SummaryMycobacteria have a unique outer membrane (OM) that is thicker than any other known biological membrane. Nutrients cross this permeability barrier by diffusion through porins. MspA is the major porin of Mycobacterium smegmatis . In this study we showed that three paralogues of MspA, namely MspB, MspC and MspD are also porins. However, only the mspA and mspC genes were expressed in the wild-type strain. None of the single deletion mutants displayed a significant OM permeability defect except for the mspA mutant. Deletion of the mspA gene caused activation of transcription of mspB and/or mspD in three independent strains by unknown chromosomal mutations. It is concluded that mspB and mspD provide backup porins for M. smegmatis . This also indicated that a minimal porin-mediated OM permeability is essential for survival of M. smegmatis . Electron microscopy in combination with quantitative image analysis of protein gels revealed that the number of pores per cell dropped from 2400 to 800 and 150 for the Δ Δ Δ Δ mspA and Δ Δ Δ Δ mspA Δ Δ Δ Δ mspC mutant (ML10) respectively. The very low number of pores correlated well with the at least 20-fold lower channel activity of detergent extracts of the ML10 strain and its 15-and 75-fold lower permeability to nutrient molecules such as serine and glucose respectively. The amount of Msp porin and the OM permeability of the triple porin mutant lacking mspA , mspC and mspD was not altered. The growth rate of M. smegmatis dropped drastically with its porin-mediated OM permeability in contrast to porin mutants of Escherichia coli . These results show that porin-mediated influx of nutrients is a major determinant of the growth rate of M. smegmatis .

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The metabolism of dietary proteins generates waste products that are excreted by the kidney, in particular nitrogen-containing urea, uric acid, ammonia, creatinine, and other metabolites such as phosphates, sulfates, and protons. Kidney adaptation includes an increase in renal plasma flow (RPF) and glomerular filtration rate (GFR) and represents a burden for diseased kidneys increasing the progression rate of CKD. The present study aimed at identifying potential differences between amino acid (AA) groups constituting dietary proteins regarding their impact on RPF, GFR, and CKD progression. We utilized the well-established 5/6 nephrectomy (5/6 Nx) CKD model in rats and submitted the animals for 5 weeks to either the control diet (18% casein protein) or to diets containing 8% casein supplemented with 10% of a mix of free amino acids, representing all or only a subset of the amino acids contained in casein. Whereas the RPF and GFR measured in free moving animals remained stable during the course of the diet in rats receiving the control mix, these parameters decreased in animals receiving the branched chain amino acid (BCAA) supplementation and increased in the ones receiving the aromatic amino acids (AAAs). In animals receiving essential amino acids (EAAs) containing both BCAAs and AAAs, there was only a small increase in RPF. The kidneys of the 5/6 Nx rats receiving the BCAA diet showed the strongest increase in smooth muscle actin and collagen mRNA expression as a result of higher level of inflammation and fibrosis. These animals receiving BCAAs also showed an increase in plasma free fatty acids pointing to a problem at the level of energy metabolism. In contrast, the animals under AAA diet showed an activation of AMPK and STAT3. Taken together, our results demonstrate that subsets of EAAs contained in dietary proteins, specifically BCAAs and AAAs, exert contrasting effects on kidney functional parameters and CKD progression.

The present study was designed to investigate the metabolism of glycylglutamine and its effects on organ balances of amino acids during intravenous infusion of this dipeptide (100 mumol.h-1.kg-1) in postabsorptive and briefly starved (84-86 h) human subjects. Arterial concentrations of glycylglutamine were not significantly different in postabsorptive (265 +/- 18 microM) and starved (241 +/- 13 microM) subjects. Among the organs examined, kidney predominated in clearance of glycylglutamine from plasma. Moreover, renal clearance of glycylglutamine was reduced by starvation (87 +/- 7 vs. 52 +/- 5 mumol/min, P less than 0.01), whereas neither splanchnic nor muscle clearance was significantly affected. Infusion of glycylglutamine raised plasma concentrations of glycine and glutamine by increasing renal release of these amino acids. In postabsorptive subjects the infusion significantly increased splanchnic balances of glycine and glutamine with little or no effect on the muscle balances; the opposite was found in starved subjects. As far as other amino acids are concerned, the infusion decreased the muscle release of alanine and increased renal release of serine. We conclude that the amino acid residues of glycylglutamine are largely metabolized by the splanchnic organs in postabsorptive subjects and by peripheral organs in starved subjects. The latter results in selective inhibition of muscle release of amino acids.