Plasminogen activator inhibitor-1 and diabetic nephropathy

SUMMARY:   Diabetic nephropathy is characterized by excessive accumulation of extracellular matrix (ECM) in the kidney. Decreased ECM degradation as well as increased ECM synthesis plays an important role in ECM remodeling that favours tissue fibrosis. Plasminogen activator (PA)/plasmin/PA inhibitor (PAI) system is involved in ECM degradation and PAI-1 plays a critical role in ECM remodeling in the kidney. Normal human kidneys do not express PAI-1 but PAI-1 is overexpressed in pathologic conditions associated with renal fibrosis including diabetic nephropathy. Reactive oxygen species mediate PAI-1 up-regulation in renal cells cultured under high glucose, hypoxia, and TGF-β1. Recent studies utilizing PAI-1 deficient mice suggest that PAI-1 induce ECM deposition in diabetic kidney through increased ECM synthesis by TGF-β1 up-regulation as well as through decreased ECM degradation by suppression of plasmin and MMP-2 activity.     

Experimental Glomerulosclerosis: Defektheilung of the Kidney

Abstract Research in the role of cytokines in experimental glomerulonephritis has increased our understanding of the mechanisms that may be involved in the development of progressive renal disease. Glomerulosclerosis, the final common pathway in a variety of underlying kidney diseases, is characterized by increased extracellular matrix formation and cell proliferation. Transforming growth factor-β (TGF-β) and monocyte chemoattractant protein-1 (MCP-1) have been identified in animal models as mediators in the processes that follow renal injury. There is evidence of similar events occurring in other fibrotic disorders, suggesting that there is a common generic pathway of fibrosis. This review summarizes our knowledge of TGF-β and MCP-1 in experimental kidney disease and compares these results with mechanisms described in other organs. We propose that glomerulosclerosis represents Defektheilung (healing by secondary intention) of the kidney after various injuries. The growing knowledge of the mechanisms involved will help advance future therapeutic interventions by directing the healing process toward primary healing.     

Expression of transforming growth factor-β type II receptor mRNA in embryonic and adult rat kidney

Summary: The transforming growth factor-β (TGF-β) family of growth factors regulates cell proliferation, differentiation, extracellular matrix synthesis and angiogenesis in many developing tissues. Transforming growth factor-β1 was recently shown to affect the branching of ureteric epithelium and nephron formation in cultured rat metanephroi. As the TGF-β type II receptor is specific for the TGF-β family, the present study used in situ hybridization to localize mRNA for this receptor in metanephroi from Sprague-Dawley rat embryos. Transforming growth factor-β type II receptor mRNA was located in ureteric duct epithelium, undifferentiated mesenchymal cells in the nephrogenic zone, vesicles, comma-shaped bodies and S-shaped bodies. In some S-shaped bodies, TGF-β type II receptor mRNA was not expressed in the lower limb, which subsequently forms the renal corpuscle. Expression was not observed in capillary loop stage glomeruli and maturing glomeruli, or in proximal tubules and interstitial cells. In adult rat kidney, TGF-β type II receptor mRNA was expressed in cortical collecting ducts and distal tubules but not in glomeruli or proximal tubules. These findings demonstrate that the prominent expression of TGF-β type II receptor mRNA decreases as glomeruli and tubules develop. Expression then remains undetectable in adult glomeruli and proximal tubules. the developmentally-regulated expression of this receptor suggests a key role in glomerular and nephron development.     

