A self-perpetuating vicious cycle of tissue damage in human hibernating myocardium.

Recently, we proposed the hypothesis that a vicious cycle exists in human hibernating myocardium (HM) between the progression of myocyte degeneration and the development of fibrosis. We now investigated the pathomechanism of this cycle in more detail and established a correlation between the severity of the morphological changes and the degree of postoperative functional recovery of HM. HM was diagnosed by dobutamine echocardiography, thallium-201 scintigraphy and radionuclide ventriculography. Functional recovery was present at 3 months after coronary bypass surgery but remained unchanged at 15 months. Forty patients were subdivided into 2 groups: A with complete and B with incomplete recovery. Biopsies taken during surgery and studied by electron microscopy, immunocytochemistry, rt-PCR, and morphometry revealed myocyte degeneration and inflammatory and fibrinogenic changes in a widened interstitial space. We report here for the first time an upregulation of TGF-beta1 evident by a 5-fold increase of fibroblasts and macrophages exhibiting a TGF-beta1 content 3-fold larger than in control, and a > 3-fold increase in TGF-beta1 mRNAby rt-PCR. The number of angiotensin converting enzyme (ACE) containing structures was increased (n/mrm2: control-11.4, A-17.6, B-19.2, control vs. A and B, p < 0.05). Fibrosis was more severe in group B than A or control (%: C-10.1; A-21.2; B-40.6; p < 0.05). Capillary density was significantly reduced (n/mm2: C-1152; A-782; B-579, p < 0.05) and intercapillary distance was widened (microm: C-29.5, A-36.1, B-43.3, p < 0.05). The number of CD 3 (n/mm2: C-5.0; A-9.6; B-9.4, ns) and CD 68 positive cells (n/mm2: C-37.2; A-80.7; B-55.0, C vs. A p < 0.05) was elevated in HM as compared to control indicating an inflammatory reaction. Cut-off points for functional recovery are fibrosis > 32%, capillary density < 660/mm2 and intercapillary distance > 39.0 microm. In HM a self-perpetuating vicious cycle of tissue alterations leads to progressive replacement fibrosis and continuous intracellular degeneration which should be interrupted by early revascularization.

Dopamine depolarizes podocytes via a D1-like receptor.

BACKGROUND: Dopamine influences glomerular haemodynamics and dopamine receptors have been demonstrated in the glomerulus, but little is known about the cellular effects of dopamine in glomerular cells. The aim of this study was to investigate the influence of dopamine on the cellular functions of podocytes. METHODS: The effect of dopamine on membrane voltage was investigated in differentiated mouse podocytes. The membrane voltage was measured using the patch clamp technique. Reverse transcribed-polymerase chain reaction (RT-PCR) studies were performed to investigate the expression of dopamine receptor mRNA in mouse glomeruli and podocytes. RESULTS: The addition of dopamine (100 nM-1000 microM) caused a concentration-dependent depolarization of podocytes (EC50 is approximate to 10 microM). Like dopamine, the selective agonist of the D1-like receptor, SKF 82958, depolarized podocytes in a concentration-dependent manner. (EC50 is approximate to 50 microM). SKF 82958 stimulated a time-and concentration-dependent accumulation of cyclic adenosine 3',5'-monophosphate (cAMP) in podocytes (EC50 is approximate to microM). RT-PCR studies with primers derived from mouse sequences amplified mouse mRNA for the D1-like and the D2-like receptor in glomeruli, which were obtained by the sieve technique, whereas only mRNA for the D1-like receptor was detected in cultured mouse podocytes. CONCLUSION: The data indicate that dopamine induces a cAMP-dependent depolarization via a D1-like receptor in podocytes.

NAD(P)H oxidase activity in cultured human podocytes: effects of adenosine triphosphate.

