Mechanical strain of glomerular mesangial cells in the pathogenesis of ...

Nephrol Dial Transplant (1999) 14: 1351–1354

Nephrology Dialysis Transplantation

Editorial Comment

Mechanical strain of glomerular mesangial cells in the pathogenesis of glomerulosclerosis: clinical implications Pedro Cortes, Bruce L. Riser, Jerry Yee and Robert G. Narins Division of Nephrology and Hypertension, Department of Medicine, Henry Ford Hospital, Detroit, Michigan, USA

Introduction The pre-glomerular vasodilation that characterizes a wide range of renal diseases allows delivery of a greater fraction of normal or elevated systemic pressure to glomerular capillaries. This glomerular hypertension is, at the very least, a strong contributing force to the glomerulosclerosis seen in many progressive nephropathies. How the physical force of high intraglomerular pressure is translated into the biochemical process of glomerular scar formation, i.e. extracellular matrix ( ECM ) accumulation, has until recently, been largely unexplored. In the following paragraphs, citing our work and that of others, we will show that the mesangial cell stretch provoked by capillaries distended by hypertension, triggers the release of cytokines, including transforming growth factor-beta ( TGF-b). The actions of these cytokines are, at least in part, responsible for net mesangial matrix synthesis. Thus, the mechanical strain of hypertension spawns the injurious accumulation of glomerular ECM which ultimately causes renal insufficiency.

Glomerular pressure: a determinant of capillary and mesangial expansion Glomeruli in kidneys perfused and fixed in situ, have a larger volume than glomeruli in immersion-fixed specimens from non-perfused kidneys [1,2]. Preservation of glomerular pressure in the former setting presumably maintains expanded capillaries, implying the presence of an intrinsic pliability that allows the glomerulus to distend and contract. The characteristics of glomerular elasticity or compliance, the property that allows pressure to regulate volume, were not defined until studies in isolated microperfused glomeruli became possible [2,3]. These studies have demonstrated that the glomerulus is indeed a highly elastic structure capable of marked volume changes within 2–3 s of Correspondence and offprint requests to: Pedro Cortes, MD, Division of Nephrology and Hypertension, CFP-519, Henry Ford Hospital, 2799 West Grand Boulevard, Detroit, MI 48202, USA.

altering intraglomerular pressure. Further, the attainable degree of glomerular distension can be extensive. Increasing capillary pressure from zero to physiological levels expands normal glomeruli up to 30% of their basal volume [4]. However under normal conditions, because glomeruli are exposed to only small pulse pressure variations and not to the low frequency, moment-to-moment oscillations in systemic pressure, volume remains stable [5]. This tight control of intraglomerular pressure is due to the very effective autoregulation provided by the contractile activity of the afferent arteriole [6 ]. This autoregulatory glomerular protection is characteristically impaired in many models of progressive renal disease, including the remnant and diabetic kidney [6,7]. Continuous systemic blood pressure monitoring has shown that in addition to the increasing mean arterial pressure, subtotal nephrectomy causes marked augmentation of the moment-to-moment variations in pressure [8]. Therefore, impaired autoregulation allows for the intraglomerular transmission of systemic pressure, resulting in wide swings in glomerular volume, an alteration which is further magnified by arterial hypertension. Normal glomeruli with intact autoregulation vary their moment-to-moment volume by a mere 0.4%, while glomeruli from remnant kidneys of hypertensive animals demonstrate variations of up to 7.3% [4]. In addition, it has been shown that changes in overall volume are associated with parallel variations in all the glomerular structural components, including the mesangial areas [4].

Mechanical strain imposed on mesangial cells Located at the centre of the glomerular lobule, mesangial cells extend cytoplasmic projections which attach to the peripheral basement membrane at the points where it deflects from its pericapillary course and at the perimesangeal areas [9]. With pressure-induced glomerular expansion, the outward displacement of these anchoring points caused by distending capillaries and mesangium results in intense mesangial cell stretch. It is therefore, anticipated that cyclic changes in

© 1999 European Renal Association–European Dialysis and Transplant Association

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glomerular volume are associated with repeated episodes of mesangial cell stretch and relaxation. The effects of cyclic stretch–relaxation of mesangial cells has been investigated in tissue culture [2,10]. These studies demonstrated that mechanical strain induces profound changes in the synthesis and catabolism of ECM components leading to their accumulation in the cell layer and in the incubation medium. Furthermore, the intensity of this metabolic change is proportional to the degree of mechanical strain imposed on the mesangial cell. Interestingly, the accumulation of ECM induced by cell stretch is markedly enhanced when the ambient glucose concentration is increased [10]. It logically follows from the above that haemodynamically induced glomerular expansion–contraction may stimulate mesangial ECM deposition and mesangial expansion with the eventual development of glomerulosclerosis [11]. In addition, the hyperglycaemia of diabetes mellitus may sensitize glomeruli to these adverse metabolic effects of mechanical strain.

