Combination Therapy with an Angiotensin-Converting Enzyme Inhibitor and a Vitamin D Analog Suppresses the Progression of Renal Insufficiency in Uremic Rats
Masahide Mizobuchi*,,
Jeremiah Morrissey*,,
Jane L. Finch*,
Daniel R. Martin*,
Helen Liapis*,,
Tadao Akizawa and
Eduardo Slatopolsky*
* Renal Division, Cell Biology and Physiology, and Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri; and Department of Nephrology, Showa University School of Medicine, Tokyo, Japan
Address correspondence to: Dr. Eduardo Slatopolsky, Renal Division, Box 8126, Washington University School of Medicine, St. Louis, MO 63110. Phone: 314-362-7208; Fax: 314-362-7875; E-mail: eslatopo{at}im.wustl.edu
Received for publication September 21, 2006.
Accepted for publication March 22, 2007.
Monotherapy with angiotensin-converting enzyme inhibitors hasbeen shown to be beneficial in suppressing the progression ofexperimentally induced kidney diseases. Whether such therapyprovides additional benefits when combined with vitamin D oran analog of vitamin D has not been established. Rats were madeuremic by 5/6 nephrectomy and treated as follows: Uremic + vehicle(UC), uremic + enalapril (30 mg/L in drinking water; E), uremic+ paricalcitol (19-nor; 0.8 µg/kg, three times a week),and uremic + enalapril + paricalcitol (E + 19-nor). A groupof normal rats served as control (NC). BP was significantlyelevated in the UC and 19-nor groups compared with the NC groupbut was indistinguishable from normal in the E and E + 19-norgroups. The decrease in creatinine clearance and the increasein the excretion of urinary protein that were observed in theUC group were ameliorated by the use of E alone or by E + 19-nor(P < 0.05 versus UC). The glomerulosclerotic index was significantlydecreased in both the 19-nor (P < 0.01) and E + 19-nor groups(P < 0.01) compared with the UC group. Tubulointerstitialvolume was significantly decreased in both the E (P < 0.05)and E + 19-nor groups (P < 0.01) compared with the UC group.Both macrophage infiltration (ED-1–positive cells) andproduction of the chemokine monocyte chemoattractant protein-1were significantly blunted in E + 19-nor compared with E group.TGF-1 mRNA and protein expression were increased in the UC group(mRNA: 23.7-fold; protein: 29.1-fold versus NC). These increaseswere significantly blunted in the 19-nor group (mRNA: 7.1-fold;protein: 8.0-fold versus NC) and virtually normalized in theE + 19-nor group (protein: 0.8-fold versus NC). Phosphorylationof Smad2 was also elevated in the UC group (7.6-fold versusNC) but less so in the 19-nor–treated rats (5.5-fold versusNC). When rats were treated with E + 19-nor, the phosphorylationof Smad2 was normal (1.1-fold versus NC). Thus, 19-nor can suppressthe progression of renal insufficiency via mediation of theTGF- signaling pathway, and this effect is amplified when BPis controlled via renin-angiotensin system blockade.
The progression of chronic kidney disease (CKD) leads to end-stagekidney disease and consequent renal replacement therapy. Therenin-angiotensin-aldosterone system (RAAS) regulates extracellularvolume homeostasis, which contributes to BP stability. Overactivityof this system is involved in the pathophysiology of kidneydisease. Experimental studies in animals suggest that glomerularhypertension, profibrotic factors, and proteinuria contributeto the kidney damage that is associated with an activated RAAS(1). The initial stage in the development of kidney diseaseis due to a pathologic process that produces nephron injury.The loss of these functioning units results in systemic andglomerular hypertension, with the damage caused by glomerularhypertension being key in the pathophysiology of kidney disease.The hyperfiltration state that is caused by glomerular hypertensioninduces the local stimulation of the RAAS. This local stimulationcauses glomerular injury by further raising glomerular capillarypressure through angiotensin II (AngII)-driven efferent arteriolarvasoconstriction (2). AngII also stimulates cell proliferationand tissue remodeling by enhancing the synthesis of profibroticcytokines and growth factors, among these, TGF-1 (3,4). Becausethe RAAS has an important role in renal pathophysiology, agentsthat inhibit this system, such as angiotensin-converting enzymeinhibitor (ACEI) or AngII receptor 1 blocker, have been shownto have beneficial effects in patients with CKD (5–14).
Calcitriol, 1,25-dihydroxyvitamin D3, and its analogs have beenshown to have therapeutic potential in attenuating experimentallyinduced kidney disease (15–18). Treatment with calcitriolsuppresses the progression of glomerulosclerosis and albuminuriain subtotally nephrectomized rats (18). Calcitriol (17) andone of its analogs, 22-oxa-calcitriol (16), have been shownto reduce both mesangial cell proliferation and the degree ofglomerulosclerosis in the rat model of anti–Thy-1.1 glomerulonephritis.Calcitriol also alleviates chronic allograft nephropathy inrats by altering TGF-1 and matrix-regarding molecules (19).Although most of these studies focused on the effect of calcitriolor one of its analogs on glomerular damage, the suppressiveeffect of calcitriol on renal interstitial myofibroblasts indicatesthat calcitriol has a potential role in suppressing the developmentof renal interstitial fibrosis (20). Recently, Tan et al. (21)demonstrated that the vitamin D analog paricalcitol attenuatesrenal interstitial fibrosis in a mouse model of obstructivenephropathy. Agarwal et al. (22) reported that paricalcitolalso has an antiproteinuric effect on patients with CKD.
Activation of TGF-1 has a crucial role in matrix expansion andin the development of fibrosis in experimentally induced kidneydisease (23–26). Binding of TGF-1 to its cell-surfacereceptors results in the phosphorylation of receptor-activatedTGF- signaling proteins known as Smads. Individually, Smad2or Smad3 can interact with the co-Smad Smad4 to form binarycomplexes that translocate to the nucleus and regulate targetedgene expression. There is a direct link between the vitaminD receptor and Smads, indicating an interaction between calcitrioland the TGF- signaling pathway (27,28). Because clinical studiessuggest that not only RAAS blockers but also calcitriol or itsanalogs have beneficial effects on the progression of kidneydisease (22,29), we tested the effect of combination therapywith the ACEI enalapril and the vitamin D analog paricalcitolon the progression of renal insufficiency in the 5/6 nephrectomyuremic rat model.
