Complications and Their Prevention in Experimental Renal Transplantation in Rats.

OBJECTIVES: Experimental rat models of renal transplant have played a pivotal role in renal transplant research. Both intraoperative and postoperative complications during donor nephrectomy and implantation in the recipient can be associated with significant morbidity and mortality. The aim of this paper is to discuss the incidence, pathophysiology, and prevention of complications that occurred in the process of establishment of a rat model of chronic allograft injury at our institution. MATERIALS AND METHODS: The complications observed while performing 67 consecutive donor nephrectomies and 61 renal transplants were recorded prospectively, and appropriate measures were taken to prevent these complications in the subsequent transplant procedures. RESULTS: Donor-related complications included failure of the kidney to clear of blood by the kidney perfusion solution and intraoperative deaths. The recipient-related complications included intraoperative hemorrhage, inadequately perfused kidneys with dusky appearance, congested and paralyzed hind limbs, urine leak, necrosis of the kidneys, renal and bladder calculi formation, and death during and after kidney transplant. CONCLUSIONS: Complications during donor nephrectomy and renal transplant can lead to significant loss of kidneys and animals. Proper recognition can allow appropriate measures to be taken to prevent these complications, thus achieving high-quality transplants and prolonged graft and animal survival.

Surgical Anatomy and Microvascular Surgical Technique Relevant to Experimental Renal Transplant in Rat Employing Aortic and Inferior Venacaval Conduits.

OBJECTIVES: Rat models of renal transplant are used to investigate immunologic processes and responses to therapeutic agents before their translation into routine clinical practice. In this study, we have described details of rat surgical anatomy and our experiences with the microvascular surgical technique relevant to renal transplant by employing donor inferior vena cava and aortic conduits. MATERIALS AND METHODS: For this study, 175 rats (151 Lewis and 24 Fisher) were used to establish the Fisher-Lewis rat model of chronic allograft injury at our institution. Anatomic and technical details were recorded during the period of training and establishment of the model. RESULTS: A final group of 12 transplanted rats were studied for an average duration of 51 weeks for the Lewis-to-Lewis isografts (5 rats) and 42 weeks for the Fisher-to-Lewis allografts (7 rats). Functional measurements and histology confirmed the diagnosis of chronic allograft injury. CONCLUSIONS: Mastering the anatomic details and microvascular surgical techniques can lead to the successful establishment of an experimental renal transplant model.

Review of Surgical Techniques of Experimental Renal Transplantation in Rats.

Microvascular surgical techniques of renal transplant in rats have evolved over the past 5 decades to achieve successful rat renal transplant; these modifications have included surgical techniques to address the anatomic variations in the renal blood vessels and those to reduce ischemic and operation durations. Here, we review the surgical techniques of renal transplant in rats and evaluate the advantages and disadvantages of individual techniques of vascular and ureteric anastomoses. For this review, we performed a systematic literature search using relevant medical subject heading terms and included appropriate publications in the review. Since the first description of a rat model of renal transplant by Bernard Fisher and his colleagues in 1965, which used end-to-side anastomosis between the renal vein and renal artery to the recipient inferior vena cava and aorta, several vascular and ureteric anastomosis techniques have been modified. Vascular anastomosis techniques now include end-to-end anastomosis, use of donor aortic and inferior vena cava conduits, sleeve and cuff anastomoses, and application of fibrin glue. Likewise, restoration of the urinary tract can now be achieved by direct anastomosis of the donor ureter to the recipient bladder, end-to-end anastomosis between the donor and recipient ureters, and donor bladder cuff to the recipient bladder. There are advantages and disadvantages attributable to individual techniques. The range of vascular and ureteric anastomosis techniques that has emerged reflects the need for mastering more than one technique to suit the vascular anatomy of individual animals and to reduce operating time for achieving successful outcomes after renal transplant.

Evolution of the Concept and Pathogenesis of Chronic Renal Allograft Injury: An Updated Review.