Mediators of the mechanically-induced metabolic change How mesangial cell stretch leads to the deposition of ECM is not precisely known, but recent studies have identified the participation of specific growth factors. Cyclic stretch of mesangial cells in culture is associated with the over-expression and activation of the cytokine, transforming growth factor-b1 ( TGF-b1) and the upregulation of its specific receptors on mesangial cells [12,13]. This is a highly relevant finding because abundant in vivo and in vitro evidence points to the participation of this cytokine in various forms of glomerulosclerosis [14,15]. TGF-b1 neutralization experiments during cycles of stretch–relaxation of mesangial cells incubated in media with high glucose concentrations, have demonstrated that the accumulation of ECM products is mainly ascribable to this growth factor [16 ]. However, the metabolic effects of mechanical strain do not appear to be a result of TGFb1 action under conditions of physiological glucose concentration, suggesting that other, as yet undefined mediators may be at play. More recently a newly identified prosclerotic cytokine, connective tissue growth factor (CTGF ), has also been shown to be upregulated in stretched mesangial cells, although it remains unknown if its participation is essential for the accumulation of ECM [17]. Since the actions of TGF-b1 and CTGF appear to be intimately related, concurrently stimulating the synthesis and deposition of collagenous tissue, it is likely that they may act in concert. In relation to the relevance of mesangial cell mechanical strain in the pathogenesis of diabetic glomerulosclerosis, it is interesting that stretch-induced upregulation of TGF-b1, its receptors and CTGF are all accentuated in cultures exposed to a high glucose concentration. The cellular sensing mechanism that translates

Nephrol Dial Transplant (1999) 14: Editorial Comments

mechanical force into biochemical changes, remains largely unknown. Possible key components of this translating system have been identified in terms of their sequence of activation. Within 30 min following cyclic stretch, mesangial cell phosphorylation of the focal adhesion kinase, pp125FAK, is increased, suggesting that the points at which the cell anchors to its substrate and possibly the cytoskeleton, are involved in the early steps of the translating mechanism [18]. In addition, after only 5–30 min a significant stimulation of signalling cascades controlled by protein kinase C [19] and mitogen-activated protein kinases [20] is detectable. Therefore, even brief periods of glomerular distension and mesangial cell strain may effectively initiate a chain of events which, if perpetuated, alters the deposition of ECM.

Control of glomerular volume Because restriction of glomerular distension may be pivotal in avoiding the progression of glomerular sclerosing processes, studies of the pathophysiologic factors which regulate glomerular volume have become critical. Since intraglomerular pressure is an obvious and major factor [4], it follows that perfusion pressure, the autoregulatory capacity of the afferent arteriole and the tone of the efferent arteriole, all play key roles in determining the state of glomerular distension. In this regard, it is important to note that calcium channel blockers, while diminishing systemic pressure, tend to dilate afferent arterioles [21]. The balance struck by these drugs between diminishing perfusion pressure and its oscillations and the prevalent afferent arteriolar resistance will determine whether glomerular pressures are favourably or unfavourably altered. Converting enzyme inhibitors and angiotensin II-receptor blockers may lower intraglomerular pressure by both decreasing systemic pressure and inducing efferent arteriolar vasodilatation. In addition, these agents may also inhibit the direct- or TGF-b1-mediated angiotensin II enhancement of collagen deposition [22,23]. While differences in action between these antihypertensive agents exist, settings where afferent arteriolar autoregulation is totally absent, a major factor in alleviating glomerular strain is the decrease in systemic pressure and its oscillations, regardless of how this is achieved. A second factor determining glomerular expansion is the basal glomerular volume [4]. At any given intraglomerular pressure, larger, hypertrophied glomeruli are more distensible than smaller glomeruli. Although this effect could be the mere result of capillary lengthening, it is more likely related to increased capillary wall tension resulting from the increased radius of hypertrophied capillaries [24]. Consequently, whether induced by nephron loss or diabetes, glomerular hypertrophy magnifies the deleterious effects of altered glomerular haemodynamics. The only maneuvers known to ameliorate this hypertrophy are strict metabolic control of diabetes and protein restriction [25,26 ]. Whether calcium channel blockers may


also limit glomerulosclerosis by reducing glomerular hypertrophy, remains controversial [27,28]. A third factor influencing glomerular distension is the intrinsic rigidity of the glomerular scaffold, primarily provided by the composition and distribution of the collagenous components in the peripheral basement membrane and mesangium. Remnant glomeruli undergoing incipient sclerosis demonstrate a paradoxical diminution in rigidity, rendering them more compliant and prone to distension, independently of their basal size [4]. In contrast, glomerular rigidity is normal in glomeruli of diabetic animals, even after prolonged periods of the disease [10]. Finally, mesangial cell tone may also contribute to glomerular rigidity by opposing the pressure-driven expansion. However, direct assessment of this component has shown that its role is probably unimportant because in angiotensin II-perfused glomeruli, only 4% of the total glomerular rigidity could be attributed to the associated mesangial cell contraction [4].

