Capillary/myocyte mismatch in the heart in renal failure—a role for erythropoietin?

Kerstin Amann1,2,, Moriz Buzello1, Aurelia Simonaviciene1,4, Gabriel Miltenberger-Miltenyi1,4, Andreas Koch1, Alexander Nabokov1,4, Marie-Luise Gross1, Bernhard Gless5, Gerhard Mall3 and Eberhard Ritz4

1 Department of Pathology, University of Heidelberg, Heidelberg, 2 Department of Pathology, University of Erlangen-Nürnberg, Erlangen, 3 Department of Pathology, Darmstadt, Darmstadt, 4 Department of Nephrology, Internal Medicine, University of Heidelberg, Heidelberg and 5 Department of Physiology I, University of Regensburg, Regensburg, Germany



   Abstract
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Background. Chronic renal failure is characterized by remodeling of the heart with left ventricular hypertrophy (increasing oxygen demand) and capillary deficit leading to capillary/myocyte mismatch (decreasing oxygen supply). Erythropoietin (Epo) has known angiogenic properties causing endothelial cell activation, migration and sprouting, mediated at least in part via the JAK/STAT (Janus kinase/signal transducers and activators of transcription) pathway. In uraemic cardiac hypertrophy the presence of diminished capillary supply implies that capillary growth does not keep pace with development of hypertrophy. To investigate whether this was due to a deficit of the angiogenic hormone Epo we examined whether Epo levels are altered and whether an increase in haematocrit by administration of rhEpo influences capillary supply, i.e. capillary/myocyte mismatch in experimental renal failure.

Method. Male Spraque–Dawley rats were either subjected to partial renal ablation or sham operation. Only modest amounts of renal tissue were removed so that the rats were not anemic. Subgroups of rats received either human (rh)Epo alone or in combination with unspecific antihypertensive treatment (dihydralazine plus furosemide) in order to control the Epo induced rise in blood pressure. Capillary supply was measured stereologically as capillary length per volume myocardium using the orientator method.

Results. Capillary length density was reduced by approximately 25% after partial renal ablation (3237±601 vs 4293±501 mm/mm3 in controls). It was not statistically different in animals with partial renal ablation+rhEpo+antihypertensive treatment (3620±828 mm/mm3) compared to partial ablation alone.

Conclusion. The study shows that lack of Epo does not cause, or contribute to, the deficit of capillary growth in the hypertrophied left ventricle of rats with renal failure. In addition, a rise in haematocrit is not accompanied by beneficial effects on alterations of cardiovascular structure in experimental renal failure.

Keywords: experimental renal failure; heart; capillarization; rhEpo



   Introduction
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
In subtotally nephrectomized rats and in uraemic patients [14] left ventricular hypertrophy (LVH) develops and this is partly independent of elevated blood pressure. LVH is accompanied by complex structural changes comprising hypertrophy of cardiomyocytes, expansion of interstitial tissue, arteriolar thickening and diminished capillary supply [5,6]. Myocyte/capillary mismatch is important because diminished capillary supply in the face of increased cardiomyocyte mass must interfere with ischaemia tolerance of the heart.

The genesis of this abnormality is certainly complex. Studies with the sympatholytic agent Moxonidine showed that the deficit of capillary length density was improved, but not completely normalized, by sympathetic blockade [7]. The latter point is consistent with the idea that factors other than sympathetic overactivity also play a role in the genesis of the capillary abnormalities [3].

Endothelial cells express erythropoietin (Epo) receptors [8,9] and are activated through the JAK/ STAT (Janus kinase/signal transducers and activators of transcription) pathway [10] similar to what is seen in erythroid cells [11]. Activation involves tyrosine phosphorylation of membrane bound peptides [12] and activation of receptor operated Ca2+ channels and of phosphorylase C gamma [13], increase of cytosolic calcium in some [13,14] but not in all studies [12], and stimulation of endothelin-1 (ET-1) synthesis [1417]. Interaction of Epo with mesangial, and possibly also interstitial cells [15] leads to stimulation of endothelial cell proliferation [17], migration [8] and sprouting in an ET-1 dependent fashion [18]. The above stages in the activation sequence were not confirmed in all studies, however [19].

The observation that Epo causes sprouting of capillaries [18] is consistent with the idea that it is an angiogenic substance. This notion together with other findings [20] raises the possibility that lack of Epo plays a role in the genesis of the capillary/myocyte mismatch in the heart in renal insufficiency.



