This study explored the tissue-protective properties of thecontinuous erythropoietin receptor activator (CERA) in an experimentalmodel of (nonischemic) diabetic kidney injury (i.e., the db/dbmouse). Mice were randomly treated with placebo (n = 25), low-dosageCERA (n = 25), and high-dosage CERA (n = 25). Also studied were25 nondiabetic db/m mice. Hematocrit was comparable in placeboand low-dosage CERA–treated mice but increased significantlywith high-dosage CERA (P < 0.01 versus both). Significantlyreduced expression of TGF-, vascular endothelial growth factor,and collagen IV was found in glomeruli and the tubulointerstitialarea with CERA treatment, and these beneficial molecular effectswere clearly dosage dependent (both P < 0.05 versus placebo).Similarly, CERA treatment caused a dosage-dependent increasein p-Akt, nephrin, and perlecan tissue expression (all P <0.05 versus placebo). However, the accelerated mesangial expansionthat was observed in placebo-treated db/db mice (versus db/mcontrols) was significantly reduced only in low-dosage CERA–treatedmice (P < 0.01). Moreover, albuminuria was significantlyreduced in low- but not high-dosage CERA–treated micecompared with placebo treatment (P < 0.05). In an ancillarystudy, phlebotomy was performed in high-dosage CERA–treateddb/db mice to keep hematocrit within normal (baseline) levels.This procedure resulted in significantly (P < 0.05) lessalbuminuria as compared with high-dosage CERA–treatedmice without phlebotomy, thus preserving the tissue-protectivepotential of CERA. Long-term CERA treatment has beneficial dosage-dependenteffects on molecular pathways of diabetic kidney damage. Low-dosageCERA does not affect hematocrit and therefore may be a feasiblemethod of tissue protection in this setting.
Recent experimental studies revealed that erythropoietin (EPO)has numerous tissue-protective effects apart from its actionon erythropoiesis and that it prevents vascular and tissue damageas a result of ischemia in the heart, the brain, and also thekidney.1,2 EPO maintains normal red blood cell mass by exertinga continuous antiapoptotic activity in erythrocyte precursorsvia stimulation of crucial surviving intracellular signalingcascades such as JAK2/STAT5 and phosphatidylinositol 3-kinase/Aktpathways.2 This signaling subsequently leads to phosphorylationof the proapoptotic factor Bad, which in turn dissociates froma cell survival factor, Bcl-XL, resulting in protection fromprogrammed cell death. We recently demonstrated that long-termtreatment with the recombinant human EPO (rHuEPO) analogue darbepoetin conferred renal vascular and tissue protection, preserved renalfunction, and significantly improved survival in the remnantrat kidney model (five-sixths nephrectomy).3 Importantly, weused a hematologically noneffective dosage of darbepoetin toobviate potential adverse effects of rHuEPO therapy. In thisexperimental setting of chronic renal ischemia, darbepoetinpersistently activated the Akt pathway and reduced apoptoticcell death in renal tissue.
In this study, we set out to explore the therapeutic potentialof another erythropoiesis-stimulating agent—the continuouserythropoietin receptor activator (CERA)—for tissue protectionin an animal model of nonischemic chronic kidney injury (i.e.,the leptin receptor knockout db/db mouse). In this animal modelfor type 2 diabetes, the mice develop characteristic molecularand clinical features of diabetic nephropathy.4 We administereda hematologically noneffective (low) and effective (high) dosageof CERA and studied several molecular pathways of diabetic kidneydamage. CERA is chemically synthesized through integration ofamide bonds between amino groups of rHuEPO and methoxy polyethyleneglycol-succinimidyl butanoic acid. This modification resultsin a substantial prolongation of duration of action.
