Delayed branching of endothelial capillary-like cords in glycated collagen I is mediated by early induction of PAI-1.

Development of micro- and macrovascular disease in diabetes mellitus (DM) warrants a thorough investigation into the repertoire of endothelial cell (EC) responses to diabetic environmental cues. Using human umbilical vein EC (HUVEC) cultured in three-dimensional (3-D) native collagen I (NC) or glycated collagen I (GC), we observed capillary cord formation that showed a significant reduction in branching when cells were cultured in GC. To gain insight into the molecular determinants of this phenomenon, HUVEC subjected to GC vs. NC were studied using a PCR-selected subtraction approach. Nine different genes were identified as up- or downregulated in response to GC; among those, plasminogen activator inhibitor-1 (PAI-1) mRNA was found to be upregulated by GC. Western blot analysis of HUVEC cultured on GC showed an increase in PAI-1 expression. The addition of a neutralizing anti-PAI-1 antibody to HUVEC cultured in GC restored the branching pattern of formed capillary cords. In contrast, supplementation of culture medium with the constitutively active PAI-1 reproduced defective branching patterns in HUVEC cultured in NC. Ex vivo capillary sprouting in GC was unaffected in PAI-1 knockout mice but was inhibited in wild-type mice. This difference persisted in diabetic mice. In conclusion, the PCR-selected subtraction technique identified PAI-1 as one of the genes characterizing an early response of HUVEC to the diabetic-like interstitial environment modeled by GC and responsible for the defective branching of endothelial cells. We propose that an upregulation of PAI-1 is causatively linked to the defective formation of capillary networks during wound healing and eventual vascular dropout characteristic of diabetic nephropathy.

Is there a common mechanism for the progression of different types of renal diseases other than proteinuria? Towards the unifying theme of chronic hypoxia.

The question of why chronic renal diseases progress is a topic only recently investigated. Putative causes such as proteinuria do not account for all aspects of progressive renal disease. An alternative mechanism, chronic hypoxia, is proposed that might better explain certain elements of progressive renal disease, but elements of the hypothesis remain subject to further study.

Hypoxia-induced changes in extracellular matrix metabolism in renal cells.

The mechanisms underlying the progressive fibrosis that characterises end-stage renal disease in vivo remain to be established but hypoxia, as a result of microvascular injury and loss, has been suggested to play an important role. In support of this hypothesis, in vitro studies show that hypoxia (1% O(2)) induces a fibrogenic phenotype in human renal tubular endothelia, interstitial fibroblasts and microvascular endothelial cells, simultaneously increasing extracellular matrix (ECM) production and decreasing turnover via effectors on matrix-degrading enzymes and their inhibitors. The effects of hypoxia on ECM metabolism are independent of hypoxia-induced growth factors and are mediated by a haem-protein sensor and activation of both protein kinase C- and tyrosine kinase-mediated signal transduction pathways. De novo gene transcription is regulated by both hypoxia-inducible factor-1-dependent and -independent mechanisms. Further understanding of the molecular mechanisms by which decreased oxygen alters expression of genes involved in ECM metabolism in renal cells may provide new insights into the pathogenesis of fibrosis and identify novel avenues for intervention.

The concept of glomerular self-defense.

The balance between local offense factors and defense machinery determines the fate of tissue injury: progression or resolution. In glomerular research, the most interest has been on the offensive side, for example, the roles of leukocytes, platelets, complement, cytokines, eicosanoids, and oxygen radical intermediates. There has been little focus on the defensive side, which is responsible for the attenuation and resolution of disease. The aim of this review is to address possible mechanisms of local defense that may be exerted during glomerular injury. Cytokine inhibitors, proteinase inhibitors, complement regulatory proteins, anti-inflammatory cytokines, anti-inflammatory eicosanoids, antithrombotic molecules, and extracellular matrix proteins can participate in the extracellular and/or cell surface defense. Heat shock proteins, antioxidants, protein phosphatases, and cyclin kinase inhibitors may contribute to the intracellular defense. This article outlines how the glomerulus, when faced with injurious cells or exposed to pathogenic mediators, defends itself via the intrinsic machinery that is brought into play in resident glomerular cells.

British contributions to renal physiology: Of dynasties and diuresis.

