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Nephrology beyond JASN |
Despite a great deal of recent progress in the treatment of heart failure (1), the prognosis of this condition continues to be poor and new approaches to its treatment are most welcome. One such new approach was originally based on the recognition that anemia is a strong predictor of outcome (2,3). The observation was made (4,5) that quality of life and NYHA (New York Heart Association) category as well as hospitalization for heart failure is remarkably improved by administration of erythropoietin (EPO). This issue is currently the object of prospective controlled trials.
Why is heart failure of interest to the nephrologists? Canadian studies, meanwhile confirmed by others, clearly documented that heart failure conferred the worst prognosis to patients on hemodialysis (6,7). Heart failure is also a major contributor to the excess cardiac mortality of patients with chronic kidney disease (CKD). Therefore, any progress in the treatment of heart failure could potentially have a substantial impact on cardiovascular outcome in renal patients.
A new perspective is opened by the experimental study of van der Meer et al. (8). They studied the acute effect of administration of EPO on infarct size and the chronic effects on hemodynamics, myosin heavy chain (MHC) isoforms, myocyte hypertrophy, and particularly capillary density in the rat ischemia-reperfusion model (ligation of the left descending coronary artery).
For the interpretation of the study it is important to know that EPO receptors are present not only on hematopoietic cells, but also, among others, on endothelial cells (9,10), cardiomyocytes (11), and neurons (12). In the ischemia reperfusion model after ligation of the descending left coronary artery, it had also been shown that acute administration of high doses of EPO diminished infarct size and improved cardiac function (13–15). EPO also rescued hypoxic cultured cardiomyocytes from apoptosis (13). Using this model of myocardial infarction, Dikow had provided evidence that in uremic animals the size of myocardial infarcts is excessive, pointing to reduced hypoxia tolerance (16). In a preliminary report he also found that EPO reduced the infarct size in uremic animals (17).
What had not been studied so far was the effect of delayed administration of EPO on long-term cardiac function and specifically on angiogenesis in the heart. Endothelial cells exhibit EPO receptors (9). In the chick embryo EPO stimulated angiogenesis and the angiogenic potential of EPO was confirmed in models such as the aortic ring model (18), the hind limb model (19), and the stroke model (20). EPO also stimulated proliferation, chemotaxis, and differentiation into vascular structures in cultured endothelial cells, and was equipotent with vascular EGF in stimulating angiogenesis in endothelial cells derived from the myocardium (21,22). Finally, EPO increases the number of circulating EPO progenitor cells; thus, angiogenesis may be favored both by an effect on the resident and by an effect on circulating precursor cells (19).
In a previous study, improved left ventricular function and blood flow had been observed in long-term follow-up of EPO treated animals surviving the acute infarct in the cardiac ischemia reperfusion model (15). Against this background it was a reasonable hypothesis to investigate whether such benefit was related to formation of new capillaries, i.e., angiogenesis. The authors confirmed that EPO treatment immediately after coronary artery ligation reduced infarct size by 30%; these animals had received one single bolus of 8000 U/kg darbepoietin-
intraperitoneally. Without the initial bolus, the infarct size was no longer influenced by delayed EPO treatment, i.e., administration of the same dose once every 3 wk, which increased hematocrit from 44 to 56%. In animals without the initial bolus of EPO, despite no effect on infarct size, substantial benefit was seen from delayed EPO treatment. Such benefit included higher left ventricular systolic pressures, lower left ventricular end-diastolic pressures, improved myocardial relaxation and contractility, lower lung weight/body weight ratio as an index of pulmonary congestion, less expression of 
-MHC, and a trend to lower N-ANP (N-terminal atrial natriuretic peptide) and lower cardiomayocyte cross-sectional area.
The finding of main interest, however, was the documentation of the decrease in capillary density seen in the group without EPO. This was prevented by EPO and the density of capillaries was restored to the values seen in sham-operated, untouched animals. A causal relationship is suggested, but not proven, by the observation that the capillary density was strongly related to the expression of -MHC and myocardial contractility as well as myocardial relaxation.