Clinical and Laboratory Data
The db/db mice became obese and developed frank hyperglycemiaduring the observation period when compared with their nondiabeticdb/m littermates (Table 1). Blood glucose levels were alreadyslightly higher in db/db mice in comparison with db/m mice atthe start of the observation period, and in the next 2 wk, bloodglucose increased significantly and remained consistently higherin db/db mice than in db/m controls. We observed no significantdifference in blood glucose levels between untreated and CERA-treateddb/db mice. Similarly, body weight was not affected by CERAtreatment. Hematocrit increased in all four groups during thestudy period (Table 1), but this increase was more pronouncedin the db/db mice. During the study period, we observed no significantdifference in hematocrit levels between placebo- and low-dosageCERA–treated db/db mice. However, hematocrit increasedsignificantly in high-dosage CERA–treated mice in comparisonwith all other treatment groups. At the end of the treatmentperiod, systolic BP (SBP) was significantly higher in placebo-treateddb/db mice (149 ± 3 mmHg) in comparison with db/m controlmice (135 ± 3 mmHg). BP was not affected by treatmentwith low-dosage (146 ± 3 mmHg) and high-dosage (148 ±4 mmHg) CERA (NS versus placebo-treated db/db mice). Kidneyweight increased markedly in db/db mice and was significantlyhigher than in db/m mice (Table 2). It is interesting that inboth CERA-treated groups, we observed a less marked increasein kidney weight as compared with placebo-treated db/db mice.Similarly, the kidney-to-body weight ratio differed significantlybetween nondiabetic db/m and diabetic db/db mice. In placebo-treateddb/db mice, urinary albumin excretion was significantly higherthan in db/m controls at baseline, week 6, and week 13. Albuminexcretion was significantly reduced compared with placebo-treateddb/db mice at both early and late time points with low-dosageCERA treatment. In the high-dosage group, albuminuria was significantlylower after 6 wk of treatment, whereas later, it returned tothe level that was observed in placebo-treated db/db mice (Table 1).We observed no significant differences in serum creatinine levelsbetween placebo-treated and low-dosage and high-dosage CERA–treatedmice (data not shown).
Table 2. Parameters of kidney function in control mice (db/m) and in db/db mice treated with saline (placebo) and low- and high-dosage CERAa
Renal Histology and Immunohistochemistry
The glomerular appearance in placebo-treated db/db mice showedaccelerated mesangial expansion characterized by an increasein periodic acid-Schiff–positive mesangial matrix arearelative to that observed in db/m mice at the end of the studyperiod (Figure 1). In mice that were treated with low-dosageCERA, the score was significantly reduced (P < 0.01). Incontrast, in mice that were treated with high-dosage CERA, weobserved an even higher score than in placebo-treated mice.Furthermore, we analyzed small arteries for evidence of arteriolarhyalinosis as a marker for diabetic vascular damage (Figure 1).We found no arteriolar hyalinosis in db/m control mice, whereasin placebo-treated db/db mice, a mild degree of arteriolar hyalinosiswas present. Arteriolar hyalinosis was less frequent in micethat were treated with low-dosage CERA, but similar to the previousfinding on mesangial expansion, we observed a higher degreeof arteriolar hyalinosis in mice that were treated with high-dosageCERA.
Figure 1. Mesangial expansion and arteriolar hyalinosis in renal tissue of db/db mice. Diabetic db/db mice were treated with saline (placebo) or continuous erythropoietin receptor activator (CERA) for 14 wk. Db/m nondiabetic mice were used as control. After the perfusion with Ringer solution, kidneys were excised, decapsulated, weighed, immersed in 4% formalin, and embedded in paraffin. Sections were stained with periodic acid-Schiff. (A) Db/m control mice. (B) Db/db placebo-treated mice. (C) Db/db mice that were treated with 0.4 µg CERA/kg per wk (low dosage CERA). (D) Db/db mice that were treated with 1.2 µg CERA/kg per wk (high dosage CERA). A semiquantitative analysis on a scale from 0 to 3 for presence of mesangial expansion and arteriolar hyalinosis (arrows) was performed (E). **P 0.01 versus db/m control; #P 0.05 versus db/db placebo treatment; P 0.05 versus low-dosage CERA.