Reactive oxygen species contribute to glomerular damage and proteinuria. In this study, we show that cultured human podocytes produce superoxide in response to extracellular adenosine triphosphate (ATP), and we identified the oxidases involved in this process. Adenosine triphosphate (10-4 M for 4 hr) raised superoxide production from 1.28 +/- 0.15 to 2.67 &/- 0.34 nmol/mg protein/min. Studies with podocyte homogenates revealed activation of both nicotinamide adenine dinucleotide (NADH; from 2.65 +/- 0.23 to 7.43 +/- 0.57) and nicotinamide adenine dinucleotide phosphate (NADPH) dependent oxidases [from 1.74 +/- 0.13 to 4.05 +/- 0.12 (nmol O2/mg protein/min)] by ATP. Activity of xanthine-oxidases was low and unchanged by ATP. Activation of the plasma-membrane bound NAD(P)H oxidases by ATP was time and dose dependent. Reverse transcribed-polymerase chain reaction (RT-PCR) studies with primers derived from monocyte sequences amplified mRNA for the NADPH oxidase subunits p22phox, p47phox, gp91phox, and p67phox, and the latter was transiently increased by ATP. Experiments with actinomycin D and cycloheximide suggested that ATP modulates enzyme activity at the transcriptional and translational levels. In conclusion, NAD(P)H dependent, membrane associated oxidases represent the major superoxide source in human podocytes. Activation of NAD(P)H oxidase by ATP might be secondary to increased mRNA expression of the NADPH oxidase subunit gp67phox.

Lipoprotein(a) induces glomerular superoxide anion production.

BACKGROUND: Lipoprotein(a) (Lp(a)) is considered to accelerate glomerular injury in various forms of renal disease. Several tissue culture studies suggested that biological effects of Lp(a) are inhibitable by oxygen radical scavengers. Since reactive oxygen metabolites (ROM) are important mediators of renal disease, we studied the effects of native and oxidized Lp(a) on generation of the ROM superoxide anion in isolated glomeruli and compared them with the effects of native (nLDL) and oxidized LDL cholesterol (oxLDL). METHODS: The effect of native and oxidized Lp(a) and LDL on ROM production in isolated rat glomeruli was investigated with a lucigenin chemiluminescence assay. RESULTS: Native Lp(a) caused a moderate, dose dependent stimulation of glomerular ROM production: Maximum ROM production to 159 +/- 9% of control glomeruli was induced by nLp(a) 20 micrograms/ml. Lp(a)-induced chemiluminescence was completely inhibited by the cell permeable oxygen radical scavenger Tiron (10 Mm). Oxidized Lp(a) (20 micrograms/ml) caused a more pronounced stimulation of ROM production to 204 +/- 12% of control values. Interestingly, only oxLDL, but not nLDL had a significant effect on glomerular ROM production (ox LDL 50 micrograms/ml: 192 +/- 19% of control). Lp(a) stimulated ROM production was completely inhibited by the protein kinase C inhibitor bis- indolyl malemide (BIM): BIM 10(-6) M inhibited 52 +/- 3%, BIM 10(-5) M inhibited 94 +/- 5% of Lp(a)-induced ROM production. ROM production was also inhibited, when intracellular CAMP levels were elevated by forskolin. CONCLUSION: Lp(a) and oxLp(a) induce the activation of ROM in glomeruli by a pathway that is sensitive to inhibition of protein kinase C and elevation of intracellular CAMP levels.

Activated clotting time is not a sensitive parameter to monitor anticoagulation with low molecular weight heparin in hemodialysis.

To study whether the activated clotting time (ACT) is a sensitive parameter to monitor anticoagulation with low molecular weight heparin (LMWH) during hemodialysis, ACT, polymorphonuclear granulocyte-elastase, and anti-factor Xa activity were studied during 30 dialysis treatments with LMWH (35 IU/kg body weight bolus; 10 IU/h/kg). Twenty treatments were performed with Hemophan, ten with polysulfone dialyzers. No clinically relevant clotting of dialyzers was observed, but minimal fibrin deposition was found more often in the Hemophan group (50 vs. 30%). Despite continuously elevated anti-factor Xa levels (Hemophan 0.49 +/- 0.03, polysulfone 0.62 +/- 0.01 IU/ml), a significant increase of ACT was only demonstrated 10 min after bolus application in the Hemophan group. Elevated polymorphonuclear granulocyte-elastase levels were demonstrated in the Hemophan group but were linked to the presence of minimal fibrin deposits and not to the dialyzer material. We conclude that ACT is not a sensitive parameter to monitor anticoagulation with standard doses of LMWH.