This observation identifies the capillaries of the heart as a novel, potential, therapeutic target for nonhematopoietic effects of EPO. It is not without interest that previous studies had shown that apoptosis of endothelial cells preceded myocyte apoptosis (23), thus raising the possibility that endothelial cells may rescue surrounding cardiomyocytes as originally postulated by van der Meer et al. (15). This mechanism may be particularly relevant in the so-called "penumbra", i.e., the part of the myocardium that is hypoxic but not yet necrotic. In uremic animals Dikow had shown that the zone of necrosis was increased at the expense of the potentially salvageable tissue in the penumbra (16,17). The issue of myocardial capillary supply is particularly relevant in renal failure, because here capillary supply does not keep pace with the enlargement of cardiomyocytes when the heart undergoes hypertrophy. Such cardiomyocyte/capillary mismatch was found both in uremic patients (24) and in subtotally nephrectomized rats (25). In the latter, doses of EPO substantially lower than in the above experiment had failed to raise capillary density (26). The much higher doses of EPO required in the study of van der Meer et al. (8) may indicate that the EPO compound acting in vivo is some congener of EPO, perhaps acting via a nonclassical receptor (27). Certainly at this time the extremely high doses used in these experiments are a serious obstacle to translate the findings into clinical use, but the situation may become brighter if clinically useful EPO analogues become available in the future.
In the past, the capillaries had been the Cinderella not only in heart disease but also in kidney disease (28,29). With the availability of agents enabling us to deal with the capillary deficit, this area will hopefully become the object of intensified research in the future.
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The unraveling of the Toll receptor and the Toll-like receptor (TLR) family is a fascinating story full of surprises. It all started with the observation of the Nobel laureate Nüsslein-Volhard (related to the family of the nestor of nephrology Franz Volhard [1]) that the dorsal-ventral polarity in Drosophila resulted from the action of a specific gene product; because the resulting shape of the disorganized body looked "crazy" (in German, "toll") the gene was called Toll (2). It was later found that the Toll gene encodes an interleukin1 receptor-like protein triggering synthesis of bactericidal and fungicidal peptides in blood cells of Drosophila (3,4), linking the gene to the innate immune response—obviously leading to the question of whether analogues existed in mammalian species. In the search for human homologues of Drosophila Toll, the group of Medzhitov et al. (5) identified the human Toll-like receptor 4 (TLR4), which was shown to activate NF
B-controlled genes such as IL-1, IL-6, and IL-8, and to cause the induction of members of the B7 family required for the activation of naïve T cells. This observation pointed to a potentially important link between pathogen detection and the induction of the adaptive immune response suggesting that TLR functioned as a link between innate and acquired immunity. It was soon recognized that the underlying common principle of the rapidly expanding family of TLR, currently comprising 10 human isoforms, was the recognition of phagocyte-related chemical patterns, e.g., mannans, lipopolysaccharide (LPS), teichoic acids, etc. Such recognition is obviously important to distinguish potential pathogens from self to preserve tolerance to self and defend against foreign (6). The specificity for the different ligands is provided by heterodimerization of a given TLR with cytoplasmic adaptor molecules (7). What has also become clear in recent years, however, is that not only exogenous microbial products but also endogenous ligands released after cell injury or inflammation may activate the TLR (8)—in other words, TLR sense danger signals of exogenous or endogenous origin. It has further been recognized that TLR are not only expressed by antigen-presenting cells, as originally thought, but also by cells that are not normally involved in host defense, but are involved in tissue damage. Thus TLR have a Janus-like aspect: They are beneficial by defending against microbes, but they may also be also deleterious by promoting tissue damage.
Against this background it may not come as a complete surprise that TLR are also expressed in the kidney: Constitutive expression of TLR2 mRNA has been documented by Wolfs et al. (9) in tubular epithelial cells as well as in epithelial cells of Bowman’s capsule, and it had also been shown that TLR2 mRNA is upregulated by ischemia, although the endogenous cellular signals had remained undefined.
A new twist is now provided by the experiments of Leemans et al., which document a causal role in acute renal dysfunction after ischemia reperfusion injury—and this may have ramifications far beyond acute renal failure.