To examine the underlying molecular mechanism of the clinicaland laboratory observation, we first analyzed the expressionof TGF-1 as an important mediator of diabetic kidney damage(Figure 2, A through D). Not unexpected, we found significantlyhigher TGF-1 expression in db/db mice as compared with db/mcontrols. It is interesting that we observed a dosage-dependentand significant reduction of TGF-1 expression in mice that weretreated with CERA. When we analyzed the collagen IV expressionin glomeruli and the tubulointerstitial area, we found a similarpattern in the different treatment groups (Figure 2, E throughH). Next, we analyzed the expression of nephrin (Figure 3, Athrough D). We found a significant reduction of the nephrinexpression in db/db mice in comparison with db/m mice, whereasin tissue of CERA-treated mice, higher nephrin levels were detectable.The effect was dosage dependent. We found similar results whenanalyzing glomerular perlecan expression (Figure 3, E throughH), a heparan sulphate proteoglycan. It is postulated that suchproteoglycans play an important role in holding up the anioniccharge of the filtration barrier and thereby prevent loss ofalbumin. Moreover, we observed an increase in glomerular vascularendothelial growth factor (VEGF) expression in the db/db versusdb/m mice. This was reduced in a dosage-dependant manner bytreatment with CERA (Figure 3, I through L). Finally, we founda significant reduction of p-Akt expression in renal tissueof placebo-treated db/db mice in comparison with db/m controls(data not shown). This reduction of p-Akt in renal tissue wassignificantly ameliorated in CERA-treated mice. Again, the effectof CERA was dosage dependent.
Figure 2. TGF-1 and collagen IV expression in renal tissue of db/db mice. Diabetic db/db mice were treated with saline (placebo) or CERA for 14 wk. Db/m nondiabetic mice were used as control. Immunochemistry was performed on paraffin sections. Representative pictures are shown for TGF-1 (A through D) and collagen IV (E through H) staining. (A and E) Db/m control mice. (B and F) Db/db placebo-treated mice. (C and G) Db/db mice that were treated with 0.4 µg CERA/kg per wk (low-dosage CERA). (D and H) Db/db mice that were treated with 1.2 µg CERA/kg per wk (high-dosage CERA). TGF-1 expression in glomeruli and the tubulointerstitial collagen IV expression were graded on a scale from 0 to 3. **P 0.01 versus db/m control; #P 0.05 versus db/db placebo treatment; ##P 0.01 versus db/db placebo treatment; P 0.01 versus low-dosage CERA.
Figure 3. Nephrin, perlecan, and vascular endothelial growth factor (VEGF) expression in renal tissue of db/db mice. Diabetic db/db mice were treated with saline (placebo) or CERA for 14 wk. Db/m nondiabetic mice were used as control. Immunochemistry was performed on cryosections. Representative pictures are shown for nephrin (A through D), perlecan (E through H), and VEGF (I through L). (A, E, and I) Db/m control mice. (B, F, and J) Db/db placebo-treated mice. (C, G, and K) Db/db mice that were treated with 0.4 µg CERA/kg per wk (low-dosage CERA). (D, H, and L) Db/db mice that were treated with 1.2 µg CERA/kg per wk (high-dosage CERA). Nephrin, perlecan, and VEGF expression in glomeruli was graded on a scale from 0 to 3. **P 0.01 versus db/m control; #P 0.05 versus db/db placebo treatment; ##P 0.01 versus db/db placebo treatment; P 0.01 versus low-dosage CERA.
