Renin-Angiotensin System Blockade Is Renoprotective in Immune Complex–Mediated Glomerulonephritis
Shunhua Guo,
Jolanta Kowalewska,
Tomasz A. Wietecha,
Masayuki Iyoda,
Li Wang,
Kenneth Yi,
Min Spencer,
Miriam Banas,
Sanda Alexandrescu,
Kelly L. Hudkins and
Charles E. Alpers
Department of Pathology, University of Washington School of Medicine, Seattle, Washington
Correspondence: Dr. Charles E. Alpers, Department of Pathology, University of Washington, 1959 NE Pacific Avenue, Box 357470, Seattle, WA 98195. Phone: 206-598-6409; Fax: 206-543-6678; E-mail: calp{at}u.washington.edu
Received for publication May 24, 2007.
Accepted for publication January 3, 2008.
Blockade of the renin-angiotensin system is renoprotective ina variety of chronic nephropathies, but the direct effect ofsuch treatment in active, immune complex–mediated glomerulonephritisis unknown. This study investigated the short- and long-termeffects of an angiotensin-converting enzyme inhibitor (enalapril)and an angiotensin II type 1 receptor blocker (losartan) inthymic stromal lymphopoietin transgenic (TSLPtg) mice, whichdevelop mixed cryoglobulinemia and severe cryoglobulinemia-associatedmembranoproliferative glomerulonephritis. Enalapril and losartaneach reduced hypertension, proteinuria, glomerular extracellularmatrix deposition, and mesangial cell activation in TSLPtg mice.These renoprotective effects were not observed with hydralazinetreatment, despite a similar antihypertensive effect. Treatmentwith enalapril or losartan also decreased renal plasminogenactivator inhibitor-1 in TSLPtg mice, assessed by immunohistochemistryand quantitative real-time reverse transcriptase–PCR.None of the treatments affected immune complex deposition ormacrophage infiltration. Overall, enalapril- and losartan-treatedTSLPtg mice survived significantly longer than untreated TSLPtgmice. These studies demonstrate that angiotensin blockade mayprovide renoprotective benefits, independent of its BP-loweringeffect, in the treatment of active immune complex–mediatedglomerulonephritis.
Effective treatment for most forms of glomerulonephritis remainsan elusive goal. In cases of immune complex–mediated glomerulonephritis,specific treatment options are often limited to immunosuppressiveagents such as glucocorticoids and cytotoxic agents, which havethe dual burdens of limited efficacy and multiple severe toxicities.An exception is the case of membranoproliferative glomerulonephritis(MPGN) consequent to cryoglobulinemia associated with longstandinghepatitis C virus (HCV) infection. In that setting, antiviraltherapy directed at the underlying HCV infection, typicallyIFN based, can cause remission of cryoglobulinemia and the MPGNif HCV viremia is eradicated.1 Unfortunately, this occurs inonly a minority of patients with this disorder, and other efficacioustherapies either have not been identified or have been testedin only a small number of patients.
In recent years, blockade of the renin-angiotensin system (RAS)with angiotensin-converting enzyme inhibitors (ACEI) or angiotensinII type 1 receptor blockers (ARB) has shown compelling renoprotectiveeffects in chronic renal diseases of humans and animal models.2,3Most experimental studies of RAS blockade have used chronicnephropathy models resulting from diabetes,4,5 hypertension,6,7nephrotoxicity,8 reduction of renal mass,6,9 or chronic renalinjury subsequent to acute mesangiolysis induced in rats byanti–Thy-1 antibody10,11 or subsequent to establishmentof an immune complex–induced glomerulonephritis.12,13Similarly, the efficacy of RAS blockade in human kidney diseaseshas been best demonstrated in patients with diabetic nephropathy,14,15hypertension,16,17 or chronic kidney disease (CKD) arising frommultiple causes.18–20 These studies largely focused ongeneral effects of RAS blockade on renal function and proteinuriaand did not focus on active immune complex–induced renaldisease. ACEI was tested in an active immune complex depositionmodel of glomerulonephritis in nonhuman primates.13 That study,in which BP was not measured, failed to demonstrate a benefitduring acute injury but showed structural improvements in glomeruliwhen ACEI commenced subsequent to the termination of immunecomplex stimulus. We address this limited information base byexamining the effect of ACEI and ARB in a unique mouse modelof immune complex–mediated MPGN.