The advantages conferred by renal transplant, such as the improved quality of life and survival, are compromised by the reduced half-life of the transplanted kidney to a decade because of chronic allograft injury, which is the leading cause of transplant loss. There has been a significant evolution in the concept of the nomenclature, grading of histologic changes, diagnostic markers, and the theories of the pathogenesis of chronic allograft injury in the past decade. This review sought to consolidate the published literature that contributes toward understanding the changing concepts and pathogenesis of the chronic allograft injury, which has implications to managing and preventing chronic allograft injury in experimental and clinical settings.

Inhibiting ERK Activation with CI-1040 Leads to Compensatory Upregulation of Alternate MAPKs and Plasminogen Activator Inhibitor-1 following Subtotal Nephrectomy with No Impact on Kidney Fibrosis.

Extracellular-signal regulated kinase (ERK) activation by MEK plays a key role in many of the cellular processes that underlie progressive kidney fibrosis including cell proliferation, apoptosis and transforming growth factor ß1-mediated epithelial to mesenchymal transition. We therefore assessed the therapeutic impact of ERK1/2 inhibition using a MEK inhibitor in the rat 5/6 subtotal nephrectomy (SNx) model of kidney fibrosis. There was a twentyfold upregulation in phospho-ERK1/2 expression in the kidney after SNx in Male Wistar rats. Rats undergoing SNx became hypertensive, proteinuric and developed progressive kidney failure with reduced creatinine clearance. Treatment with the MEK inhibitor, CI-1040 abolished phospho- ERK1/2 expression in kidney tissue and prevented phospho-ERK1/2 expression in peripheral lymphocytes during the entire course of therapy. CI-1040 had no impact on creatinine clearance, proteinuria, glomerular and tubular fibrosis, and α-smooth muscle actin expression. However, inhibition of ERK1/2 activation led to significant compensatory upregulation of the MAP kinases, p38 and JNK in kidney tissue. CI-1040 also increased the expression of plasminogen activator inhibitor-1 (PAI-1), a key inhibitor of plasmin-dependent matrix metalloproteinases. Thus inhibition of ERK1/2 activation has no therapeutic effect on kidney fibrosis in SNx possibly due to increased compensatory activation of the p38 and JNK signalling pathways with subsequent upregulation of PAI-1.

Historical perspectives in kidney transplantation: an updated review.

The present state of success in kidney transplantation, including its benefits to patients with end-stage renal failure, was achieved through relentless research, both in experimental animal models and human volunteers. Kidney transplantation has evolved during the past century thanks to various milestones in surgical techniques, immunology, immunosuppressive drugs, expansion of donor sources, organ preservation, transplant against immunological barriers (ABO blood group-incompatible and positive crossmatch transplants), and research on induction of tolerance, xenotransplants, and stem cell technology. Despite significant improvements in graft and patient survival, several issues still must be addressed to reduce the growing number of patients with kidney failure waiting to receive organs. This article provides an up-to-date review of the milestones in the history of kidney transplantation and highlights strategies to resolve current problems faced by patients and the transplant community.

Upregulation of transglutaminase and ε (γ-glutamyl)-lysine in the Fisher-Lewis rat model of chronic allograft nephropathy.

