Cardiac resistance to growth hormone in uremia.

BACKGROUND: Cardiovascular disease is a major cause of death in end-stage renal disease (ESRD). Since growth hormone is required for maintaining normal cardiac structure and function and as growth hormone has a salutary effect on cardiac remodeling in disease, we postulated that if cardiac resistance to growth hormone develops in chronic renal failure (CRF) this may predispose to the cardiomyopathy of uremia. We set out to test whether in CRF there is resistance to the cardiac action of growth hormone and whether this defect might be caused by altered growth hormone signaling. METHODS: Growth hormone-deficient (dw/dw) rats and growth hormone-intact Sprague-Dawley rats underwent a subtotal nephrectomy or sham operation and pair feeding. RESULTS: In dw/dw rats treated with growth hormone for 8 days there was a significant increase in insulin-like growth factor-1 (IGF-1) mRNA levels in controls but this response was attenuated in CRF. Next, growth hormone-stimulated Janus kinase-signal transducers and activators of transcription (JAK2-STAT5) signaling was studied 15 minutes after intravenous growth hormone in dw/dw and Sprague-Dawley rats. Growth hormone receptor, JAK2, STAT5a, and STAT5b protein levels were unaltered in CRF. Growth hormone-induced JAK2, growth hormone receptor (GHR), and STAT5 tyrosine phosphorylation was significantly depressed in CRF as was nuclear translocation of phosphorylated STAT5. When rats were treated with pharmacologic dose growth hormone, STAT5 phosphorylation increased similarly in CRF and control rats. CONCLUSION: Uremic rats develop cardiac resistance to growth hormone caused at least, in part, by a postreceptor defect in growth hormone-induced signaling that is characterized by impaired phosphorylation and nuclear translocation of STAT5. These findings raise the question whether growth hormone resistance contributes to the cardiac changes of uremia.

Growth hormone resistance in uremia, a role for impaired JAK/STAT signaling.

Resistance to growth hormone (GH) is a significant complication of advanced chronic renal failure. Thus while the circulating GH levels are normal or even elevated in uremia, resistance to the hormone leads to stunting of body growth in children and contributes to muscle wasting in adults. Insensitivity to GH is the consequence of multiple defects in the GH/insulin-like growth factor-1 (IGF-1) system. Expression of the GH receptor may be reduced, although this is not a consistent finding, GH activation of the Janus kinase 2-signal transducer (JAK2) and activator of transcription (STAT) signal transduction pathway is depressed and this leads to reduced IGF-1 expression, and finally there is resistance to IGF-1, a major mediator of GH action. We review these various defects with an emphasis on the GH-activated JAK2-STAT5 pathway, since this pathway is essential for normal body growth and there has been recent progress in our understanding of the perturbations that occur in uremia.

Chronic uremia attenuates growth hormone-induced signal transduction in skeletal muscle.

Malnutrition and muscle wasting are common in chronic renal failure (CRF) and adversely affect morbidity and mortality. Contributing to the muscle wasting is resistance to growth hormone (GH). For testing whether impaired GH signaling is a cause of the skeletal muscle GH resistance and for elucidating its mechanisms, muscle GH signaling and action were studied in GH-deficient rats with surgically induced CRF and sham-operated pairfed control rats. GH treatment increased gastrocnemius muscle IGF-1 mRNA levels significantly in control but not in CRF rats. GH-activated Janus-associated kinase 2 (JAK2)-signal transducers and activators of transcription 5 (STAT5) signaling was impaired in CRF rats, despite normal GH receptor (GHR), JAK2, and STAT5 protein levels. Phosphorylation of the GHR, JAK2, and STAT5 in response to GH was depressed by nearly half in CRF (P < 0.05), and nuclear phospho-STAT5 levels were depressed by approximately one third (P < 0.01). GH-stimulated suppressors of cytokine signaling 2 mRNA levels were significantly higher in CRF. This may be related to inflammatory cytokine activity because C-reactive protein levels were elevated. Muscle protein-tyrosine phosphatase activity was also increased significantly by twofold. In conclusion, rats with CRF acquire skeletal muscle resistance to GH that is caused at least in part by impaired JAK2-GHR-STAT5 phosphorylation and nuclear STAT5 translocation. Furthermore, it seems that the attenuated JAK2-STAT5 phosphorylation may be caused by at least two different processes. One involves depressed phosphorylation of the signaling proteins because of increased suppressors of cytokine signaling 2 expression that may be linked to low-grade inflammation. The other may involve increased signaling protein dephosphorylation because of heightened protein-tyrosine phosphatase activity.