Effects of therapeutic agents on the inflammatory and fibrogenic factors in IgA nephropathy

SUMMARY:   It is desirable in the treatment of IgA nephropathy (IgAN) to prevent the downstream events after the immune response has involved the glomerulus. We and others observed that IgA itself could directly activate mesangial cells to produce monocyte chemotactic peptide-1 (MCP-1), interleukin-6 (IL-6) and transforming growth factor-β (TGF-β) and this was suppressed by the treatment with steroid or angiotensin receptor blocker (ARB). It was shown in mesangial cells that the increased expression of TGF-β and plasminogen activator inhibitor-1 induced by angiotensin II was suppressed by the treatment with ARB, calcium channel blocker (CCB), spironolactone or peroxisome proliferator-activated-receptor-γ (PPAR-γ) agonist. It was well known in the patients with IgAN that renal or intraglomerular TGF-β1 gene expression was increased. Interestingly, treatment with angiotensin-converting enzyme (ACE) inhibitors induced significantly lower renal TGF-β1 gene expression in patients with IgAN. It was reported in several studies that urinary levels of IL-6, IL-8, MCP-1 or TGF-β were increased in patients with IgAN. The increase was suppressed by the treatment with steroid, ARB or ACE inhibitor. More effective agents are necessary to ameliorate pathogenetic abnormalities and so to prevent the progression of IgAN.     

End-stage renal failure in New Zealand Maori: an analysis of circulating transforming growth factor-β1

SUMMARY: There is a high incidence of end-stage renal disease in New Zealand Maori. Reasons for this have not been established. Transforming growth factor-β, (TGF-β₁) is a profibrogenic cytokine, which stimulates the secretion of extracellular matrix components, and has been implicated in the pathogenesis of kidney failure. the aim of this study was to examine TGF-β₁ in the serum of haemodialysis patients at our institution, in order to determine whether there was an upregulation of TGF-β₁ in Maori. A TGF-Prspecific sandwich enzyme-linked immunosorbant assay was used to measure active TGF-β from the sera of 74 haemodialysis patients, and 19 healthy Maori without renal disease, diabetes or hypertension. In addition, clinical and laboratory markers were examined in the haemodialysis patients studied. There was no association between TGF-β₁ and ethnicity in the groups studied. Transforming growth factor-β₁ protein appeared to be inversely related to age. but was not associated with parameters of survival on dialysis such as serum albumin or measures of dialysis adequacy. Although there was a significantly higher incidence of type II diabetes mellitus in the Maori ( P < 0.001) in comparison to European patients, the glycaemic control was comparable between the groups, as were all other laboratory and clinical parameters studied. This is the first study to examine the fibrogenic growth factor TGF-β₁ in New Zealand Maori. Thus, an endogenous increase in TGF-β₁ in Maori does not appear to be implicated in the increased incidence of end-stage renal disease in this population.     

Stimulation of PAI-1 in rabbit anti-GBM glomerulonephritis

SUMMARY: It has previously been shown in human disease and animal models of glomerulonephritis (GN) that fibrin deposition is associated with a net reduction of glomerular fibrinolytic activity as a result of reduced expression of plasminogen activators and increased expression of plasminogen activator inhibitor type 1 (PAI-1). Conditioned media (CM) prepared from cultured glomeruli of normal rabbits and rabbits 24 (Day 1) and 96 (Day 4) h after induction of anti-GBM GN were compared for their effects on the synthesis of fibrinolytic molecules in human endothelial cells (EC). Only CM from Day 4 GN rabbits showed PAI-1 protein stimulatory activity of up to 148% ( P <0.05; n = 3) above that of untreated EC. This was also seen at the mRNA level. Glomerulonephritis Day 4 CM showed significantly higher amounts of tumour necrosis factor (TNF) and thrombin and transforming growth factor-β (TGF-β) bioactivity in comparison to glomerular CM from normal rabbits. After high performance liquid chromatography (HPLC) of Day 4 GN CM, PAI-1 stimulatory activity was found to correlate with the presence of interleukin 1 (IL-1), TNF and TGF-β. These results suggest a correlation between severity of anti-GBM GN in a rabbit model, increased PAI-1 synthesis and increased expression of TNF and TGF-β. This may potentiate glomerular fibrin and extracellular matrix deposition in anti-GBM GN, leading to glomerular crescent formation and eventual renal failure.     