BACKGROUND: Tissue transglutaminase (TG2), a cross-linking enzyme, modulates deposition of extracellular matrix protein in renal fibrosis. This study aimed to examine TG2 and its cross-link product ε(γ-glutamyl)-lysine in the Fisher-Lewis rat renal transplantation (RTx) model of chronic allograft nephropathy (CAN). MATERIALS AND METHODS: Left renal grafts from male Fisher and Lewis were transplanted into Lewis rats, generating allografts and isografts, respectively. Blood pressure, renal function, and proteinuria were monitored for up to 52 weeks. At termination, CAN was assessed in the renal tissue by light and electron microscopy, TG2 and ε(γ-glutamyl)-lysine by immunofluorescence, and the urinary ε(γ-glutamyl)-lysine by high performance liquid chromatography. RESULTS: Compared to the isograft, the allografts were hypertensive, proteinuric, and uraemic and developed CAN. Extracellular TG2 (glomerulus: 64.55 ± 17.61 versus 2.11 ± 0.17, P < 0.001; interstitium: 13.72 ± 1.62 versus 3.19 ± 0.44, P < 0.001), ε(γ-glutamyl)-lysine (glomerulus: 21.74 ± 2.71 versus 1.98 ± 0.37, P < 0.01; interstitium: 37.96 ± 17.06 versus 0.42 ± 0.11, P < 0.05), TG2 enzyme activity (1.09 ± 0.13 versus 0.41 ± 0.03 nmol/h/mg protein, P < 0.05), TG2 mRNA (20-fold rise), and urinary ε(γ-glutamyl)-lysine (534.2 ± 198.4 nmol/24 h versus 57.2 ± 4.1 nmol/24 h, P < 0.05) levels were significantly elevated in the allografts and showed a positive linear correlation with tubulointerstitial fibrosis. CONCLUSION: CAN was associated with upregulation of renal TG2 pathway, which has a potential for pharmacological intervention. The elevated urinary ε(γ-glutamyl)-lysine, measured for the first time in RTx, is a potential biomarker of CAN.

Experimental rat models of chronic allograft nephropathy: a review.

Chronic allograft nephropathy (CAN) is the leading cause of late allograft loss after renal transplantation (RT), which continues to remain an unresolved problem. A rat model of CAN was first described in 1969 by White et al. Although the rat model of RT can be technically challenging, it is attractive because the pathogenesis of CAN is similar to that following human RT and the pathological features of CAN develop within months as compared with years in human RT. The rat model of RT is considered as a useful investigational tool in the field of experimental transplantation research. We have reviewed the literature on studies of rat RT reporting the donor and recipient strain combinations that have investigated resultant survival and histological outcomes. Several different combinations of inbred and outbred rat combinations have been reported to investigate the multiple aspects of transplantation, including acute rejection, cellular and humoral rejection mechanisms and their treatments, CAN, and potential targets for its prevention.

Biological pathways and potential targets for prevention and therapy of chronic allograft nephropathy.

Renal transplantation (RT) is the best option for patients with end-stage renal disease, but the half-life is limited to a decade due to progressive deterioration of renal function and transplant failure from chronic allograft nephropathy (CAN), which is the leading cause of transplant loss. Extensive research has been done to understand the pathogenesis, the biological pathways of fibrogenesis, and potential therapeutic targets for the prevention and treatment of CAN. Despite the advancements in the immunosuppressive agents and patient care, CAN continues to remain an unresolved problem in renal transplantation. The aim of this paper is to undertake a comprehensive review of the literature on the pathogenesis, biological pathways of RT fibrogenesis, and potential therapeutic targets for the prevention and therapy of CAN.

Inhibition of collagen I accumulation reduces glomerulosclerosis by a Hic-5-dependent mechanism in experimental diabetic nephropathy.

Glomerulosclerosis of any cause is characterized by loss of functional glomerular cells and deposition of excessive amounts of interstitial collagens including collagen I. We have previously reported that mesangial cell attachment to collagen I leads to upregulation of Hic-5 in vitro, which mediates mesangial cell apoptosis. Furthermore, glomerular Hic-5 expression was increased during the progression of experimental glomerulosclerosis. We hypothesized that reducing collagen I accumulation in glomerulosclerosis would in turn lower Hic-5 expression, reducing mesangial cell apoptosis, and thus maintaining glomerular integrity. We examined archive renal tissue from rats undergoing experimental diabetic glomerulosclerosis, treated with the transglutaminase-2 inhibitor NTU281. Untreated animals exhibited increased glomerular collagen I accumulation, associated with increased glomerular Hic-5 expression, apoptosis, and mesangial myofibroblast transdifferentiation characterized by α-smooth muscle actin (α-SMA) expression. NTU281 treatment reduced glomerular collagen I accumulation, Hic-5 and α-SMA expression, and apoptosis. Proteinurea and serum creatinine levels were significantly reduced in animals with reduced Hic-5 expression. In vitro studies of Hic-5 knockdown or overexpression show that mesangial cell apoptosis and expression of both α-SMA and collagen I are Hic-5 dependent. Together, these data suggest that there exists, in vitro and in vivo, a positive feedback loop whereby increased levels of collagen I lead to increased mesangial Hic-5 expression favoring not only increased apoptosis, but also mesangial myofibroblast transdifferentiation and increased collagen I expression. Prevention of collagen I accumulation interrupts this Hic-5-dependent positive feedback loop, preserving glomerular architecture, cellular phenotype, and function.