Lipoprotein(a) in nephrotic syndrome and end-stage renal disease.

Lipoprotein(a) [Lp(a)] may be elevated in patients with the nephrotic syndrome and patients on hemodialysis or continuous ambulatory peritoneal dialysis. High levels of Lp(a) are due to proteinuria or an activated acute-phase response. Serum concentrations greater than 30 mg/dl are independently associated with coronary heart disease. Data from cell culture studies suggest that it is not uptake of Lp(a) by mesangial cells but trapping by matrix proteins that contributes to the generation of glomerular apo(a) deposits. Lp(a) alters mesangial cell DNA synthesis and stimulates the generation of reactive oxygen species. Prolonged exposure to Lp(a) causes mesangial cell death in vitro culture' experiments. Lp(a) does not alter autocrine transforming growth factor-beta transcription in human mesangial cells and has, unlike low-density lipoprotein, no effect on the production of the extracellular matrix protein fibronectin. Future cell culture studies on the role of Lp(a) in renal disease have to address whether Lp(a) induces cell death via apoptosis and to what extent the generation of oxygen radicals is involved in this process.

Receptor-mediated lipoprotein uptake by human glomerular cells: comparison with skin fibroblasts and HepG2 cells.

BACKGROUND: Currently the mechanisms of glomerular lipid accumulation are not completely understood. The present study characterizes the mechanisms of lipid uptake by glomerular cells. Since renal diseases are frequently associated with an accumulation of apoE-containing triglyceride-rich lipoproteins, we were interested to investigate whether glomerular epithelial or mesangial cells possess VLDL receptors besides the well established LDL receptors. METHODS: Uptake kinetics of 125I-labelled very-low-density lipoproteins (VLDL) and low-density lipoproteins (LDL) in human glomerular epithelial and mesangial cells were compared to lipid uptake in cells with established receptor status, i.e. human skin fibroblasts and HepG2 cells. RESULTS: Glomerular epithelial cells, mesangial cells, and skin fibroblasts as well as hepatocytes express VLDL receptor mRNA, indicating that they exhibit VLDL receptors. VLDL uptake in glomerular epithelial cells, mesangial cells and skin fibroblasts occurred with a lower specificity than in HepG2 cells (-25%). No differences were found for the specificity of LDL uptake. VLDL uptake in HepG2 cells was inhibited more effectively with VLDL than with LDL. In skin fibroblasts, glomerular epithelial and mesangial cells, VLDL and LDL were equally effective inhibitors of VLDL uptake. The degradation-uptake ratio of VLDL in glomerular cells was elevated 50% compared to HepG2 cells, suggesting highly efficient intracellular lipoprotein turnover in these cells. CONCLUSION: We conclude that glomerular epithelial and mesangial cells as well as skin fibroblasts and HepG2 exhibit VLDL receptors additionally to their LDL receptors, even though the regulation of the VLDL receptor in HepG2 cells seems to differ from the regulation in glomerular epithelial and mesangial cells. The high degradation-uptake-ratio in these renal cells suggests the presence of an effective clearance pathway which might serve as protection against lipoprotein accumulation.

Lipids and progression of renal disease: role of modified low density lipoprotein and lipoprotein(a).