Effect of Phlebotomy in High-Dosage CERA–Treated Mice
Phlebotomy in high-dose CERA–treated mice resulted inhematocrit levels that were comparable to baseline values beforethe start of CERA treatment (Table 3). SBP at the end of thetreatment period in high-dosage CERA–treated mice andin phlebotomized mice was comparable (142 ± 6 versus146 ± 8 mmHg; NS). In phlebotomized mice, kidney weightas well as the kidney-to-body weight ratio were not significantlydifferent from those in high-dosage CERA–treated db/dbmice (data not shown). In contrast, in phlebotomized, high-dosageCERA–treated mice, urinary albumin excretion after 6 and13 wk of treatment was significantly lower as compared withmice that were treated with high-dose CERA only (Table 3). Ingeneral, albumin excretion was lower in both groups of db/dbmice as compared with those in the first experimental seriesbecause in this experiment, we studied younger animals (4 wkof age). The immunohistochemical analysis of renal tissue revealedsimilar patterns of TGF-1, nephrin, and perlecan expressionin both groups of db/db mice as compared with the high-dosageCERA–treated mice in the first experiment (Figure 4).Above that, TGF-1 expression was comparable in mice that weretreated with high-dosage CERA and in high-dosage CERA–treated,phlebotomized mice (Figure 4, A and B). This was also true fornephrin (Figure 4, C and D) and perlecan (Figure 4, E and F)tissue expression. In summary, TGF-1, nephrin, and perlecanexpression was not significantly different in mice that weretreated with high-dosage CERA (Figure 4, A, C, and E) and high-dosageCERA–treated, phlebotomized db/db mice (Figure 4, B, D,and F). Finally, we found a comparable expression of p-Akt expressionin renal tissue of both groups of mice (data not shown).
Figure 4. TGF-1, nephrin, and perlecan in renal tissue of phlebotomized db/db mice. Diabetic db/db mice were treated with 1.2 µg CERA/kg per wk (high-dosage CERA) and high-dosage CERA plus phlebotomy for 14 wk. Immunochemistry was performed on paraffin sections. Representative pictures are shown for TGF-1 (A and B), nephrin (C and D), and perlecan (E and F) staining. (A, C, and E) Db/db mice that were treated with high-dosage CERA. (B, D, and F) Phlebotomized db/db mice that were treated with high-dosage CERA. TGF-1, nephrin, and perlecan expression in glomeruli was graded on a scale from 0 to 3. *P 0.05, **P 0.01 versus high-dosage CERA.
In this study, we documented that long-term treatment with CERAhas striking effects on different molecular pathways in an experimentalmodel of diabetic kidney damage: The db/db mouse. Long-termtreatment with CERA not only reduced mediators of accumulationof extracellular matrix in diabetic nephropathy, such as TGF-1,but also prevented the loss of nephrin in glomeruli of treatedmice. To our knowledge, this is the first description of a positiveeffect of an erythropoiesis-stimulating agent in a model ofnonischemic renal tissue injury. So far, positive effects ofrHuEPO administration have been documented only in experimentalmodels of acute ischemia/reperfusion kidney injury5–8or chronic vascular injury to the kidney,3 similar to what hasbeen shown in numerous studies in the heart.2 In these experimentalsettings, the main action of rHuEPO was inhibition of apoptosisin renal and cardiac tissue. However, in most of these studies,very high dosages of rHuEPO have been administered only onceor for a short period to obviate potential adverse effects ofrHuEPO therapy. Particularly, effects on the number and activationstate of thrombocytes and the stimulation of platelet adherenceto endothelium could mitigate the beneficial effects of a long-termhigh-dosage therapy on the kidney.9,10 In addition, changesin blood viscosity because of marked erythrocytosis and tissuegeneration of endothelin may work against the positive effectsof rHuEPO.11 In line with these experimental data are our findingswith high-dosage CERA treatment in db/db mice. We could clearlyshow beneficial dosage-dependent effects of CERA on molecularpathways of diabetic kidney damage, yet mesangial expansionwas not reduced after 14 wk of high-dosage CERA treatment. Asa consequence, urinary albumin excretion rate, which decreasedby week 6 of treatment, increased again with ongoing high-dosagetherapy. This result is in contrast with low-dosage CERA, forwhich the increase in albuminuria during the observation periodwas significantly reduced compared with placebo treatment, althoughthis renoprotective effect of low-dosage CERA was not complete.
To explore further the confounding effect of increased hematocritlevels in this experimental setting, we performed an additionalseries of experiments in which high-dosage CERA–treatedmice were phlebotomized to achieve hematocrit levels that werecomparable to those at baseline (i.e., before the start of high-dosageCERA treatment). We could indeed show that this procedure preventedthe adverse effects of increased hematocrit levels (i.e., reducedthe increase in microalbuminuria in high-dosage CERA–treatedanimals, thereby exposing the tissue-protective potential ofCERA). Further studies are warranted to elucidate the molecularmechanism(s) by which a rise in hematocrit with hematologicallyeffective CERA dosages (and presumably also with rHuEPO) lessensthe beneficial effects on the molecular pathways of diabeticnephropathy. Moreover, establishing a minimal effective dosagemay also provide a new strategy to prevent progression in diabetickidney disease as well as other conditions of chronic kidneyinjury.
