Gene expression of the renin-angiotensin system in human kidney.

BACKGROUND: Immunocytochemical studies have revealed that all components of the renin-angiotensin system are widely distributed in human tissues yet the information on the gene expression of the renin-angiotensin system in various types of cell remains scarce. OBJECTIVE: We explored the presence of a local renin-angiotensin system in human kidney. METHODS: We sought to determine the presence of messenger RNA (mRNA) encoding for renin, angiotensinogen, and angiotensin converting enzyme (ACE) in cultured human glomerular cells and human umbilical vein endothelial cells using a two-step polymerase chain reaction. The gene expression of the renin-angiotensin system in normal human kidney and in diseased kidney was studied by in-situ hybridization using synthetic oligonucleotides. RESULTS: By using a two-step polymerase chain reaction, renin, angiotensinogen, and ACE mRNA were found in cultured mesangial and epithelial cells but only ACE mRNA was present in human umbilical vein endothelial cells. Renin mRNA was detected in juxtaglomerular granular cells and also in glomerular and tubular epithelia in normal kidney by in-situ hybridization. A similar tubular, but not mesangial, distribution was found with angiotensinogen and ACE mRNA. In contrast, stronger signals for renin, angiotensinogen and ACE mRNA were detected in mesangial and epithelial cells of kidney tissues from hypertensive patients and from patients with renal pathology characterized by mesangial proliferation (immunoglobulin A nephropathy, diabetes mellitus, or lupus nephritis). CONCLUSIONS: That gene expression of the renin-angiotensin system occurs in resident glomerular cells supports the hypothesis that there is a local renin-angiotensin system in human kidney. Our findings support the previous speculation that the renin-angiotensin system could be a local factor involved in the progression of chronic renal failure and consequent development of hypertension.

Identification and characterization of an activated 20S proteasome in Trypanosoma brucei.

Recently, we have reported the isolation and purification of 20S proteasomes from both the procyclic and bloodstream forms of Trypanosoma brucei, but no 26S proteasome was identified under those experimental conditions (Hua et al., Mol. Biochem. Parasitol. (1996) 78, 33-46). Subsequent attempts to identify a 26S proteasome in T. brucei led to the discovery of another form of the 20S proteasome designated the activated 20S proteasome because it exhibited much higher peptidase activities than the original 20S proteasome on all the fluorogenic peptides tested, and it crossreacted with the rabbit antisera against the 20S proteasomes purified from T. brucei. The activated 20S proteasome can be isolated from both procyclic and bloodstream forms of T. brucei and has a slightly higher molecular weight than the 20S proteasome. It is stable in the absence of ATP but susceptible to elution through a DE52 column. Analysis of the activated 20S proteasome in SDS-PAGE showed the presence of all the subunit proteins from the 20S proteasome plus an extra protein with an estimated molecular mass of 26 kDa. This protein, designated PA26, is not a degradation product of the 20S proteasomal subunit proteins. It could be a homolog of the bovine PA28 and human 11S regulator protein which form complexes with the 20S proteasomes resulting in activation of their peptidase activities. This likelihood was confirmed in a reconstitution experiment in which PA26 separated from the proteasome by a DE52 column chromatography was re-introduced into the purified 20S proteasome, and resulted in the emergence of a new protein band with the same mobility and peptidase activities as the activated 20S proteasome in native polyacrylamide gel electrophoresis. The presence of an activated 20S proteasome rather than a homolog of the 26S proteasome in T. brucei suggests that PA26 may play an important role in regulating the proteasome-mediated protein degradations in trypanosomes.

Inhibition of proteasome activity blocks cell cycle progression at specific phase boundaries in African trypanosomes.