Atherogenic lipoproteins accumulate in the arterial wall as well as within the glomerulus and may accelerate vascular and glomerular injury. We therefore assessed whether oxidized low density lipoprotein (LDL) and lipoprotein(a) [Lp(a)] influence three major systems: (i) endothelium-dependent vasodilation, (ii) renin release of juxtaglomerular (JG) cells, and (iii) proliferation and viability of mesangial cells (MC). Lipoproteins were prepared from human plasma. Renal arteries were obtained from rabbits and JG as well as MC cells from mouse, rat and human kidneys. Dilator responses were detected in isolated arterial segments by a photoelectric device. Renin activity of JG cells was measured in culture supernatants and cells and DNA synthesis by 3H-thymidine incorporation in MC. Acetylcholine-induced, endothelium-dependent dilator responses of renal arteries were not significantly attenuated after incubation with native Lp(a). However, exposure to in vitro oxidized Lp(a) suppressed dilator responses in a dose-dependent manner. Using a chemiluminescence assay, we could detect increased O2- production by arteries pretreated with oxidized Lp(a), which suggested that enhanced nitric oxide (NO) inactivation by O2- might be the underlying mechanism of impairment of endothelium-dependent dilations. In general, oxidized Lp(a) was far more potent than oxidized LDL in this effect. In JG cells, both oxidized LDL and Lp(a) dose-dependently stimulated renin release. Coincubation with HDL significantly suppressed oxidized LDL and Lp(a) stimulated renin release and O2- production. In MC native and oxidized Lp(a) were poor ligands for the LDL receptor, but bound more tightly to extracellular matrix than native LDL. Native and oxidized Lp(a) elicited proliferation or toxicity of MC in a dose-dependent fashion. Stimulation of DNA synthesis in MC or renin release in JG cells was partly blunted or eliminated when cells were incubated with oxidized LDL and Lp(a) in the presence of superoxide dismutase and catalase, enzymes removing O2- and H2O2. These dat suggest a common underlying mechanism. Atherogenic lipoproteins induce formation of oxygen radicals not only in arteries, but also in glomeruli and JG cells, causing an inhibition of nitric oxide mediated vasodilation, stimulation of renin release, and modulation of mesangial cell growth and proliferation. The damaging effect of the lipoproteins can be prevented by antioxidative enzymes and HDL.

Potential role of lipids in the progression of diabetic nephropathy.

Dyslipoproteinemia in non-insulin-dependent diabetes mellitus (NIDDM) is an important risk factor in the development of atherosclerosis and glomerulosclerosis. The lipid profile of NIDDM patients is characterized by elevated serum triglycerides and VLDL levels and reduced HDL cholesterol levels. Serum LDL levels may be elevated as well in some patients with NIDDM, but several alterations in the biochemical and physical properties of LDL particles are more characteristic resulting in reduced receptor specific uptake of these lipoproteins. Non-enzymatic glycosylation of LDL and augmented oxidation is common in diabetic patients making lipoproteins susceptible for uptake by the macrophage scavenger receptors and thus leading to foam cell formation and further glomerular damage. A reduction in the progression of diabetic nephropathy by lowering proteinuria and thereby serum cholesterol during treatment with ACE-inhibitors demonstrates the importance of such a therapy. The multiple factors involved in the pathogenesis of diabetic nephropathy are difficult to evaluate in regard to their individual contribution. Nevertheless antiproteinuric and lipid lowering therapy can be expected to reduce vascular damage and the progression of diabetic nephropathy.

Interaction of native and oxidized lipoprotein(a) with human mesangial cells and matrix.