   Material and methods
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Animals
Forty-eight male Sprague–Dawley rats (SD, 200 g; Invanovas Co., Kisslegg, Germany) were housed in single cages at constant temperature (20°C) and humidity (25%). The animals were given a high protein, low salt diet containing 40% protein and 0.6% NaCl (Altromin C 1002/C 1036, Altromin Co., Lage, Germany). After 3 days of adaptation, the animals were randomly allocated to moderate partial nephrectomy (SNX) or sham operation (sham).

Blood pressure (BP) was measured at the beginning of the experiment and then every 2 weeks by tail plethysmography.

Renal ablation
First, the right kidney was removed under ketamin/diazepam anaesthesia (100 mg/kg or 2.5 µg/kg, respectively). Seven days later, the left kidney was partially resected by removing a quantity corresponding to 2/3 of the weight of the resected right kidney from the left cortex. The control animals were sham operated by decapsulating the kidney. Special care was taken to avoid damage to the adrenals [7,21].

Experimental protocol
The animals were randomly allocated to the following experimental groups (n=8 animals per group).

1. Sham operated controls.
2. Sham operated controls+human (rh)Epo.
3. Sham operated controls+rhEpo+antihypertensive treatment.
4. Untreated SNX.
5. SNX+rhEpo.
6. SNX+rhEpo+antihypertensive treatment.

rhEpo and antihypertensive treatment
rhEpo was given at a dose of 20 IE s.c. twice per week. In addition, once a week 5 mg iron s.c. were given. Antihypertensive treatment consisted of dihydralazine and furosemide in the drinking fluid designed to deliver 20 mg/kg body weight dihydralazine and 5 mg/kg body weight furosemide per day. Serum Epo levels were measured in four animals of groups 1, 2, 4, 5 at baseline and 24 h after s.c. injection as described in [22].

Perfusion fixation and tissue sampling
Eight weeks after the second operation the experiment was terminated by perfusion fixation [7]. At the end of the experiment, the abdominal aorta was catheterized under ketamin/diazepam anaesthesia (doses as above), blood samples were taken and the viscera were rinsed with 10% dextran solution containing 0.5 g/l Procain–HCl for 2 min. Ten seconds after starting the aortic perfusion, the vena cava was incised to drain the blood. After dextran infusion, the vascular system was perfused with 0.2 M phosphate buffer containing 3% glutaraldehyde for 12 min.

After the perfusion, the heart of each animal was taken out for determination of weight and volume, tissue sampling and section stainining was performed according to the orientator method [23]. Uniformly random sampling was achieved by preparing a set of equidistant slices of the left ventricle and the interventricular septum with a random start. Two slices were selected by area weighted sampling and processed accordingly [23]. Eight pieces of the left ventricular muscle including the septum were prepared and afterwards embedded in Epon-Araldite. Semithin sections (1 µm) were stained with methylene-blue and basic fuchsin and examined by light microscopy with oil immersion and phase contrast at a magnification of x1000.

In three animals per group ultrathin sections (0.08 µm) were stained with uranyl acetate and lead citrate and examined qualitatively with a Zeiss EM 10 electron microscop (Zeiss Co., Oberkochen, Germany).

Stereological analysis, quantitative stereology
All investigations were performed in a blinded manner, i.e. the observer was unaware of the study group the animal belonged to. Stereological analysis was performed on eight random samples of differently orientated sections of the left ventricular myocardium per animal according to the orientator method [7,23].

In brief, the length density (LV) of capillaries, i.e. the length of capillaries per unit tissue volume, and the volume density (VV) of cardiac capillaries, i.e. the volume of capillaries per unit myocardial tissue volume, were measured in eight systematically subsampled areas per section (57 600 µm2) using a Zeiss eyepiece (Zeiss Co., Oberkochen, Germany) with 100 points for point counting.

The length density of myocardial capillaries (LV) was determined using the equation LV=2 QA (with QA is area density, e.g. the number of capillary transsects per area of myocardial reference tissue) [7,23]. Intercapillary distance, i.e. the distance between the centres of two adjacent intramyocardial capillaries, was calculated according to a modification of the formula of Henquell and Honig [7].

Volume density (VV) of capillaries, interstitial tissue and myocytes was obtained using the point counting method [24,25] according to the equation PP=VV (with PP is point density). Reference volume was the total myocardial tissue (exclusive of non-capillary vessels, i.e. arterioles and veins).

Statistics
All data are expressed as mean±standard deviation. After testing for normality distribution one-way analysis of variance (ANOVA) was performed, followed by the least significant difference test to assess the differences between the groups. Results were considered significant when the probability of error (P) was less than 0.05.