Plasma protein extravasation and vascular endothelial growth factor expression with endothelial nitric oxide synthase induction in gentamicin-induced acute renal failure in rats.

Microvascular hyperpermeability to plasma proteins via vascular endothelial growth factor (VEGF) with endothelial nitric oxide synthase (eNOS) induction may contribute to wound healing through matrix remodeling. However, vascular hyperpermeability is not examined in acute renal failure (ARF), a unique form of wound healing. Subcutaneous injection of gentamicin (400 mg/kg per day for 2 days in divided doses every 8 h) in rats increased serum creatinine levels and induced tubular damage, which peaked at day 6, after the last gentamicin injection. Ki67-positive regenerating proximal tubules (PTs) peaked in number at day 6 and almost covered the bare tubular basement membrane (TBM) by day 10. Staining of fibrinogen and plasma fibronectin began to increase in the peritubular regions as early as day 0, steadily increased in TBM and tubular lumen until day 6 and then decreased. Hyperpermeable peritubular capillaries were identified by extravasation of perfused-fluoresceinated dextran (both 70 kDa and 250 kDa) into peritubular regions as early as day 0 and prominently into TBM and tubular lumen at day 6. Electron microscopy further suggested the intraendothelial pathway of dextran. Immunoreactive VEGF increased in the damaged and regenerating PTs. Immunoreactive VEGF receptors-1 and -2 did not change, but immunoreactive eNOS increased in the peritubular capillaries after induction of ARF. Western blotting for VEGF and eNOS supported the immunostaining findings. In addition, we assessed the effects of NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME) on vascular hyperpermeability during the recovery phase of this model. Treatment with L-NAME (s.c. at a dose of 100 mg/kg/day from day 3 to day 6) decreased extravasation of perfused-250-kDa dextran and significantly inhibited the regenerative repair of PTs at day 6 when compared with vehicle-treated rats. In conclusion, plasma protein extravasation occurred, leading to matrix remodeling, such as the process of wound healing during the tubular repair in gentamicin-induced ARF. Since VEGF-induced vascular hyperpermeability may depend on NO production, VEGF/VEGF receptor system with eNOS induction might be responsible for this process.

Important role for fibronectin-EIIIA during renal tubular repair and cellular recovery in uranyl acetate-induced acute renal failure of rats.

The present study was designed to identify the source and kinetics of an alternatively spliced "embryonic" cellular fibronectin EIIIA (cFn-EIIIA) in relation to regenerating renal tubules in uranyl acetate (UA)-induced acute renal failure (ARF) in rats. Damage of the proximal tubules was found as early as day 2 after induction of ARF, peaked at day 5, and was almost substituted by epithelial relining by day 7. Immunohistochemistry showed de novo deposition of cFn-EIIIA in peritubular regions as early as day 2, then on the tubular basement membrane (TBM) after day 4. beta1 Integrin, the receptor for Fn, was mainly found at the basal side of tubules in the normal control and increased in the interstitium after induction of ARF, but the staining pattern gradually returned to the control after day 7. Immunoelectron microscopy revealed that cFn-EIIIA was produced initially by the peritubular endothelium and later by fibroblastic cells and was deposited to the TBM, on which regenerating tubules proliferated, probably with cFn-EIIIA production. beta1 Integrin was expressed in cFn-EIIIA-producing cells, especially in regenerating tubular cells, suggesting that cFn-EIIIA signal transduction affects regenerating tubules. Transforming growth factor (TGF)-beta1 was found in some damaged proximal tubules and interstitial cells after induction of ARF and later in the regenerating tubules. CFn-EIIIA and beta1 integrin mRNA levels were upregulated as early as day 2. TGF-beta1 mRNA level significantly increased after day 3, suggesting a modulatory role for TGF-beta1 on cFn-EIIIA production, but not by day 2. Our data suggest that cFn-EIIIA production by the endothelium during the very early response to tubular injury and by fibroblastic cells and regenerating tubules may play an important role in the cellular recovery of UA-induced ARF in rats.

