A mechanism regulating proteolysis of specific proteins during renal tubular cell growth.

Growth factors suppress the degradation of cellular proteins in lysosomes in renal epithelial cells. Whether this process also involves specific classes of proteins that influence growth processes is unknown. We investigated chaperone-mediated autophagy, a lysosomal import pathway that depends on the 73-kDa heat shock cognate protein and allows the degradation of proteins containing a specific lysosomal import consensus sequence (KFERQ motif). Epidermal growth factor (EGF) or ammonia, but not transforming growth factor beta1, suppresses total protein breakdown in cultured NRK-52E renal epithelial cells. EGF or ammonia prolonged the half-life of glyceraldehyde-3-phosphate dehydrogenase, a classic substrate for chaperone-mediated autophagy, by more than 90%, whereas transforming growth factor beta1 did not. EGF caused a similar increase in the half-life of the KFERQ-containing paired box-related transcription factor, Pax2. The increase in half-life was accompanied by an increased accumulation of proteins with a KFERQ motif including glyceraldehyde-3-phosphate dehydrogenase and Pax2. Ammonia also increased the level of the Pax2 protein. Lysosomal import of KFERQ proteins depends on the abundance of the 96-kDa lysosomal glycoprotein protein (lgp96), and we found that EGF caused a significant decrease in lgp96 in cellular homogenates and associated with lysosomes. We conclude that EGF in cultured renal cells regulates the breakdown of proteins targeted for destruction by chaperone-mediated autophagy. Because suppression of this pathway results in an increase in Pax2, these results suggest a novel mechanism for the regulation of cell growth.

Catabolism in uremia: the impact of metabolic acidosis.

Chronic metabolic acidosis stimulates the catabolism of bone and muscle in experimental animals and humans. The toxicity caused by acidosis involves changes in endocrine function and toxicity arising from the homeostatic responses that are activated by the body to maintain pH near normal levels. Glucocorticoids, insulin, insulin-like growth factor-1, and parathyroid hormone play important roles in the homeostatic responses of bone and muscle to acid. Bone buffering of acid and the resulting increase in renal calcium excretion leads to negative calcium balance. Activation of the ubiquitin-proteasome proteolytic system and branched-chain ketoacid dehydrogenase in muscle, along with hepatic glutamine synthesis in the liver and renal glutamine uptake, are homeostatic mechanisms that cause negative nitrogen balance and loss of muscle mass. Treating the acidosis of chronic renal insufficiency improves both bone and muscle metabolism by reducing the loss of calcium and protein and amino acids in the two organs, respectively. Thus, treating acidosis suppresses both bone and muscle catabolism in patients with normal and reduced renal function.

Media acidification inhibits TGF beta-mediated growth suppression in cultured rabbit proximal tubule cells.

Chronic metabolic acidosis induces both hyperplastic and hypertrophic renal growth and is associated with progressive loss of renal function. These studies examine the direct effect of media acidification on the growth of rabbit proximal tubule cells in primary culture. The results demonstrate that media acidification has a direct antiproliferative (hypoplastic) effect on both quiescent and mitogen-stimulated [epidermal growth factor (EGF)-stimulated] cells and does not induce hypertrophy. This direct antiproliferative effect of acid is associated with inhibition of EGF-induced phosphorylation of the retinoblastoma protein (pRB), which maintains pRB activity and inhibits cell cycle progression from G1 to S phase. Transforming growth factor-beta (TGF-beta) alone has an antiproliferative effect in these cells. TGF-beta converts EGF-induced hyperplasia to hypertrophy and inhibits EGF-induced pRB phosphorylation. Media acidification inhibits both the antiproliferative effect of TGF-beta and the ability of TGF-beta to convert EGF-induced hyperplasia to hypertrophy. This activity is associated with inhibition of TGF-beta-mediated retention of pRB in the active, hypophosphorylated state. These results demonstrate that metabolic acidosis has a direct growth-suppressive effect on renal epithelial cells but inhibits the growth-suppressive effects of TGF-beta. Inhibition of the antiproliferative effect of cytokines, such as TGF-beta, may be responsible for acidosis-induced hyperplasia in vivo.

Mechanisms of renal tubular cell hypertrophy: mitogen-induced suppression of proteolysis.