   Results
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Description of the model (Table 1Go, Figure 1Go)
In order to avoid confounding effects of anaemia per se resulting from partial nephrectomy, only a modest amount of renal tissue was removed so that only a minor increase in S-creatinine and no change in haemoglobin (Hb) occurred. Systolic BP was increased even in such animals with modest partial renal ablation. A remarkably higher left ventricular weight and left ventricular/body weight ratio was noted after partial nephrectomy. The increase in left ventricular weight was almost competely prevented by the rise in Hb which was induced by rhEpo treatment even though in the untreated animals with partial nephrectomy the Hb was perfectly within the normal range (Figure 1Go).


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Table 1. Characterization of the experimental model of partial renal ablation

 


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Fig. 1. Effect of Epo on left ventricular hypertrophy in subtotally nephrectomized rats. *P<0.05 vs untreated SNX.

 
Serum Epo levels were comparable at baseline (18.3±1.53 mU/ml in untreated and 21.7±2.5 mU/ml in rhEpo treated sham, 26.3±9.3 mU/ml in untreated and 23.0±7.8 mU/ml rhEpo treated SNX). The values 24 h after the injection of rhEpo were significantly higher in rhEpo treated sham (55±8 mU/ml) and SNX (66.7±7.5 mU/ml) compared to untreated sham (18.7±2.1 mU/ml) and SNX (35±1.4 mU/ml).

Capillary density of the heart (Table 2Go, Figure 2Go)
The length density of capillaries, i.e. the total length of capillaries per unit volume of myocardial tissue, was significantly decreased in animals with partial renal ablation compared to sham op. No significant changes were seen after administration of rhEpo whether or not antihypertensive treatment was administered (Table 2Go).


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Table 2. Capillary density and interstitial volume density in the left ventricle

 


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Fig. 2. Ultrastructural findings in sham operated control rats (A) and subtotally nephrectomized rats (B). Electron micrographs, magnification: x3500. The bar indicates 1 µm. (A) Normal ultrastructure of the myocardium in a sham operated control animal. (B) Ultrastructure of the myocardium of a subtotally nephrectomized untreated animal with renal failure of 8 weeks duration. A marked increase in myocyte diameter and intercapillary distance as well as enlargement of interstitial cells (arrow) is noted.

 
The differences in volume density of capillaries were analogous to those of length density (data not shown). As a consequence of diminished capillary supply the mean intercapillary distance was significantly increased in animals with partial renal ablation (18.3±3.16 vs 16.7±3.32 µm in sham operated rats, P<0.025). This finding is illustrated in Figure 2Go. Mean intercapillary distance was not affected by rhEpo treatment whether or not antihypertensive treatment was administered.

Volume density of the cardiac interstitial tissue was significantly higher after partial renal ablation than in the control groups. This increase in interstitial tissue which was not prevented by rhEpo or unspecific antihypertensive treatment, respectively, was accompanied by activation and enlargement of interstitial cells on the ultrastructural level (Figure 2Go).



   Discussion
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
The main finding of the present study was the documentation that treatment with rhEpo, despite its well documented angiogenic properties [8,17,18], fails to prevent the reduction of capillary supply in the left ventricle of rats with partial renal ablation (SNX). The presumed structural basis for the imbalance between oxygen demand and oxygen supply in the heart of the uraemic organism [3] is therefore not affected by rhEpo. This finding is supported by the notion that serum Epo levels were not different in SNX and controls at baseline. However, as a result of the s.c. rhEpo injection they were significantly and comparably increased in sham and SNX.

Nevertheless, the increase in left ventricular weight in SNX was prevented by rhEpo treatment. Thus, the rhEpo induced increase in Hb with the attendant increase in oxygen transport capacity may have a beneficial effect on ischaemia tolerance of the left ventricle in uraemia despite no effect on capillary density [20,26]. The apparently paradoxical increase of left ventricular mass with antihypertensive treatment (Table 1Go) may be due to the fact that BP was lowered with dihydralazine and furosemide, a manoeuvre which may be associated with reflex sympathetic activation. Activation of the sympathetic nervous system can lead to left ventricular hypertrophy or at least may prevent its regression [27].