Inflammatory lymphangiogenesis in a rat transplant model of interstitial fibrosis and tubular atrophy.

We have previously reported de novo lymphangiogenesis in human renal allograft nephrectomy specimens that exhibited interstitial fibrosis and tubular atrophy (IFTA). This study examined whether a similar pathology developed in an experimental model of renal transplantation in the rat. Renal transplants were carried out in rats comprising both isografts (Lewis kidneys → Lewis rats) and allografts (Fisher kidneys → Lewis rats). Animals were immunosuppressed in the immediate postoperative period and sacrificed at 12 months. Experimental readouts included lymphatic vessel number and location, inflammatory cell infiltration, interstitial fibrosis, renal function, blood pressure and proteinuria. Rat allografts demonstrated the characteristic features of IFTA with increased macrophage and T cell infiltration and scattered B cells aggregates. Rat allografts exhibited impaired renal function and proteinuria. Although there was no difference in the number of perivascular lymphatic vessels, there was a striking 18-fold increase in the number of interstitial lymphatic vessels in renal allografts. Furthermore, the lymphatic vessel number correlated with the extent of interstitial fibrosis. This rat allograft model of IFTA demonstrates a marked increase in the number of interstitial lymphatic vessels and mirrors previous work in failing human renal allografts.

Skin gadolinium following use of MR contrast agents in a rat model of nephrogenic systemic fibrosis.

PURPOSE: To detect the ultrastructural site of gadolinium retention in skin by using an animal model of nephrogenic systemic fibrosis and compare a linear, low-stability gadolinium chelate (formulated gadodiamide) with a macrocylic, high-stability gadolinium chelate (gadoterate meglumine). MATERIALS AND METHODS: Experimental procedures were performed according to rules and regulations laid down by the UK Home Office (Animal Procedures Act of 1986). Male Wistar rats were subjected to 5/6 subtotal nephrectomy (creatinine clearance, 25% normal). Gadolinium-based contrast agents, formulated gadodiamide (n = 9) and gadoterate meglumine (n = 11), were administered intravenously (2.5 mmol/kg for 5 days). After 28 days, skin was analyzed by means of morphometric and immunohistochemical techniques and electron microscopy. Data were compared with the Student t test. Skin gadolinium was located by means of energy-filtered transmission electron microscopy. RESULTS: Formulated gadodiamide produced a 40-fold greater increase in gadolinium in skin than did gadoterate meglumine. An electron-dense filamentous material, detected within extracellular matrix, displayed a "halo" appearance, associated with collagen fibrils and electron-dense intracellular fragments of collagen fibrils within activated fibroblasts. Both electron-dense features demonstrated the presence of gadolinium but were much less apparent following gadoterate meglumine administration, where the presence of gadolinium was not detected. Formulated gadodiamide increased dermal cell count, dermal thickness, and collagen bundle density with enhanced immunostain for CD34, fibroblast-specific protein 1,4-hydroxy-prolyl-hydroxylase, and factor XIIIa. Circular staining for α-smooth muscle actin indicated new blood vessel formation. Skin of rats receiving gadoterate meglumine remained unchanged. CONCLUSION: Gadolinium retention in skin following formulated gadodiamide administration was located to the collagen fibril, in both the extracellular matrix and within activated fibroblasts.

Atorvastatin improving renal ischemia reperfusion injury via direct inhibition of active caspase-3 in rats.

