Most of the increased protein degradation in muscle atrophy caused by starvation and denervation is due to activation of a non-lysosomal ATP-dependent proteolytic process. To determine whether expression of the ubiquitin-proteasome-dependent pathway is activated in atrophying muscles, we measured the levels of mRNA for ubiquitin (Ub) and proteasome subunits, and Ub content. After rats had been deprived of food for 1 or 2 days, the concentration of the two polyubiquitin (polyUb) transcripts increased 2-4-fold in the pale extensor digitorum longus muscle and 1-2.5-fold in the red soleus, whereas total muscle RNA and total mRNA content fell by 50%. After denervation of the soleus, there was a progressive 2-3-fold increase in polyUb mRNA for 1-3 days, whereas total RNA content fell. On starvation or denervation, Ub concentration in the muscles also rose by 60-90%. During starvation, polyUb mRNA levels also increased in heart, but not in liver, kidney, spleen, fat, brain or testes. Although the polyUb gene is a heat-shock gene that is induced in muscles under certain stressful conditions, the muscles of starving rats or after denervation did not express other heat-shock genes. On starvation or denervation, mRNA for several proteasome subunits (C-1, C-3, C-5, C-8 and C-9) also increased 2-4-fold in the atrophying muscles. When the food-deprived animals were re-fed, levels of Ub and proteasome mRNA in their muscles returned to control values within 1 day. In contrast, no change occurred in the levels of muscle mRNAs encoding cathepsin L, cathepsin D and calpain 1 on denervation or food deprivation. Thus polyUb and proteasome mRNAs increased in atrophying muscles in co-ordination with activation of the ATP-dependent proteolytic process.

Metabolic acidosis often leads to loss of body protein due mainly to accelerated protein breakdown in muscle. To identify which proteolytic pathway is activated, we measured protein degradation in incubated epitrochlearis muscles from acidotic (NNH4CI-treated) and pair-fed rats under conditions that block different proteolytic systems. Inhibiting lysosomal and calcium-activated proteases did not reduce the acidosis-induced increase in muscle proteolysis. However, when ATP production was also blocked, proteolysis fell to the same low level in muscles of acidotic and control rats. Acidosis, therefore, stimulates selectively an ATP-dependent, nonlysosomal, proteolytic process.We also examined whether the activated pathway involves ubiquitin and proteasomes (multicatalytic proteinases). Acidosis was associated with a 2.5-to 4-fold increase in ubiquitin mRNA in muscle. There was no increase in muscle heat shock protein 70 mRNA or in kidney ubiquitin mRNA, suggesting specificity of the response. Ubiquitin mRNA in muscle returned to control levels within 24 h after cessation of acidosis. mRNA for subunits of the proteasome (C2 and C3) in muscle were also increased 4-fold and 2.5-fold, respectively, with acidosis; mRNA for cathepsin B did not change. These results are consistent with, but do not prove that acidosis stimulates muscle proteolysis by activating the ATP-ubiquitin-proteasomedependent, proteolytic pathway. (J. Clin. Invest. 1994. 93: 2127-2133

Expression of HSP-27, HSP-60 and HSP-70 was estimated in the cortex, outer medulla and inner medulla (papilla) of rats undergoing water diuresis or water restriction for two days. The mRNAs for HSP-27 and HSP-60 in renal papilla were two- to threefold greater in rats during water restriction than in those excreting a dilute urine, but levels of mRNA for HSP-70 were not reduced by water diuresis and Western analysis for HSP-70 protein showed no difference between water-loaded and water-restricted animals.

The possible protective effect of heat-shock proteins (HSPs) on ischemic injury to renal cells was assessed in two different experimental models: ischemia-reflow in intact rats and medullary hypoxic injury as seen in the isolated perfused rat kidney. Heat shock was induced by raising the core temperature of rats to 42 degrees C for 15 minutes. Following this, Northern blots showed enhanced gene expression of HSP70, HSP60 and ubiquitin at one hour and reaching a maximum by six hours after heat shock in all regions of the kidney, but most prominently in medulla and papilla. The HSP70 protein in the kidney, estimated by immunohistochemical means, was detectable 24 hours following heat shock and further increased at 48 hours following heat shock. In the first set of experiments, the animals underwent uninephrectomy followed by cross clamping of the remaining renal artery for 40 minutes prior to reflow. Serum creatinine and urea nitrogen rose to 3.15 +/- 0.98 and 126.4 +/- 62.5 mg/dl at 24 hours. No significant differences were observed at 24, 48 and 72 hours after reflow between these values in control rats and rats pretreated with heat shock 48 hours earlier. Severe morphological damage to proximal tubules of the renal cortex was observed to the same extent in both groups. In a second set of experiments, the right kidney was removed either 24 or 48 hours after heat shock and perfused in isolation for 90 minutes. Functional and morphological parameters were compared with those of isolated perfused kidneys obtained from animals that had not been subjected to heat shock.(ABSTRACT TRUNCATED AT 250 WORDS)