We provide evidence for different mechanisms by which CERA mayexert its protective effect on diabetic kidney damage. CERAtreatment significantly reduced TGF-1 expression in renal tissueof db/db mice. Overexpression of TGF-1 plays an important integralrole in the development of diabetic nephropathy, because thisfibrogenic cytokine stimulates podocyte expression of collagenIV and VEGF, and the latter action in turn may increase theactivity of the VEGF autocrine loop.12,13 Inhibition of TGF-1with a specific antibody almost completely prevented the mesangialexpansion in db/db mice.14,15 Increased TGF-1 levels can alsocause podocyte detachment and/or apoptosis.16 Thus, inhibitionof TGF-1–induced production of collagen IV by CERA mayreduce mesangial matrix expansion. In addition, decreased podocyteloss may be another beneficial effect of CERA-induced downregulationof TGF-1. CERA treatment could also directly diminish the activityof the VEGF autocrine loop. It has been documented that neutralizationof VEGF with a systemically administered anti-VEGF antibodymarkedly reduced the urinary albumin excretion in db/db miceand the streptozotocin type 1 diabetes model.17,18 Expressionof VEGF in the glomerulus is most pronounced in podocytes, whereVEGF mRNA and protein expression is stimulated by high glucoseconcentrations and TGF-1.12 Increased activity of the VEGF autocrineloop plays an important role in podocyte biology because VEGFby itself stimulates podocytes to produce collagen IV.13,19Theoretically, treatment with CERA could have mitigated intrarenalVEGF activity indirectly by ameliorating diabetic tissue damageand/or through a direct effect on the VEGF/VEGF receptor pathway.Recently published experimental data provide evidence for sucha cross-link between the EPO/EPO receptor and the VEGF/VEGFreceptor system, at least in the vascular system.20
Finally, CERA may also directly act on podocytes, possibly byactivating Akt, similar to what has been shown for rHuEPO inendothelial glomerular and tubular kidney cells.21 In addition,the CERA-induced increase in nephrin and perlecan cell contentmay result in reduced glomerular basement membrane permeabilityand albuminuria. Perlecan is a negatively charged heparan-sulfateproteoglycan that is responsible for permselectivity of thenegatively charged glomerular basement membrane, whereas nephrin,a transmembrane protein with a large extracellular portion,forms the molecular substrate of the slit diaphragm.22 It isinteresting that an important target of nephrin-induced signalingis Bad, the proapoptotic protein of the Bcl-2 family that isalso involved in erythrocyte proliferation.23 Thus, CERA-inducedsignaling and maintenance of adequate nephrin content couldprevent podocyte apoptosis, a truly beneficial effect giventhat terminally differentiated podocytes do not proliferate.23In diabetes, nephrin protein production is downregulated, andthe decrease in nephrin correlates with the broadening of thefoot process widths.24,25 In addition, the podocyte number andtheir density have been reported to be markedly reduced in glomeruliof patients with type 1 and type 2 diabetes.26,27 In this regard,protection of podocytes seems to be of paramount importancein preventing the development and progression of human diabeticnephropathy.22,28,29
Long-term treatment with a long-acting erythropoiesis-stimulatingagent has striking dosage-dependent effects on molecular pathwaysof diabetic kidney damage. However, in contrast to high-dosageCERA treatment, which resulted in a significant increase inhematocrit that abrogated these beneficial effects, low-dosagetherapy (as well as phlebotomy- in high-dosage–treatedanimals) fully exposed the tissue-protective potential of CERA.Therefore, treatment with low-dosage CERA may be a feasiblemethod of long-term tissue protection, and further studies areneeded to establish minimal effective therapeutic dosages thatare not associated with potentially harmful consequences ofan increase in hematocrit. This proposal is corroborated bythe results of recently published large trials in patients withchronic kidney disease, in which (almost) complete correctionof anemia with an erythropoiesis-stimulating agent did not retardprogression.30,31