Experimental Protocol
All studies were approved by the Washington University AnimalStudies Committee in accordance with federal regulations. Renalinsufficiency was induced by 5/6 nephrectomy in a group of femaleSprague-Dawley rats that weighed 225 to 250 g. The 5/6 nephrectomyinvolves the ligation of several branches of the left renalartery and excision of the right kidney. All animals were feda diet that contained 0.9% phosphorus and 0.6% calcium (Dyets,Bethlehem, PA). At the onset of renal insufficiency, uremicrats were given the ACEI enalapril in their drinking water (30mg/L) and treated three times a week for 4 mo with 100 µlof vehicle (propylene glycol) or 0.8 µg/kg 19-nor as follows:Uremic + vehicle (UC), uremic + enalapril (E), uremic + paricalcitol(19-nor), and uremic + E + 19-nor (E + 19-nor). A group of normalrats that were treated with vehicle served as control (NC).
After 5/6 nephrectomy, BP was measured monthly using the Non-InvasiveBlood Pressure System XBP1000 (Kent Scientific Corp., Torrington,CT). During the last 3 d of the 4-mo treatment period, the ratswere placed in metabolic cages and 24-h urine samples were collected.The first 24-h period was discarded, and the average 24-h urinaryexcretion of protein, calcium (Ca), and phosphorus (P) and thecreatinine clearance (Ccr) for the last 2 d of treatment weremeasured. After 4 mo, rats were killed by exsanguination viathe dorsal aorta, and blood was retained for analytical determinations.The remnant kidney was removed and divided into two sectionsperpendicular to the longitudinal axis. One piece was rinsedin 1% PBS and fixed in 10% formalin for histologic examination.The renal cortex was dissected from the remaining piece, frozenimmediately in liquid nitrogen, and stored at –80°Cuntil analysis.
Analytical Determinations
Urine samples were acidified and analyzed for 24-h excretionof creatinine (Cr), total protein, Ca, and P. Serum and urinarylevels of P and Cr, as well as urinary protein, were measuredby autoanalyzer (COBAS-MIRA Plus, Branchburg, NJ). Total serumand urinary Ca were measured by atomic absorption spectrophotometry(1100B; Perkin-Elmer, Norwalk, CT). Ionized Ca was measuredusing a Nova 8 electrolyte analyzer (Nova Biomedical, Waltham,MA). Ccr was calculated as previously reported (30). Intactparathyroid hormone (PTH) was measured by an immunoradiometricassay specific for intact rat PTH (Immutopics, San Clemente,CA).
Histologic Examination
Kidney tissue was embedded in paraffin, sectioned, and stainedwith Masson trichrome reagent. One hundred glomeruli were randomlyselected for determination of glomerulosclerosis. Glomerulithat exhibited adhesion of the capillary tuft to the Bowman'scapsule, capillary obliteration, mesangial expansion, or fibroticcrescents were defined as glomerulosclerotic. The extent ofglomerular damage was expressed as the percentage of glomerulithat exhibited sclerosis. For evaluation of the extent of renalinterstitial expansion, the fraction of renal cortex that wasoccupied by interstitium that stained positively for extracellularmatrix components by Masson Trichrome was quantitatively evaluatedby a point-counting technique in 10 randomly selected microscopicfields, at a final magnification of x200 under a 100-point grid(31). Blind analysis was done on all sections by the same observer.
Real-Time PCR
Total RNA from kidney was extracted using TRIzol in accordancewith the manufacturer's instructions (Invitrogen, Carlsbad,CA). Five micrograms of total RNA was reverse-transcribed tofirst-strand cDNA using Superscript II Reverse transcriptase(Invitrogen). Synthesized cDNA was amplified by a standard PCRprotocol using SYBR Green Jump Start TaqReady Mix (Sigma, St.Louis, MO) and rat-specific primers for TGF-1 and monocyte chemoattractantprotein-1 (MCP-1). Parallel amplifications with primers forglyceraldehyde-3-phosphate dehydrogenase (GAPDH) were performed.The primer set for TGF-1, MCP-1, and GAPDH primers were purchasedfrom Qiagen (Germantown, MD; assay ID of TGF-1 Rn_Tgfb1_1_SG,MCP-1 Rn_Ccl2_1_SG, and GAPDH Rn_Gapd_2_SG). Cycling conditionswere 10 min of preincubation at 95°C, 15 s of denaturationat 95°C, and 1 min of annealing at 56°C for 40 cyclesusing a 5700 Sequence Detection System (Applied Biosystems,Foster City, CA). The relative amounts of TGF-1, MCP-1, andGAPDH mRNA in each sample were determined compared with serialdilutions of a normal control and computed by the GeneAmp system.For correction for the amount of mRNA, the ratios of TGF-1 andMCP-1 to GAPDH mRNA were calculated in each sample. The amountof TGF-1, MCP-1, and GAPDH mRNA in the UC, E, 19-nor, and E+ 19-nor samples was normalized to the value of the NC samples.All measurements were performed in duplicate.