Stimulation of PAI-1 in rabbit anti-GBM glomerulonephritis

It has previously been shown in human disease and animal models of glomerulonephritis (GN) that fibrin deposition is associated with a net reduction of glomerular fibrinolytic activity as a result of reduced expression of plasminogen activators and increased expression of plasminogen activator inhibitor type 1 (PAI-1). Conditioned media (CM) prepared from cultured glomeruli of normal rabbits and rabbits 24 (Day 1) and 96 (Day 4) h after induction of anti-GBM GN were compared for their effects on the synthesis of fibrinolytic molecules in human endothelial cells (EC). Only CM from Day 4 GN rabbits showed PAI-1 protein stimulatory activity of up to 148% ( P <0.05; n =3) above that of untreated EC. This was also seen at the mRNA level. Glomerulonephritis Day 4 CM showed significantly higher amounts of tumour necrosis factor (TNF) and thrombin and transforming growth factor-β (TGF-β) bioactivity in comparison to glomerular CM from normal rabbits. After high performance liquid chromatography (HPLC) of Day 4 GN CM, PAI-1 stimulatory activity was found to correlate with the presence of interleukin 1 (IL-1), TNF and TGF-β. These results suggest a correlation between severity of anti-GBM GN in a rabbit model, increased PAI-1 synthesis and increased expression of TNF and TGF-β. This may potentiate glomerular fibrin and extracellular matrix deposition in anti-GBM GN, leading to glomerular crescent formation and eventual renal failure.     

Radical approach to diabetic nephropathy

There is increasing evidence that reactive oxygen species (ROS) play a major role in the development of diabetic complications. Oxidative stress is increased in diabetes and in chronic kidney disease (CKD). High glucose upregulates transforming growth factor-beta1 (TGF-beta1) and angiotensin II (Ang II) in renal cells and high glucose, TGF-beta1, and Ang II all generate and signal through ROS. ROS mediate high glucose-induced activation of protein kinase C and nuclear factor-kappaB in renal cells. Intensive glycemic control and inhibition of Ang II delay the onset and progression of diabetic nephropathy, in part, through antioxidant activity. Conventional and catalytic antioxidants were shown to prevent or delay the onset of diabetic nephropathy. Transketolase activators and poly (ADP-ribose) polymerase inhibitors were shown to block major biochemical pathways of hyperglycemic damage. Combination of strategies to prevent overproduction of ROS, to increase the removal of preformed ROS, and to block ROS-induced activation of biochemical pathways leading to cellular damage may prove to the effective in preventing the development and progression of CKD in diabetes.     

Angiotensin IV stimulates plasminogen activator inhibitor-1 expression in proximal tubular epithelial cells.

BACKGROUND: Angiotensin II (Ang II) has been shown to be implicated in the development of renal fibrosis in several forms of chronic glomerulonephritides, but the precise mechanisms of its effects remain unclear. It has recently been reported that Ang II stimulates the expression of plasminogen activator inhibitor-1 (PAI-1) in several cell lines. PAI-1 is a major physiological inhibitor of the plasminogen activator/plasmin system, a key regulator of fibrinolysis and extracellular matrix (ECM) turnover. PAI-1 induction by Ang II in endothelial cells seems to be mediated by Ang IV via a receptor that is different from Ang II type 1 and 2 receptors (AT1 and AT2). METHODS: In this study, we sought to evaluate the effects of Ang IV on PAI-1 gene and protein expression in a well-characterized and immortalized human proximal tubular cell line (HK2) by Northern blot and enzyme-linked immunosorbent assay. RESULTS: Ang IV stimulated PAI-1 mRNA expression, whereas it did not induce a significant increase in tritiated thymidine uptake after 24 hours of incubation. This effect was dose and time dependent. Ang IV (10 nM) induced a 7.8 +/- 3.3-fold increase in PAI-1 mRNA expression. The PAI-1 antigen level was significantly higher in conditioned media and the ECM of cells treated with Ang II and Ang IV than in control cells (both P < 0.02). Although Ang II induced a 4.2 +/- 2. 1-fold increase in PAI-1 mRNA expression, its effect underwent a dose-dependent reduction when amastatin, a potent inhibitor of the endopeptidases that catalyzes the conversion of Ang II to Ang IV, was added. In contrast, amastatin was not able to prevent the expression of PAI-1 mRNA induced by Ang IV. Finally, pretreatment of HK2 cells with losartan and N-Nicotinoyl-Tyr-N3-(Nalpha-CBZ-Arg)-Lys-His-Pro-Ile, the specific antagonists of AT1 and AT2 receptors, failed to modify PAI-1 mRNA expression as induced by Ang II. CONCLUSIONS: Our results demonstrate that Ang II stimulates PAI-1 mRNA expression and the production of its protein in human proximal tubular cells. This is mainly-if not exclusively-due to Ang IV, which acts on a receptor that is different than AT1 or AT2. Therefore, it can be hypothesized that the induction of PAI-1 by Ang IV may be implicated in the pathogenesis of renal interstitial fibrosis in several forms of chronic glomerulonephritides.     

