No abstract

Human Alpha 2-HS glycoprotein (AHSG), a glycoprotein synthesized by hepatocytes, was expressed in insect cells using the recombinant baculovirus system. The protein was purified from the cell supernatant, and appeared as a single band at about 52 kDa. Western blot using a specific antibody to the B-chain of AHSG indicated that the connecting peptide was present in the protein. When incubated with solubilized insulin receptors, recombinant AHSG inhibited the tyrosine kinase activity of the receptors in a dose-dependent fashion at concentrations in the range of those of the circulating protein. AHSG did not interfere with the binding of insulin to its receptor. These results indicate that human AHSG represents a natural inhibitor of the insulin receptor tyrosine kinase, is active as a single-chain protein and possesses a biological role similar to that of its homologue in rats, pp63, described by Auberger et al. (1).

Insulinlike growth factors (IGF) and epidermal growth factor (EGF) are produced in renal tissue, as are specific receptors for these hormones. To evaluate the significance of these observations to regulation of renal tubular cell proliferation, we have examined the interaction of IGF and EGF with cultured human proximal tubular epithelial cells (HPT). HPT cells showed specific binding of IGF-1, insulin, and EGF. IGF-1 binding was inhibited by antibody to the type 1 IGF receptor (alpha-IR3). Insulin receptors and type 1 IGF receptors were identified by bifunctional cross-linking. IGF-1, insulin, and EGF stimulated [3H]thymidine incorporation by 77, 73, and 87%, respectively. Half maximal stimulation by IGF-1, insulin, and EGF were produced with 4 X 10(-9) M, 2.5 X 10(-8) M, and 8 X 10(-10) M concentrations of these hormones. Alpha-IR3 inhibited stimulation of thymidine incorporation by IGF-1 and insulin but had no effect in EGF-stimulated thymidine incorporation. EGF and high concentrations of insulin both stimulated cell proliferation by 83 and 79%, respectively. These data are consistent with regulation of tubular epithelial proliferation by IGF-1, insulin, and EGF and suggest that the mitogenic activity of both insulin and IGF-1 is mediated by the type 1 IGF receptor.

Skeletal muscle wasting and dysfunction is a common characteristic of non-dialysis dependent chronic kidney disease (NDD-CKD). The mechanisms by which this occurs are not clearly understood and one reason for this is a lack of well controlled in-vitro methodologies to simulate NDD-CKD induced muscle wasting for mechanistic investigation at the cellular level. Here we sought to conduct the initial investigations into developing a CKD-induced skeletal muscle model for use as a mechanistic analysis tool as well as a test bed for potential novel therapeutics in this population. Human derived muscle cells (HDMCs) were isolated from n=5 NDD-CKD patients and n=3 matched healthy controls (HC) and taken through proliferation and differentiation phases in cell culture. Upon comparison of the 2 donor types, significantly greater mRNA expression of myogenic markers was noted in the NDD-CKD cultures in comparison to HC cultures, which was carried through to greater mRNA expression of myosin heavy chains (MyHCs) post differentiation. However, this was not carried over to protein expression where Pax7 and MyoD were seen to be expressed to a greater extent in HC cultures. mRNA expression markers of protein degradation were noted to be elevated in NDD-CKD cultures in comparison to HC cultures. In light of our findings, future work should seek to investigate the role of the ‘CKD environment’ as well as mechanisms implicated in transcription regulation to further advance the current model development as well as the mechanistic understanding of skeletal muscle wasting in CKD.