The trapping of apolipoprotein(a) and apolipoprotein B-100 in glomeruli of patients with the nephrotic syndrome seems to be linked to a less favorable course of renal disease. To evaluate the potential role of lipoprotein(a) as a mediator of glomerular injury, we measured uptake of native lipoprotein(a) [Lp(a)] and oxidatively modified Lp(a) by cultured human mesangial cells and matrix and studied the effects of Lp(a) on mesangial cell DNA-synthesis and cellular proliferation. Uptake of Lp(a) by mesangial cells occurred at a significantly lower affinity (KD 16 micrograms/ml vs. 39 micrograms/ml) and a lower maximum degradative capacity (6.7-fold) than for LDL. Specificity of receptor mediated uptake was 50% for Lp(a) compared to 84% for LDL. Oxidative modification of both Lp(a) and LDL was accompanied by a significant decrease in uptake and degradative capacities. Due to the limited uptake, native and oxidatively modified Lp(a) had only marginal effects on intracellular cholesterol metabolism, which was measured as inhibition of sterol synthesis and stimulation of cholesterol esterification. However, binding of Lp(a), oxidized Lp(a) and oxidized LDL to extracellular mesangial matrix was enhanced compared to LDL. Furthermore, incubation of mesangial cells with Lp(a) and oxLp(a) in concentrations of 2.5 micrograms/ml and higher resulted in a decrease of DNA synthesis. Regardless of the oxidative status, a maximal suppression of DNA synthesis was observed at 20 micrograms/ml Lp(a). Native Lp(a) also blunted the stimulatory effects of PDGF on mesangial cell DNA-synthesis. Lp(a) did not alter basal TGF-beta transcription in human mesangial cells. The avid interaction of Lp(a) and modified lipoproteins with mesangial matrix provides a concept for the enhanced entrapment of these lipoproteins in the diseased glomerulum. Native Lp(a) is a poor ligand for the LDL receptor; oxidation of Lp(a) even lowers the affinity towards this receptor. Further studies must be carried out to clarify the pathophysiological significance of Lp(a) trapping in the mesangial matrix.

Effects of lipoprotein(a) on mesangial cell proliferation and viability.

BACKGROUND: Lipoprotein abnormalities are considered to accelerate glomerular injury in various forms of renal disease, probably affecting mesangial proliferation. Serum levels of the atherogenic Lipoprotein(a) (Lp(a)) are elevated in patients with nephrotic syndrome and Lp(a) deposits have been identified in diseased glomeruli. So far, the influence of Lp(a) on mesangial cell function has not been defined. METHODS: The influence of Lp(a) on mesangial cell proliferation was assessed in a rat mesangial cell culture model by direct measurement of cell growth as well as analysis of DNA-synthesis and mRNA levels of c-fos and c-myc, two growth-associated 'immediate early response genes'. Results. Lp(a) triggered a biphasic response on DNA synthesis: 3H-thymidine uptake was increased when cells were incubated with Lp(a) (2.5-10 microg/ml) for 24 h. The response was dose dependent, a maximal effect was seen for Lp(a) 5 microg/ml. The stimulatory properties of Lp(a) were comparable to 10% fetal calf serum (FCS). No additive effect of 10% FCS and Lp(a) on DNA synthesis was observed. Cell proliferation was moderately stimulated (120+/-9% of control) by low levels of Lp(a) in the presence of small amounts of FCS. Messenger RNA levels for c-fos and c-myc were upregulated as early as 15 min after incubation with Lp(a) 5 microg/ml, a maximum response was observed after 20 and 240 min respectively. Stimulation of DNA synthesis was partly blunted when cells were incubated with Lp(a) in the presence of catalase 100 U/ml and superoxide dismutase 10(-7)M (SOD) but not in the presence of SOD alone. Lp(a) in concentrations above 10 microg/ml depressed DNA-synthesis and elicited signs of cytotoxicity. The cytotoxic effects of Lp(a) were not blunted by oxygen radical scavengers. The stimulatory and cytotoxic effects of Lp(a) were not restricted to specific isoform. CONCLUSION: Low concentrations of Lp(a) stimulated growth of mesangial cells, whereas higher concentrations had antiproliferative or toxic effects. The stimulation on mesangial cell proliferation as well as the cytotoxic effects caused by Lp(a) are both likely to have a negative impact on the course of renal disease.

Lipoprotein(a) in patients with the nephrotic syndrome: influence of immunosuppression and proteinuria.