Anti-thrombin Therapy During Warm Ischemia and Cold Preservation Prevents Chronic Kidney Graft Fibrosis in a DCD Model

Ischemia reperfusion injury (IRI) is pivotal for renal fibrosis development via peritubular capillaries injury. Coagulation represents a key mechanism involved in this process. Melagatran® (M), a thrombin inhibitor, was evaluated in an autotransplanted kidney model, using Large White pigs. To mimic deceased after cardiac death donor conditions, kidneys underwent warm ischemia (WI) for 60 min before cold preservation for 24 h in University of Wisconsin solution. Treatment with M before WI and/or in the preservation solution drastically improved survival at 3 months, reduced renal dysfunction related to a critical reduction in interstitial fibrosis, measured by Sirius Red staining. Tissue analysis revealed reduced expression of transforming growth factor-β (TGF-β) and activation level of its effectors phospho-Smad3, Smad4 and connective tissue growth factor (CTGF) after M treatment. Fibrinolysis activation was also observed, evidenced by downregulation of PAI-1 protein and gene expression. In addition, M reduced S100A4 expression and vimentin staining, which are markers for epithelial mesenchymal transition, a major pathway to chronic kidney fibrosis. Finally, expression of oxidative stress markers Nox2 and iNOS was reduced. We conclude that inhibition of thrombin is an effective therapy against IRI that reduces chronic graft fibrosis, with a significantly positive effect on survival.     

Angiotensin II regulates the synthesis of proinflammatory cytokines and chemokines in the kidney

BACKGROUND: Emerging evidence suggests that angiotensin II (Ang II) is not only a vasoactive peptide, but also a true cytokine that regulates cell growth, inflammation and fibrosis. Many studies have demonstrated that this peptide plays an active role in the progression of renal injury. Some of Ang II-induced effects are mediated by the production of a large array of growth factors. The aim of this study was to investigate whether Ang II could regulate the expression of cytokines and chemokines in the kidney and its correlation with the Ang II-induced renal damage. METHODS: The model of Ang II-induced renal damage was done by systemic Ang II infusion into normal rats (50 ng/kg/min; subcutaneous osmotic minipumps). In addition, the implication of Ang II was investigated in a model of immune complex nephritis in rats treated with the angiotensin converting enzyme (ACE) inhibitor quinapril. The mRNA expression was analyzed by RT-PCR and/or Northern blot, and protein levels by Western blot and/or immunohistochemistry. RESULTS: Rats infused with Ang II for 3 days caused elevated renal expression of tumor necrosis factor-alpha (TNF-alpha; gene and protein levels). TNF-alpha positive cells were observed in glomeruli (mainly in endothelial cells), tubules and vessels. In rats with immune complex nephritis, the renal overexpression of TNF-alpha was diminished by the ACE inhibitor quinapril. Systemic infusion of Ang II also increased renal synthesis of cytokines (interleukin-6, IL-6) and chemokines (monocyte chemoattractant protein-1; MCP-1) that were associated with elevated tissue levels of activated nuclear factor-kappaB (NF-kappaB) and the presence of inflammatory cell infiltration. CONCLUSIONS: Ang II in vivo increases TNF-alpha production in the kidney. Ang II also up-regulates other proinflammatory mediators, including IL-6, MCP-1 and NF-kappaB, coincidentally associated to the presence of glomerular and interstitial inflammatory cells in the kidney. All these data further strengthen the idea that Ang II plays an active role in the inflammatory response in renal diseases.     