Caspase-3 is a key molecule involved in the inflammation and apoptosis of ischemia reperfusion (IR) injury. Statins are known to inhibit IR injury, but the mechanism of action remains uncertain. In the present study, the effect and underlying mechanism of ischemia alone, and reperfusion with or without atorvastatin (AT) as a timed intervention were examined, since clinically the kidney is only exposed to drug delivery during reperfusion. Male Sprague-Dawley rats were subjected to 45-min clamping of the left renal hilus followed by four hours reperfusion with a right nephrectomy. AT 10 mg/kg was intravenously administered after clamping the renal hilus, but prior to kidney reperfusion. Ischemia alone did cause tubulointerstitial damage (TID), protein carbonylation and caspase-3 activation with an increase in 12 kDa subunit, while reperfusion further enhanced TID, monocyte (ED-1+ cell) infiltration, apoptosis and necrosis together with caspase-3 activity and 17 kDa subunit, but reversed protein carbonylation. AT significantly reduced TID (26%), ED-1+ cell infiltration (74%), tubular apoptosis (47%) and necrosis (73%), and interstitial apoptosis (64%), as well as caspase-3 activity (26%), but did not change serum creatinine and cholesterol. Importantly, without affecting either caspase-3 active protein cleavage or S-nitrosylation, AT directly inhibited caspase-3 active enzyme in a dose-dependent manner in vitro. In conclusions, IR and AT exerted opposing effects on caspase-3 activity by differing mechanisms, with IR stimulating caspase-3 proteolytic cleavage and AT inhibiting active caspase-3 enzyme. This new inhibitory mechanism of AT may improve reperfusion tolerance in ischemic kidneys and benefit transplant recipients.

Pathophysiology of nephrogenic systemic fibrosis: A review of experimental data.

Since the association between nephrogenic systemic fibrosis (NSF) and gadolinium contrast agents (Gd-CAs) was suggested in 2006, several experimental studies have been published to elucidate the role of these agents in the pathogenesis of NSF. Low stability Gd-CAs have a stimulant effect on human skin and fibroblasts in culture and modulate the production of collagen by these cells. Low stability agents have also induced NSF-like skin changes in a rat model with normal renal function after multiple repeat administrations. The role of the 5/6 subtotal nephrectomy rat model in investigating NSF remains under evaluation.

Nephrogenic gadolinium biodistribution and skin cellularity following a single injection of Omniscan in the rat.

OBJECTIVES: The development of nephrogenic systemic fibrosis (NSF) following MRI contrast examination has been associated with gadolinium (Gd) toxicity. Animal models should show the key features of NSF in man where, the only immutable epidemiological feature is renal impairment. A rat model of chronic renal insufficiency has been employed to establish whether tissue gadolinium retention and increased skin cellularity following a gadolinium based contrast agent (GBCA) can be correlated with a reduction in renal function. The GBCA chosen for investigation was Omniscan, the least stable of the commercially available agents. MATERIALS AND METHODS: Wistar rats were subjected to 5/6 subtotal nephrectomy (SNx) under isoflurane anesthesia. The glomerular filtration rate (GFR) was assessed from serum creatinine and creatinine clearance. Two SNx rats groups were established, following either 75% or 80% resection of the kidney, which reduced the GFR down to 40% and down to 20%, respectively, of sham-operated controls. Three months after surgery, rats received a single intravenous injection of either saline or Omniscan (gadodiamide 2.5 mmol/kg). Four weeks later, the Gd content of serum, skin, liver, and bone was measured by inductively coupled plasma mass spectrometry and skin cellularity determined. RESULTS: In sham-operated rats, Gd was detected in skin < liver < bone. SNx rats with the GFR reduced down to 20% normal, had an increased tissue Gd concentration in bone (2.5-fold), skin (3-fold), and liver (10-fold) compared with sham-operated controls. The Gd concentration in all 3 tissues showed a positive linear correlation with serum creatinine (P < 0.01). No external skin lesions were observed. The skin cellularity of rats with the GFR reduced down to 20% of normal was increased following Omniscan, together with positive immunostain for CD34 and prolyl-4-hydroxylase. CONCLUSIONS: The SNx rat is a sensitive model for investigating the pathophysiology of NSF. A positive linear correlation was obtained between tissue Gd and serum creatinine, the major clinical marker of renal function. An increase in skin cellularity, a feature of human NSF, was demonstrated in rats with a level of renal impairment equivalent of stage 4 chronic kidney disease following just a single intravenous dose of Omniscan. This response was obtained in the absence of ulcerogenic skin lesions, at skin Gd concentrations as low as 50 nmol/g.

