward normal in dw/dw rats given GH but not when given IGF-1. Cancellous bone area and osteoblast number were significantly (P < 0.005) lower in dw/dw rats, though alkaline phosphatase (ALP) activity in individual osteoblasts was unchanged. GH treatment increased % osteoblast covered bone surface without affecting individual cell ALP activity. Ovariectomy in normal or dw/dw rats had no affect on marrow adipocyte number nor size, although estrogen treatment in ovariectomized (Ovx) normal rats did increase adipocyte number. Ovx decreased tibial cancellous bone area in normal rats (64%; P < 0.05) and decreased osteoblast ALP-activity (P < 0.01) but did not affect the percentage of osteoblast-covered bone surface. Estrogen replacement reversed these changes. While treatment with PTH by continuous sc infusion decreased cancellous bone (P < 0.05) and high fat feeding increased the size of BM adipocytes (P < 0.01), they did not affect BM adipocyte number. These results suggest that GH has a specific action on BM adipocytes that is not simply due to altered bone or fat metabolism. We conclude that the marrow adipocyte lineage is an important and specific target for GH action. The inverse relationship between adipocyte number and osteoblast covered bone surface, together with the well-known effects of GH on epiphysial chondrocytes leads us to propose that GH plays two important roles on cells of all three lineages. During differentiation, it regulates the numbers of each cell type that are maintained from the common precursor lineage. Subsequently it has cell-specific effects on the metabolic activities of the differentiated cells. In the case of marrow adipocytes, GH-dependent lipolysis could provide an important hormonally regulated local high energy source in bone.     

Melanin-concentrating hormone receptor is a target of leptin action in the mouse brain

Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide that is important in the regulation of energy homeostasis. MCH signals via a seven-transmembrane G protein-coupled receptor, which is coupled to Galpha(i). This receptor was initially cloned in rat and human and designated SLC-1 because of its homology to the somatostatin receptor. In rat brain, it is expressed in a pattern that mirrors the previously described pattern of projections of MCH-immunoreactive fibers. In the present study we cloned the mouse MCH receptor (MCH-R) ortholog by a rapid amplification of 5'- and 3'-cDNA ends approach and have found it to be 98% homologous with the rat sequence. We have characterized MCH-R messenger RNA distribution in the mouse brain by in situ hybridization and have shown MCH-R to be expressed in diverse brain areas implicated in the regulation of feeding, body adiposity, and sensory integration of smell and gustatory inputs, including the hypothalamus [paraventricular nucleus (magnocellular part) and dorsomedial, ventromedial, and arcuate nucleus], areas of the olfactory pathway, and the nucleus of the solitary tract. We also studied MCH-R regulation and found that MCH-R expression is increased 7-fold by 48-h fasting or genetic leptin deficiency (ob/ob mice) and is completely blunted by leptin administration. In contrast, MCH-R messenger RNA expression remains unaltered in genetic MCH deficiency. Our findings suggest that MCH-R constitutes a central target of leptin action in the mammalian brain.     

Identification of the angiogenic endothelial-cell growth factor-1/thymidine phosphorylase as a potential target for immunotherapy of cancer

Characterization of the antigens recognized by tumor-reactive T cells isolated from patients successfully treated with allogeneic HLA-matched hematopoietic stem cell transplantation (SCT) can lead to the identification of clinically relevant target molecules. We isolated tumor-reactive cytotoxic CD8(+) T-cell (CTL) clones from a patient successfully treated with donor lymphocyte infusion for relapsed multiple myeloma after allogeneic HLA-matched SCT. Using cDNA expression cloning, the target molecule of an HLA-B7-restricted CTL clone was identified. The CTL clone recognized a minor histocompatibility antigen produced by a single nucleotide polymorphism (SNP) in the angiogenic endothelial-cell growth factor-1 (ECGF1) gene also known as thymidine phosphorylase. The SNP leads to an Arg-to-His substitution in an alternatively translated peptide that is recognized by the CTL. The ECGF1 gene is predominantly expressed in hematopoietic cells, although low expression can also be detected in other tissues. The patient from whom this CTL clone was isolated had mild graft-versus-host disease despite high numbers of circulating ECGF-1-specific T cells as detected by tetramer staining. Because solid tumors expressing ECGF-1 could also be lysed by the CTL, ECGF-1 is an interesting target for immunotherapy of both hematologic and solid tumors.     