Western Blot Analysis
Protein expression of TGF-1, Smad2, phosphorylated Smad2, Smad7,and -actin were determined by Western blot analysis. Briefly,renal cortex tissue was homogenized in 2 ml of Cell Lysis Buffer(Cell Signaling Technology, Beverly, MA). Samples were centrifugedat 3000 x g for 15 min, and the supernatants were assayed. Afterbeing mixed with SDS-PAGE sample buffer and boiled for 5 min,samples (8 µg per lane) were electrophoresed on 4 to 12%SDS polyacrylamide gels and transferred to nitrocellulose membranesfor 2 h at 30 V. Membranes were blocked for 30 min with Tris-bufferedsaline that contained 5% BSA (5% BSA/TBS) and incubated withdiluted primary antibody overnight at room temperature in 5%BSA/TBS that contained 0.05% Tween 20. The source and the concentrationof each antibody were as follows: Rabbit anti–TGF-1 (SantaCruz Biotechnology, Santa Cruz, CA; 1:200), anti-Smad2 and anti–phosphorylatedSmad2 (Cell Signaling Technology; 1:500), goat anti-Smad7 (SantaCruz Biotechnology; 1:500), and rabbit anti–-actin antibody(Santa Cruz Biotechnology; 1:500). The membranes were washed,and diluted secondary horseradish peroxidase (HRP)-conjugatedantibodies were added (anti-rabbit IgG-HRP 1:1000; anti-goatIgG-HRP 1:1000; Santa Cruz Biotechnology). The membranes wereagain washed and developed using the enhanced chemiluminescencesystem (LumiGLO Reagent; Cell Signaling Technology). Changesin TGF-1 and Smad7 expression were normalized by correctionfor the densitometric intensity of -actin for each sample.
Immunohistochemistry
Immunohistochemical staining for ED-1 was performed by usinga mouse anti-rat ED-1 mAb (MCA341R; Serotec, Oxford, UK) anda commercial staining kit (Histostain-Plus, mouse; Zymed Laboratories,South San Francisco, CA). Negative control was obtained by substitutingmouse preimmune IgG for the primary antibody. The sections weredeparaffinized, rehydrated, and microwaved in 0.01 mol/L citratebuffer (pH 6.0) for 10 min to retrieve the antigens. The sectionswere then treated with 0.6% hydrogen peroxide in methanol for10 min at room temperature to block endogenous peroxidase andsubsequently blocked with 10% preimmune goat serum for 30 minat room temperature. The primary ED-1 antibody (1:100 dilution)or preimmune IgG was added followed by incubation at room temperaturefor 2 h. Biotinylated secondary antibody was applied, followedby a streptavidin-HRP conjugate. The immune complexes were visualizedwith 3-amino-9-ethylcarbazole substrate-chromagen. Finally,all sections were counterstained with hematoxylin. The positivecell numbers that were stained with ED-1 were counted manuallyin the 20 renal cortical areas including the glomeruli at amagnification of x400. The positive cells were expressed asthe number per field.
Statistical Analyses
All results were expressed as means ± SEM. One-way ANOVAwas used to assess the statistical differences between groups.A post hoc Scheffe test was used to assess the statistical significancein the differences between all possible two-group comparisons.P < 0.05 was considered significant.
BP Figure 1 depicts the BP values from all groups of rats. Uremicrats developed significant hypertension (systolic BP [SBP] 176± 8 mmHg). 19-Nor alone did not control the BP (SBP 171± 15 mmHg). Uremic rats that were treated with E hadnormal BP (SBP 104 ± 3 mmHg), and rats that were treatedwith E + 19-nor also showed BP levels that were indistinguishablefrom the NC (SBP 114 ± 4 mmHg).
Figure 1. Time course of systolic BP in normal and in treated rats after 5/6 nephrectomy. NC, normal controls; UC, uremic controls; E, enalapril; 19-nor, paricalcitol; E + 19-nor, enalapril + paricalcitol. Data are means ± SEM (n = 6 to 14). P < 0.0001 by ANOVA; #P < 0.01 versus NC by post hoc Scheffe test.
Serum Chemistries
Serum chemistries are shown in Table 1. Treatment with 19-noralone did not affect serum Cr, Ca, and P compared with UC rats.Treatment with E resulted in lower serum Cr levels. This renoprotectiveeffect resulted in lower P and PTH levels in both groups thatwere treated with E. Markedly elevated PTH levels (Figure 2)were seen in UC rats (1899 ± 522 pg/ml) compared withthe other uremic groups (E 213 ± 74 pg/ml, 19-nor 146± 78 pg/ml, E + 19-nor 145 ± 65 pg/ml).
Figure 2. Effect of 4 mo of combination therapy of E + 19-nor on serum parathyroid (PTH) level in uremic rats. Data are means ± SEM (n = 6 to 14). P < 0.0001 by ANOVA; *P < 0.01 versus NC; #P < 0.01 versus UC by post hoc Scheffe test.
Urinary Findings
UC rats had a significantly lower Ccr (0.32 ± 0.05 ml/min;P < 0.01) and increased urinary protein excretion (254 ±27 mg/d; P < 0.01) compared with NC rats (Ccr 1.31 ±0.05 ml/min; urinary protein excretion 30 ± 9 mg/d; Table 2).Treatment with 19-nor inhibited the decrease in Ccr by 25% (0.40± 0.06 ml/min) and the increase in urinary protein excretionby 29% (180 ± 24 mg/d) compared with UC rats. The E +19-nor proved more efficacious (Ccr 0.55 ± 0.06 ml/min[P < 0.01]; urinary protein excretion 96 ± 11 mg/d[P < 0.01]). None of the therapeutic interventions had asignificant effect on the urinary excretion of Ca or P.
Renal Histology
Morphologic evaluations and representative images are shownin Figures 3 and 4. Varying degrees of glomerular sclerosis,from global to segmental sclerosis of the glomerular tuft, wereobserved in the UC group and affected 61% of glomeruli (P <0.01 versus NC). 19-Nor alone produced a significant decreasein the percentage of glomeruli that exhibited sclerotic changes(33%) compared with UC rats (P < 0.01). The E + 19-nor furtherdecreased this parameter (12%). An increase in interstitialvolume (23%) was also observed in UC rats (P < 0.01 versusNC). E significantly limited the increase in interstitial volume(15%; P < 0.05 versus UC) and, when combined with 19-nor,produced a further improvement (10%; P < 0.01 versus UC).
Figure 3. Representative microphotograph of Masson trichrome–stained sections from NC (A) and 5/6 nephrectomized rats that were treated with vehicle (B), E (C), 19-nor (D), or a E + 19-nor (E) for 4 mo. Arrow shows FSGS. Magnification, x200.