Lipoprotein(a) [Lp(a)] is a plasma lipoprotein whose structure and composition closely resemble that of low-density lipoproteins, but contains an additional protein called apolipoprotein(a) [apo(a)]. Factors which modulate plasma Lp(a) concentrations are poorly understood. The influence of nephrotic syndrome on Lp(a) levels was investigated in 103 patients with nephrotic syndrome: 72 with primary kidney disease and 31 with diabetic nephropathy. Nephrotic patients had significantly higher Lp(a) levels (mean 63 +/- 7 mg/dl; median 42 mg/dl) compared with controls (mean 22 +/- 2 mg/dl; median 8 mg/dl). Fifty-seven percent of the patients and 22% of the controls had values greater than 30 mg/dl. Within all apo(a) isoform classes, higher concentrations of Lp(a) were seen in the nephrotic patients compared with controls. In 17 patients with primary kidney disease remission of the nephrotic syndrome was induced by immunosuppressive treatment and Lp(a) concentration dropped in parallel with the reduction of proteinuria (pretreatment mean, 98 +/- 9 mg/dl vs. remission mean, 25 +/- 5 mg/dl). In 9 patients where multiple measurements were done, multiple regression analysis showed a strong relation of Lp(a) with the amount of proteinuria (p < 0.01). We conclude that most patients with the nephrotic syndrome have Lp(a) concentrations which are substantially elevated compared with control subjects of the same apo(a) isoform. Because Lp(a) concentrations are substantially reduced when remission of the nephrotic syndrome is induced by immunosuppressive drugs, it is likely that nephrotic syndrome directly results in elevation of Lp(a). The high levels of Lp(a) in nephrotic syndrome could potentially cause glomerular injury as well as increase the risk of atherosclerosis and thrombotic events associated with this disorder.

Rat muscle branched-chain ketoacid dehydrogenase activity and mRNAs increase with extracellular acidemia.

The rate-limiting enzyme in branched-chain amino acid catabolism is branched-chain ketoacid dehydrogenase (BCKAD). In rats fed NH4Cl to induce acidemia, we find increased basal BCKAD activity as well as maximal activity in skeletal muscle. Concurrently, there is a > 10-fold increase in mRNAs of BCKAD subunits in skeletal muscle plus an increase in cardiac muscle but not in liver or kidney. There was no increase in mRNA for malate dehydrogenase or for cytosolic glyceraldehyde-3-phosphate dehydrogenase. Evaluation of the translation capacity of BCKAD mRNAs in muscle of acidemic rats yielded more immunoreactive BCKAD whether the proteins were synthesized from muscle RNA using rabbit reticulocyte lysate or directly using postmitochondrial homogenates. Although the RNA from muscle of acidemic rats yielded twice as much BCKAD protein, we found no net increase in mitochondrial BCKAD protein in muscle by Western blotting. Because there is increased proteolysis in muscle of rats with acidemia, the increase in mRNA might be a mechanism to augment BCKAD synthesis and activity in muscle.

[Atherogenic lipoprotein(a) as a progression factor in glomerular diseases: studies in cultured rat mesangial cells]. / Das atherogene Lipoprotein(a) als Progressionsfaktor glomerulärer Erkrankungen: Untersuchungen an kultivierten Mesangialzellen der Ratte.

Lipoprotein abnormalities are a risk factor for atherosclerotic disease and considered to accelerate glomerular injury in kidney disease. Serum levels of Lp(a) are elevated in patients with nephrotic syndrome and Lp(a) deposits are found in diseased glomeruli. Since mesangial hypercellularity is a prominent feature in a variety of glomerular diseases, we studied the effects of Lp(a) on proliferation of cultured rat mesangial cells. DNA synthesis was stimulated in cells incubated in the presence of Lp(a) as were the mRNA levels for c-fos and c-myc, two "early genes" that serve as transcription factors. Lp(a) also accelerated cell growth by 42 +/- 6% compared to control cells. Increased DNA synthesis was partially blunted, when cells were incubated with Lp(a) in the presence of oxygen radical scavengers CAT and SOD. We conclude that Lp(a) abnormalities are likely to contribute to glomerular injury in kidney disease. The mechanism by which Lp(a) alters the proliferation rate of mesangial cells involves the formation of reactive oxygen species.

Impaired cation transport in thymocytes of rats with chronic uremia includes the Na+/H+ antiporter.