The present study confirms previous observations of diminished capillary supply in the left ventricle of animals with partial renal ablation [5] and of uraemic patients [6]. As early as 8 weeks after modest partial renal ablation marked cardiac structural abnormalities were seen in the heart, i.e. left ventricular hypertrophy, interstitial expansion, arteriolar wall thickening and capillary deficit. The capillary deficit has previously been shown to be specific for the heart, i.e. it was not seen in other organs of subtotally nephrectomized rats [28]. The most important functional consequence of the reduction in myocardial capillary supply and the concomitant increase in cardiomyocyte diameter and volume [5,6], is an increase in intercapillary oxygen diffusion distance from the capillary to the centre of the cardiomyocyte [7], leading to capillary/myocyte mismatch and thus rendering the myocardium more susceptible to hypoxia [3].

Since BP measuraements were performed by tail plethysmography we cannot formally exclude minor changes in the BP profile which may have affected left ventricular mass. It is remarkable, however, that the left ventricular weight was significantly lower despite higher BP when Hb was raised in rats with partial renal ablation. This finding illustrates the important role of Hb concentration in the development of left ventricular hypertrophy of renal failure [29] and confirms findings in uraemic patients [30].

We acknowledge that the ß-error of the study, i.e. the failure to recognize an actually existing difference, is not neglectable and may account, at least in principle, for our negative finding. Whilst for logistic reasons the sample size is somewhat limited, the precision of the measurements is extremely high because of the many individual counts per heart. Furthermore, in past intervention studies [7] the percent changes of capillary length density with intervention was substantially higher than the minute differences noted in the present study (see Table 2Go).

Although Epo-mRNA is not expressed in most tissues, the upstream regulatory element of the Epo gene which is sensitive to partial pressure of oxygen via the hypoxia induced factor (HIF) is expressed almost ubiquitously [3133]. An important role in the control of capillary supply has been ascribed to HIF [34]. The present study excludes a role of Epo in the development of cardiac capillary abnormalities of experimental renal failure, but obviously not malfunction of this oxygen sensor. We have to acknowledge, however, that the rise in haematocrit resulting from rhEpo treatment may have reduced the hypoxic drive for angiogenesis in the heart of rats. Changes induced by rhEpo treatment may have indirectly affected other angiogenic systems or factors, i.e. angiopoietin, fibroblast growth factor-2 (FGF-2) or vascular endothelial growth factor (VEGF). Potential alternative mechanism(s) which may be involved in the genesis of capillary/myocyte mismatch in renal failure include also ET-1 since treatment with a selective ET-1 receptor A antagonists prevented capillary reduction in SNX [35].

In addition, a potential role of VEGF has also to be considered, since increased expression of VEGF is found in the walls of intramyocardial arterioles after subtotal nephrectomy [35]. VEGF is known to interact with ET-1, e.g. ET-1 and ET-3 stimulate VEGF gene and protein expression in cultured human vascular smooth muscle cells [36]. The effect is similar in magnitude to that of hypoxia and is mediated via the ETA- and ETB-receptor. VEGF is known to stimulate the growth of endothelial cells in vitro [37] and in vivo [38] in many situations of increased angiogenesis.

Whether diminished capillary growth despite increased VEGF expression points to diminished VEGF responsiveness requires further studies.



   Acknowledgments
 
The study was performed with Else-Kröner-Fresenius, Stiftung grants from Deutsche Forschungsgemeinschaft (Am 93/ 2–2/3), Baxter Healthcare Co., USA, and with a financial support of Janssen Cilag Company, Germany. Dr Moriz Buzello, Dr Marie-Luise Gross and Dr Andreas Koch are recipient of a scholarship of the Deutsche Forschungsgemeinschaft (Graduiertenkolleg ‘Kreislauf- und Nierenregulation’ Heidelberg). Dr Gabriel Miltenberger-Miltenyi (Department of Pediatric Nephrology, Semmelweis University Budapest) is recipient of a scholarship of the Deutsche Akademische Austauschdienst (DAAD). Dr Alexander Nabokov is recipient of the International Fellowship Training Award granted by the International Society of Nephrology. The skilful technical assistance of Zlata Antoni, Harald Derks, Gudrun Gorsberg, Diana Lutz, Gaby Merkel, Peter Rieger and Heike Ziebart is gratefully acknowledged.



   Notes
 
Correspondence and offprint requests to: Prof. Dr Kerstin Amann, Department of Pathology, University Erlangen-Nürnberg, Krankenhausstr. 8–10, D-91054 Erlangen, Germany. E-mail:kerstin. amann{at}patho.imed. uni\|[hyphen]\|erlangen. de. Back



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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 

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Received for publication: 6.10.99
Revision received 18. 2.00.