Do changes in transglutaminase activity alter latent transforming growth factor beta activation in experimental diabetic nephropathy?

BACKGROUND: Diabetic nephropathy is the leading cause of end-stage kidney failure worldwide. It is characterized by excessive extracellular matrix accumulation. Transforming growth factor beta 1 (TGF-ß1) is a fibrogenic cytokine playing a major role in the healing process and scarring by regulating extracellular matrix turnover, cell proliferation and epithelial mesanchymal transdifferentiation. Newly synthesized TGF-ß is released as a latent, biologically inactive complex. The cross-linking of the large latent TGF-ß to the extracellular matrix by transglutaminase 2 (TG2) is one of the key mechanisms of recruitment and activation of this cytokine. TG2 is an enzyme catalyzing an acyl transfer reaction leading to the formation of a stable ε(γ-glutamyl)-lysine cross-link between peptides. METHODS: To investigate if changes in TG activity can modulate TGF-ß1 activation, we used the mink lung cell bioassay to assess TGF-ß activity in the streptozotocin model of diabetic nephropathy treated with TG inhibitor NTU281 and in TG2 overexpressing opossum kidney (OK) proximal tubular epithelial cells. RESULTS: Application of the site-directed TG inhibitor NTU281 caused a 25% reduction in kidney levels of active TGF-ß1. Specific upregulation of TG2 in OK proximal tubular epithelial cells increased latent TGF-ß recruitment and activation by 20.7% and 19.7%, respectively, in co-cultures with latent TGF-ß binding protein producing fibroblasts. CONCLUSIONS: Regulation of TG2 directly influences the level of active TGF-ß1, and thus, TG inhibition may exert a renoprotective effect by targeting not only a direct extracellular matrix deposition but also TGF-ß1 activation and recruitment.

Pkd2 dosage influences cellular repair responses following ischemia-reperfusion injury.

Autosomal dominant polycystic kidney disease (ADPKD) results from mutations in either PKD1 or PKD2 and accounts for 10% of all patients on renal replacement therapy. The kidney disease phenotype is primarily characterized by cyst formation, but there are also prominent interstitial changes (inflammation, apoptosis, proliferation, and fibrosis). Using a model of unilateral ischemia-reperfusion injury, we tested the hypothesis that Pkd2 heterozygous kidneys are more sensitive to injury and that this could lead to interstitial inflammation and fibrosis. Baseline tubular proliferation in heterozygous kidneys was twofold higher than in wild-type kidneys. The magnitude and duration of tubular and interstitial proliferative responses was consistently greater in injured heterozygous compared with wild-type kidneys at all time points. Conversely, tubular p21 expression in heterozygotes was lower at baseline and following injury at all time points. Significantly more neutrophils and macrophages were detected in injured Pkd2 heterozygous kidneys at 2 days, correlating with increased expression of the cytokines interleukin (IL)-1beta and keratinocyte-derived chemokine and resulting in interstitial fibrosis at 28 days. We conclude that Pkd2 dosage influences both susceptibility and nature of the repair responses following injury. Polycystin-2 is therefore likely to play multiple roles in regulating tubular cell viability, repair, and remodeling in the mature kidney.

Transglutaminase inhibition ameliorates experimental diabetic nephropathy.