We characterized a mouse model of cryoglobulinemia-associatedMPGN resulting from overexpression of thymic stromal lymphopoietin(TSLP), a cytokine that causes abnormalities in B cell development.21,22TSLP transgenic (TSLPtg) mice develop mixed cryoglobulinemiaand a systemic inflammatory disease that involves the kidney,lung, spleen, liver, and skin. These mice develop renal diseasethat closely resembles human cryoglobulinemia-associated MPGNin which the glomeruli show extensive subendothelial and mesangialimmune deposits, marked macrophage influx, mesangial cell proliferation,and mesangial matrix expansion.21 The disease is fully establishedat 50 d of age in females and at 120 d of age in males.
In this study, female TSLPtg and wild-type (WT) mice were treatedfor 4 and 8 wk and male TSLPtg and WT mice were treated for12 and 24 wk with enalapril (ACEI) or losartan (ARB), and additionalgroups of female mice were treated with hydralazine. Both enalapriland losartan but not hydralazine treatment resulted in remarkableand rapid amelioration of this immune complex–mediatedglomerulonephritis, pointing to potentially new uses for thesetherapeutic agents in the treatment of humans with similar typesof glomerulonephritis.
Enalapril and Losartan Reduced Glomerular Matrix Deposition
TSLPtg mice showed marked enlargement of the glomerular tuftarea (GTA) and hypercellularity (Figure 1). Neither enalaprilnor losartan altered the overall glomerular cell number. TSLPtgmice had significant expansion of mesangial matrix that progressedfrom 4- to 8-wk time points in females and from 12- to 24-wktime points in males (Figures 1 and 2). At each time point,enalapril and losartan treatment significantly decreased mesangialmatrix expansion, whereas TSLPtg mice treated with hydralazinefor 4 and 8 wk showed similar mesangial matrix expansion asuntreated TSLPtg mice (percentage of silver-stained area/GTA:females at 8 wk: TSLPtg untreated 24.8 ± 2.0, hydralazine24.2 ± 1.4, enalapril 15.4 ± 2.2, losartan 16.2± 1.2, WT 9.6 ± 1.5; males at 24 wk: TSLPtg untreated26.9 ± 2.4, enalapril 18.1 ± 1.7, losartan 20.6± 1.6, WT 12.7 ± 2.5; P < 0.01 versus matcheduntreated or hydralazine-treated TSLPtg mice).
Figure 1. Representative photographs of glomeruli with hematoxylin and eosin (A through E), silver methenamine (F through J), and type IV collagen (K through O) immunohistochemistry staining of 8-wk groups of untreated WT (A, F, and K) and TSLPtg mice (B, G, and L), and hydralazine-treated (C, H, and M), enalapril-treated (D, I, and N), or losartan-treated (E, J, and O) TSLPtg mice. Magnification, x400.
Figure 2. Morphometric analysis of glomerular silver staining matrix and type IV collagen immunohistochemistry staining in untreated and hydralazine-, enalapril-, or losartan-treated WT and TSLPtg mice of female 4- and 8-wk and male 12- and 24-wk groups. Data are means ± SEM (n = 4 to 6 in each group). ***P < 0.001, **P < 0.01, *P < 0.05 versus matched untreated TSLPtg control.
Type IV collagen, an important component of glomerular extracellularmatrix, was used as a second measure of matrix expansion. Therewas an increase in type IV collagen deposition comparable inextent and amount to increased silver staining matrix in TSLPtgmice (Figures 1 and 2). The deposition of type IV collagen inglomeruli decreased significantly in enalapril- or losartan-but not hydralazine-treated TSLPtg groups (percentage of typeIV collagen staining area/GTA: females at 8 wk: TSLPtg untreated16.7 ± 0.9, hydralazine 17.1 ± 0.5, enalapril10.7 ± 1.0, losartan 12.5 ± 1.5, WT 7.4 ±0.9; males at 24 wk: TSLPtg untreated 18.3 ± 3.5, enalapril13.4 ± 1.3, losartan 14.3 ± 1.2, WT 8.1 ±0.5; P < 0.05 versus matched untreated or hydralazine-treatedTSLPtg mice). There was no alteration of type IV collagen distributionin treated versus untreated WT mice.
Enalapril and Losartan Reduced Glomerular Mesangial Cell Activation
Glomerular -smooth muscle actin (-SMA) expression, an indicatorof mesangial cell activation, was dramatically increased inthe TSLPtg mice. Treatment with enalapril or losartan but nothydralazine significantly decreased mesangial -SMA expression(Supplemental Figures 1 and 2).