Proteasomes are one of the cellular complexes controlling protein degradation from archaebacteria to mammalian cells. We recently purified and characterized the catalytic core of the proteasome, the 20S form, from Trypanosoma brucei, a flagellated protozoa which causes African trypanosomiasis. To identify the role of proteasomes in African trypanosomes, we used lactacystin, a specific inhibitor of proteasome activity. Lactacystin showed potent inhibition of the activity of 20S proteasomes purified from both bloodstream and procyclic (insect) forms of T. brucei (IC50 = 1 microM). It also inhibited proliferation of T. brucei cells in culture assays, with 1 microM inhibiting growth of bloodstream forms, whereas 5 microM was required to block proliferation of procyclic forms. Analysis of the DNA content of these cells by flow cytometry showed that 5 microM lactacystin arrested procyclic cells in the G2 + M phases of the cell cycle. Fluorescence microscopy revealed that most of the cells had one nucleus and one kinetoplast each, indicating that the cells had replicated their DNA, but failed to undergo mitosis. This suggests that transition from G2 to M phase was blocked. On the other hand, incubation of bloodstream forms with 1 microM lactacystin led to arrest of 30-35% of the cell population in G1 and 55-60% of the cells in G2, indicating that both transition from G1 to S and from G2 to M were blocked. These observations were also confirmed by using another inhibitor of proteasome, N-carbobenzoxy-L-leucyl-L-leucyl-L-norvalinal (LLnV), which arrested procyclic forms in G2, and bloodstream forms in both G1 and G2. These results suggest that proteasome activity is essential for driving cell cycle progression in T. brucei, and that proteasomes may control cellular functions differently in bloodstream and procyclic forms of T. brucei.

Purification and characterization of proteasomes from Trypanosoma brucei.

Proteasomes are multisubunit proteases that exist universally among eukaryotes. They have multiple proteolytic activities, and are believed to have important roles in regulating cell cycle, selective intracellular proteolysis, and antigen presentation. To determine the possible role that proteasomes may play in controlling the life cycle of African trypanosomes, we have isolated proteasomes from the bloodstream and the insect (procyclic) forms of Trypanosoma brucei by DEAE-cellulose chromatography and glycerol gradient fractionation in the presence of ATP. No 26 S proteasome homologs was identified in T. brucei under these experimental conditions. The proteasomes isolated from these two forms of T. brucei are very similar to the rat blood cell 20 S proteasome in their general appearance under the electron microscope. The profile of trypanosome proteasome subunits in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) has eight visible protein bands with molecular weights ranging from 23 to 34 kDa, and cross-reacted very poorly with the anti-human 20 S proteasome antibodies on immunoblots. Two-dimensional gel electrophoresis of the parasite proteasomes shows a similar number of major subunits with pI's ranging from 4.5 to 7. Using a variety of fluorogenic peptides as substrates, the trypanosome proteasomes exhibited unusually high trypsin-like, but somewhat lower chymotrypsin-like activities, as compared to the rat 20 S proteasome. These proteolytic activities were, however, insensitive to phenylmethylsulfonyl fluoride (PMSF), tosyl-phenylalanine chloromethylketone (TPCK), tosyl-lysine chloromethylketone (TLCK) and trans-epoxy succinyl-L-leucylamido-(4 guanidino) butane (E-64), but the trypsin-like activity of trypanosome proteasomes was inhibited by leupeptin, an aldehyde known to inhibit the trypsin-like activity of mammalian proteasomes, thus ruling out possible contamination by other serine or cysteine proteases. Some quantitative differences in the substrate specificities between the proteasomes from bloodstream and procyclic forms were indicated, which may play a role in determining the differential protein turnovers at two different stages of development of T. brucei.

Serologic study of immunoglobulin A-fibronectin aggregates in immunoglobulin A nephropathy.

The immunoglobulin A (IgA)-fibronectin aggregates, detected by enzyme-linked immunosorbent assay using either antifibronectin or collagen I as binding protein, were previously found to be raised in the circulation of patients with IgA nephropathy (IgAN). It has been suggested that IgA-fibronectin aggregates are involved in the pathogenesis and that the plasma IgA-fibronectin level may even be of diagnostic value in IgAN. Nevertheless, a recent report has questioned the specificity of these assays as plasma IgA may interact with immobilized IgG and these assays detect not only IgA-fibronectin, but also total plasma IgA. These doubts render the interpretation of raised IgA-fibronectin aggregates in IgAN impossible. We isolated total IgA, in plasma by jacalin-agarose. Monomeric and polymeric IgA1 were distinctly separated by fast protein liquid chromatography. When the fast protein liquid chromatography fractions were analyzed for IgA-fibronectin using the antifibronectin capture assay, increased optical density values were predominantly observed in polymeric IgA but not in monomeric IgA. Similar findings were found when the fast protein liquid chromatography fractions were studied using a novel gelatin-anti-IgA assay that avoided nonspecific interaction between plasma IgA and immobilized IgG used as the capture antibody in antifibronectin capture assay. Using our gelatin-anti-IgA assay, we failed to demonstrate a diagnostic increase in IgA-fibronectin aggregates in polymeric IgA from patients with IgAN compared with controls. Our finding of circulating IgA-fibronectin aggregates in patients with IgAN comparable to those of healthy controls did not support the notion that these aggregates may have a pathogenetic role or diagnostic value in IgAN.

