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Effects of lisinopril administration on blood bcl-2 concentrations in patients with immunoglobulin A nephropathy. Clin Pharmacol Ther 1999; 65: 649652 25. Onozato ML, Tojo A, Leiper J, Fujita T, Palm F, Wilcox CS. Expression of NG, NG-dimethylarginine dimethylaminohydrolase and protein arginine N-methyltransferase isoforms in diabetic rat kidney: effects of angiotensin II receptor blockers. Diabetes 2008; 57: 172180 26. Gonzalez-Cuadrado S, Lorz C et al. Agonistic anti-Fas antibodies induce glomerular cell apoptosis in mice in vivo. Kidney Int 1997; 51: 17391746 27. Jeremias I, Kupatt C, Martin-Villalba A et al. Involvement of CD95/ Apo1/Fas in cell death after myocardial ischemia. Circulation 2000; 102: 915920 28. Nagata S, Golstein P. The Fas death factor. Science 1995; 267: 14491456 29. Yang W, Zhang Y, Li Y, Wu Z, Zhu D. Myostatin induces cyclin D1 degradation to cause cell cycle arrest through a phosphatidylinositol 3-kinase/AKT/GSK-3 beta pathway and is antagonized by insulinlike growth factor 1. J Biol Chem 2007; 282: 37993808 30. Yilmaz MI, Axelsson J, Sonmez A et al. Effect of renin angiotensin system blockade on pentraxin 3 levels in type 2 diabetic patients with proteinuria. Clin J Am Soc Nephrol 2009; 4: 535541

M.C.J. Slagman et al. 31. Yilmaz MI, Saglam M, Carrero JJ et al. Serum visfatin concentration and endothelial dysfunction in chronic kidney disease. Nephrol Dial Transplant 2008; 3: 959965 32. Wei Y, Sowers JR, Nistala R et al. Angiotensin II-induced NADPH oxidase activation impairs insulin signaling in skeletal muscle cells. J Biol Chem 2006; 281: 3513735146 33. Wei Y, Sowers JR, Clark SE, Li W, Ferrario CM, Stump CS. Angiotensin II-induced skeletal muscle insulin resistance mediated by NFkappaB activation via NADPH oxidase. Am J Physiol Endocrinol Metab 2008; 294: E345E351 34. Boulanger CM, Amabile N, Guerin AP et al. In vivo shear stress determines circulating levels of endothelial microparticles in end-stage renal disease. Hypertension 2007; 49: 902908 35. Benigni A, Corna D, Zoja C et al. Disruption of the Ang II type 1 receptor promotes longevity in mice. J Clin Invest 2009; 119: 524530 36. Yilmaz MI, Saglam M, Carrero JJ et al. Normalization of endothelial dysfunction following renal transplantation is accompanied by a reduction of circulating visfatin/NAMPT. A novel marker of endothelial damage? Clin Transplant 2009; 23: 241248 Received for publication: 17.12.09; Accepted in revised form: 26.2.10

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Nephrol Dial Transplant (2010) 25: 32563260 doi: 10.1093/ndt/gfq149 Advance Access publication 25 March 2010

Erythropoietin is reduced by combination of diuretic therapy and RAAS blockade in proteinuric renal patients with preserved renal function
Maartje C.J. Slagman1, Steef J. Sinkeler1, Marc H. Hemmelder2, Femke Waanders1, Liffert Vogt1, Hanneke C. Kluin-Nelemans3, Gerjan Navis1 and Gozewijn D. Laverman1
1 2

Division of Nephrology, Department of Internal Medicine, University Medical Center Groningen, Groningen, The Netherlands, Division of Nephrology, Department of Internal Medicine, Medical Center Leeuwarden, Leeuwarden, The Netherlands and 3 Department of Hematology, University Medical Center Groningen, Groningen, The Netherlands Correspondence and offprint requests to: Gozewijn D. Laverman; E-mail: g.d.laverman@int.umcg.nl

Abstract Background. Reninangiotensinaldosterone system (RAAS) blockade improves prognosis in renal patients, but usually requires diuretic co-treatment. RAAS blockade can decrease erythropoietin (EPO) and/or haemoglobin (Hb) levels. Diuretics decrease EPO in rodents, but their effect on EPO and Hb in humans is unknown. Methods. Proteinuric renal patients with preserved renal function were treated during 6-week periods with placebo, losartan 100 mg/day (LOS) and LOS plus hydrochlorothiazide 25 mg/day (LOS/HCT), in random order. Results. Hb was inversely related to proteinuria, and EPO levels were inappropriately low in relation to Hb. Hb was lowered by LOS with and without HCT. EPO was decreased by LOS/HCT, but not by LOS. Conclusions. EPO and Hb are reduced by HCT added to LOS in proteinuric renal patients with preserved renal

function. We hypothesize that EPO reduction by HCT is caused by a decrease in renal oxygen requirement, which is the main stimulus for EPO production, due to the inhibition of active tubular sodium reabsorption. Further studies should explore the exact mechanism of this phenomenon and its clinical impact.
Keywords: diuretics; erythropoietin; haemoglobin; proteinuria; renal disease; reninangiotensinaldosterone system blockade