Summary Due to their elasticity, glomeruli will undergo excessive expansion and repetitive cycles of distension–contraction under conditions of impaired glomerular pressure autoregulation and systemic arterial hypertension. These alterations in glomerular volume are associated with mesangial cell stretch which in turn stimulates the synthesis and deposition of ECM with eventual mesangial expansion and glomerulosclerosis. Hyperactivity of growth factors with prosclerotic activity is an important component in the translation of cellular mechanical strain into the abnormal metabolism of ECM components. Although mesangial cell mechanical strain is expected to occur in both remnant glomeruli and in glomeruli of diabetic kidneys, quantitatively different factors will determine the resultant metabolic consequences. In remnant glomeruli, the mechanical stretch is intense, being accounted for largely by the marked glomerular hypertrophy and increased glomerular compliance. In diabetic glomeruli, however, the mechanical stretch is less prominent but its effect on ECM synthesis is markedly aggravated by the presence of hyperglycaemia. There are presently no methods clinically available to diminish the prosclerotic action of growth factors at the glomerular level. In addition, there are no effective means to specifically improve glomerular pressure autoregulation. Therefore, current therapies must be aimed at decreasing systemic arterial pressure, blocking angiotensin II action and reducing glomerular hypertrophy. While there are effective drugs for the treatment of hypertension and for angiotensin II inhibition, protein restriction is the only measure available to diminish glomerular hypertrophy. Finally, in diabetes correction of systemic and glomerular hypertension should be coupled with strict glycaemic control

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to correct both glomerular autoregulation and increased ECM deposition.

References 1. Miller PL, Meyer TW. Methods in laboratory investigation. Effects of tissue preparation on glomerular volume and capillary structure in the rat. Lab Invest 1990; 63: 862–866 2. Riser BL, Cortes P, Zhao X, Bernstein J, Dumler F, Narins RG. Intraglomerular pressure and mesangial stretching stimulate extracellular matrix formation in the rat. J Clin Invest 1992; 90: 1932–1943 3. Osgood RW, Patton M, Hanley MJ, Venkatachalam M, Reineck HJ, Stein JH. In vitro perfusion of the isolated dog glomerulus. Am J Physiol 1983; 244: F349–F354 4. Cortes P, Zhao X, Riser BL, Narins RG. Regulation of glomerular volume in normal and partially nephrectomized rats. Am J Physiol 1996; 270: F356–F370 5. Brenner BM, Troy JL, Daugharty TM. The dynamics of glomerular ultrafiltration in the rat. J Clin Invest 1971; 50: 1776–1780 6. Pelayo JC, Westcott JY. Impaired autoregulation of glomerular capillary hydrostatic pressure in the rat remnant nephron. J Clin Invest 1991; 88: 101–105 7. Hayashi K, Epstein M, Loutzzenhiser R, Forster H. Impaired myogenic responsiveness of the afferent arteriole in streptozotocin-induced diabetic rats: role of eicosanoid derangements. J Am Soc Nephrol 1992; 2: 1578–1586 8. Bidani AK, Griffin KA, Picken M, Lansky DM. Continuous telemetric blood pressure monitoring and glomerular injury in the rat remnant kidney model. Am J Physiol 1993; 265: F391–F398 9. Kriz W, Elger M, Lemley K, Sakai T. Structure of the glomerular mesangium: A biomechanical interpretation. Kidney Int 1990; 38 [Suppl 30]: S2–S9 10. Cortes P, Zhao X, Riser BL, Narins RG. Role of glomerular mechanical strain in the pathogenesis of diabetic nephropathy. Kidney Int 1997; 51: 57–68 11. Cortes P, Riser BL, Zhao X, Narins RG. Glomerular volume expansion and mesangial cell mechanical strain: mediators of glomerular pressure injury. Kidney Int 1994; 45 [Suppl 45]: S11–S16 12. Riser BL, Cortes P, Heilig C et al. Cyclic stretching force selectively up-regulates transforming growth factor-b isoforms in cultured rat mesangial cells. Am J Pathol 1996; 148: 1915–1923 13. Riser BL, Landson-Wofford S, Sharba A et al. TGF-b receptor expression and binding in rat mesangial cells: modulation by glucose and cyclic mechanical strain. Kidney Int (in press) 14. Yamamoto T, Nakamura T, Noble NA, Ruoslahti E, Border WA. Expression of transforming growth factor b is elevated in human and experimental diabetic nephropathy. Proc Natl Acad Sci USA 1993; 90: 1814–1818 15. Isaka Y, Fujiwara Y, Ueda N, Kaneda Y, Kamada T, Imai E. Glomerulosclerosis induced by in vivo transfection of transforming growth factor-b or platelet-derived growth factor gene into the rat kidney. J Clin Invest 1993; 92: 2597–2601 16. Riser BL, Cortes P, Yee J et al. Mechanical strain-and high glucose-induced alterations in mesangial cell collagen metabolism: role of TGF-b. J Am Soc Nephrol 1998; 9: 827–836 17. Riser BL, DeNichilo M, Grondin JM et al. Connective tissue growth factor (CTGF ) as a determinant of extracellular matrix (ECM ) deposition in diabetic glomerulosclerosis. J Am Soc Nephrol 1998; 9: 640A [Abstract] 18. Hamasaki K, Mimura T, Furuya H et al. Stretching mesangial cells stimulates tyrosine phosphorylation of focal adhesion kinase. Biochem Biophys Res Comm 1995; 212: 544–549 19. Akai Y, Homma T, Burns K, Yasuda T, Bdar KF, Harris RC. Mechanical stretch/relaxation of cultured rat mesangial cells induces protooncogenes and cyclooxygenase. Am J Physiol 1994; 267: C482–C490 20. Ishida T, Haneda M, Isono M et al. Pivotal role of mitogen activated protein kinases in stretch-induced fibronectin synthesis in mesangial cells. J Am Soc Nephrol 1997; 8: 517A [Abstract] 21. Carmines PK, Mitchell KD, Navar LG. Effects of calcium