Diabetic nephropathy is characterized by excessive extracellular matrix accumulation resulting in renal scarring and end-stage renal disease. Previous studies have suggested that transglutaminase type 2, by formation of its protein crosslink product epsilon-(gamma-glutamyl)lysine, alters extracellular matrix homeostasis, causing basement membrane thickening and expansion of the mesangium and interstitium. To determine whether transglutaminase inhibition can slow the progression of chronic experimental diabetic nephropathy over an extended treatment period, the inhibitor NTU281 was given to uninephrectomized streptozotocin-induced diabetic rats for up to 8 months. Effective transglutaminase inhibition significantly reversed the increased serum creatinine and albuminuria in the diabetic rats. These improvements were accompanied by a fivefold decrease in glomerulosclerosis and a sixfold reduction in tubulointerstitial scarring. This was associated with reductions in collagen IV accumulation by 4 months, along with reductions in collagens I and III by 8 months. This inhibition also decreased the number of myofibroblasts, suggesting that tissue transglutaminase may play a role in myofibroblast transformation. Our study suggests that transglutaminase inhibition ameliorates the progression of experimental diabetic nephropathy and can be considered for clinical application.

Modulation of tissue transglutaminase in tubular epithelial cells alters extracellular matrix levels: a potential mechanism of tissue scarring.

The up-regulation and trafficking of tissue transglutaminase (TG2) by tubular epithelial cells (TEC) has been implicated in the development of kidney scarring. TG2 catalyses the crosslinking of proteins via the formation of highly stable epsilon(gamma-glutamyl) lysine bonds. We have proposed that TG2 may contribute to kidney scarring by accelerating extracellular matrix (ECM) deposition and by stabilising the ECM against proteolytic decay. To investigate this, we have studied ECM metabolism in Opossum kidney (OK) TEC induced to over-express TG2 by stable transfection and in tubular cells isolated from TG2 knockout mice. Increasing the expression of TG2 led to increased extracellular TG2 activity (p<0.05), elevated epsilon(gamma-glutamyl) lysine crosslinking in the ECM and higher levels of ECM collagen per cell by (3)H-proline labelling. Immunofluorescence demonstrated that this was attributable to increased collagen III and IV levels. Higher TG2 levels were associated with an accelerated collagen deposition rate and a reduced ECM breakdown by matrix metalloproteinases (MMPs). In contrast, a lack of TG2 was associated with reduced epsilon(gamma-glutamyl) lysine crosslinking in the ECM, causing reduced ECM collagen levels and lower ECM per cell. We report that TG2 contributes to ECM accumulation primarily by accelerating collagen deposition, but also by altering the susceptibility of the tubular ECM to decay. These findings support a role for TG2 in the expansion of the ECM associated with kidney scarring.

Lack of effect of IGF-I on the glomerular filtration rate in non-diabetic patients with advanced chronic kidney disease.

Recombinant human insulin-like growth factor I (rhIGF-I) acutely increases the glomerular filtration rate (GFR) in human volunteers and patients with advanced chronic kidney disease (CKD). However, on chronic administration, rhIGF-I induces tolerance to its renal effects attributed to a fall in serum IGF-binding protein 3 (IGFBP-3) enhancing its systemic clearance. Tolerance may be avoided by the use of an intermittent dosage regimen of rhIGF-I. A randomised, double-blind, placebo-controlled study was undertaken in non-diabetic patients with advanced CKD to establish whether intermittent subcutaneous injections of rhIGF-I (50 microg/kg, four days/week) could increase GFR over a 24 week period and thereby have the potential to delay the onset of renal replacement therapy. Twenty-seven patients were randomised into rhIGF-I/placebo groups using a 2:1 treatment ratio. GFR was determined by inulin clearance. RhIGF-I therapy produced a sustained increase serum total and free IGF-I elevating IGFBP-1 without decreasing IGFBP-3. Inulin clearance however, was not increased after either four weeks or over the 24 week observation period. Only 4/18 rhIGF-I treated patients compared to 6/9 placebo patients completed the study, the major reason being the requirement for dialysis. Compared with healthy volunteers, advanced CKD patients had elevated serum levels of IGFBP-1, IGFBP-2, tumour necrosis factor-alpha and asymmetric dimethylarginine, all factors proposed to mediate IGF-I resistance. In conclusion, although intermittent rhIGF-I therapy elevated serum total IGF-I and prevented any fall in serum IGFBP-3, it failed to increase GFR in non-diabetic patients with advanced CKD. The lack of efficacy was attributed to the presence of renal IGF-I resistance in CKD.