Introduction
Blockade of the reninangiotensinaldosterone system (RAAS) with angiotensin-converting enzyme inhibitors (ACEI) or angiotensin receptor blockers (ARB) reduces

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EPO reduction by diuretics and RAAS blockade Table 1. Baseline characteristics of renal patients Male General parameters Number Age (years) Caucasian race (%) Body mass index (kg/m2) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Creatinine clearance (mL/min) Proteinuria (g/24 h) Haematological parameters Hb (mmol/L) Haematocrit (L/L) Ferritin (g/L) EPO (U/L) O/PEPO Female

3257 Hb, the observed/predicted log EPO ratio (O/PEPO) was calculated as proposed by Westenbrink et al. [14]. O/PEPO in healthy reference subjects (aged 50 5 years) was 0.90 0.029.

24 52 2 100 28 1 144 5 87 3 92 6 3.6 0.5 9.4 0.2 0.45 0.01 153 22 14.8 [12.317.7] 0.65 0.02

9 46 5 100 27 2 140 10 85 4 81 9 4.5 0.08 8.6 0.3* 0.41 0.01* 76 28 12.9 [7.422.5] 0.59 0.05

Data analysis Data obtained during PLA plus HS (HS/PLA) were taken as baseline values. Data are given as the mean standard error or geometric mean [interquartile range] when skewed. Before statistical testing, skewed variables were natural log transformed to obtain normality. Associations between variables were evaluated with Pearsons correlation tests. Therapy effects were determined using paired T-tests, with a Bonferroni correction for multiple testing. P < 0.05 was considered statistically significant. SPSS 16.0 for Windows (SPSS Inc., Chicago, Illinois, USA) was used for all analyses.

Results
General parameters Baseline characteristics are shown in Table 1. During the six different treatment periods, proteinuria decreased from 3.8 0.4 g/day at baseline (HS/PLA) to 1.1 0.2 g/day during RAAS blockade with maximal volume intervention (LS/LOS/HCT, P < 0.001; Figure 2A). Mean arterial pressure decreased accordingly (105 3 mmHg at baseline versus 90 1 mmHg during LS/LOS/HCT, P < 0.001), as previously described in more detail [13]. Creatinine clearance (89 5 mL/min at baseline versus 75 5 mL/min during LS/LOS/HCT, P = 0.001) and body weight (91 3 kg at baseline versus 88 3 kg during LS/LOS/HCT, P < 0.001) decreased as well, consistent with a negative fluid balance during LS and HCT. Haematological parameters At baseline (HS/PLA), Hb was inversely related to proteinuria, but was not related to creatinine clearance (Figure 3). O/PEPO was decreased (0.64 0.02 versus 0.90 0.029 in healthy reference subjects, P < 0.001), indicating that EPO levels were inappropriately low in relation to Hb levels. Hb was decreased by LOS with and without HCT (Figure 2B). EPO levels were reduced by the addition of HCT on top of LOS, but not by LOS monotherapy, compared to PLA (Figure 2C). There was no statistical difference, however, between the effect of LOS monotherapy and the effect of HCT on top of LOS on EPO levels. O/PEPO was further decreased by LOS with and without HCT
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Characteristics of renal patients during HS/PLA. EPO, erythropoietin. * P < 0.05 versus males.

hypertension and proteinuria and improves renal and cardiovascular outcome in chronic kidney disease (CKD) [1,2]. For optimal therapeutic efficacy, co-treatment with diuretics is often required [3,4]. ACEI and ARB decrease erythropoietin (EPO) and/or haemoglobin (Hb) levels in different populations [58] by blocking the effects of angiotensin II on erythropoiesis [9,10]. Diuretics reduce EPO levels in rodents [11,12], but their effect on EPO and Hb in humans is unknown. We report the effects of the diuretic hydrochlorothiazide (HCT) and the ARB losartan (LOS) on EPO and Hb levels in proteinuric CKD patients with preserved renal function.
Materials and methods
Patients and protocol This is a post hoc analysis of a randomized, double-blind, placebo (PLA)controlled cross-over study. The protocol was described in detail elsewhere [13]. In short, 33 non-diabetic CKD patients with overt proteinuria and preserved renal function (Table 1) were included. Patients were treated during 6-week periods with PLA, LOS 100 mg/day, and LOS plus HCT 25 mg/day (LOS/HCT), consecutively combined with a low sodium diet (LS, 92 8 mmol/day) and a high sodium diet (HS, 196 9 mmol/day), in random order (Figure 1). Measurements and calculations EPO levels were measured by chemiluminescence immunoassay (Siemens, Los Angeles, CA, USA). To relate the EPO level to the actual