To examine how uremia changes sodium, potassium, and proton transport, thymocytes from chronic renal failure (CRF) rats were studied. If alterations in cation transport associated with chronic uremia (CRF) extend to intracellular pH regulation, the susceptibility to the catabolic effects of acidosis might be increased. To evaluate the influence of acidosis, cation transport in thymocytes from normal rats with NH4Cl-induced acidosis was also studied. Ouabain-sensitive 86Rb influx in thymocytes from acidotic CRF rats was 32% lower than in control cells (P < 0.05), but intracellular sodium concentration was unchanged. This may be related to a 47 +/- 22% reduction in 22Na influx. In thymocytes from nonuremic, acidotic rats, ouabain-sensitive 86Rb influx was decreased 39% (P < 0.025), similar to the change in CRF. In CRF thymocytes, Na(+)-H+ antiporter activity in response to cell acidification (7.13 +/- 0.8 versus 9.42 +/- 0.8 mmol of H+/L per min; CRF versus control), or to osmotic shrinkage (0.43 +/- 0.09 versus 0.82 +/- 0.11 mmol of H+/L per min; CRF versus control), was significantly (P < 0.01) reduced. Buffering capacity at resting and acidic intracellular pH was unchanged by uremia, but Na+/H+ antiporter activity in response to acid loading or osmotic shrinkage was unchanged in thymocytes of nonuremic rats with metabolic acidosis. Thus, CRF reduces both Na/K-ATPase and Na+/H+ antiporter activities in rat thymocytes. The former may be secondary to reduced sodium influx. Impaired Na+/H+ antiporter activity is not caused by metabolic acidosis alone, whereas reduced Na/K-ATPase activity is found in both acidosis and uremia.

Na pump defects in chronic uremia cannot be attributed to changes in Na-K-ATPase mRNA or protein.

We have found abnormalities in Na-K-adenosine-triphosphatase (Na-K-ATPase) function in different tissues of rats with chronic renal failure (CRF). A potential mechanism for these findings is a change in Na-K-ATPase alpha- and/or beta-gene expression. To evaluate this possibility, we compared CRF with pair-fed, sham-operated rats to determine whether chronic uremia changes the expression of Na-K-ATPase alpha 1-, alpha 2-, beta 1-, and beta 2-isoform mRNAs or protein in different types of skeletal muscle, heart, liver, adipose, and kidney tissue. In CRF rats, alpha 1-mRNA in heart tended to be higher and beta 2-mRNA was lower in fat and kidney. There were no other statistically significant differences in isoform mRNAs in tissues of CRF compared with the control rats. Western blot analysis revealed a 38% increase in alpha 1-protein in adipocytes and a 61% decrease in kidney of CRF rats but no significant differences in the amounts of isoform protein in other tissues. Thus, in uremia, posttranslational events or inhibitors of the enzyme are more likely causes of defects in Na-K-ATPase than changes in mRNA or protein abundance.

Mechanisms for protein catabolism in uremia: metabolic acidosis and activation of proteolytic pathways.

Accelerated protein catabolism in uremia occurs in animals and patients with acute (ARF) and chronic renal failure (CRF). Possible causes include resistance to both insulin-induced inhibition of protein-degradation and insulin-induced stimulation of protein synthesis. The mechanisms for these effects are unknown. However, metabolic acidosis has been shown to increase proteolysis in rat skeletal muscle even in the presence of insulin and this effect is absent in adrenalectomized rats. Similarly, metabolic acidosis accounts for increased muscle proteolysis in rats with CRF. Metabolic acidosis also stimulates branched-chain amino acid (BCAA) breakdown by increasing the activity of branched-chain keto acid decarboxylase. Uremia causes high corticosterone levels in ARF and CRF and this hormone could contribute significantly to increased proteolysis, BCAA-breakdown and possibly, the inhibition of protein synthesis. Besides changing glucocorticoids, uremia could inhibit the activity of transporters which regulate intracellular pH and ultimately, the metabolism of protein and amino acids. For example, uremia inhibits ion transporters including Na/H exchange in a variety of tissues and therefore, could increase the susceptibility to metabolic acidosis. Research directed at identifying specific, proteolytic pathways stimulated by metabolic acidosis has excluded a major role for Ca2+ activated and lysosomal proteases and suggests activation of an ATP- and ubiquitin-dependent proteolytic pathway.