Figure 4. Quantification of interstitial volume (A) and glomerulosclerosis (B) in rat kidneys from NC and 5/6 nephrectomized rats that were treated with vehicle (UC), E, 19-nor, or a combination of E + 19-nor for 4 mo. Data are means ± SEM (n = 6 to 14). P = 0.0003 for interstitial volume and P < 0.0001 for glomerulosclerosis by ANOVA; *P < 0.01 versus NC; #P < 0.01 and ##P < 0.05 versus UC; •P < 0.01 and ••P < 0.05 versus 19-nor by post hoc Scheffe test.
Effect on Inflammatory Cell Infiltration
For determination of the effect on renal inflammation, the infiltrationof ED-1–positive macrophages (Figure 5), quantificationof the macrophage infiltration (Figure 6), and MCP-1 mRNA expression(Figure 7) in renal tissue were analyzed. The infiltration ofED-1–positive macrophages in UC rats were significantlyincreased compared with NC rats (P < 0.01). This increasewas significantly abrogated in uremic rats that were treatedwith E (P < 0.05 versus UC). When combined with 19-nor, however,the infiltration of ED-1–positive cells was further reduced(P < 0.05 versus E; Figure 6). MCP-1 mRNA expression in UCrats was also significantly increased compared with NC rats(P < 0.01). Treatment with E alone had no suppressive effect,whereas treatment with E + 19-nor significantly suppressed MCP-1mRNA expression compared with E alone (Figure 7).
Figure 5. Representative microphotograph of ED-1–stained sections from NC (A) and 5/6 nephrectomized rats that were treated with vehicle (B), E (C), 19-nor (D), or E + 19-nor (E) for 4 mo. Magnification, x400.
Figure 6. Quantification of ED-1–positive cells in each group. Data are means ± SEM (n = 6 each). P < 0.0001 by ANOVA; *P < 0.01 and **P < 0.05 versus NC; #P < 0.01 and ##P < 0.05 versus UC; •P < 0.01 versus 19-nor; ¶¶P < 0.05 versus E by post hoc Scheffe test.
Figure 7. Effect of 4 mo of combination therapy of E + 19-nor on monocyte chemoattractant protein-1 (MCP-1) mRNA expression level in uremic rats. MCP-1 mRNA expression levels were analyzed by the real-time PCR technique and normalized for the amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA for the NC, UC, E, 19-nor, and E + 19-nor groups. Data are means ± SEM (n = 6 each). P = 0.0114 by ANOVA; *P < 0.01 and **P < 0.05 versus NC; ##P < 0.05 versus UC; ¶¶P < 0.05 versus E by post hoc Scheffe test.
TGF-1 mRNA and Protein Expression
TGF-1 mRNA expression in the kidneys of UC rats was significantlyincreased (23.7-fold) relative to NC rats (P < 0.01; Figure 8).This increase was significantly blunted in uremic rats thatwere treated with 19-nor (P < 0.05; Figure 8). A similartrend was observed in TGF-1 protein expression (Figure 9). Inaddition, with E + 19-nor, TGF-1 protein level was essentiallynormalized (0.8-fold versus NC; Figure 9).
Figure 8. Effect of 4 mo of combination therapy of E + 19-nor on TGF-1 mRNA expression level in uremic rats. TGF-1 mRNA expression levels were analyzed by the real-time PCR technique and normalized for the amount of GAPDH mRNA for the NC, UC, E, 19-nor, and E + 19-nor groups. Data are means ± SEM (n = 6 each). P = 0.0423 by ANOVA; *P < 0.01 versus NC; ##P < 0.05 versus UC by post hoc Scheffe test.
Figure 9. Effect of 4 mo of combination therapy of E + 19-nor on TGF-1 protein level in uremic rats. (A) TGF-1 at 12.5 kD of protein and -actin as a control were analyzed by Western blotting for the NC, UC, E, 19-nor, and E + 19-nor groups. (B) Densitometric quantification of the corresponding bands was performed using an image analyzer. The data are presented after normalization to -actin expression and depicted as times of respective controls. Data are means ± SEM (n = 5 each). P = 0.0050 by ANOVA; *P < 0.01 versus NC; #P < 0.01 versus UC by post hoc Scheffe test.
Effect on the TGF- Signaling Pathway
To examine the effect of the combined therapy of E + 19-noron the intracellular TGF- signaling pathway, we investigatedthe expression of the following components of the TGF- signalingpathway: Total and phosphorylated Smad2 (Figure 10) and Smad7(Figure 11). Phosphorylation of Smad2, however, was markedlyincreased (7.6-fold; P < 0.01) in UC rats compared with NCrats (Figure 10). Phosphorylation of Smad2 was reduced by 28%by treatment with 19-nor alone (P < 0.05), and E + 19-norresulted in normal phosphorylation (1.1-fold versus NC). Thesame effect was observed in Smad7 (Figure 11). UC rats had an11.1-fold increase in Smad7 expression relative to the NC rats.19-Nor treatment alone resulted in decreased Smad7 expression(62%; P < 0.05). Even more efficacious was the combinationof E + 19-nor, which resulted in lower Smad7 expression (0.94-foldversus NC).
Figure 10. Effect of 4 mo of combination therapy of E + 19-nor on the phosphorylation of Smad2 in uremic rats. (A) The expression of total and phosphorylated Smad2 at 58 kD was analyzed by Western blotting for the NC, UC, E, 19-nor, and E + 19-nor groups (B) Densitometric quantification of the corresponding bands was performed using an image analyzer. The data are presented after normalization to total Smad2 expression and expressed as means ± SEM (n = 5 each). P < 0.0001 by ANOVA; *P < 0.01 versus NC; #P < 0.01 and ##P < 0.05 versus UC; •P < 0.01 versus 19-nor by post hoc Scheffe test.
Figure 11. Effect of 4 mo of combination therapy of E + 19-nor on Smad7 protein level in uremic rats. (A) Smad7 at 51 kD and -actin as a control were analyzed by Western blotting for the NC, UC, E, 19-nor, and E + 19-nor groups. (B) Densitometric quantification of the corresponding bands was performed using an image analyzer. The data are presented after normalization to -actin expression and depicted as times of respective controls. Data are means ± SEM (n = 5 each). P = 0.0098 by ANOVA; *P < 0.01 and **P < 0.05 versus NC; #P < 0.01 and ##P < 0.05 versus UC; ••P < 0.05 versus 19-nor by post hoc Scheffe test.