Increased binding of polymeric lambda-IgA to cultured human mesangial cells in IgA nephropathy.

IgA nephropathy (IgAN) is characterized by raised plasma lambda-IgA1 and mesangial polymeric lambda-IgA1 deposits. It remains uncertain whether the predominant glomerular lambda-IgA1 deposits represent a selective uptake of polymeric IgA or a non-specific uptake due to elevated circulating lambda-IgA1 levels in response to an unidentified antigen. In this study, we explored whether there is an increased binding of monomeric IgA1 (mIgA1) or polymeric IgA1 (pIgA1) from patients with IgAN to cultured human mesangial cells (HMC). Total IgA1 in plasma from patients or healthy controls was isolated by jacalin-agarose column as jacalin-bound proteins (JBP). Monomeric IgA1 and pIgA1 were distinctly separated by FPLC. HMC were incubated with IgA preparations and IgA bound to HMC was determined by flow cytometry analysis using standard curves constructed by known concentrations of kappa-IgA1 or lambda-IgA1. In order to avoid any increased binding of IgA to HMC due to elevated kappa- or lambda-IgA concentrations in JBP samples from patients, JBP samples from patients or controls were appropriately diluted to achieve comparable levels of total IgA1. No differences in the total mIgA1 or pIgA1 concentration, percentage of mIgA1 or pIgA1, or the kappa/lambda ratio of mIgA1 or pIgA1 were found between adjusted JBP samples from patients or healthy controls. We found a sharp rise in percentage of pIgA1 among IgA1 bound to HMC (70%), despite the fact that only 3% of the IgA1 in the adjusted JBP samples were polymeric, suggesting that pIgA1 had a higher affinity to HMC than mIgA1. Furthermore, the kappa/lambda ratios of pIgA1 bound to HMC were significantly lower than the kappa/lambda ratios of pIgA1 in adjusted JBP only with IgAN patients but not healthy controls (P = 0.0026). Our data suggest a preferential mesangial binding of polymeric lambda-IgA1 from patients with IgAN. These polymeric lambda-IgA immune complexes are likely to be "pathogenic" and are important in the pathogenesis of IgAN.

Identification and characterization of human serum alpha2-HS glycoprotein as a jacalin-bound protein.

We recently adopted immobilized jacalin as an affinity adsorbent to purify human serum IgA for laboratory study. In the course of our investigation, we detected a serum protein that co-eluted with IgA from jacalin-agarose affinity column. It constituted in significant quantity (24.0 +/- 0.9%, n = 30) of total jacalin-bound protein (JBP) and the yield was equivalent to 0.4 +/- 0.1 mg per ml serum. The molecular mass of this protein was 55 kDa with electromobility in the alpha 2 region as demonstrated by SDS-PAGE and immunoelectrophoresis. N-terminal microsequencing of this 55 kDa protein revealed that it is human alpha 2-HS glycoprotein (alpha 2HSG). The molecular interaction of alpha 2HSG with jacalin was characterized by competitive ELISA: human serum IgA, human colostrum secretory IgA (sIgA), and monosaccharides including D-galactose and melibiose exhibited strong inhibitory effect on its binding to jacalin. Accordingly, we propose that human alpha 2HSG binds in a similar manner as that of the bovine fetuin to jacalin. In addition, alpha 2HSG displays similar binding property to jacalin from different geographic area (India and Malaysia) and from different laboratory preparations (Sigma, Pierce and 'homemade' jacalin).