PLACEBO

LOSARTAN

LOSARTAN / HCT

LOSARTAN

LOSARTAN / HCT

PLACEBO

PLACEBO

LOSARTAN / HCT

LOSARTAN

LOSARTAN / HCT

LOSARTAN

PLACEBO

= high sodium diet

= low sodium diet

Fig. 1. Study design. Renal patients were consecutively treated with PLA, LOS, and LOS plus HCT (LOS/HCT), combined with HS and LS, in random order.

3258

M.C.J. Slagman et al.

A
5

*
4

= high sodium diet = low sodium diet

9.4

= high sodium diet = low sodium diet

hemoglobin (mmol/L)

proteinuria (g/24h)

9.2
@ @ @ @

9.0

*
1 0
PLACEBO LOSARTAN LOSARTAN/HCT

8.8

8.6
PLACEBO LOSARTAN LOSARTAN/HCT

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C
17 16
= high sodium diet = low sodium diet

D
0.68
= high sodium diet = low sodium diet

erythropoietin (umol/L)

15
@ @

0.64

@ @ @

O/PEPO
PLACEBO LOSARTAN LOSARTAN/HCT

14 13 12

0.60

0.56

11 10
0.52
PLACEBO LOSARTAN LOSARTAN/HCT

Fig. 2. Proteinuria and haematological parameters in renal patients during different regimes. LOS/HCT, losartan plus hydrochlorothiazide; O/PEPO, observed/predicted log erythropoietin ratio, i.e. EPO level in relation to Hb level. *P < 0.05 versus all periods, @P < 0.05 versus HS/PLA, P < 0.05 versus LS/PLA, #P < 0.05 versus same treatment on HS (effect of LS), P < 0.05 versus LOS on same diet (effect of HCT). Panel A has been previously published in the Journal of the American Society of Nephrology [13], permission for reproduction was obtained.

(Figure 2D). No clear-cut effect of LS on Hb, EPO or O/ PEPO was observed.

Discussion
We found that Hb levels are inversely related to proteinuria, and EPO levels are inappropriately low in relation to Hb (low O/PEPO) in untreated non-diabetic CKD patients with overt proteinuria and preserved renal function. HCT added to LOS decreases EPO, O/PEPO and Hb in these patients. Hb was inversely related to proteinuria, but was not related to renal function, which strongly suggests an effect of proteinuria per se on Hb levels. This is a new finding. Proteinuria can reduce circulating EPO levels through

urinary EPO loss [1517], which may explain the inappropriately low circulating EPO in our patients at baseline. No relationship was, however, found between circulating EPO and proteinuria in these patients, suggesting that other factors such as inflammation may be involved as well [1820]. Remarkably, HCT added to LOS, while reducing proteinuria, decreased EPO and O/PEPO levels compared to PLA. Although RAAS blockade is known to reduce EPO levels [68], effects of (add-on) diuretics on EPO were not reported before, besides HCT added to enalapril reducing haematocrit in hypertensive patients [21]. In rodents, diuretics reduce renal EPO production [11,12] via the inhibition of tubular sodium reabsorption which reduces renal oxygen consumption and increases renal oxygen pressure [22,23], causing decreased EPO production

EPO reduction by diuretics and RAAS blockade

3259

A
11 r = -0.57, p = 0.001

hemoglobin (mmol/L)

10

Effects of RAAS blockade on EPO and Hb usually become evident 312 weeks after initiation of therapy [5]. Therefore, our treatment periods may have been too short to evaluate the full haematological effect of the treatment regimens. Other limitations are the small sample size, the overall small changes and large variation of EPO levels and the lack of information on HCT monotherapy.

Conclusion
To conclude, Hb levels are inversely related to proteinuria, and EPO levels are inappropriately low in relation to Hb in renal patients with overt proteinuria and preserved renal function. EPO and Hb levels are reduced by HCT added to LOS in these patients. We hypothesize that EPO reduction by add-on HCT is caused by a decrease in renal oxygen requirement, which is the main stimulus for EPO production, due to the inhibition of active tubular sodium reabsorption. Further studies should explore the exact mechanism of this phenomenon and its clinical impact.

