Tandem antifibrotic actions of L-arginine supplementation and low protein diet during the repair phase of experimental glomerulonephritis

Tandem antifibrotic actions of L-arginine supplementation and low protein diet during the repair phase of experimental glomerulonephritis.BackgroundBased upon the central role transforming growth factor-beta (TGF-β) overexpression appears to play in renal fibrotic diseases, we have recently advocated reduction of TGF-β as a therapeutic target. As part of efforts to determine the strength of this approach, we have undertaken studies to quantitate the effects of currently used and promising therapies in terms of their potential to reduce markers of disease in anti-thymocyte-serum (ATS)-glomerulonephritis in the rat. Here we assess the therapeutic effect of L-arginine supplementation, which has been shown to reduce fibrosis in a number of hypertensive models, given alone or in combination with low protein diet and started 24 hours after disease induction.MethodsGlomerulonephritis was induced by intravenous injection of OX-7 monoclonal antibody into 200g Sprague-Dawley rats. Twenty-four hours later animals were placed in groups that were either untreated, treated with 1% L-arginine in drinking water or 6% protein diets or both. On the fifth day of disease 24-hour urine specimens were collected and systemic blood pressure was measured. On the sixth day rats were anesthetized. Kidneys were perfused, tissue was taken for PAS staining and glomeruli were isolated. Aliquots of glomeruli were used for RNA preparation and for culture to determine 72-hour production of TGF-β, fibronectin and plasminogen activator-type 1 (PAI-1), which were assayed by ELISA on culture supernatants. Measures of nitrate and nitrite (NOx) production included plasma NOx, urinary NOx and glomerular production of NOx in culture.ResultsAll disease measures except proteinuria and including matrix accumulation, TGF-β, fibronectin and PAI-1 production and mRNA expression for TGF-β, fibronectin and PAI-1 were significantly and similarly reduced by about 50% in groups treated with L-arginine or with low protein diet. Proteinuria was reduced in low protein treated but not in L-arginine supplemented rats. Neither systemic blood pressure nor measures of NO synthesis showed differences between groups that could be attributed to L-arginine supplementation. In contrast, disease-related increases in glomerular production of NOx were markedly reduced by low protein. Combined therapy resulted in small, but statistically significant decreases in most measures of disease.ConclusionsL-arginine supplementation reduces fibrotic disease in ATS-induced glomerulonephritis if started after disease induction. The absence of evidence for increased NO production related to L-arginine supplementation suggests that L-arginine is acting here through different pathways from those demonstrated in hypertensive models of disease. The data support the ideas that TGF-β reduction is a valid therapeutic target and that quantitation of TGF-β reduction is a useful approach for comparing antifibrotic drug candidates

Glomerular matrix accumulation is linked to inhibition of the plasmin protease system

Glomerular matrix accumulation is linked to inhibition of the plasmin protease system. TGF-β plays a pivotal role in the pathological accumulation of extracellular matrix in experimental glomerulonephritis. Increased TGF-β expression leads to increased synthesis and deposition of extracellular matrix components while administration of antiserum to TGF-β suppresses the major manifestations of the disease. We hypothesized that TGF-β might also enhance matrix accumulation by decreasing matrix turnover via effects on protease/protease inhibitor balance. Plasmin is a potent protease capable of degrading a variety of matrix molecules. Plasmin generation from plasminogen is regulated by plasminogen activator(s) (PA) and plasminogen activator inhibitor(s) (PAI). In this study PA activity was markedly reduced and PAI-1 synthesis dramatically increased when TGF-β was added to normal glomeruli. Diseased glomeruli also showed decreased PA activity, increased PAI-1 synthesis and increased PAI-1 deposition into matrix. Administration of anti-TGF-β serum to glomerulonephritic rats blocked the expected increase in glomerular PAI-1 deposition. Thus changes in the PA/PAI balance favoring accumulation of matrix are induced by TGF-β in normal glomeruli and are present in nephritic glomeruli when endogenous TGF-β production is high. Our findings implicate the plasmin protease system in tissue repair following acute glomerular injury and suggest another mechanism by which TGF-β enhances the matrix accumulation characteristic of many glomerular diseases