Animal Model and Study Protocol
The Animal Care Committee of Lower Saxony approved the study.We studied 75 male diabetic db/db (BKS.Cg-m+/+Leprdb) and 25male nondiabetic db/m control mice (Charles River Laboratories,Sulz-feld, Germany). All mice were 6 wk of age at the beginningof the study. They were housed with 12:12-h light-dark cyclesand had free access to food and water. Using a parallel-groupstudy design, we randomly allocated the db/db mice to receivea weekly injection of (1) 0.9% NaCl (placebo; n = 25), (2) 0.4µg CERA/kg body wt (low dosage; n = 25), and (3) 1.2 µgCERA/kg body wt (high dosage; n = 25). CERA was provided byRoche Diagnostics (Penzberg, Germany). It is chemically synthesizedthrough integration of amide bonds between amino groups of rHuEPOand methoxy polyethylene glycol-succinimidyl butanoic acid.The resulting molecular weight of approximately 60,000 Da causesa considerably prolonged elimination half-life, thereby prolongingthe action of CERA on its target tissues. Recently publishedreports have specified that the half-life of action of CERAin humans is approximately 130 h.32 We determined hematologicallynoneffective (low) and effective (high) dosages of CERA in anancillary dosage-finding experiment.
The main study duration was 14 wk, and we assessed body weightof mice at baseline and every 2 wk after the start of treatment.In addition, SBP was measured in conscious mice with an occlusivetail-cuff plethysmograph attached to a pneumatic pulse transducer(TSE BP system, Bad Homburg, Germany). For these measurements,we kept mice at 37°C. Furthermore, we assessed blood glucosewith an Ascensia glucometer (Bayer Diagnostics, Leverkusen,Germany) and hematocrit with a vet scil abc (SCIL, Vierheim,Germany) at baseline and at regular intervals throughout thestudy. Finally, for the assessment of 24-h albuminuria, we placedmice in separate mouse diuresis cages (Tecniplast, Hohenpeissenberg,Germany) with access to water (but not food) for 24 h. We measuredurinary albumin concentration at baseline and after 6 and 13wk with an ELISA specific for mouse albumin (Albuwell M; Exocell,Philadelphia, PA). SBP was measured by a noninvasive tail-cuffmethod with the BP-2000 BP analysis system (Visitech Systems,Apex, NC). Ten mice from each group underwent repeated measurementson two different days 2 wk before the study end.
Renal Histology and Immunohistochemistry
After 14 wk of follow-up, we obtained renal tissue for morphologicand immunohistochemical analyses. For this purpose, 10 micefrom each group were narcotized and the kidneys were perfusedwith Ringer lactate via the left ventricle. After the mice werekilled, sagittal sections of the kidney were fixed in 4% neutralbuffered formalin, embedded in paraffin, sectioned at 2 µm,and stained with the periodic acid-Schiff reagent. Coded tissuesections were analyzed by an investigator who was blinded withrespect to the allocation of mice to the treatment groups. Fortyglomeruli from each mouse were examined, and the amount of mesangialexpansion was graded as follows: 0, no changes; 1, minor, mesangialexpansion up to the diameter of two nuclei; 2, moderate, mesangialexpansion with a diameter of 3 to 4 nuclei; 3, severe, mesangialexpansion with a diameter of more than 4 nuclei. Similarly,arteriolar hyalinosis in the tissue sections was graded as follows:0, no changes; 1, minor, few arterioles involved; 2, moderate,<50% of arterioles involved; 3, severe, >50% of arteriolesinvolved.