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antagonists on renal hemodynamics and glomerular function. Kidney Int 1992; 41 [Suppl 36 ]: S43–S48 Wolf G, Haberstroh U, Neilson EG. Angiotensin II stimulates the proliferation and biosynthesis of type I collagen in cultured murine mesangial cells. Am J Pathol 1992; 140: 95–107 Kagami S, Border WA, Miller DE, Noble NA. Angiotensin II stimulates extracellular matrix protein synthesis through induction of transforming factor-b expression in rat glomerular mesangial cells. J Clin Invest 1994; 93: 2431–2437 Daniels B, Hostetter TH. Adverse effects of growth in the glomerular microcirculation. Am J Physiol 1990; 258: F1409–F1416 Stackhouse S, Miller PL, Park SK, Meyer TW. Reversal of

Nephrol Dial Transplant (1999) 14: Editorial Comments glomerular hyperfiltration and renal hypertrophy by blood glucose normalization in diabetic rats. Diabetes 1990; 39: 989–995 26. Savin VJ, Seaton RD, Richardson WP, Duncan K, BeasonGriffin C, Ahnemann J. Dietary protein and glomerular response to subtotal nephrectomy in the rat. J Lab Clin Med 1989; 113: 41–49 27. Dworkin LD, Benstein JA, Parker M, Tolbert E, Feiner HD. Calcium antagonists and converting enzyme inhibitors reduce renal injury by different mechanisms. Kidney Int 1993; 43: 808–814 28. Irzyniec T, Mall G, Greber D, Ritz E. Beneficial effect of nifedipine and moxonidine on glomerulosclerosis in spontaneously hypertensive rats. A micromorphometric study. Am J Hypertens 1992; 5: 437–443


Aptamers: novel tools for specific intervention studies Ju¨rgen Floege, Tammo Ostendorf and Nebojsˇa Janjic´1 Division of Nephrology, Medical School, Hannover, Germany, and 1NeXstar Pharmaceuticals, Boulder, Colorado, USA

Specific intervention studies are a crucial requirement to demonstrate the pathogenetic relevance of particular molecules and to ultimately develop novel therapeutic approaches to renal disease. Inhibition studies of cytokines or growth factors provide a good example of the various approaches that have been employed in such studies in vivo. (i) Neutralizing antibodies. Generation of such antibodies is probably the easiest way to accomplish intervention studies. Problems of neutralizing antibodies relate to their immunogenicity in heterologous systems and, more importantly, to non-specific effects of immunoglobulins per se, which may be difficult to control. (ii) Natural or designed antagonists (e.g. extracellular domains of receptors or receptor antagonists). An elegant, yet laborious way, which requires the expression of considerable amounts of recombinant protein. (iii) Interference with cytokine or growth factor signalling. An attractive approach since many compounds that inhibit molecules involved in signalling are simple chemical structures that can easily be synthesized. A frequent problem of this approach, however, is non-specificity of the intervention given the convergence of many signalling pathways and/or toxic in vivo effects. (iv) Targeted gene deletion. A laborious way to investigate the function of a mediator, which also suffers from the potential problem that the ontogenetic lack of a single gene might have induced compensatory mechanisms that blunt the effects of the deficiency. Correspondence and offprint requests to: Ju¨rgen Floege MD, Division of Nephrology 6840, Medizinische Hochschule, D-30623 Hannover, Germany.