Many clinical studies have shown the beneficial effects of ACEIon delaying the progression of kidney disease (5–8). Thishas led to the widespread use of ACEI in patients with kidneydisease. However, the mechanisms by which the ACEI exert theirbeneficial effects are not yet fully understood. Despite thebenefits gained by the use of ACEI or AngII receptor 1 blocker,renal function continues to decline in the majority of patients,eventually leading to end-stage kidney disease. However, a rolefor abnormal activation of vitamin D receptor (VDR) in the presentationof diseases such as diabetes, cardiovascular and muscular dysfunction,infectious diseases, autoimmune diseases, and cancer cannotbe ignored (32–34). Evidence suggests that calcitrioland the VDR have a crucial role in the regulation of cell proliferationand differentiation and immunomodulation as well as mineralmetabolism. This study demonstrates that treatment with thevitamin D analog 19-nor, which has shown therapeutic potentialin arresting CKD (22), results in an additional renoprotectiveeffect in 5/6 nephrectomized rats when coupled with ACEI therapy.
The most important factors in the progression of kidney diseaseare matrix expansion, interstitial fibrosis, tubular atrophy,and glomerulosclerosis. Because overexpression of TGF-1 is involvedin each of these, the TGF- signaling pathway is considered tobe an important modulator of the progression of renal insufficiency.Neutralizing TGF-1 activity by the use of anti–TGF- antibodieshas been shown to ameliorate experimentally induced kidney diseases(35–38). Calcitriol and its analogs have also been shownto possess a renoprotective effect in ameliorating glomerularsclerotic injury and fibrogenesis of interstitial tissue (15–21).Schwarz et al. (18) reported that calcitriol treatment reducesglomerulosclerosis and albuminuria in subtotally nephrectomizedrats and that this is accompanied by a lower expression of TGF-1in renal tubules. Makibayashi et al. (16) reported that calcitriolor its analog 22-oxa-calcitol attenuates mesangial expansion,glomerulosclerosis, and albuminuria, which are associated withthe reduction in glomerular TGF-1 expression in rats with anti–Thy-1.1glomerulonephritis. Hullett et al. (19) reported that a highdosage of calcitriol preserves renal function and induces expressionof Smad7, an inhibitor of TGF- signaling, in a rat model ofchronic allograft nephropathy. These reports suggest that calcitriolattenuates the progression of experimentally induced kidneydiseases by downregulating the TGF- signaling pathway. Calcitrioltreatment, however, can lead to hypercalcemia and hyperphosphatemia,increasing the risk for the progression of kidney disease. Inthis study, we used an analog of calcitriol, 19-nor, which haslower calcemic and phosphatemic activities than the parent compound(39–42). We show that treatment with 19-nor alone producesa modest but significant renoprotective effect in 5/6 nephrectomizedrats, including inhibition of the TGF- signaling pathway througha reduction of TGF-1 mRNA levels, and that the effect occurswithout affecting serum Ca or P. Because no consensus vitaminD responsive element T but multiple Sp1 binding sites existin the rat TGF-1 promoter, it is likely that 19-nor reductionof TGF-1 mRNA levels involves the inhibition of TGF- gene transcriptionthrough binding of a Sp1/VDR complex to Sp1 sites, as demonstratedfor calcitriol-induced downregulation of p45/Skp2 (43).
Overexpression of TGF-1 is a characteristic feature in manyhuman kidney diseases as well as in the 5/6 nephrectomy ratmodel (44). Although ACEI attenuate the overexpression of TGF-1,the effect of ACEI therapy in combination with vitamin D analogshas not been reported. When 19-nor was combined with E, totalsuppression of TGF-1 and its signaling pathway was observed.Vitamin D has been shown to have an anti-inflammatory effectin experimental kidney disease (17). Macrophages are presentin the experimentally induced kidney diseases, where they areassociated with fibrosis (45). An important chemokine for macrophagesin kidney disease is MCP-1, and MCP-1 is known to be expressedin the 5/6 nephrectomized rat model (46–48). In one ofthese studies (46), E treatment significantly decreased MCP-1expression. In that instance, the E concentration in the drinkingwater was at least three-fold higher than the concentrationthat was used in our study. This may account for the lack ofeffect on MCP-1 expression of E monotherapy in our study butan effect in the previous (46) study. In this study, combinationtherapy with E + 19-nor caused an additional reduction in macrophageinfiltration and chemokine production. These results suggestedthat controlling BP by blocking the RAAS is vital for renoprotectionin the 5/6 nephrectomized rats and that when BP is normallycontrolled, 19-nor exerts an additional beneficial effect inpart because of its anti-inflammatory effects on the progressionof renal insufficiency by mediating TGF- signaling.
One interesting observation in this study is that Smad7, whichhas an inhibitory effect on the TGF- signaling pathway, wasnot induced in the E or E + 19-nor rats. Smad7 has been shownto be downregulated in some kidney disease models (49,50), andits overexpression ameliorates renal fibrosis in rat modelsof ureteral obstruction (51,52) or in a remnant kidney model(53). Conversely, upregulation of Smad7 in podocytes has beenobserved in some human kidney diseases (54). Moreover, Smad7transcription is activated by binding of Smad3 or Smad4, whichare activators of the TGF- signaling pathway, to the Smad7 promoter.This indicates that Smad7 expression is regulated by the TGF-signaling pathway (55). These studies support our data thatSmad7 expression was not induced in the E, E + 19-nor, or NCgroups when the activation of the TGF- signaling pathway wassuppressed and when BP was decreased. It is possible that Smad7is induced during renal disease as a counterregulatory mechanismto the elevated TGF- signaling system. When Smad7 is overexpressedas in the ultrasound-microbubble–mediated gene transferstudies (53), Smad7 expression leads to a demonstrative decreasein renal fibrosis. When E alone or E + 19-nor are used in thetreatment, the suppression of other pathophysiologic factorsmakes it unnecessary for Smad7 expression as a counterregulatoryfactor.