British contributions to renal physiology trace back to the seminal contribution of William Bowman, who defined the structure of mammalian nephron and its blood supply and pointed out its likely mode of function. A series of contributions followed, which stemmed from a distinguished lineage of physiologists based at University College London and which had, as its high point, the establishment by Ernest H. Starling, in 1899, of a method for measuring oncotic pressure and the subsequent demonstration that glomerular filtration requires a hydrostatic pressure in excess of oncotic pressure in the glomerular capillaries. Arthur Cushny published a monograph on renal function in 1917 which had wide influence. The homeostasis of water and sodium chloride were the subjects of important contributions by Verney and De Wardener, respectively. Little contribution was made by British renal physiologists to the understanding of single nephron function with the emphasis being largely on integrative physiology.

Inference of the existence of high blood pressure as a cause of renal disease in the mid-19th century: observations on vascular structures in the kidney.

Histological examination of the kidney was well under way by the mid-19th century. Pathological changes noted to be present in Bright's disease gave rise to considerable debate in the literature of the time. Toynbee was perhaps the first to note medial hypertrophy and intimal narrowing of blood vessels in the kidney, while Johnson, around the same time, thought that kidney disease was the cause of compressed vessels. Although he later proposed a causal relationship between contraction of vessels and hypertrophy, Johnson never went beyond the insights articulated by Bright himself and failed to make the link between hypertrophy of vessels and persistently raised blood pressure. Traube considered the possibility that cardiac and renal disease could be the consequences of the same unknown disease, but rejected hypertrophy per se as a causal factor. Gull and Sutton disagreed strongly with Johnson and proposed the presence of a general disease which leads to both cardiac hypertrophy and renal disease. But it was Ewald, writing in Germany, who was able to ascribe both cardiac and vascular hypertrophy to increasing tension in the arterial system and he was the first to articulate the effect of hypertension on the kidney.

Progressive renal disease: fibroblasts, extracellular matrix, and integrins.

Progressive renal disease is characterized by expansion of the tubulo-interstitium and accumulation of extracellular matrix within this tissue compartment. Interstitial fibroblasts are the primary producers of the interstitial matrix, and in the evolution of tubulo-interstitial fibrosis these cells undergo changes, namely increased proliferation, differentiation to myofibroblasts, and altered extracellular matrix metabolism, all of which, in other cell types, have been shown to be regulated by the major family of extracellular matrix receptors, the integrins. In the normal kidney, interstitial fibroblasts express alpha1, alpha4, alpha5, and beta1 integrins, and fibrosis is associated with increased expression of alpha1, alpha2, alpha5, alphav, and beta1 integrins. In particular, alpha5, beta1, and alphav are suggested to be linked with the fibrotic process. In vitro, renal fibroblasts express a similar range of integrins, and ligation of selected receptors is associated with specific functions. Ligation of alpha6 stimulates proliferation, while alpha5 promotes expression of myofibroblastic phenotype, and beta1 integrin has been implicated in cell contraction. Recent studies suggest that renal fibroblasts also express the non-integrin matrix receptors, discoidin domain receptors, and that changes in activation of these receptors may be associated with fibrogenic events. Thus the current, albeit limited, data suggest an important role for receptors for extracellular matrix molecules in the pathogenesis of progressive renal fibrosis.

Gene transfer into the adult kidney for unravelling disease processes.

Insights into the pathogenesis of human disease must be based on an understanding of the molecular mechanisms that regulate the structure and function of individual organs. For this purpose, gene transfer technologies provide powerful and attractive tools. In principle, two different approaches are feasible to identify pathophysiological roles of certain genes: 'gain-of-function', and 'loss-of-function'. The former examines consequences of overexpression of an exogenous gene, and the latter investigates the outcomes of inhibition of a particular molecule via antisense, decoy, ribozyme and dominant-negative strategies. Gene transfer to specific renal structures allows evaluation of in vivo effects of certain molecules on the structure and function of each nephron segment. It would also be useful for therapeutic intervention in renal diseases through introduction of therapeutically relevant genes into affected sites. This article summarizes current experience with renal gene transfer and addresses its potential impacts on the understanding of renal function in vivo.

Evidence for TGF-beta-mediated 'defense' of the glomerulus: a blackguard molecule rehabilitated?

Transforming growth factor beta (TGF-beta) has been regarded as a 'blackguard molecule' that induces glomerular diseases. During the process of glomerulonephritis, upregulated TGF-beta stimulates the production of extracellular matrix and inhibits its degradation, leading to excessive matrix deposition. On the other hand, TGF-beta has the potential to be anti-inflammatory via inhibition of mitogenesis and production of inflammatory mediators by glomerular cells. This molecule strongly inactivates infiltrating cells, especially macrophages, which play a pivotal role in the generation of glomerular injury. The aim of this article is to summarize the potentially beneficial action of TGF-beta in the glomerulus and to address its 'bright side' in glomerular inflammation.