(v) Antisense or ribozyme studies. While the compounds are relatively easy to synthesize, they unfortunately have to enter the cell in order to act. This renders in vivo studies difficult as the compounds usually have to be transfected in vivo using liposomes or viral vectors. (vi) Specific interventions with aptamers.

What is an aptamer? The term ‘aptamer’ was originally proposed by Ellington and Szostak based on the latin word aptus, i.e. to fit [1]. It describes the property of DNA or RNA oligonucleotides to specifically bind to other molecules outside or inside of cells. Thus, in this case biological effects of the oligonucleotide are not a function of the DNA or RNA code (as involved in transcription and translation) but rather reside in the specific three-dimensional structure of the oligonucleotide [1,2]. Aptamers are usually 20–50mers with distinct structural elements (such as stems, loops, pseudoknots, helix junctions, G-quartets etc.) that create a stable framework in which some invariant nucleotides are precisely arranged [3–5]. It is these nucleotides at specific positions in the threedimensional structure that are central to the physicochemical interaction of the aptamer with other molecules. K -values for the interaction of aptamers with d their targets extend from the micromolar to low picomolar range and are therefore comparable in terms of binding affinity to antibody–antigen interactions [3–5]. An impressive example of how seemingly minor changes in an oligonucleotide sequence can have dramatic effects on the specificity of an aptamer is given


in Figure 1, which shows how the exchange of three nucleotides in a 42-mer determines whether the aptamer will bind L-citrulline or L-arginine with high strength [6 ]. Aptamers have been produced against very diverse molecules ranging from organic dyes to nucleotides, such as ATP, to amino acids, peptides and complex proteins (both extracellular and intracellular) and even disaccharides [3–5,7].

How is an aptamer produced? In many ways the generation of a particular aptamer resembles evolution in that it starts from a random mixture of oligonucleotides and then selects for the ‘best’ sequences in a process called systematic evolution of ligands by exponential enrichment (SELEX ) [2,5]. The starting point is the synthesis of single-stranded nucleic acid libraries (RNA, DNA or modified nucleic acids) containing 20–40 randomized positions. This creates an enormous diversity of possible sequences (and hence different structures). For example, four different nucleotides at 40 randomized positions will theoretically yield 440 (=1.24×1024) combinations, although in practice starting libraries usually contain no more than about 1015 different sequences. These random sequence regions are flanked by fixed sequences, which are essential for subsequent PCR amplification ( Figure 2). This vast array of different sequences is next incubated with a given target molecule, followed by removal of unbound oligonucleotides. Binding sequences are then amplified by PCR and regenerated, either by in vitro transcription (for RNA-

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based libraries) or strand separation (for DNA-based libraries). These amplified sequences are then employed for further rounds of the SELEX process, usually 5–12, after which high affinity sequences have been sufficiently enriched. These sequences are cloned and can then be evaluated specifically for their binding properties. Once a binding sequence has been identified, it can be modified further, for example, by testing whether particular substitutions might further increase binding affinity [8]. Other modifications are aimed at improving nuclease resistance, for example by incorporating nucleotides with 2∞-amino-, 2∞-fluoro- or 2∞-O-alkylmoieties instead of natural nucleotides into the sequence [8,9]. Another interesting approach to improving nuclease resistance is the production of mirror-image L-oligonucleotides, which are not recognized by natural nucleases [10]. However, for protein targets this requires the prior synthetic preparation of D-polypeptides. Finally, in order to increase the in vivo residence time of aptamers in plasma (usually they are excreted quickly via the kidney because of their small size), their effective molecular size can be increased by anchoring them in liposome bilayers or by coupling them to inert large molecules such as polyethylene glycol (PEG) [11–13].

In vivo applications of aptamers There are now several reports on the use of aptamers in vivo. The first, a 15-mer ssDNA antagonist against thrombin was infused into monkeys at doses of up to 0.6 mg/kg/min [14] and led to a two-fold increase in

Fig. 1. Structure of an high affinity aptamer for L-citrulline as opposed to a high affinity aptamer for L-arginine. Note how changes at three nucleotide positions (circles) completely change the specificity of the aptamer. Modified from [6 ].