Temporary changes in macrophages and MHC class-II molecule-expressing cells in the tubulointerstitium in response to uranyl acetate-induced acute renal failure in rats.

The present study was designed to asses the dynamic changes in macrophages (Møs) with or without expression of major histocompatibility complex (MHC) class-II molecule in response to uranyl acetate-induced acute renal failure (ARF) in rats. ED1+ monocytes/Møs infiltrated into the interstitium as early as day 2, peaked in number on day 5 after uranyl acetate-induced ARF. ED1+ cells did not correlate with necrotic tubules but accumulated abundantly in the vicinity of the Ki67+ regenerating proximal tubules around days 4-5. Afterward, regeneration of proximal tubules was accelerated. After day 5, some ED1+ cells entered the tubular lumen, and became ED1+ giant cells, which had features of phagocytic Møs by immunoelectron microscopy, peaking in number on day 7. Most ED1+ cells did not incorporate [(3)H]-thymidine, indicating lack of active proliferation. The number of OX6+ cells (directed to MHC class-II molecule) in the interstitium significantly increased on day 4 and peaked on day 5. Double staining revealed that ED1+OX6- cells entered the tubular lumen while ED1+OX6+ cells remained in the peritubular regions. Osteopontin (OPN) protein and mRNA were significantly upregulated. No specific relationship could be found between OPN+ regenerating proximal tubules and ED1+ cells, but most ED1+ giant cells were OPN+ and intermingled among OPN+ cell debris. Our findings suggest that ED1+ Møs are actively associated with regenerating proximal tubules and, thus, might promote proximal tubular regeneration. ED1+OX6- Møs may function as scavengers and phagocytose cellular debris in the tubular lumen, cleaning the wound site. OPN might be involved in this process. ED1+OX6+ Møs in the peritubular regions may act as outpost of the defense system to monitor incoming antigens. Our data indicate that Møs with or without expressing MHC class-II molecule contribute to the defense and repair of injured proximal tubules in this ARF.

Relation of distal nephron changes to proximal tubular damage in uranyl acetate-induced acute renal failure in rats.

AIMS: To elucidate the pathophysiological roles of the changes of distal nephron in uranyl acetate (UA)-induced acute renal failure (ARF), we investigated the relation of changes of constituent molecules in distal nephron to proximal tubular damage and repair in UA-treated rats. METHODS: ARF was induced in rats by intravenous injection of UA, and all rats received bromodeoxyuridine (BrdU) intraperitoneally 1 h before sacrifice. RESULTS: Proximal tubular damage with necrosis appeared as early as day 2, mainly in the outer stripe of outer medulla and reached a peak level at day 5. Slight cellular damage was evident in the distal nephron as early as day 3, reaching a peak level around day 9. Immunoreactive BrdU- or vimentin-positive regenerating proximal tubules (PT) appeared at day 2 and regenerating PT relining was almost completed by day 7. Immunostaining for EGF, which was constitutively expressed in the thick ascending limb (TAL) and distal convoluted tubule (DCT), diminished significantly as early as day 2, when PT regeneration became evident, and remained below normal levels until day 21. In contrast, slight immunoreactivity for EGF was observed in regenerated PT accompanying brush-border formation mainly after day 9, suggesting newly expressed EGF might contribute to PT maturation. Lectin staining or immunostaining for representative constituent molecules of the thin descending limb, TAL, DCT and collecting duct demonstrated marked and transient reduction after day 5. CONCLUSIONS: EGF was not associated with regenerating PT, but may be involved in the maturation of PT. Transient reduction in expression of constituent molecules of the distal nephron following the reduction in EGF could reflect dedifferentiation or phenotypic simplification during regenerative repair of PT in UA-induced ARF in rats.