The investigators studied renal ischemia reperfusion injury comparing wild-type (TLR2+/+) and TLR2 knockout mice (TLR2–/–). To distinguish whether the beneficial effect seen in TLR2–/– mice was the result of deficient TLR2 expression by circulating blood cells or by intrinsic renal cells, they created and studied chimeric mice with deficient expression of TLR2 either by blood cells or by renal cells. Finally, to confirm the results by an independent methodological approach and to investigate a potential modality of intervention, they studied knockdown of TLR2 in cultured tubular epithelial cells as well as in vivo using antisense oligonucleotides.
In a first step, the authors showed in primary cultures of tubular epithelial cells of TLR–/– mice that ischemia simulated by immersion into mineral oil caused less production of cytokines and chemokines, such as KC (granulocyte chemotactic keratinocyte chemoattractant), MIP-2 (macrophage inflammatory protein2), MCP1 (monocyte chemotactic monocyte chemoattractant protein-1), and IL-6. The same pattern was seen when the cultures were stimulated by homogenates of kidneys subjected to ischemia-reperfusion. This finding suggests that ischemia produces some stimulatory molecule(s).
In a second step, the concentrations of these cytokines and chemokines in kidney homogenates were assessed after ischemia-reperfusion injury in wild-type and TLR2 knockout animals. In agreement with the results in the tubular cell cultures, the cytokine and chemokine concentrations were lower in the knockout mice. In addition, the influx of granulocytes and macrophages into the kidney was studied and again the transient infiltration was less pronounced in the TLR2 knockout mice.
In a third step, the authors tried to provide evidence that deletion of TLR2 protected the function and the morphology of the kidney after ischemia-reperfusion injury. Indeed, lower early increase of serum creatinine or urea and less morphologic damage (tubular cell necrosis, tubular dilation, brush border loss, cast formation) were noted in TLR2 knockout mice. In addition, less caspase 3 as an index of apoptosis was detected by immunohistochemistry in TLR2 knockout mice. This finding is in line with recent observations that TLR2 activates apoptotic signaling pathways (10). More apoptosis in the wild-type mice was also associated with more cell regeneration as measured by incorporation of BrdU (brom-deoxy-uridine).
In a fourth step, the authors compared mice in which the expression of TLR2 was selectively deficient in either blood cells only or in kidney cells only. Protection against injury, assessed as increased serum creatinine concentration, neutrophil infiltration, and apoptosis, was provided by absence of TLR2 in renal tubular epithelial cells, but not by absence of TLR2 in blood cells.
In a final step, the potential protection against ischemia reperfusion injury was studied by administering antisense oligonucleotides in vivo to invalidate the TLR2 gene. These oligonucleotides were taken up by tubular epithelial cells and ameliorated the ischemia reperfusion injury as assessed by blood chemistry, apoptotic cells, neutrophil infiltration, and recovery of renal function.
The results indicate that the proinflammatory cascade triggered by stimulation of TLR2 and presumably NFB plays an important role in the genesis of acute renal failure after ischemia reperfusion injury. This role is all the more plausible as TLR2-mediated activation of NFB and oxidative stress has been shown before in other organs, e.g., cardiomyocytes (11). What remains unclear, however, is which endogenous signal activates TLR2 in kidney cells. It has been shown that TLR2-stimulating endogenous ligands are released by necrotic cells, but not by apoptotic cells (12). Among the substantial number of molecules known to stimulate TLR2 (13), the authors discuss as candidates heat-shock protein 70 (14) and debris including cellular matrix components (15).
It is also clear that the pathogenetic role of TLR2 (and of other members of the TLR family) in the genesis of renal disease may well extend beyond renal failure and comprise infection- and injury-associated renal damage and kidney diseases ranging from immune complex glomerulonephritis, e.g., infection-associated glomerulonephritis or lupus nephritis (15), to urinary tract infection (16,17) and malfunction of renal transplants (18–20). What is particularly intriguing with respect to the latter possibility is the observation that, in TLR2 knockout mice, skin allograft rejection was delayed, pointing to a role of TLR2 in transplantation immunology (20).
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