In vivo transfer of engineered macrophages into the glomerulus: endogenous TGF-beta-mediated defense against macrophage-induced glomerular cell activation.

Communication between resident glomerular cells and infiltrating macrophages plays a crucial role in the pathogenesis of glomerular disease. Using matrix metalloproteinase-9 (MMP-9) as an indicator molecule, we examined the interaction between mesangial cells and macrophages. Mesangial cells cocultured with activated macrophages or exposed to macrophage-conditioned media produced abundant MMP-9. We identified the stimulator secreted by macrophages as IL-1 because mesangial cells overexpressing IL-1 receptor antagonist protein showed a blunted expression of MMP-9 in response to the macrophage-conditioned medium. In contrast, culture supernatants of mesangial cells inhibited MMP-9 production by macrophages in a dose-dependent fashion. This inhibitor was identified to be TGF-beta1, since neutralization of TGF-beta1 abrogated the inhibitory effect of the mesangial cell-conditioned medium. To investigate whether activated macrophages induce glomerular MMP-9 expression, and if so, how endogenous TGF-beta1 modulates the induction, stimulated reporter macrophages were transferred into normal rat glomeruli or glomeruli in the regeneration phase of acute anti-Thy-1 glomerulonephritis. In the normal glomeruli, MMP-9 expression was up-regulated in resident cells after the transfer of activated macrophages. This induction was substantially repressed in the regenerating glomeruli that produced active TGF-beta1. These results point to potential mechanisms involved in glomerular control of MMP-9. Based upon the in vitro evidence, TGF-beta1 was identified as an endogenous "defender" that attenuates certain actions of infiltrating macrophages in the glomerulus.

Hypoxia stimulates proximal tubular cell matrix production via a TGF-beta1-independent mechanism.

Tubulointerstitial fibrosis is characterized by tubular basement membrane thickening and accumulation of interstitial extracellular matrix (ECM). Since chronic low-grade hypoxia has been implicated in the pathogenesis of fibrosis and proximal tubular epithelial cells (PTE) are sensitive to oxygen deprivation, we hypothesized that hypoxia may stimulate ECM accumulation. In human PTE, hypoxia (1% O2, 24 hr) increased total collagen production (15%), decreased MMP-2 activity (55% +/- 13%; control = 100%) and increased tissue inhibitor of metalloproteinase-1 (TIMP-1) protein. Collagen IV mRNA levels decreased while collagen I mRNA increased, suggesting induction of interstitial collagen. Hypoxia-induced changes persisted on re-oxygenation with increased expression of TIMP mRNAs. A potential mediator for these effects is transforming growth factor-beta1 (TGF-beta1), a major pro-fibrogenic factor produced by PTE. Although hypoxia stimulated TGF-beta production (2- to 3-fold), neutralizing anti-TGF-beta1 antibody did not abolish the hypoxia-induced changes in gelatinase activity, TIMP-1, collagen IV or collagen I mRNA expression, implying that TGF-beta1 is not the mediator. Furthermore, exogenous TGF-beta1 (0 to 10 ng/ml) did not mimic hypoxia, as it stimulated MMP-2 activity and increased the expression of collagen IV, collagen I and TIMP-1 mRNA. The data suggest that hypoxia may be an important pro-fibrogenic stimulus independent of TGF-beta1.

Unravelling dropsy: from Marcello Malpighi's discovery of the capillaries (1661) to Stephen Hales' production of oedema in an experimental model (1733).

A modern understanding of oedema formation traditionally begins with Starling's description in 1898 of hydrostatic and oncotic forces acting on the capillary membrane. Clearly, hypotheses of oedema formation predating the knowledge of the existence of capillaries must have been incomplete. Marcello Malpighi first described capillaries in 1661, but although he displayed a good grasp of the principles of the Harveian circulation and believed that oedema fluid (the clinical entity dropsy) was derived from the blood rather than the tissues, we have found no evidence that he realised the central role played by his discovery. However, only 60 years later, Stephen Hales' Haemastaticks reveals the creation of an experimental model for dropsy which led him towards an understanding of oedema formation not far behind Starling.

Genetic manipulation of the kidney.