The combination of epidermal growth factor (EGF) plus transforming growth factor-beta 1 (TGF-beta 1) causes hypertrophy in renal epithelial cells. One mechanism contributing to hypertrophy is that EGF induces activation of the cell cycle and increases protein synthesis, whereas TGF-beta 1 prevents cell division, thereby converting hyperplasia to hypertrophy. To assess whether suppression of proteolysis is another mechanism causing hypertrophy induced by these growth factors, we measured protein degradation in primary cultures of proximal tubule cells and in cultured NRK-52E kidney cells. A concentration of 10(-8) M EGF alone or EGF plus 10(-10) M TGF-beta 1 decreased proteolysis by approximately 30%. TGF-beta 1 alone did not change protein degradation. Using inhibitors, we examined which proteolytic pathway is suppressed. Neither proteasome nor calpain inhibitors prevented the antiproteolytic response to EGF + TGF-beta 1. Inhibitors of lysosomal proteases eliminated the antiproteolytic response to EGF + TGF-beta 1, suggesting that these growth factors act to suppress lysosomal proteolysis. This antiproteolytic response was not caused by impaired EGF receptor signaling, since lysosomal inhibitors did not block EGF-induced protein synthesis. We conclude that suppression of lysosomal proteolysis contributes to growth factor-mediated hypertrophy of cultured kidney cells.

NH4Cl-induced hypertrophy is mediated by weak base effects and is independent of cell cycle processes.

Renal hypertrophy occurs in a number of clinical conditions, some of which are associated with increases in ambient ammonia concentrations. NH4Cl induces hypertrophy in cultured renal epithelial cells. The present studies examined the mechanism of NH4Cl-induced hypertrophy in NRK-52E cells. Hypertrophy was also induced by methylammonium chloride, a related weak base, but not by tetramethylammonium chloride, a weak base analogue that can neither accept nor donate protons. Bafilo-mycin A1, an inhibitor of vacuolar proton pumps, also induced hypertrophy. Together, these studies suggest that NH4Cl-induced hypertrophy is mediated by its weak base property, allowing it to enter and alkalinize acid vesicular compartments. Additional studies demonstrated that NH4Cl-induced hypertrophy is not mediated by modulation of cell cycle processes. NH4Cl addition had no effect on the following: c-fos mRNA abundance, typically associated with entrance into the cell cycle; cyclin E protein abundance, which increases as cells progress through G1; or protein synthesis, which also increases during G1. In addition, inactivation of pRB by overexpression of human papilloma virus-16 carrying the E7 gene, which inhibits cell cycle-dependent hypertrophy, had no effect on the ability of NH4Cl to induce hypertrophy. Based on these data, we postulate that, in hypertrophic conditions associated with increased ammoniagenesis, hypertrophy is mediated by vesicular alkalinization and occurs independently of processes that govern progression through the cell cycle.

Renal epithelial cell hyperplasia and hypertrophy.

Renal epithelial cells that are part of an intact tubule epithelium divide at a very slow rate. However, in response to physiological signals or pathological processes, their rate of growth can rapidly increase. In these situations, the growth response can be hyperplasic (an increase in cell number) and/or hypertrophic (an increase in cell size). This article reviews our current understanding of the signaling pathways involved in renal epithelial cell hyperplasia and hypertrophy. Hyperplasia involves an initiating mitogenic stimulus, followed by the synthesis of a number of proteins that regulate a cascade of events governing progression through each of the phases of the cell cycle (G1, S, G2, and M phases). Renal epithelial cell hypertrophy can occur by cell cycle-dependent or -independent mechanisms. Cell cycle-dependent hypertrophy involves signals that cause cells to enter the first phase of the cell cycle (G1), but become arrested before leaving this phase. The consequence of these two sequential events is cell growth without DNA replication and, thus, cell hypertrophy. pRB plays a key role is the development of this form of hypertrophy. Cell cycle-independent hypertrophy probably involves inhibition of pH-sensitive lysosomal enzymes, leading to decreased protein degradation, and consequently an increase in cell protein content and cell hypertrophy.

Involvement of pRB family in TGF beta-dependent epithelial cell hypertrophy.

