Tanny Tsao

Impaired JAK-STAT signal transduction contributes to growth hormone resistance in chronic uremia

Chronic renal failure (CRF) is associated with resistance to the growth-promoting and anabolic actions of growth hormone (GH). In rats with CRF induced by partial renal ablation, 7 days of GH treatment had a diminished effect on weight gain and hepatic IGF-1 and IGFBP-1 mRNA levels, compared with sham-operated pair-fed controls. To assess whether GH resistance might be due to altered signal transduction, activation of the JAK-STAT pathway was studied 10 or 15 minutes after intravenous injection of 5 mg/kg GH or vehicle. Hepatic GH receptor (GHR) mRNA levels were significantly decreased in CRF, but GHR protein abundance and GH binding to microsomal and plasma membranes was unaltered. JAK2, STAT1, STAT3, and STAT5 protein abundance was also unchanged. However, GH-induced tyrosine phosphorylation of JAK2, STAT5, and STAT3 was 75% lower in the CRF animals. Phosphorylated STAT5 and STAT3 were also diminished in nuclear extracts. The expression of the suppressor of cytokine signaling-2 (SOCS-2) was increased twofold in GH-treated CRF animals, and SOCS-3 mRNA levels were elevated by 60% in CRF, independent of GH treatment. In conclusion, CRF causes a postreceptor defect in GH signal transduction characterized by impaired phosphorylation and nuclear translocation of GH-activated STAT proteins, which is possibly mediated, at least in part, by overexpression of SOCS proteins.

Effect of Prolonged Uremia on Insulin-Like Growth Factor-I Receptor Autophosphorylation and Tyrosine Kinase Activity in Kidney and Muscle

Recently, based on a study in rats with chronic renal failure (CRF), it has been suggested that IGF-I resistance in uremia may be caused in part by defective IGF-I receptor autophosphorylation and tyrosine kinase activity. Thus if such a defect were to develop in prolonged acute renal failure (ARF), this may explain why IGF-I therapy, effective in rats, has failed to promote recovery from ARF in patients. Accordingly, we examined IGF-I receptor function in rats with uremia of increasing duration and in pair-fed sham-operated controls. After 6 days of prolonged ARF, kidney IGF-I receptor binding increased twofold, while IGF-I stimulated receptor phosphorylation and tyrosine kinase activity were unchanged. Muscle receptor binding, autophosphorylation and tyrosin kinase activity were similar to control values after 6 or even 21 days of uremia. Taking all these findings together it appears that IGF-I resistance in uremia cannot be attributed to a receptor defect. This in turn argues against altered receptor function as a cause of the failure of IGF-I to modify clinical ARF.

Effect of Bacitracin on Retroendocytosis and Degradation of Insulin in Cultured Kidney Epithelial Cell Line

In an earlier study, we described the presence of a retroendocytotic pathway for insulin in a cultured kidney epithelial cell line. Derived from the opossum kidney (OK), these cells possess many features of proximal tubule epithelium, which is the major site of kidney insulin metabolism. We studied the interaction between the retroendocytotic and the degradative pathways with bacitracin as a pharmacological probe. Monolayers of OK cells were loaded with 125I-labeled insulin over 30 min, acid washed to remove membrane-bound insulin, then incubated in fresh medium for 60 min while the release of intracellular radioactivity was monitored. In experiments carried out in the presence of bacitracin (2 mM), there was a twothirds increase in intracellular radioactivity at the end of the loading phase. Measurements made during the subsequent release phase showed that bacitracin reduced the release of degradation products. Thus, although controls released 72.1 ± 8.1% of the internalized radioactivity as trichloroacetic acid (TCA)-soluble products, bacitracin-treated cells released 59.2 ± 9.4% (P < 0.02). In contrast, release of TCAprecipitable insulin increased from 15.2 ± 4.6% in controls to 25.8 ± 3.7% in bacitracin-treated cells (P < 0.01). In separate experiments analyzed by gelexclusion chromatography, 6.4 ± 0.6% of radioactivity released from preloaded control cells into medium over 60 min was insulin sized compared to 29.7 ± 1.4% in bacitracin-treated cells. High-performance liquid chromotography revealed that 61.5 ± 3.5% of this insulin-sized material released from control cells preloaded with A14-insulin eluted as intact insulin and the remainder as unidentified intermediate degradation products. In the bacitracin group, 48.0 ± 2.1% of the radioactivity eluted with insulin, and 18.5 ± 0.6% eluted under a series of peaks representing intermediate products. Included in this material was a doublet, which has been identified in previous experiments with OK cells, other cell types, and liver endosomes. Previous characterization of this doublet indicates that it results from cleavage of insulin by insulin protease or a similar enzyme. We conclude that, by inhibiting the initial steps in the degradation of insulin, bacitracin diverts intact insulin and early degradation products from the degradative to the retroendocytotic pathway. These findings suggest that insulin degradation commences in an extralysosomal site, probably recycling endosomes.