Enalapril and low protein reverse chronic puromycin aminonucleoside nephropathy

Enalapril and low protein reverse chronic puromycin aminonucleoside nephropathy. The effects of dietary protein and converting enzyme inhibition (CEI) on chronic puromycin aminonucleoside nephropathy (PAN) were studied. PAN was induced with seven SQ injections of puromycin aminonucleoside 20 mg/kg over 10 weeks in male Sprague-Dawley rats. The rats were divided into a 22.5% protein diet group (Gr 1), a 6% protein diet group (Gr 2), and an enalapril-treated group on 22.5% protein diet (Gr 3). Group 4 animals served as age-matched controls. Both diets were isocaloric and had the same phosphorus content. Rats from groups 1, 2, and 4 were sacrificed at 12, 18 and 24 weeks. Five rats of group 3 were sacrificed at 12 weeks, and the others were divided in subgroups 3A (diet changed to 6% protein) and 3B (no changes); half of each subgroup was sacrificed at 18 and 24 weeks, respectively. Group 2 had significantly less proteinuria than group 1 at all times. Group 3 had the same proteinuria as group 1 until 12 weeks and then began to decrease. In group 3A proteinuria decreased to group 2 levels, while in group 3B the decrease was slower but still prominent. Early lesions of focal and segmental glomerular sclerosis/hyalinosis (FSH) were present in groups 1, 2, 3 at 12 weeks (16 ± 1.2%, 15 ± 1.3%, 7 ± 1.3%, respectively, versus 1.3 ± 0.4% in controls), but by 18 weeks a reversal in FSH was seen in groups 2 and 3A/B (3 ± 1.6%, 2 ± 0.4%, and 3 ± 0.9%, respectively, vs. 14 ± 1.5% in group 1). This reversal persisted at 24 weeks (5 ± 2.5%, 3 ± 0.8%, 4 ± 0.8% vs. 18 ± 2.6%). At 24 weeks mean glomerular diameter was significantly less in group 2 compared to group 1, 100.7 ± 2.0 µ versus 112.2 ± 2.7 µ, P = 0.009. In summary, both low protein diet and CEI for 24 weeks reversed both proteinuria and early FSH lesions in chronic PAN after cessation of PA injections

Combining TGF-β inhibition and angiotensin II blockade results in enhanced antifibrotic effect

Combining TGF-β inhibition and angiotensin II blockade results in enhanced antifibrotic effects.BackgroundAlthough angiotensin II (Ang II) blockade is rapidly becoming standard antifibrotic therapy in renal diseases, current data suggest that Ang II blockade alone cannot stop fibrotic disease. New therapies, such as antibodies to transforming growth factor-β (TGF-β), or drug combinations will be required to further slow or halt disease progression. Here, using the anti-Thy1 model of glomerulonephritis, the maximally therapeutic dose of the TGF-β neutralizing mouse monoclonal antibody (1D11) was determined and compared with the maximally effective dose of enalapril. Then, the effect of combining both treatments at maximal doses was determined.MethodsAfter disease induction with the anti-Thy1 antibody, OX-7, increasing doses of 1D11 were given intraperitoneally (IP) on days 1, 3, and 5. Enalapril was administered in drinking water from day 1. The fibrotic response was assessed at day 6.Results1D11 dose-dependently reduced fibrosis, with the 0.5 and 5 mg/kg doses showing maximal therapeutic effects, reducing period-acid Schiff (PAS) staining by 56% and 45%, respectively. Fibronectin and collagen I staining was reduced by 32% to 36%, respectively. Glomerular mRNA and production of fibronectin, plasminogen activator inhibitor-1 (PAI-1), TGF-β1, and p-Smad2 protein were also reduced. The maximal therapeutic effects of 1D11 and enalapril alone were very similar. However, combination therapy led to further reduction in disease. Notably, matrix deposition was reduced by 80%.ConclusionWhile 1D11 or enalapril at maximal doses reduce fibrosis equally, simultaneous blockade of Ang II and TGF-β reduces fibrotic disease considerably more, offering hope that such drug combinations may confer a therapeutic advantage over angiotensin blockade alone

A Qualitative View of Drug Use Behaviors of Mexican Male Injection Drug Users Deported from the United States

Deportees are a hidden yet highly vulnerable and numerous population. Significantly, little data exists about the substance use and deportation experiences of Mexicans deported from the United States. This pilot qualitative study describes illicit drug use behaviors among 24 Mexico-born male injection drug users (IDUs), ≥18 years old, residing in Tijuana, Mexico who self-identified as deportees from the United States. In-person interviews were conducted in Tijuana, Mexico in 2008. Content analysis of interview transcripts identified major themes in participants’ experiences. Few participants had personal or family exposures to illicit drugs prior to their first U.S. migration. Participants reported numerous deportations. Social (i.e., friends/family, post-migration stressors) and environmental factors (e.g., drug availability) were perceived to contribute to substance use initiation in the U.S. Drugs consumed in the United States included marijuana, heroin, cocaine, methamphetamine, and crack. More than half of men were IDUs prior to deportation. Addiction and justice system experiences reportedly contributed to deportation. After deportation, several men injected new drugs, primarily heroin or methamphetamine, or a combination of both drugs. Many men perceived an increase in their substance use after deportation and reported shame and loss of familial social and economic support. Early intervention is needed to stem illicit drug use in Mexican migrant youths. Binational cooperation around migrant health issues is warranted. Migrant-oriented programs may expand components that address mental health and drug use behaviors in an effort to reduce transmission of blood-borne infections. Special considerations are merited for substance users in correctional systems in the United States and Mexico, as well as substance users in United States immigration detention centers. The health status and health behaviors of deportees are likely to impact receiving Mexican communities. Programs that address health, social, and economic issues may aid deportees in resettling in Mexico

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead