A robust route to enzymatically functional, hierarchically self-assembled peptide frameworks.

The addition of enzyme biofunctionality to self-assembling peptide nanofibers is challenging since such additions can inhibit functionality or self-assembly. We introduce a method for peptide nanofiber enzyme functionalization, demonstrated by the attachment of a polymerization synthase to peptide nanofibers. The enzyme generates a biocompatible, biodegradable biopolyester coat on the fibers with applicablity in medical engineering. This approach provides a template for generation of functional bionanomaterials.

Dysregulated growth factor gene expression is associated with tubulointerstitial apoptosis and renal dysfunction.

Chronic renal disease is characterized by declining renal function, loss of intrinsic renal cells, and their replacement with fibrotic tissue. This study investigates apoptosis and its regulation in the context of chronic renal disease. RNA was extracted from renal biopsies from patients with various forms of chronic renal disease. Expression of genes of the Bcl-2 family, death receptor pathway, and growth factors were measured by reverse-transcription real-time polymerase chain reaction. Apoptosis was detected by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling technique. Tubulointerstitial apoptosis was positively associated with tubulointerstitial injury and renal dysfunction and increased 2.3-fold per unit (U) increase in transforming growth factor beta(1) (TGFbeta(1)) mRNA (P<0.05). Conversely, a 1 U increase in epidermal growth factor (EGF) mRNA was associated with a 47% decrease in tubulointerstitial apoptosis (P<0.05). Tubulointerstitial injury was correlated with increased TGFbeta(1) and tumour necrosis factor alpha (TNFalpha) mRNA (P<0.005) and decreased EGF mRNA (P<0.05). Additionally, for a 10 U decrease in the glomerular filtration rate there was an estimated increase of 5 and 10% in TGFbeta(1) and TNFalpha mRNA, respectively (P<0.05), whereas EGF mRNA decreased by an estimated 15% (P<0.005). Therefore dysregulation of cytokine/growth factor expression plays a central role in the progression of chronic renal disease through contribution to renal cell loss, tubulointerstitial injury, and renal dysfunction.

Apoptosis and the expression of genes of the Bcl-2 family and TGF-beta1 in rat renal allografts transplanted after donor-specific blood transfusion.

UNLABELLED: Factors involved in "operational" tolerance in animal models induced by recipient pre-treatment with donor-specific blood transfusion (DSBT) need elucidation. This study examined apoptosis, expression of genes of the Bcl-2 family and of TGF-beta(1) in isografts, rejecting and tolerant allografts. METHODS: Adult inbred Dark Agouti (DA) kidneys were transplanted, with immediate nephrectomy of recipient kidneys, to (1) ALLO, inbred Albino Surgery (AS) rats; (2) DSBT ALLO, AS rats who received two DA blood transfusions under cover of cyclosporine prior to transplantation; or (3) ISO, DA rats. Grafts were retrieved on day 1, 3, or 5. Apoptosis was assessed by TUNEL. RNA was extracted and reverse transcribed to cDNA for quantification by real-time PCR, relative to the 18s housekeeping gene. RESULTS: Apoptosis was negligible in ISO while it increased in allograft groups from day 1. On day 5, apoptosis in ALLO (114.0 +/- 30.6), involved renal tubular cells and leukocytes compared to DSBT ALLO (9.7 +/- 4.0) and ISO (0.9 +/- 0.3) involving leukocytes only. On day 1, DSBT ALLO had higher expression of Bax than ALLO or ISO. On day 3, DSBT ALLO and ALLO had higher TGF-beta(1) mRNA than ISO. On day 5, Bcl-2 expression was significantly decreased (P < .001) in ALLO compared to DSBT ALLO and ISO. Bad and Bid were higher in DSBT ALLO than in ALLO. TGF-beta(1) was higher in DSBT ALLO compared to ISO. CONCLUSIONS: Decreased expression of anti-apoptotic Bcl-2 gene may be implicated in increased apoptosis in rejecting allograft while expression of pro-apoptotic genes may be involved in the establishment of operational tolerance.

Proteinuria and the expression of the podocyte slit diaphragm protein, nephrin, in diabetic nephropathy: effects of angiotensin converting enzyme inhibition.

AIMS/HYPOTHESIS: Proteinuria, reflecting increased glomerular permeability to macromolecules is a characteristic feature of diabetic nephropathy. Nephrin, a 1241-residue transmembrane protein is a key component of the podocyte slit pore membrane and a major contributor of the glomerular filtration barrier. We investigated the expression of nephrin in human kidney tissue from patients with diabetic nephropathy to elucidate its relationship with proteinuria and the effects of anti-proteinuric therapy with angiotensin converting enzyme inhibition. METHODS: Renal biopsies were examined from 14 patients with Type II (non-insulin-dependent) diabetes mellitus and proteinuria who had been randomised to receive treatment with the ACE inhibitor, perindopril (4 mg/day) or placebo for the preceding 2 years. These specimens were compared with control human tissue sections, obtained from areas of normal renal cortex following nephrectomy for malignancy. Proteinuria was measured, specimens were examined histologically for injury and the expression of nephrin messenger RNA was assessed by quantitative in situ hybridisation. RESULTS: Glomeruli from placebo-treated patients with diabetic nephropathy, showed a 62% reduction in nephrin expression compared with control subjects (p=0.0003). In contrast, nephrin RNA in glomeruli from perindopril treated patients was similar to that in the non-diabetic control group. In both placebo and perindopril treated patients, a close inverse correlation was noted between the magnitude of nephrin gene expression and the degree of proteinuria (placebo: r=0.86, p=0.013, perindopril: r=0.91, p=0.004). CONCLUSION/INTERPRETATION: Modulation in nephrin expression is related to the extent of proteinuria in diabetic nephropathy. These changes define, at a molecular level alterations in the glomerulus that occur in relation to proteinuria in diabetes and the effects of anti-proteinuric treatment with ACE inhibition.

Transforming growth factor-beta1 and tumor growth factor-beta-inducible gene-H3 in nonrenal transplant cyclosporine nephropathy.

Cyclosporine nephropathy (CyAN) is a major limiting factor in the otherwise successful widespread use of cyclosporine in solid organ transplant. Transforming growth factor-beta1 (TGF-beta1) has been implicated as an important fibrogenic cytokine in the development of this disease. TGF-beta-inducible gene-H3 (beta(ig)-H3) is a TGF-beta1- induced gene product, which acts as a marker for biologically active TGF-beta1. This study reports TGF-beta1 gene expression and beta(ig)-H3 tissue distribution in non-renal allograft CyAN. Renal tissue from nine patients who had developed CyAN after successful heart or heart-lung transplantation and from four kidneys removed for tumour were analyzed for TGF-beta1 gene expression beta(ig)-H3 protein with reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry, respectively. TGF-beta1 gene expression was increased in CyAN compared to nephrectomy (P<0.0001). Beta(ig)-H3 protein expression was identified in distal convoluted tubular epithelium and parietal glomerular epithelium in CyAN, and not in nephrectomy samples. Expression of TGF-beta1 mRNA was significantly higher in renal tissue from patients not receiving angiotensin converting enzyme inhibitor (ACEI) therapy for hypertension (P<0.05). These findings support the hypothesis that TGF-beta1 is an important cytokine in the development of CyAN, independent of its role in chronic rejection in renal allografts.

Interactions between methylating and pyridyloxobutylating agents in A/J mouse lungs: implications for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis.

The tobacco-specific nitrosamine, 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone, is activated to lung DNA methylating and pyridyloxobutylating intermediates. It is likely that both pathways play a role in lung tumor initiation by this nitrosamine. Previous studies indicated that O(6)-methylguanine (O(6)-mG) persistence is critical for lung tumor formation in A/J mice. The model pyridyloxobutylating agent, 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc), enhanced the tumorigenic activity of a model methylating agent, acetoxymethylmethylnitrosamine (AMMN), presumably by increasing O(6)-mG persistence in lung DNA. We have been testing the hypothesis that the pyridyloxobutylation pathway increases the mutagenic activity of the DNA methylation pathway by preventing the repair of O(6)-mG by O(6)-alkylguanine-DNA alkyltransferase (AGT). In this study, we report that NNKOAc depletes AGT in lungs but not livers of A/J mice. The consequences of AGT depletion by NNKOAc were then compared with those observed with a known AGT inhibitor, O(6)-benzylguanine (O(6)-bG). NNKOAc and O(6)-bG had similar effects on the levels of AMMN-derived O(6)-mG at 4 and 96 h postinjection. This increase in O(6)-mG levels correlated to increased lung tumor multiplicity in animals simultaneously treated with AMMN (0.75 or 1 micromol) and NNKOAc or O(6)-bG. Only NNKOAc significantly increased lung tumor multiplicity at doses of 0.25 or 0.5 micromol AMMN. The results from these studies indicate that the pyridyloxobutylating agent, NNKOAc, can influence the tumorigenic activity of methylating agents in two ways. At low AMMN doses, the increase in tumor multiplicity is dominated by the additive tumorigenic properties of AMMN and NNKOAc. At higher AMMN doses, NNKOAc appears to enhance the tumorigenic activity of AMMN through enhanced depletion of the repair protein, AGT, leading to increased O(6)-mG persistence. It is likely that similar interactions are important for the organospecific effects of 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone.

Functional, histological, and inflammatory changes in chronically rejecting small bowel transplants.

Our aim was to develop a model of chronic rejection (CR) in small bowel allografts, and to study the changes occurring in these grafts. Small bowel transplantation was performed using the DA to AS rat strain combination. Short-term (5 mg/kg intramuscular, from days -2 to +9), or long-term cyclosporin treatment (5 mg/kg, 3 times a week until day 50) was given to prevent acute rejection. Controls were untreated allografts, DA isografts with and without cyclosporin, and normal DA and AS rats. They were followed for 50 and 100 days after transplantation. Recipients of a syngeneic graft lost weight during the first week after transplantation, but started to regain weight and kept growing thereafter. Histology showed normal bowel architecture with normal mesenteric lymph nodes and Peyers patches. Vigorous acute rejection occurred in the untreated allografts. Animals had persistent weight loss, and were killed between 6-13 days after transplantation. No clinical signs of graft-versus-host disease were seen. Histology showed end-stage acute rejection. In both cyclosporin-treated allografted groups the postoperative course was as in the isografted animals. However, all animals had histologic signs of CR by 50 and 100 days after transplantation. Changes were most prominent in the mesentery. Serositis with increased vascularity, inflammation with sclerosis, and patchy myointimal proliferation with endothelialitis of the mesenteric vessels were found. Changes in the bowel were patchy and included some thickening of the muscle coat, crypt hyperplasia, scattered necrotic cells in the crypts, slight blunting of villi and loss of goblet cells. Infiltrating cells in the mesentery and bowel consisted mainly of CD 4+ cells, CD 8- T-cells and monocytes/macrophages. Lactulose-mannitol urinary excretion ratio was significantly increased in short-term cyclosporin treated allografts at days 50 and 100 posttransplant. Serum albumin levels were significantly lowered in this group at both time points examined. We developed two models in which CR occurs after small bowel transplantation. Long-term cyclosporin treatment delayed the development of CR, since functional abnormalities were only seen in the animals that were treated with short-term cyclosporin.

EGF and TGF-beta1 gene expression in chronically rejecting small bowel transplants.

Long-term survival of small bowel transplants is hampered by chronic rejection. Epidermal growth factor (EGF) and transforming growth factor beta (TGF-beta) have opposing, regulatory roles in normal intestinal physiology and may be involved in the pathogenesis of chronic intestinal rejection. Our aim was to investigate the expression of EGF and TGF-beta1 in chronically rejecting small bowel transplants. Orthotopic small bowel transplantation was performed in the allogeneic DA-to-AS rat combination; Cyclosporin was administered temporarily to prevent acute rejection. Controls were DA isografts and normal DA rats. PreproEGF and TGF-beta1 gene expression was evaluated by northern blot analysis of the ileum RNA and standardized against glyceraldehyde-3-phosphate-dehydrogenase expression. Allografts demonstrated functional impairment and histological features of chronic rejection, whereas isografts appeared normal. Allografts demonstrated a significant reduction of EGF mRNA when compared to DA isografts. No significant changes were detected in TGF-beta1 expression in either allogeneic or syngeneic grafts. In conclusion, this study demonstrates reduced preproEGF and preserved TGF-beta1 gene expression in chronically rejecting small bowel transplants.

Development of injury in a rat model of chronic renal allograft rejection: effect of dietary protein restriction.

Non-allogeneic factors such as increased nephron "workload" may contribute to chronic renal allograft rejection. Reducing dietary protein from 20% to 8% was tested in a model of chronic rejection: Dark Agouti kidney to Albino Surgery recipient, "tolerised" by previous donor blood transfusions. Survival, weight gain, serum creatinine concentration and creatinine clearance were similar for both groups at all times. Urinary protein was significantly (P < 0.05) lower in the low-protein (LP) group 1 month after transplantation. After 3 and 6 months, both groups demonstrated mild chronic rejection. After 6 months, tubular atrophy was significantly (P < 0.05) less in the LP group and interstitial fibrosis was marginally reduced. Glomerular hypertrophy, glomerular sclerosis, tubular dilatation, leucocyte infiltration, adhesion molecule expression and TGF-beta1 mRNA expression were similarly increased in both groups. Thus, reducing dietary protein to 8% lowered urinary protein, but did not significantly affect the development of chronic rejection in renal allografts beyond affording a degree of protection from tubulointerstitial damage.

Increased expression of basic fibroblast growth factor during chronic rejection in intestinal transplants is associated with macrophage infiltrates.

Long-term survival of intestinal transplants is hampered by chronic rejection (CR). Since transplants with CR demonstrate fibrotic changes, the cytokine basic fibroblast growth factor (bFGF) may be involved in the tissue remodelling of chronic intestinal rejection. The aim of this study was to investigate the bFGF gene and protein expression and distribution in chronically rejecting intestinal allografts. Orthotopic small bowel transplantation was performed in the allogeneic DA-to-AS rat combination. Cyclosporin was administered temporarily to prevent acute rejection. Controls were DA isografts and normal DA. bFGF gene expression was evaluated using reverse transcriptase polymerase chain reaction (RT-PCR) of the ileum RNA and was standardized against Glyceraldehyde-3-phosphate-dehydrogenase (GAP-DH) expression. bFGF protein was determined using immunohistochemistry. To identify the bFGF-positive cell type, sequential sections were stained for cell markers. Allografts showed histological features of CR, whereas isografts preserved normal architecture. bFGF gene expression was present in normal ileum and significantly upregulated in allografts. Immunohistochemical staining showed a significant increase in bFGF protein compared to isografts. Most bFGF-positive cells were localized in the submucosa and muscularis, particularly around the neural plexus. bFGF-positive cells appeared to be ED-2-positive macrophages, strongly suggesting that the site of bFGF production is the activated macrophage. This study demonstrates increased bFGF mRNA and protein in chronically rejecting intestinal allografts that appear to be produced by macrophages.

Primary T-cell-rich B-cell lymphoma in the kidney presenting with acute renal failure and a second malignancy.