Mechanisms and kinetics of Bowman's epithelial-myofibroblast transdifferentiation in the formation of glomerular crescents.

BACKGROUND: We investigated the mechanisms and kinetics of Bowman's epithelial-myofibroblast transdifferentiation in the formation of glomerular crescents. METHODS: Crescentic glomerulonephritis was induced by i.v. injection of rabbit anti-rat glomerular basement membrane antiserum in WKY rats. RESULTS: Cellular crescents (83.5% of glomeruli) were first observed at day 7 after disease induction. Immunostaining of alpha-smooth muscle actin (alpha-SMA), as a marker for the myofibroblast phenotype, was found in some periglomerular regions as early as day 3, when it was also seen in parietal epithelial cells (PEC) of Bowman's capsule at day 5 and in crescent formation at day 7. Proliferation marker Ki67-positive PEC was found at day 3, and double Ki67- and alpha-SMA-positive PEC could be seen at day 5. The migratory figure of PEC with the expression of alpha-SMA was found by immunoelectron microscopy. At day 7, some crescent cells were stained positive for PEC marker, protein gene product 9.5, in association with alpha-SMA or Ki67. Expression of transforming growth factor (TGF)-beta receptor types I and II, as well as platelet-derived growth factor (PDGF) receptor beta and PDGF-B increased in PEC as early as day 3. At day 5 marked deposition of cellular and common fibronectin, but not other extracellular matrix components examined was found in Bowman's spaces where ED 1-positive macrophages infiltrated. CONCLUSIONS: PEC may be stimulated to proliferate and/or transdifferentiate into myofibroblast phenotype possibly by action of TGF-beta and PDGF and/or binding of fibronectin to PEC, then migrate and/or proliferate, participating in glomerular crescents.

Mycophenolate mofetil inhibits regenerative repair in uranyl acetate-induced acute renal failure by reduced interstitial cellular response.

We recently reported that transient appearance of interstitial myofibroblasts and infiltrating macrophages might play a role in cellular recovery in uranyl acetate (UA)-induced acute renal failure (ARF). Here we tested the effects of mycophenolate mofetil (MMF), which attenuates infiltration of lymphocytes, macrophages, and myofibroblasts, but does not suppress epithelial regeneration, on renal tissue repair. Rats treated with MMF (20 mg/kg/day) or vehicle were sacrificed at 2, 5, and 7 days after induction of ARF by injection of 5 mg/kg UA. Renal tissues were immunostained for bromodeoxyuridine (BrdU) and Ki67, alpha-smooth muscle actin (alpha-SMA), ED1, and CD43. The expression levels of alpha-SMA mRNA were examined by reverse transcription-polymerase chain reaction. Body weight loss or serum albumin levels were similar in MMF and vehicle rats during the experiment. In vehicle group, serum creatinine (Scr) significantly increased after day 5, but proximal tubular (PT) damage score increased as early as day 2 after UA injection. BrdU- or Ki67-positive regenerating tubular cells, ED1-positive macrophages and alpha-SMA-positive myofibroblasts significantly increased in the interstitium after day 5. In MMF-treated rats, Scr and PT damage score significantly increased at day 7 and the number of regenerating PT were significantly reduced compared with vehicle-treated rats at days 5 and 7. The numbers of macrophages and myofibroblasts and the expression of alpha-SMA mRNA were significantly lower in MMF than in vehicle rats at day 5, indicating that reduced interstitial cellular response is linked to the inhibition of regenerative repair. CD43-positive lymphocytes were significantly reduced in MMF group than in vehicle group at day 7, suggesting that lymphocyte infiltration does not seem to contribute to early regenerative response of proximal tubules. The transient appearance of myofibroblasts and macrophages in the interstitium may promote regenerative repair in UA-induced ARF in rats.