Growth Hormone–Mediated Janus Associated Kinase–Signal Transducers and Activators of Transcription Signaling in the Growth Hormone–Resistant Potassium-Deficient Rat

Potassium deficiency (KD) is associated with severe growth failure, in part caused by growth hormone (GH) resistance. This study set out to determine whether the resistance could be caused by a defect in GH-mediated janus associated kinase-signal transducers and activators of transcription (STAT) signaling as occurs in uremia. To this end, rats were fed a K-deficient diet for 8 d and pair-fed controls received a K-replete diet. Animals from each group received GH or vehicle, and during this period, KD rats were GH resistant; GH induced body and liver weight gain and linear body growth were severely attenuated in these rats. In addition, signal transduction was studied in the liver of rats that were killed 10 or 15 min after an intravenous GH bolus or vehicle. When the rats were killed, GH receptor mRNA and protein levels were similar in the two groups. The abundance of STAT5, STAT3, and STAT1, proteins that mediate GH signaling, was significantly increased by 40 to 130% in KD. Furthermore, GH induced a far greater increase in STAT5 and STAT3 phosphorylation in this group. STAT5 phosphorylation was enhanced fourfold even when normalized for total STAT5 content. Phosphorylated STAT5 and STAT3 proteins were also increased in nuclear extracts, suggesting normal nuclear translocation of the activated signaling proteins. DNA binding of nuclear STAT5 was unaltered. Thus, in KD, there is resistance to the growth-promoting action of GH despite hyperactivation of the janus associated kinase-STAT signaling pathway. This suggests the presence of a defect distal to the nuclear binding of STAT or, alternatively, a defect in a STAT-independent GH-activated signaling pathway.

PARADOXICAL BODY AND KIDNEY GROWTH IN POTASSIUM DEFICIENCY

In the growing animal, K deficiency (KD) retards body growth, but paradoxically stimulates renal growth. If KD persists, interstitial infiltrates appear and eventually tubulointerstitial fibrosis develops. In patients with chronic KD, renal cysts may form and with time tubulointerstitial disease with renal failure develops. Since early in KD, kidney IGF-I levels increase and may be a cause of the renal hypertrophy, and as TGF-β promotes hypertrophy and fibrosis, we examined the expression of these growth factors in chronic KD. Rats were given a KD diet or pair or ad-lib fed a normal K diet. After 21 days, KD rats weighed less than pair fed controls, while the kidneys were 49 % larger Serum IGF-I and kidney IGF-I protein levels were depressed, as were IGF-I mRNA levels, and is largely attributable to decreased food intake. Kidney IGFBP-1 and TGF-β mRNA levels were increased (p < 0.05). There was marked hypertrophy and adenomatous hyperplasia of outer medullary collecting ducts, hypertrophy of thick ascending limbs of Henle (TALH) and interstitial infiltrates. Both nephron segments stained strongly for IGF-I and IGFBP-l. Only the non-hyperplastic TALH was strongly TGF-β positive. Interstitial infiltrates containing monocytes/macrophages were prominent. These findings are consistent with a sustained role for IGF-I in promoting the renal hypertrophy of KD and appear to be caused by local trapping of IGF-I by the over-expressed IGFBP-1. Localization of TGF-β to the hypertrophied non-hypoplastic tubules containing IGF-I, suggests that TGF-β may be acting to convert the proliferative action of IGF-I into a hypertrophic response. TGF-β may also contribute to the genesis of the tubulointerstitial infiltrate. Finally, the reduced levels of serum IGF-1 levels may be a cause of the blunted body growth

Response to growth hormone therapy in experimental ischemic acute renal failure

In acute renal failure (ARF), the gene and peptide expression of insulin-like growth factor-I (IGF-I) falls. Because IGF-I is regulated by growth hormone (GH) and because kidney GH receptor expression is also attenuated in ARF, the impaired IGF-I expression may partly reflect local GH resistance. Because IGF-I treatment accelerates recovery from ARF, we determined whether high-dose GH therapy could overcome this putative GH resistance, stimulate IGF-I production, and enhance recovery. Rats with ARF were given 2.5 mg GH or vehicle (V) over 2 days, beginning 24 hours before the onset of ARF. GH prevented weight loss but did not modify the course of ARF. Next we determined whether the failure of GH to modify kidney recovery could reflect a failure to stimulate renal IGF-I gene expression. Rats were treated with GH or V over an 18-hour period beginning 1 day after the induction of ARF. Hepatic IGF-I mRNA and serum IGF-I peptide levels rose significantly with GH treatment, but the low kidney IGF-I mRNA levels did not respond. We conclude that the failure of GH to enhance recovery from ARF is caused by impaired GH-stimulated renal IGF-I production, while the maintenance of body weight likely reflects the systemic effects of the increase in hepatic IGF-I production.

The insulin-like growth factor-I axis in acute renal failure.

We have examined the response of the renal insulin-like growth factor (IGF-I) axis to acute ischemic injury in the rat Key findings included a decrease in IGF-I mRNA and peptide levels, a decrease in GH receptor gene plus protein expression and a decrease in the IGF binding proteins except for IGF binding protein I. Administration of GH to compensate for the reduced GH receptor binding corrected the IGF-I mRNA levels suggesting a relative GH deficiency. Interestingly, IGF-I receptor mRNA levels were unchanged while plasma membrane IGF-I receptor number increased two fold. This appeared to be due to a redistribution of receptors to a membrane location. IGF-I receptor autophosphorylation and tyrosine kinase activity were intact despite severe uremia for up to 6 days. We propose that this increase of functional IGF-I receptors following acute tubular necrosis will sensitize the kidney to the administration of exogenous IGF-I.