Infiltration of the kidney is commonly found in lymphoma, but acute renal failure arising from bilateral renal infiltration is uncommon. Primary renal lymphoma may occur and is usually of B-cell lineage. It is rare for patients with lymphoma to develop acute renal failure as their initial clinical presentation. Recently, an association between primary renal lymphoma and a second primary malignancy has been reported. We describe the first case of a renal T-cell-rich B-cell lymphoma presenting as acute renal failure, which was associated with a second primary pulmonary malignancy.

Collagen induced arthritis in rats. Contrasting effect of subcutaneous versus intradermal inoculation of type II collagen.

OBJECTIVE: To determine the effect of subcutaneous (s.c.) as compared to intradermal (i.d.) inoculation of collagen type II (CII) in induction of collagen induced arthritis (CIA). METHODS: Dark Agouti (DA) and Lewis rats were injected with CII either i.d. or s.c.. A group of s.c. inoculated DA rats was re-injected with CII intradermally 45 days after first injection (s.c./i.d.). Arthritis was assessed by macroscopic scoring, histology, and immunohistochemistry. Levels of anti-CII antibody subtypes were measured by ELISA. RESULTS: Intradermal but not s.c. inoculation of CII resulted in histologically confirmed erosive arthritis in both Lewis and DA strains. Subcutaneous/intradermal inoculated DA rats developed mild CIA with lower arthritic scores and delayed onset. Lewis rats injected s.c. had lower levels of total Ig, IgG, IgG2a, and IgG2b and similar titers of IgG1 compared to i.d. inoculated rats. In contrast, only IgG2b levels were lower in s.c./i.d. compared to i.d. rats. CONCLUSION: Our data suggest that s.c. administration of CII tolerises animals against autoimmune CIA.

The effect of ureteric stenting on the function and morphology of long-term rat renal allografts.

In the development of a reliable model for chronic rejection in rat renal allografts, the effect of modifying the ureteric anastomosis was tested. Rats, tolerized by pretreatment with two donor blood transfusions under Cyclosporin A, received renal allografts with either sewn or stented ureter. Control groups received isografts or underwent uninephrectomy with insertion of ureteric stents. For the first 6 days after transplantation, serum creatinine and urea values were lower in allograft recipients with stented ureters than in the group with sewn ureters. The method of ureteric anastomosis did not affect the long-term incidence of abnormal function. Allograft morphology was extremely variable from minor to extensive tubular atrophy, interstitial fibrosis, glomerular hypertrophy, focal and segmental glomerulosclerosis as well as vascular changes. Glomerulosclerosis was absent in controls and increased with time in the allografts. Two hundred days after transplantation all allograft recipients with sewn ureters exhibited some glomerulosclerosis, in half of these kidneys more than 25% of glomeruli were affected. Only 33% recipients of allografts with stented ureters exhibited some glomerulosclerosis and less than 20% of glomeruli were affected. The stented ureteric anastomosis provides a reliable method, a reduction of the technical failure rate, a reduction of the incidence of hydronephrosis, allows more accurate assessment of early renal function and may be of importance in reducing the occurrence and prevalence of glomerulosclerosis in the long-term allografts.

Drugs and the kidney in the elderly.

The high rates of drug-induced acute renal failure, worsening chronic renal dysfunction and systemic toxicity of renally excreted drugs in the elderly can be minimised by carefully assessing renal function, avoiding potentially nephrotoxic drugs as much as possible and closely monitoring drug concentrations and renal function when they must be used. The co-existence of impaired renal function, degenerative vascular disease or cardiac failure in the elderly substantially increases the risk of renal toxicity. When in doubt about potential nephrotoxicity or an increased risk of systemic toxicity from renally excreted drugs in the elderly, the practitioner should consult the numerous published guidelines.

A reproducible model of chronic rejection in rat renal allografts.