7 0 3 6 9 12

proteinuria (g/24h)

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12

Authorship
11

10

n.s.
9

7 0 50 100 150 200

M.C.J.S. performed the analyses and wrote the manuscript. S.J.S. contributed to the study design. M.H.H. supplied the research facilities and wrote the manuscript. F.W. performed the clinical intervention. L.V. performed the clinical intervention. H.C.K.-N. wrote the manuscript. G.N. designed the study, supplied the research facilities and wrote the manuscript. G.D.L. wrote the manuscript.
Acknowledgements. The original study was supported by Merck Sharp & Dohme (grant MSGP NETH-15-01). The funding source had no role in the analysis or interpretation of the results. Conflict of interest statement. None declared.

hemoglobin (mmol/L)

creatinine clearance (mL/min)

Fig. 3. Hb is related to proteinuria, but not to creatinine clearance, in renal patients during HS/PLA.

[24]. This mechanism might also be involved in our patients. Of note, the effects of LS added to LOS were similar to add-on HCT for proteinuria, blood pressure, renal function and body weight, but LS did not affect EPO levels, suggesting a direct pharmacological effect of HCT on EPO rather than a volume-mediated effect. This notion is supported by the finding that the negative fluid balance with an anticipated reduction of the distribution volume of EPO [16] during HCT was associated with a decrease, instead of an increase, in EPO levels. At present, no direct effects of diuretics on erythroid precursor cells are known. EPO production was compromised in our patients, as shown by the low O/PEPO at baseline. Whether HCT can affect uncompromised EPO production cannot be ascertained. It would be relevant to explore this issue in other populations, as (combinations of) diuretics and RAAS blockade are widely used in non-renal conditions such as essential hypertension and heart failure [25,26].

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C. Hotu et al. 18. Stehouwer CD, Gall MA, Twisk JW, Knudsen E, Emeis JJ, Parving HH. Increased urinary albumin excretion, endothelial dysfunction, and chronic low-grade inflammation in type 2 diabetes: progressive, interrelated, and independently associated with risk of death. Diabetes 2002; 51: 11571165 19. Faquin WC, Schneider TJ, Goldberg MA. Effect of inflammatory cytokines on hypoxia-induced erythropoietin production. Blood 1992; 79: 19871994 20. Iversen PO, Woldbaek PR, Tonnessen T, Christensen G. Decreased hematopoiesis in bone marrow of mice with congestive heart failure. Am J Physiol Regul Integr Comp Physiol 2002; 282: R166R172 21. Griffing GT, Melby JC. Enalapril (MK-421) and the white cell count and haematocrit. Lancet 1982; 1: 1361 22. Kiil F, Aukland K, Refsum HE. Renal sodium transport and oxygen consumption. Am J Physiol 1961; 201: 511516 23. Redfors B, Sward K, Sellgren J, Ricksten SE. Effects of mannitol alone and mannitol plus furosemide on renal oxygen consumption, blood flow and glomerular filtration after cardiac surgery. Intensive Care Med 2009; 35: 115122 24. Bauer C, Kurtz A. Oxygen sensing in the kidney and its relation to erythropoietin production. Annu Rev Physiol 1989; 51: 845856 25. Mancia G, Laurent S, gabiti-Rosei E et al. Reappraisal of European guidelines on hypertension management: a European Society of Hypertension Task Force document. J Hypertens 2009 26. Jessup M, Abraham WT, Casey DE et al. 2009 focused update: ACCF/AHA Guidelines for the Diagnosis and Management of Heart Failure in Adults: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines: developed in collaboration with the International Society for Heart and Lung Transplantation. Circulation 2009; 119: 19772016 27. Bahlmann FH, Fliser D. Erythropoietin and renoprotection. Curr Opin Nephrol Hypertens 2009; 18: 1520 Received for publication: 18.1.10; Accepted in revised form: 24.2.10

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Nephrol Dial Transplant (2010) 25: 32603266 doi: 10.1093/ndt/gfq168 Advance Access publication 6 April 2010

A community-based model of care improves blood pressure control and delays progression of proteinuria, left ventricular hypertrophy and diastolic dysfunction in Mori and Pacific patients with type 2 diabetes and chronic kidney disease: a randomized controlled trial
Cheri Hotu1, Warwick Bagg1, John Collins2, Lorraine Harwood1, Gillian Whalley1, Robert Doughty1, Greg Gamble1, Geoffrey Braatvedt1 on behalf of the DEFEND investigators
1 Department of Medicine, University of Auckland, Auckland, New Zealand and 2Department of Renal Medicine, Auckland District Health Board, Auckland, New Zealand

Correspondence and offprint requests to: Geoffrey Douglas Braatvedt; E-mail: g.braatvedt@auckland.ac.nz

Abstract Background. In this study, our main goal was to determine whether an integrated, community-based model of care using culturally appropriate health-care assistants to manage hyper-

tension in Mori and Pacific patients with diabetes and chronic kidney disease (CKD) is more effective than conventional care in achieving blood pressure (BP) targets and delaying progression of cardiac and renal end-organ damage.

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