Immunohistochemistry was performed on cryostat sections of thefrozen kidneys or on paraffin sections as described previously.33The following primary antibodies were used: Anti–TGF-1(Santa Cruz Biotechnology, Santa Cruz, CA; cat. no. sc-146),anti-VEGF (Santa Cruz Biotechnology; cat. no. sc-152), anti–typeIV collagen (Southern Biotechnology, Birmingham, AL; cat. no.1340-01), polyclonal rabbit anti-rat phospho-Akt (Ser473), polyclonalguinea pig anti-nephrin (Research Diagnostics, Concord, MA),and monoclonal rat anti-perlecan (Research Diagnostics). Forindirect immunofluorescence, nonspecific binding sites wereblocked with 10% normal donkey serum (Jackson ImmunoResearch,West Grove, PA) for 30 min. Sections were then incubated withthe primary antibody for 1 h. For fluorescence visualizationof bound primary antibodies, sections were further incubatedwith Cy3-conjugated secondary antibodies (Jackson ImmunoResearch)for 1 h. Specimens were analyzed using a Zeiss Axioplan-2 imagingmicroscope with the computer program AxioVision 3.0 (Zeiss,Jena, Germany). Semiquantitative analysis of nephrin, perlecan,TGF-1, and VEGF expression in 40 glomeruli per animal; of collagenIV and p-AKT expression in the kidney; and TGF-1, collagen I,and p-AKT expression in the heart was done by using the followingscoring system: 0, no; 1, weak; 2, moderate; and 3, strong expression.The investigator performing these immunohistochemical analyseshad no knowledge of the treatment group assignment.
Effect of Phlebotomy in High-Dosage CERA–Treated Mice
In an additional experiment, we investigated the effect of phlebotomyin high-dosage CERA–treated db/db mice. Using a parallel-groupstudy design, we randomly allocated 4-wk-old db/db mice to receivea weekly injection of (1) 1.2 µg CERA/kg body wt (highdosage; n = 20) and (2) 1.2 µg CERA/kg body wt (n = 20)plus phlebotomy. Phlebotomy was performed in narcotized miceat regular intervals throughout to keep hematocrit within normal(i.e., baseline) levels. The assessment of clinical and laboratorydata and the immuno-histochemical evaluation of renal tissuewere identical as described previously. In these additionalexperiments, we analyzed only the expression of key moleculesof diabetic injury in renal tissue (TGF-1, nephrin, and perlecan).
Statistical Analyses
We used the InStat statistical program (InStat, San Jose, CA).The statistical significance was set at P < 0.05, and dataare presented as means ± SEM. We compared baseline andend point characteristics with ANOVA and appropriately correctedt test for random data.
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Abstract
Introduction
, vascular endothelial growth factor, and collagen IV was found in glomeruli and the tubulointerstitial area with CERA treatment, and these beneficial molecular effects were clearly dosage dependent (both P < 0.05 versus placebo). Similarly, CERA treatment caused a dosage-dependent increase in p-Akt, nephrin, and perlecan tissue expression (all P < 0.05 versus placebo). However, the accelerated mesangial expansion that was observed in placebo-treated db/db mice (versus db/m controls) was significantly reduced only in low-dosage CERA–treated mice (P < 0.01). Moreover, albuminuria was significantly reduced in low- but not high-dosage CERA–treated mice compared with placebo treatment (P < 0.05). In an ancillary study, phlebotomy was performed in high-dosage CERA–treated db/db mice to keep hematocrit within normal (baseline) levels. This procedure resulted in significantly (P < 0.05) less albuminuria as compared with high-dosage CERA–treated mice without phlebotomy, thus preserving the tissue-protective potential of CERA. Long-term CERA treatment has beneficial dosage-dependent effects on molecular pathways of diabetic kidney damage. Low-dosage CERA does not affect hematocrit and therefore may be a feasible method of tissue protection in this setting. 
conferred renal vascular and tissue protection, preserved renal function, and significantly improved survival in the remnant rat kidney model (five-sixths nephrectomy).3 Importantly, we used a hematologically noneffective dosage of darbepoetin to obviate potential adverse effects of rHuEPO therapy. In this experimental setting of chronic renal ischemia, darbepoetin persistently activated the Akt pathway and reduced apoptotic cell death in renal tissue. 
0.01 versus db/m control; #P 
P 