A reproducible animal model is essential for the study of the pathogenesis of chronic rejection. This study investigates: (i) the optimal pre-transplant blood transfusion conditions to induce tolerance in a strongly rejecting rat kidney allograft model (Dark Agouti to Albino-Surgery) and avoiding post-transplant immunosuppression; (ii) the functional and histological changes that occur in long-term surviving kidneys and their similarity to chronic rejection; and (iii) the maintenance of tolerance. Prolonged survival occurred after administration of at least two donor blood transfusions with concomitant cyclosporin A (5 mg/kg per day). The time-span between transfusions appeared to be critical: 4 days was more effective than 2 or 7 days. Ineffective treatment led to death within the first 2 weeks post-transplant with histological evidence of acute graft rejection. Seventy-five per cent of long-term survivors experienced impaired renal function in the first week which improved spontaneously and remained stable in 93% of the surviving animals after 100 days and in 66% after 200 days. The morphology of long-term allografts was extremely variable from minor to extensive tubular atrophy, interstitial fibrosis, glomerular hypertrophy, focal and segmental glomerulosclerosis and vascular changes. Glomerular hypertrophy occurred in uninephrectomized controls and probably denoted a response to uninephrectomy. Glomerulosclerosis increased with time and was absent in controls. Although chronic damage was evident, the rats remained tolerant to fresh donor skin. Replacement of the original kidney allograft with a fresh donor kidney resulted in 70% survival. These second grafts showed less severe renal dysfunction and morphological damage than the original allografts in the long-term follow up.

Expression and distribution of epidermal growth factor in acute and chronic renal allograft rejection.

Epidermal growth factor (EGF) is a fibrogenic cytokine with a possible role in chronic damage. EGF is also involved in tubular regenerative response to injury. This study investigates the expression and distribution of EGF in a rat model of renal allograft rejection. EGF was localised in control kidneys to distal convoluted tubules (DCT) and thick ascending loop of Henle (TAL). Five days post-transplantation EGF was diffusely distributed. In chronic rejection at one, three and six months, damaged areas of allografts demonstrated faint diffuse EGF staining, while well-preserved areas exhibited the normal distribution pattern. PreproEGF mRNA was significantly reduced (P < 0.01) in acute rejection and in chronic rejection at three months to 28% and 51% of normal, respectively. At six months values ranged from 16% to 166% of normal kidneys, and were inversely correlated with tubular damage (P < 0.01). PreproEGF mRNA was localized to DCT and TAL in controls and in well preserved areas of the tissue in chronic rejection. Thus, EGF would not appear to contribute to the development of injury in chronic renal rejection. It may instead exert a protective effect on tubular structures.

Development of chronic injury and nature of interstitial infiltrate in a model of chronic renal allograft rejection.

A model of chronic renal rejection in the Dark-Agouti to Albino-Surgery rat combination is described. In a number of cases, the original allograft was replaced by a second Dark-Agouti allograft. Seventy-five percent of rats experienced early episodes of rejection that subsided spontaneously. Second allografts had better initial renal function. Variable degrees of tubular atrophy, interstitial fibrosis, vascular damage, glomerulosclerosis, deposition of humoral mediators, and mononuclear leukocyte infiltrate were observed in all long-term allografts. Chronic damage increased with time, and was less severe in second allografts. At 5 days, total interstitial infiltrate was similar to that seen in unmodified rejection, but there was a significant increase in CD4+ cells and a decrease in ED2 and IL-2R expression. Subsequently, the total interstitial infiltrate decreased with time, although it remained significantly higher than in isografts and residual kidneys from uninephrectomized rats. No significant decrease over time was seen in numbers of CD4+ and CD45RC+ cells. The latter had a marked focal distribution after 100 days. Total leukocyte infiltrate was similar in original and second allografts, but there were changes in the proportions of leukocyte subpopulations, including significantly lower numbers of CD45RC+ cells in the latter. The persistence of CD45RC+ cells throughout the course of chronic rejection and their lower numbers in the second allografts favors a role for these cells in the development of chronic injury. The model of chronic renal allograft rejection characterized in this study will be valuable in further studies of the mechanisms of injury in this pathology.