Although renal hypertrophy is often associated with the progressive loss of renal function, the mechanism of hypertrophy is poorly understood. In both primary cultures of rabbit proximal tubules and NRK-52E cells (a renal epithelial cell line), transforming growth factor beta 1 (TGF beta) converted epidermal growth factor (EGF)-induced hyperplasia into hypertrophy. TGF beta did not affect EGF-induced increases in c-fos mRNA abundance or cyclin E protein abundance, but inhibited EGF-induced entry into S, G2, and M phases. EGF alone increased the amount of hyperphosphorylated (inactive) pRB; TGF beta blocked EGF-induced pRB phosphorylation, maintaining pRB in the active form. To determine the importance of active pRB in TGF beta-induced hypertrophy, NRK-52E cells were infected with SV40 large T antigen (which inactivates pRB and related proteins and p53), HPV16 E6 (which degrades p53), HPV16 E7 (which binds and inactivates pRB and related proteins), or both HPV16 E6 and E7. In SV40 large T antigen expressing clones, the magnitude of EGF + TGF beta-induced hypertrophy was inhibited and was inversely related to the magnitude of SV40 large T antigen expression. In the HPV16-infected cells, EGF + TGF beta-induced hypertrophy was inhibited in E7- and E6E7-expressing, but not E6-expressing cells. These results suggest a requirement for active pRB in the development of EGF + TGF beta-induced renal epithelial cell hypertrophy. We suggest a model of renal cell hypertrophy mediated by EGF-induced entry into the cell cycle with TGF beta-induced blockade at G1/S, the latter due to maintained activity of pRB or a related protein.

Ventricular atrial natriuretic factor in the cardiomyopathic hamster model of congestive heart failure.

Cardiac atria are thought to be the principle source of plasma atrial natriuretic factor (ANF), a potent natriuretic and diuretic peptide. Whether other ANF production sites are recruited in disease states exhibiting elevated plasma ANF levels is not known. Accordingly, in the cardiomyopathic hamster, an animal model of congestive heart failure with high circulating levels of ANF, contribution of ventricular tissue to total cardiac ANF production and storage was investigated. Measurements were made of immunoreactive ANF in plasma and in atrial and ventricular extracts as well as ANF mRNA levels in the atria and ventricles from normal and cardiomyopathic golden Syrian hamsters. Plasma ANF levels were higher in cardiomyopathic than in control animals. The atrial concentration of ANF (per milligram atrial weight) was 50% and 83% lower in moderate and severe congestive heart failure, respectively, when compared with controls, while atrial ANF mRNA content of cardiomyopathic hamsters was not significantly different from normal hamsters. The ventricular concentration of ANF was 3 times and 7 times higher in animals in moderate and severe heart failure when compared with controls. In severe heart failure, ventricular ANF accounted for 23% of total cardiac stores of ANF. Ventricular ANF mRNA levels were 7 times and 13 times higher in hamsters in moderate and severe heart failure as compared with control animals. Therefore, significant increases in both ANF content and ANF mRNA in ventricles of hamsters in moderate to severe heart failure suggest that the ventricle could be an important source of ANF in congestive heart failure.

Plasma and atrial content of atrial natriuretic factor in cardiomyopathic hamsters.

Immunoreactive atrial natriuretic factor (IR-ANF) was measured in plasma and atrial extracts from normal and cardiomyopathic Syrian golden hamsters. Plasma IR-ANF was increased from 84.8 +/- 9.8 pg/ml (n = 17) to 234 +/- 23 (n = 25; P less than .0001) in hamsters with moderate failure, and to 1085 +/- 321 pg/ml (n = 10; P less than .02) in animals with severe failure. Plasma IR-ANF increased with increased atrial hypertrophy. Atrial IR-ANF content was essentially the same in normal animals and in those with moderate heart failure (3.06 +/- 0.28 vs. 3.17 +/- 0.19 microgram/100 g body wt.) and lower in the majority of those with severe failure (1.82 micrograms/100 g body wt., P less than .001). The elevations of IR-ANF in plasma are similar to those seen in patients with congestive heart failure. Our studies do not support bioassay results showing a deficiency of atrial ANF content as being important in the congestive heart failure associated with cardiomyopathy in the hamster.