Identification of a cytoplasmic linker protein as a potential target for neovascularization

Objective

Atherosclerosis and other cardiovascular diseases are serious threats to human health and become the leading cause of death in the world. Emerging evidence reveals that inhibition of plaque neovascularization could be an effective approach for the treatment of atherosclerosis. This study was conducted to identify cytoplasmic linker protein 170 as a potential target for cardiovascular diseases through modulation of neovascularization.

Methods and results

Immunofluorescence microscopy revealed that cytoplasmic linker protein 170 was ubiquitously expressed in mouse kidney, liver, lung, normal non-atherosclerotic aorta, and atherosclerotic aorta and was partly localized in the vascular endothelium. siRNAs were introduced to human umbilical vein endothelial cells (HUVECs) and the effect of knockdown was confirmed by Western blotting. Vascularization study was assessed with matrigel-based capillary assembly, branching, and in vivo matrigel plug assays. The data showed that siRNA-mediated knockdown of the cytoplasmic linker protein remarkably compromised the assembly and branching of capillary-like blood vessels and neovascularization in vivo. Cell motility and polarity properties were then analyzed using scratch wound repair, boyden chamber, and immunofluorescence assays, and the results revealed that the cytoplasmic linker protein was critical for the motility abilities of HUVECs through its actions on cell polarity.

Conclusion

Both in vitro and in vivo studies demonstrate the significance of the cytoplasmic linker protein for blood vessel formation. Mechanistic investigation reveals that its effect on neovascularization is orchestrated through its regulation of vascular endothelial cell polarity and motility. These findings provide the basis for exploring effective approaches to regulate neovascularization in cardiovascular diseases.     

人肾小球足细胞表达Nephrin的体外研究

目的：观察人足细胞在体外表达Nephrin的分子量大小、细胞内分布。方法：体外培养人肾小球足细胞，采用间接免疫荧光法、免疫蛋白印迹研究原代培养人足细胞表达Nephrin的分布、蛋白质的分子量，逆转录PCR(RT-PCR)法检测Nephrin的mRNA表达等，RT-PCR产物进行序列分析加以确定。结果：体外原代培养的人足细胞可以表达Nephrin，间接免疫荧光显示其沿足细胞的细胞膜、细胞骨架和细胞核分布，并且在细胞内有一聚集中心。免疫蛋白印迹示人足细胞表达2种分子量的Nephrin，其中135kD的分子量与预期分子量一致，180kD的分子量与肾小球蛋白提取物一致，RT-PCR从基因水平确定了该蛋白的表达。结论：人足细胞在体外培养的条件下表达不同分子量的Nephrin，并且在细胞内分布具有一定的规律，为进一步研究其在自身细胞和肾脏病中的作用提供了可靠的实验方法。     

Polyamine metabolism as a target for growth inhibition of Acanthamoeba culbertsoni

Alpha-difluoromethylornithine had no inhibitory effect on the growth of Acanthamoeba culbertsoni, while methylglyoxal bis(guanyl-hydrazone) and sinefungin completely blocked amebic growth at 2.5 mM and 5 micrograms/ml concentrations, respectively. Cyclohexylamine caused only partial inhibition of amebic growth at 50-200 microM concentrations. Methylglyoxal bis(guanyl-hydrazone) inhibited S-adenosylmethionine decarboxylase of A. culbertsoni indicating this enzyme to be the target for its antiamebic action. This enzyme was, however, not inhibited by sinefungin, suggesting an alternative target for growth inhibition by this compound.     

Loss of sulfated carbohydrate from the glomerular podocyte as a cause of albuminuria in experimental diabetic rats: Ultrastructural histochemical study

Decrease of anionic sites and heparan sulfate proteoglycan has been demonstrated in diabetic glomerular basement membrane (GBM) and causally related to albuminuria. The HID-TCH-SP-PD technique is a sensitive histochemical method for negatively charged sulfated carbohydrates. In this study, we examined the localization of HID-TCH-SP-PD reaction in the glomeruli of diabetic and control rats. In both rats, the reaction was found in assciation with the surface of podocytes and with GBM. In diabetic rats, the former-associated reaction was markedly reduced. By contrast, the latter-associated reaction did not show any differences between both animal groups. Urinary albumin excretion (UAE) increased significantly in alloxanized rats. It is suggested that a decrease of podocyte-associated sulfated carbohydrates, the primary observed change, gives rise to albuminuria in alloxan diabetic rats.