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Fig. 2. Schematic outline of the SELEX process.

Fig. 3. Glomerular immunostaining for type IV collagen in a control rat with anti-Thy 1.1 nephritis on day 9 after disease induction (A) or a rat with anti-Thy 1.1 nephritis, which had received a PDGF-B antagonistic aptamer from day 3 to 8 (B). Note how the marked mesangial expansion and matrix accumulation in the control rat is reduced to almost normal levels in the rat receiving the PDGF-B antagonist. Magnification ×400.

prothrombin time. However, the half-life of the small 15-mer was in the range of a few minutes. In other experiments a 49-mer L-selectin antagonist, coupled to 20 kDa PEG markedly reduced the influx of peripheral blood mononuclear cells (PBMC ) into lymph nodes

when given 1–5 min prior to PBMC injection into mice [15]. In vivo activity of aptamers to neutrophil elastase [16 ], vascular endothelial growth factor-165 ( VEGF ) [12,17] and other therapeutically relevant 165 proteins [unpublished results] has now been observed.


We have recently employed a 29-mer DNA-based aptamer, coupled to 40 kDa PEG, to specifically inhibit the action of platelet-derived growth factor (PDGF ) in renal disease [11]. In that study, we demonstrated that twice daily treatment with the aptamer led to a 95% reduction of the pathological mesangial cell proliferation that occurs in a mesangioproliferative glomerulonephritis model in rats (anti-Thy 1.1 nephritis). We were also able to demonstrate that the PDGF antagonist markedly reduced the mesangial matrix accumulation ( Fig. 3). This study not only confirmed the central role of PDGF-B chain in mesangioproliferative glomerular disease [18] but also demonstrated that inhibition of a growth factor is possible in vivo by systemically administered aptamer. Since aptamers, unlike neutralizing antibodies do not appear to be immunogenic, the PDGF-B chain antagonist now allows us to test the long term consequences of reduction of mesangial cell proliferation in a chronic model of anti-Thy 1.1 nephritis. In a second in vivo study, again using the anti-Thy 1.1 nephritis model, we have employed an aptamerbased antagonist to VEGF [17] to elucidate the role 165 of this growth factor in glomerular disease. We were able to demonstrate that VEGF apparently is a 165 factor of central importance for glomerular endothelial cell regeneration and survival, as its inhibition markedly augmented the glomerular capillary damage that occurs during anti-Thy 1.1 nephritis [12].

Conclusion The above data provide evidence that specific intervention trials based on aptamers are feasible and that aptamers are not only highly specific but also highly efficient in vivo. Since so far there is no evidence that aptamers are immunogenic and given the ease at which their molecular characteristics can be altered (for example by coupling them to PEG, hydrophobic groups and various other chemical moieties in a sitespecific manner), these compounds will greatly aid future studies in which specific roles of mediators are evaluated in disease. Once such roles have been clearly defined, therapeutic applications of aptamers will constitute the immediate next step. In fact, first therapeutic interventions using the VEGF antagonist are ongoing in patients with age-related macular degeneration.