Urinary transforming growth factor-β1 is an indicator of histological chronicity in IgA nephropathy

SUMMARY: This study examined urinary excretion of transforming growth factor-β1 (TGF-β1) in adult IgA nephropathy and compared this with clinical and histological parameters. TGF-β1 was measured by enzyme-linked immunosorbent assay in 24-h urine specimens from 25 patients with IgA nephropathy (17 men, eight women). Urine from eight age-matched control subjects served as the control. Serum TGF-β1 was also measured in 16 out of the 25 patients and six age-matched control subjects. TGF-β1 was detected in the urine in 72% of IgA nephropathy patients(18/25), but was not detected in any of the control subjects. Patients with decreased renal function (creatinine clearance (Ccr) < 80 mL/min) had higher urine levels of TGF-β1 than those with normal renal function (Ccr ≥ 80 mL/min) (141.8 ± 60.0 ng/day vs 39.7 ± 33.2 ng/day, P < 0.01). The level of TGF-β1 gave a negative correlation with Ccr ( r = −0.62, P = 0.001), but not with proteinuria ( r = 0.11, P = 0.58). Twenty-two of the patients were evaluated histologically. The urinary TGF-β1 levels correlated with both global sclerosis and interstitial volume ( r = 0.52, P < 0.05, and r = 0.51, P < 0.05, respectively). However, there was no correlation between TGF-β1 levels and glomerular intracapillary inflammatory cell score, mesangial proliferation score or the matrix score. No correlation was found between TGF-β1 levels and the number of glomerular or interstitial CD68⁺ cells. No significant differences in serum TGF-β1 levels were observed between patients with normal Ccr and those with decreased Ccr. Also, its levels were found to be independent of the urine levels. In conclusion, 24-h urinary TGF-β1 excretion was found to correlate with Ccr, global sclerosis and interstitial volume, demonstrating that urinary TGF-β1 is an indicator of chronicity in adult IgA nephropathy.     

Amino acids injure mesangial cells by advanced glycation end products, oxidative stress, and protein kinase C

BACKGROUND: In diabetes, high intake of dietary protein exacerbates responses associated with kidney damage. Increased levels of amino acids could injure cells by providing free amino groups for glycation reactions leading to advanced glycation end products (AGEs). METHODS: Rat mesangial cells were cultured with increased amino acids designed to resemble protein feeding, high glucose (30.5 mmol/L), and, the combination, amino acids/high glucose. AGEs, reactive oxygen species (ROS), protein kinase C (PKC) activity and production, and mitogen-activated protein (MAP) kinase-extracellular signal regulated kinase (ERK) 1,2 activity were measured. Inhibitors were used to determine roles of these processes in fibrosis and/or AGE formation. RESULTS: AGE immunostaining increased when cells were cultured in amino acids and was comparable to that observed with high glucose. In amino acids/high glucose, AGE immunostaining appeared even greater. Amino acids, high glucose, and amino acids/high glucose induced ROS production. Aminoguanidine and vitamin E prevented AGE accumulation and induction of protein and mRNA for fibrosis markers [transforming growth factor-beta1 (TGF-beta1), fibronectin, and collagen IV]. PKC and ERK 1,2 activity increased with amino acids, high glucose, and amino acids/high glucose. PKC-beta inhibition prevented ERK 1,2 activation and fibrosis induction. ERK 1,2 inhibition also blocked the fibrosis response. CONCLUSION: A profibrotic injury response occurred in mesangial cells exposed to amino acids, with or without high glucose, by formation of AGE, oxidative stress, and activation of the PKC-beta and MAP kinase-ERK 1,2 signal pathway. These observations provide new insight into cellular mechanisms of kidney damage produced by excess dietary protein, particularly in diabetes.     