We demonstrated that 19-nor can suppress the progression ofrenal insufficiency via mediation of the TGF- signaling pathway,and this effect is amplified when BP is controlled via renin-angiotensinsystem blockade. The systemic activation of the VDR is importantfor regulating not only Ca homeostasis but also hormone secretion,the immune system, cell proliferation, and cell differentiation.Combination therapy with ACEI and 19-nor may represent a noveland beneficial therapeutic strategy for arresting the progressionof CKD.
E.S. is a consultant/speaker for Genzyme and Abbott. E.S. andWashington University may receive income based on a licenseof related technology by the University of Wisconsin.
Acknowledgments
This study was supported in part by grants from research inrenal disease, Washington University, and by a grant from AbbottPharmaceutical.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
Brewster UC, Perazella MA: The renin-angiotensin-aldosterone system and the kidney: Effects on kidney disease.
Am J Med 116
: 263
–272, 2004[CrossRef][Medline]
Anderson S, Meyer TW, Rennke HG, Brenner BM: Control of glomerular hypertension limits glomerular injury in rats with reduced renal mass.
J Clin Invest 76
: 612
–619, 1985[Medline]
Luft FC: Proinflammatory effects of angiotensin II and endothelin: Targets for progression of cardiovascular and renal diseases.
Curr Opin Nephrol Hypertens 11
: 59
–66, 2002[CrossRef][Medline]
Wolf G: Angiotensin II as a mediator of tubulointerstitial injury.
Nephrol Dial Transplant 15[Suppl 6]
: 61
–63, 2000
Hannedouche T, Landais P, Goldfarb B, el Esper N, Fournier A, Godin M, Durand D, Chanard J, Mignon F, Suo JM, et al.: Randomised controlled trial of enalapril and beta blockers in non-diabetic chronic renal failure.
BMJ 309
: 833
–837, 1994[Abstract/Free Full Text]
Hou FF, Zhang X, Zhang GH, Xie D, Chen PY, Zhang WR, Jiang JP, Liang M, Wang GB, Liu ZR, Geng RW: Efficacy and safety of benazepril for advanced chronic renal insufficiency.
N Engl J Med 354
: 131
–140, 2006[Abstract/Free Full Text]
Kamper AL, Strandgaard S, Leyssac PP: Effect of enalapril on the progression of chronic renal failure. A randomized controlled trial.
Am J Hypertens 5
: 423
–430, 1992[Medline]
Maschio G, Alberti D, Janin G, Locatelli F, Mann JF, Motolese M, Ponticelli C, Ritz E, Zucchelli P: Effect of the angiotensin-converting-enzyme inhibitor benazepril on the progression of chronic renal insufficiency. The Angiotensin-Converting-Enzyme Inhibition in Progressive Renal Insufficiency Study Group.
N Engl J Med 334
: 939
–945, 1996[Abstract/Free Full Text]
Nakao N, Yoshimura A, Morita H, Takada M, Kayano T, Ideura T: Combination treatment of angiotensin-II receptor blocker and angiotensin-converting-enzyme inhibitor in non-diabetic renal disease (COOPERATE): A randomised controlled trial.
Lancet 361
: 117
–124, 2003[CrossRef][Medline]
Praga M, Andrade CF, Luno J, Arias M, Poveda R, Mora J, Prat MV, Rivera F, Galceran JM, Ara JM, Aguirre R, Bernis C, Marin R, Campistol JM: Antiproteinuric efficacy of losartan in comparison with amlodipine in non-diabetic proteinuric renal diseases: A double-blind, randomized clinical trial.
Nephrol Dial Transplant 18
: 1806
–1813, 2003[Abstract/Free Full Text]
Remuzzi A, Perico N, Sangalli F, Vendramin G, Moriggi M, Ruggenenti P, Remuzzi G: ACE inhibition and ANG II receptor blockade improve glomerular size-selectivity in IgA nephropathy.
Am J Physiol 276
: F457
–F466, 1999[Medline]
Ruilope LM, Aldigier JC, Ponticelli C, Oddou-Stock P, Botteri F, Mann JF: Safety of the combination of valsartan and benazepril in patients with chronic renal disease. European Group for the Investigation of Valsartan in Chronic Renal Disease.
J Hypertens 18
: 89
–95, 2000[Medline]
Russo D, Minutolo R, Pisani A, Esposito R, Signoriello G, Andreucci M, Balletta MM: Coadministration of losartan and enalapril exerts additive antiproteinuric effect in IgA nephropathy.
Am J Kidney Dis 38
: 18
–25, 2001[Medline]
Zucchelli P, Zuccala A, Borghi M, Fusaroli M, Sasdelli M, Stallone C, Sanna G, Gaggi R: Long-term comparison between captopril and nifedipine in the progression of renal insufficiency.
Kidney Int 42
: 452
–458, 1992[Medline]
Kuhlmann A, Haas CS, Gross ML, Reulbach U, Holzinger M, Schwarz U, Ritz E, Amann K: 1,25-Dihydroxyvitamin D3 decreases podocyte loss and podocyte hypertrophy in the subtotally nephrectomized rat.
Am J Physiol Renal Physiol 286
: F526
–F533, 2004[Abstract/Free Full Text]
Makibayashi K, Tatematsu M, Hirata M, Fukushima N, Kusano K, Ohashi S, Abe H, Kuze K, Fukatsu A, Kita T, Doi T: A vitamin D analog ameliorates glomerular injury on rat glomerulonephritis.
Am J Pathol 158
: 1733
–1741, 2001[Abstract/Free Full Text]
Panichi V, Migliori M, Taccola D, Filippi C, De Nisco L, Giovannini L, Palla R, Tetta C, Camussi G: Effects of 1,25(OH)2D3 in experimental mesangial proliferative nephritis in rats.
Kidney Int 60
: 87
–95, 2001[CrossRef][Medline]
Schwarz U, Amann K, Orth SR, Simonaviciene A, Wessels S, Ritz E: Effect of 1,25(OH)2 vitamin D3 on glomerulosclerosis in subtotally nephrectomized rats.
Kidney Int 53
: 1696
–1705, 1998[CrossRef][Medline]
Hullett DA, Laeseke PF, Malin G, Nessel R, Sollinger HW, Becker BN: Prevention of chronic allograft nephropathy with vitamin D.
Transpl Int 18
: 1175
–1186, 2005[CrossRef][Medline]
Li Y, Spataro BC, Yang J, Dai C, Liu Y: 1,25-Dihydroxyvitamin D inhibits renal interstitial myofibroblast activation by inducing hepatocyte growth factor expression.
Kidney Int 68
: 1500
–1510, 2005[CrossRef][Medline]
Tan X, Li Y, Liu Y: Paricalcitol attenuates renal interstitial fibrosis in obstructive nephropathy.
J Am Soc Nephrol 17
: 3382
–3393, 2006[Abstract/Free Full Text]
Agarwal R, Acharya M, Tian J, Hippensteel RL, Melnick JZ, Qiu P, Williams L, Batlle D: Antiproteinuric effect of oral paricalcitol in chronic kidney disease.
Kidney Int 68
: 2823
–2828, 2005[CrossRef][Medline]
Kelly DJ, Chanty A, Gow RM, Zhang Y, Gilbert RE: Protein kinase Cbeta inhibition attenuates osteopontin expression, macrophage recruitment, and tubulointerstitial injury in advanced experimental diabetic nephropathy.
J Am Soc Nephrol 16
: 1654
–1660, 2005[Abstract/Free Full Text]
Maric C, Sandberg K, Hinojosa-Laborde C: Glomerulosclerosis and tubulointerstitial fibrosis are attenuated with 17beta-estradiol in the aging Dahl salt sensitive rat.
J Am Soc Nephrol 15
: 1546
–1556, 2004[Abstract/Free Full Text]
Morath C, Dechow C, Lehrke I, Haxsen V, Waldherr R, Floege J, Ritz E, Wagner J: Effects of retinoids on the TGF-beta system and extracellular matrix in experimental glomerulonephritis.
J Am Soc Nephrol 12
: 2300
–2309, 2001[Abstract/Free Full Text]
Morrissey J, Guo G, Moridaira K, Fitzgerald M, McCracken R, Tolley T, Klahr S: Transforming growth factor-beta induces renal epithelial jagged-1 expression in fibrotic disease.
J Am Soc Nephrol 13
: 1499
–1508, 2002[Abstract/Free Full Text]
Yanagi Y, Suzawa M, Kawabata M, Miyazono K, Yanagisawa J, Kato S: Positive and negative modulation of vitamin D receptor function by transforming growth factor-beta signaling through smad proteins.
J Biol Chem 274
: 12971
–12974, 1999[Abstract/Free Full Text]
Yanagisawa J, Yanagi Y, Masuhiro Y, Suzawa M, Watanabe M, Kashiwagi K, Toriyabe T, Kawabata M, Miyazono K, Kato S: Convergence of transforming growth factor-beta and vitamin D signaling pathways on SMAD transcriptional coactivators.
Science 283
: 1317
–1321, 1999[Abstract/Free Full Text]
O'Herrin JK, Hullett DA, Heisey DM, Sollinger HW, Becker BN: A retrospective evaluation of 1,25-dihydroxyvitamin D(3) and its potential effects on renal allograft function.
Am J Nephrol 22
: 515
–520, 2002[CrossRef][Medline]
Cozzolino M, Staniforth ME, Liapis H, Finch J, Burke SK, Dusso AS, Slatopolsky E: Sevelamer hydrochloride attenuates kidney and cardiovascular calcifications in long-term experimental uremia.
Kidney Int 64
: 1653
–1661, 2003[CrossRef][Medline]
Guo G, Morrissey J, McCracken R, Tolley T, Liapis H, Klahr S: Contributions of angiotensin II and tumor necrosis factor-alpha to the development of renal fibrosis.
Am J Physiol Renal Physiol 280
: F777
–F785, 2001[Abstract/Free Full Text]
Dusso AS, Brown AJ, Slatopolsky E: Vitamin D.
Am J Physiol Renal Physiol 289
: F8
–F28, 2005[Abstract/Free Full Text]
Garland CF, Garland FC, Gorham ED, Lipkin M, Newmark H, Mohr SB, Holick MF: The role of vitamin D in cancer prevention.
Am J Public Health 96
: 252
–261, 2006[Abstract/Free Full Text]
Zittermann A: Vitamin D in preventive medicine: Are we ignoring the evidence?
Br J Nutr 89
: 552
–572, 2003[CrossRef][Medline]
Benigni A, Zoja C, Corna D, Zatelli C, Conti S, Campana M, Gagliardini E, Rottoli D, Zanchi C, Abbate M, Ledbetter S, Remuzzi G: Add-on anti-TGF-beta antibody to ACE inhibitor arrests progressive diabetic nephropathy in the rat.
J Am Soc Nephrol 14
: 1816
–1824, 2003[Abstract/Free Full Text]
Ling H, Li X, Jha S, Wang W, Karetskaya L, Pratt B, Ledbetter S: Therapeutic role of TGF-beta-neutralizing antibody in mouse cyclosporin A nephropathy: Morphologic improvement associated with functional preservation.
J Am Soc Nephrol 14
: 377
–388, 2003[Abstract/Free Full Text]
Stern JM, Chen J, Silver RB, Poppas DP, Vaughan ED Jr, Felsen D: Effect of UUO on D1aR expression reveals a link among dopamine, transforming growth factor-beta, and nitric oxide.
Am J Physiol Renal Physiol 286
: F509
–F515, 2004[Abstract/Free Full Text]
Yu L, Border WA, Anderson I, McCourt M, Huang Y, Noble NA: Combining TGF-beta inhibition and angiotensin II blockade results in enhanced antifibrotic effect.
Kidney Int 66
: 1774
–1784, 2004[CrossRef][Medline]
Brown AJ, Finch J, Slatopolsky E: Differential effects of 19-nor-1,25-dihydroxyvitamin D(2) and 1,25-dihydroxyvitamin D(3) on intestinal calcium and phosphate transport.