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References 1. Ellington AD, Szostak JW. In vitro selection of RNA molecules that bind specific ligands. Nature 1990; 346: 818–822 2. Tuerk C, Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 1990; 249: 505–510 3. Osborne SE, Ellington AD. Nucleic acid selection and the challenge of combinatorial chemistry. Chem Rev 1997; 97: 349–370 4. Eaton BE. The joys of in vitro selection: chemically dressing oligonucleotides to satiate protein targets. Curr Opin Chem Biol 1997; 1: 10–16 5. Gold L, Polisky B, Uhlenbeck O, Yarus M. Diversity of oligonucleotide functions. Annu Rev Biochem 1995; 64: 763–797 6. Famulok M. Molecular recognition of amino acids by RNAaptamers: An L-citrulline binding RNA motif and its evolution into an L-arginine binder. J Am Chem Soc 1994; 116: 1698–1706 7. Yang Q, Goldstein IJ, Mei HY, Engelke DR. DNA ligands that bind tightly and selectively to cellobiose. Proc Natl Acad Sci USA 1998; 95: 5462–5467 8. Green LS, Jellinek D, Bell C et al. Nuclease-resistant nucleic acid ligands to vascular permeability factor/vascular endothelial growth factor. Chem Biol 1995; 2: 683–695 9. Eaton BE, Gold L, Hicke BJ et al. Post-SELEX combinatorial optimization of aptamers. Bioorg Med Chem 1997; 5: 1087–1096 10. Nolte A, Klussmann S, Bald R, Erdmann VA, Furste JP. Mirror-design of L-oligonucleotide ligands binding to L-arginine. Nat Biotechnol 1996; 14: 1116–1119 11. Floege J, Ostendorf T, Janssen U et al. A novel approach to specific growth factor inhibition in vivo: antagonism of PDGF in glomerulonephritis by aptamers. Am J Pathol 1999; 154: 169–179 12. Ostendorf T, Kunter U, Loos A et al. Vascular endothelial growth factor ( VEGF165) mediates glomerular endothelial repair. Submitted 13. Willis MC, Collins B, Zhang T et al. Liposome-anchored vascular endothelial growth factor aptamers. Bioconjugate Chem 1998; 9: 573–582 14. Griffin LC, Tidmarsh GF, Bock LC, Toole JJ, Leung LL. In vivo anticoagulant properties of a novel nucleotide-based thrombin inhibitor and demonstration of regional anticoagulation in extracorporeal circuits. Blood 1993; 81: 3271–3276 15. Hicke BJ, Watson SR, Koenig A et al. DNA aptamers block Lselectin function in vivo. Inhibition of human lymphocyte trafficking in SCID mice. J Clin Invest 1996; 98: 2688–2692 16. Bless NM, Smith D, Charlton J et al. Protective effects of an aptamer inhibitor of neutrophil elastase in lung inflammatory injury. Current Biol 1997; 7: 877–880 17. Ruckman J, Green LS, Beeson J et al. 2-Fluoropyrimidine RNA-based aptamers to VEGF-165. Inhibition of receptor binding and VEGF-induced vascular permeability through interactions requiring the exon 7-encoded domain. J Biol Chem 1998; 273: 20556–20567 18. Floege J, Johnson RJ. Multiple roles for platelet-derived growth factor in renal disease. Miner Electrolyte Metab 1995; 21: 271–282

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Diuretics in congestive heart failure: new evidence for old problems Christlieb Haller Department of Internal Medicine, Division of Cardiology, University of Heidelberg, Heidelberg, Germany

When the heart fails the kidney retains sodium. Initially this is a beneficial counter-regulatory response to maintain the effective arterial volume, but the down-side is an increased preload due to volume retention, which may perpetuate and even aggravate congestive heart failure (CHF ). Diuretics counteract the sodium retention and are therefore indispensable in most patients with clinical heart failure. These drugs are clearly effective to reverse symptoms of congestion, but little information on their overall effect on prognosis is available. This point is important, since diuretics have several, potentially even life-threatening, side-effects. A recent study has shed new light on a major problem regarding diuretic treatment in patients with CHF. Discussion of this study requires a review of some of the issues concerning current diuretic therapy to put the problem into perspective.

therapy with a single agent be insufficient, combination therapy with loop diuretics and distally active agents may be effective, even in patients with advanced renal failure [3,4]. However, this so-called sequential nephron blockade can induce severe electrolyte disturbances, in particular hypokalaemia and hypomagnesaemia. In this context potassium-sparing diuretics may be useful adjuncts to therapy. The aldosterone receptor blocking agent spironolactone may have a special role in this setting, since it provides blockade at a more distal nephron segment, as will be discussed below. It should be emphasized that a reduction of sodium intake will lower urinary potassium wasting and therefore reduce the risk of hypokalaemia. This is due to the fact that less sodium is presented to the nephron segment where potassium for sodium exchange takes place. A low-sodium diet will also help achieve a negative sodium balance, which is the ultimate goal of any rational oedema-reducing therapy.

Volume overload—oedema

Electrolyte disturbances

Patients with CHF may present with massive oedema. It is notoriously difficult to overcome the sodium retention in CHF. This is a compensatory response to the decrease in effective arterial volume and any further reduction in the effective arterial volume by diuretics may precipitate renal and/or circulatory failure. Furthermore, reducing preload by diuretics may worsen cardiac function, particularly in patients with right ventricular failure, although in many patients a reduction of preload and cardiac size brings the heart into a more favourable range of the Franck–Starling curve, thus increasing stroke volume [1]. Some patients with advanced heart failure may be resistant to even large doses of loop diuretics despite apparently normal renal function [2]. One important reason for the refractoriness to loop diuretics is the so-called rebound effect after single doses of loop diuretics, which is characterized by a disproportionately increased sodium avidity of the kidney after the diuretic action has worn off. This rebound effect may be overcome by a more frequent administration or the continuous infusion of loop diuretics. Should diuretic