Successful gene transfer into specific renal structures allows for evaluation of in vivo effects of certain molecules on the structure and function of the kidney. It would also be useful for therapeutic intervention in renal diseases by introducing "beneficial" genes into the affected sites. Towards achieving these goals, several gene transfer approaches have been developed using retrovirus, adenovirus and liposome. By introducing these gene transfer vectors via particular access routes, it is feasible to selectively manipulate the function of certain renal structures. Through the renal circulation, exogenous genes can be targeted to the vasculature and glomerulus, and possibly to the proximal tubules. Using a retrograde approach via the urinary tract, access to the collecting ducts can be gained. Implantation of genetically modified cells under the capsule of the kidney allows for diffusion of transgene products into the interstitium. Transplantation of embryonic metanephric tissues also provides a biological window for genetic manipulation. Furthermore, utilisation of fertilised eggs or embryonic stem cells would enable the creation of "transgenic kidneys" or "gene knockout kidneys". This article summarises the current experience with gene transfer to the kidney and addresses the potential strategies in vivo.

Mesangial cell-derived transforming growth factor-beta 1 reduces macrophage adhesiveness with consequent deactivation.

Adhesion of macrophages is a crucial event that determines the number and function of macrophages at inflammatory sites. The aim of this study was to elucidate the role of mesangial cells in the regulation of macrophage adhesiveness. J774.2 macrophages were suspended in serial dilutions of mesangial cell conditioned medium (MC medium) and seeded on plastic tissue culture plates. MC medium did not affect the initial adhesion of macrophages but induced subsequent detachment in a concentration-dependent manner. A similar effect was observed when macrophages were plated on plastic coated with laminin, collagen type IV or Matrigel. The reduced adhesiveness was reversible, and cell viability was unaffected by MC medium, indicating that the effect is not due to cytotoxicity. Conditioned media from fibroblastic, epithelial and endothelial cell lines did not induce macrophage detachment. To identify the active component in MC medium, we examined the involvement of transforming growth factor-beta 1 (TGF-beta 1) in the process. Mesangial cells constitutively expressed TGF-beta 1 mRNA, and MC medium contained the active form of TGF-beta 1. Exogenously added TGF-beta 1 induced macrophage detachment in a dose-dependent manner, and an anti-TGF-beta 1 neutralizing antibody partially abolished the activity of MC medium, indicating the involvement of TGF-beta 1 as an active component. Compared to adherent cells, detached macrophages showed reduced mitogenic activity and blunted induction of IL-1 beta and IL-6 in response to lipopolysaccharide. These data demonstrate that TGF-beta 1 is a mesangial cell-derived factor that impairs adhesiveness of macrophages and confers blunted responses to a specific stimulus. These findings suggest one potential mechanism for macrophage clearance from inflamed glomeruli.

Transforming growth factor-beta 1 is the predominant paracrine inhibitor of macrophage cytokine synthesis produced by glomerular mesangial cells .

Cross-communication between glomerular cells and infiltrating mononuclear cells plays an important role in the generation of or recovery from glomerular diseases. We found that cultured mesangial cells secrete a factor that inhibits production of proinflammatory cytokines by activated macrophages. Treatment of J774.2 macrophages with conditioned media from rat mesangial cells blunted the transcriptional induction of IL-1 beta, IL-6, and TNF-alpha by LPS. None of the media conditioned by other fibroblastic, epithelial, or endothelial cell lines exhibited the inhibitory effect. Media conditioned by normal rat glomeruli contained a similar inhibitory activity, which was enhanced in an acute model of mesangial proliferative glomerulonephritis. To identify the active component involved, we examined the expression of known macrophage deactivators IL-10, IL-13, and TGF-beta 1 in mesangial cells. Under the basal culture conditions, strong expression of TGF-beta 1 mRNA was observed, whereas expression of neither IL-10 nor IL-13 was detected. Immunoblot analysis and a specific bioassay detected the active form of TGF-beta 1 exclusively in the mesangial cell conditioned media. The inhibitory activity was enhanced by heat treatment, consistent with the known property of TGF-beta. A specific anti-TGF-beta 1 neutralizing Ab abolished the inhibitory effect exerted by the mesangial cell media, and exogenously added TGF-beta1 suppressed macrophage cytokine expression in a dose-dependent manner. These findings demonstrate that mesangial cells and isolated glomeruli secrete a factor which suppresses cytokine expression by activated macrophages, the active entity being identified as TGF-beta 1.