J Lab Clin Med 139
: 279
–284, 2002[CrossRef][Medline]
Coyne DW, Grieff M, Ahya SN, Giles K, Norwood K, Slatopolsky E: Differential effects of acute administration of 19-Nor-1,25-dihydroxy-vitamin D2 and 1,25-dihydroxy-vitamin D3 on serum calcium and phosphorus in hemodialysis patients.
Am J Kidney Dis 40
: 1283
–1288, 2002[CrossRef][Medline]
Slatopolsky E, Cozzolino M, Finch JL: Differential effects of 19-nor-1,25-(OH)(2)D(2) and 1alpha-hydroxyvitamin D(2) on calcium and phosphorus in normal and uremic rats.
Kidney Int 62
: 1277
–1284, 2002[CrossRef][Medline]
Sprague SM, Llach F, Amdahl M, Taccetta C, Batlle D: Paricalcitol versus calcitriol in the treatment of secondary hyperparathyroidism.
Kidney Int 63
: 1483
–1490, 2003[CrossRef][Medline]
Huang YC, Hung WC: 1,25-Dihydroxyvitamin D3 transcriptionally represses p45Skp2 expression via the Sp1 sites in human prostate cancer cells.
J Cell Physiol 209
: 363
–369, 2006[CrossRef][Medline]
Wu LL, Cox A, Roe CJ, Dziadek M, Cooper ME, Gilbert RE: Transforming growth factor beta 1 and renal injury following subtotal nephrectomy in the rat: role of the renin-angiotensin system.
Kidney Int 51
: 1553
–1567, 1997[Medline]
Floege J, Burns MW, Alpers CE, Yoshimura A, Pritzl P, Gordon K, Seifert RA, Bowen-Pope DF, Couser WG, Johnson RJ: Glomerular cell proliferation and PDGF expression precede glomerulosclerosis in the remnant kidney model.
Kidney Int 41
: 297
–309, 1992[Medline]
Gong R, Rifai A, Tolbert EM, Biswas P, Centracchio JN, Dworkin LD: Hepatocyte growth factor ameliorates renal interstitial inflammation in rat remnant kidney by modulating tubular expression of macrophage chemoattractant protein-1 and RANTES.
J Am Soc Nephrol 15
: 2868
–2881, 2004[Abstract/Free Full Text]
Mu W, Ouyang X, Agarwal A, Zhang L, Long DA, Cruz PE, Roncal CA, Glushakova OY, Chiodo VA, Atkinson MA, Hauswirth WW, Flotte TR, Rodriguez-Iturbe B, Johnson RJ: IL-10 suppresses chemokines, inflammation, and fibrosis in a model of chronic renal disease.
J Am Soc Nephrol 16
: 3651
–3660, 2005[Abstract/Free Full Text]
Taal MW, Zandi-Nejad K, Weening B, Shahsafaei A, Kato S, Lee KW, Ziai F, Jiang T, Brenner BM, MacKenzie HS: Proinflammatory gene expression and macrophage recruitment in the rat remnant kidney.
Kidney Int 58
: 1664
–1676, 2000[CrossRef][Medline]
Fukasawa H, Yamamoto T, Togawa A, Ohashi N, Fujigaki Y, Oda T, Uchida C, Kitagawa K, Hattori T, Suzuki S, Kitagawa M, Hishida A: Down-regulation of Smad7 expression by ubiquitin-dependent degradation contributes to renal fibrosis in obstructive nephropathy in mice.
Proc Natl Acad Sci U S A 101
: 8687
–8692, 2004[Abstract/Free Full Text]
Omata M, Taniguchi H, Koya D, Kanasaki K, Sho R, Kato Y, Kojima R, Haneda M, Inomata N: N-acetyl-seryl-aspartyl-lysyl-proline ameliorates the progression of renal dysfunction and fibrosis in WKY rats with established anti-glomerular basement membrane nephritis.
J Am Soc Nephrol 17
: 674
–685, 2006[Abstract/Free Full Text]
Lan HY, Mu W, Tomita N, Huang XR, Li JH, Zhu HJ, Morishita R, Johnson RJ: Inhibition of renal fibrosis by gene transfer of inducible Smad7 using ultrasound-microbubble system in rat UUO model.
J Am Soc Nephrol 14
: 1535
–1548, 2003[Abstract/Free Full Text]
Terada Y, Hanada S, Nakao A, Kuwahara M, Sasaki S, Marumo F: Gene transfer of Smad7 using electroporation of adenovirus prevents renal fibrosis in post-obstructed kidney.
Kidney Int 61
: 94
–98, 2002[Medline]
Hou CC, Wang W, Huang XR, Fu P, Chen TH, Sheikh-Hamad D, Lan HY: Ultrasound-microbubble-mediated gene transfer of inducible Smad7 blocks transforming growth factor-beta signaling and fibrosis in rat remnant kidney.
Am J Pathol 166
: 761
–771, 2005[Abstract/Free Full Text]
Schiffer M, Schiffer LE, Gupta A, Shaw AS, Roberts IS, Mundel P, Bottinger EP: Inhibitory Smads and TGF-beta signaling in glomerular cells.
J Am Soc Nephrol 13
: 2657
–2666, 2002[Abstract/Free Full Text]
Nagarajan RP, Zhang J, Li W, Chen Y: Regulation of Smad7 promoter by direct association with Smad3 and Smad4.
J Biol Chem 274
: 33412
–33418, 1999[Abstract/Free Full Text]
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Top
Abstract
Introduction
1 mRNA and protein expression were increased in the UC group (mRNA: 23.7-fold; protein: 29.1-fold versus NC). These increases were significantly blunted in the 19-nor group (mRNA: 7.1-fold; protein: 8.0-fold versus NC) and virtually normalized in the E + 19-nor group (protein: 0.8-fold versus NC). Phosphorylation of Smad2 was also elevated in the UC group (7.6-fold versus NC) but less so in the 19-nor–treated rats (5.5-fold versus NC). When rats were treated with E + 19-nor, the phosphorylation of Smad2 was normal (1.1-fold versus NC). Thus, 19-nor can suppress the progression of renal insufficiency via mediation of the TGF-