Afterload reduction with angiotensin-converting enzyme (ACE) inhibitors reduces mortality in patients with symptomatic and even asymptomatic left ventricular dysfunction. Secondary hyperaldosteronism often persists, however, despite effective ACE inhibition. Consequently patients with CHF tend to develop hypokalaemia even when they are not on diuretics, particularly when the sodium intake is high. In patients with CHF severe hypokalaemia (and hypomagnesaemia) may develop, especially when high doses of diuretics are administered. Such hypokalaemia is not an innocent academic phenomenon, but a potentially lifethreatening complication: patients with reduced left ventricular function have an increased incidence of severe cardiac dysrrhythmia and this tendency is aggravated by electrolyte disturbances. When hypertensive patients are treated with high doses of diuretics the incidence of sudden cardiac death is increased [5,6 ]. At the 1998 meeting of the American Heart Association in Dallas a correlation was reported between diuretic dose and sudden death in patients with advanced heart failure; this finding was based on a retrospective analysis of the PRAISE trial [7]. In acute heart failure an association has also been found between diuretic use, clinical response and death [8]. Thus it appears that the benefit of diuretics with respect

Introduction

Correspondence and offprint requests to: Priv. Doz. Dr. C. Haller, Department of Internal Medicine III, University of Heidelberg, Bergheimerstr. 58, 69115 Heidelberg, Germany.


to prognosis may at least partially be offset by fatal cardiac dysrhythmia. In order to reduce the risk of arrhythmia, patients with CHF often receive oral potassium supplements, although this form of therapy is relatively ineffective for the prevention of potassium depletion. The cardiac risk of diuretic therapy can be reduced by prescribing low doses of diuretics and/or by the use of potassiumsparing agents [6 ]. In this context the aldosterone antagonist spironolactone has recently received much attention (RALES study; see below). With appropriate caution potassium-sparing drugs may be added to the diuretic regimen even in patients who are simultaneously treated with ACE inhibitors. Such a combination requires meticulous monitoring of the serum concentration of potassium and creatinine, since a deterioration of renal function could rapidly result in life-threatening hyperkalaemia. It is useful to remember that reduction of sodium intake, apart from decreasing oedema formation and diuretic requirements, reduces urinary potassium loss. A low-sodium diet is therefore mandatory in patients with heart failure—even if hyponatraemia is present. A detailed discussion of the complex pathophysiology of hyponatraemia in (diuretic-treated) patients with heart failure is beyond the scope of this editorial. Suffice it to say that hyponatraemia mostly results from free water excess and not from sodium depletion. The treatment of choice is therefore restriction of free water intake. When this is difficult to achieve in patients with advanced heart failure, there may be a case for vasopressin antagonists—a new class of drugs on the horizon.

Neurohumoral activation In addition to causing electrolyte disturbances, diuretics exert adverse cardiovascular effects by inducing a variety of neurohumoral alterations. Acute treatment with diuretics causes sympathetic activation [9] thus increasing the propensity to develop dysrrhythmia. Chronic treatment with furosemide, however, decreases resting (but not exercise-induced) plasma noradrenaline concentration, while the renin—angiotensin— aldosterone system is activated [10]. Regimens recently proposed for the management of CHF include ACE inhibitors and—in selected patients—low-dose beta blockers. The latter should reduce neuroendocrine activation, but ACE inhibitors do not prevent the development of secondary hyperaldosteronism (see below).

Persistent hyperaldosteronism—should all patients receive spironolactone? Evidence from the RALES study Hyperaldosteronism plays an important role in the pathophysiology of congestive heart failure, even in patients who receive therapeutic doses of ACE inhib-

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itors which have become the backbone of modern CHF therapy [11]. The mechanism(s) underlying the so-called aldosterone escape are not known. Hyperaldosteronism favours the development of hypokalaemia and aggravates oedema formation. In addition to these clinically undesirable effects, aldosterone may also lead to structural changes of the heart, particularly cardiac hypertrophy and myocardial fibrosis [12]. Aldosterone-induced structural changes may contribute to the development of arrhythmia and adversely affect survival. Therefore it seems reasonable to block the cellular actions of aldosterone with spironolactone. This approach is fraught with the risk of hyperkalaemia, especially in patients who are on ACE inhibitors. The safety of the simultaneous treatment with spironolactone and ACE inhibitors has been a major concern. This issue was addressed in a multicentre trial in patients with CHF. The randomized aldactone evaluation study (RALES ) enrolled 214 patients with congestive heart failure NYHA II-IV [13]. Spironolactone was added in a placebo-controlled fashion to the previous therapeutic regimen which included an ACE inhibitor and a loop diuretic. The doses of spironolactone ranged from 12.5 to 75 mg daily. The incidence of hyperkalaemia ( K+5.5 mmol/l ) was 5% in patients treated with 12.5 mg spironolactone per day and 13% in patients receiving 25 mg spironolactone per day; higher doses induced hyperkalaemia in up to 24% of patients. In the placebo group the incidence of hyperkalaemia was 5%. Hypokalaemia ( K+