Differential expression of platelet-derived growth factor and transforming growth factor-β in relation to progression of IgA nephropathy

SUMMARY: The role of platelet-derived growth factor (PDGF) and transforming growth factor-β1 (TGF-β1) in relation to mesangial matrix expansion and progressive glomerulosclerosis in IgA nephropathy (IgAN) is not clearly defined. Expression of PDGF B, TGF-β1, and extracellular matrix proteins in glomeruli was assessed by immunohistochemistry in 42 biopsies with IgAN and six renal biopsies with no detectable abnormalities. the mRNA expression of PDGF B, TGF-β1, α1(IV) collagen, laminin B1 and fibronectin genes was further evaluated by in situ hybridization in 25 biopsies with IgAN and six controls. In IgAN, the intensity of immunostaining for PDGF B, type IV collagen, laminin and fibronectin, but not for TGF-β1, was increased in the mesangium compared with controls. the immunoreactivity of PDGF B was closely correlated with that of type IV collagen and laminin. the number of PDGF B mRNA-, α1(IV) collagen mRNA-, laminin B1 mRNA-, and TGF-β1 mRNA-expressing cells/glomerular section, but not the number of fibronectin mRNA-expressing cells, was increased in IgAN compared with controls. the number of PDGF B mRNA-expressing cells correlated significantly with the percentage of glomerulosclerosis. In the cellular lesions of focal segmental glomerulosclerosis (FSGS), expression of TGF-β1 protein and mRNA was markedly increased in visceral glomerular epithelial cells (GEC). These results suggest that PDGF B mainly overproduced by mesangial cells may cause mesangial matrix expansion, whereas TGF-β1 produced by GEC may be related to the formation of FSGS in IgAN. Thus, PDGF B and TGF-β1 may play differential roles in the pathogenesis of renal fibrosis and the progression of IgAN.     

Transforming growth factor-β (TGF-β) activity in urine of patients with glomerulonephritis is related to their renal functional and structural changes

SUMMARY: Transforming growth factor-β (TGF-β) has been considered the principal cytokine involved in the pathogenesis of renal fibrosis. In the present study, we evaluated TGF-β activity in occasional samples from 22 normal individuals and 29 patients (11 with focal glomerulosclerosis, 11 with membranous nephropathy, five with Berger disease, one with type I membranoproliferative glomerulonephritis and one with postinfectious glomerulonephritis) using a CCL-64 mink lung cell growth inhibition assay.
A significantly increased urinary TGF-β activity (reported in relation to urine creatinine, Ucreat, and median) was observed in patients with glomerulonephritis compared with normal individuals ( P <0.01). the patients with Berger disease [median (Md) = 9.96/10 μg Ucreat.], membranous glomerulonephritis (Md = 7.23/10 μg Ucreat.) and focal glomerulosclerosis (Md = 16.6/10 μg Ucreat.) showed higher urinary TGF-β than normal individuals (Md = 1.09/10 μg Ucreat.) ( P <0.01). We found a positive correlation between the TGF-β activity in the urine of these patients and the incidence of segmental glomerulosclerosis ( r = 0.45, P <0.05) and their plasma creatinine levels ( r = 0.87, P <0.01). A negative correlation was observed between the TGF-β activity in the urine of these patients and their creatinine clearance ( r =−0.75, P <0.01).
Our data suggest that measurement of urinary TGF-β activity could be a useful non-invasive procedure for the evaluation of renal TGF-β production, permitting the assessment of prognosis and the evaluation of therapeutic efficacy in patients with renal disease.