Enalapril and Losartan Decreased Renal Plasminogen Activator Inhibitor-1 Expression
TSLPtg mice express high plasminogen activator inhibitor-1 (PAI-1)mRNA levels in the kidney (Figure 3). Enalapril decreased PAI-1mRNA expression levels in both 8- and 24-wk treatment groups(fold increase of PAI-1 mRNA expression relative to WT control:females at 8 wk: TSLPtg untreated 3.3 ± 0.4, enalapril1.4 ± 0.4, losartan 1.8 ± 0.6; males at 24 wk:TSLPtg untreated 4.5 ± 0.5, enalapril 2.3 ± 0.5,losartan 3.4 ± 0.5; P < 0.05 enalapril groups versusmatched untreated TSLPtg mice). Losartan-treated groups hada trend toward decreased expression of PAI-1 mRNA, but the differencescompared with untreated TSLPtg control were not statisticallysignificant.
Figure 3. Kidney PAI-1 mRNA expression was reduced by enalapril and losartan treatment as assessed by quantitative real-time RT-PCR. The fold change of the expression level of PAI-1 gene in TSLPtg mice compared with WT mice was normalized to the endogenous housekeeping gene glyceraldehyde-3-phosphate dehydrogenase. Data are fold change (means ± SEM) of PAI-1 mRNA expression relative to WT control (n = 4 in WT control and n = 6 in untreated and treated TSLPtg groups). ++P < 0.01, +P < 0.05 versus matched WT control; *P < 0.05 versus matched untreated TSLPtg control.
In parallel, glomerular PAI-1 protein was markedly increasedin TSLPtg mice (Figures 4 and 5). Both enalapril and losartantreatment decreased glomerular PAI-1 protein expression (percentageof PAI-1 staining area/GTA: females at 8 wk: TSLPtg untreated3.4 ± 0.1, enalapril 1.9 ± 0.1, losartan 2.3 ±0.1, WT control 1.3 ± 0.1; males at 24 wk: TSLPtg untreated3.9 ± 0.1, enalapril 2.7 ± 0.1, losartan 2.6 ±0.1, WT control 1.4 ± 0.2; P < 0.05 versus matcheduntreated TSLPtg control).
Figure 4. Representative photographs of glomerular PAI-1 immunohistochemistry staining of female 8-wk groups (A through D) and male 24-wk groups (E through H) of untreated WT (A and E), untreated TSLPtg (B and F), and enalapril-treated (C and G) and losartan-treated (D and H) TSLPtg mice. Magnification, x400.
Figure 5. Morphometric measurements of glomerular PAI-1 protein expression of female 8-wk groups and male 24-wk groups. Untreated TSLPtg control mice have markedly increased glomerular PAI-1 protein expression in both 8-wk females and 24-wk males compared with WT mice of the same age. Enalapril and losartan treatment decreased glomerular PAI-1 expression in TSLPtg mice. Data are means ± SEM (n = 4 in WT control, n = 5 in untreated and treated TSLPtg groups). +++P < 0.001, ++P < 0.01 versus WT control; **P < 0.01 versus untreated TSLPtg control.
Enalapril and Losartan Did not Change Renal Ig and Complement Deposition and Macrophage Infiltration
Immunofluorescence showed that TSLPtg mice had massive depositionof IgG, IgM, and, to a lesser extent, IgA and complement C3in glomeruli. Treatment with hydralazine, enalapril, and losartandid not change the extent or intensity of staining of depositedIg and complement C3 (Figure 6 and Supplemental Figure 3). Electronmicroscopy showed that the untreated and treated TSLPtg groupshad similarly extensive electron-dense immune deposits in subendothelialand mesangial area, as described previously (data not shown).21
Figure 6. Kidney immunofluorescence study of untreated and hydralazine-, enalapril-, or losartan-treated TSLPtg mice. Photos are representative glomerular immunofluorescence staining of deposited IgG (A through D) and complement C3 (E through H) of female 8-wk groups of untreated TSLPtg mice (A and E) and hydralazine-treated (B and F), enalapril-treated (C and G), and losartan-treated (D and H) TSLPtg mice. Magnification, x400.
TSLPtg mice had prominent monocyte/macrophage infiltration inthe glomerular tuft as assessed by Mac-2 staining. Hydralazine,enalapril, and losartan treatment did not significantly changethe extent of monocyte/macrophage influx (Supplemental Figures1 and 2).
Enalapril and Losartan Did not Change Glomerular Cell Proliferation and Apoptosis
In TSLPtg mice, the number of glomerular Ki-67–expressingcells was increased significantly compared with WT controls.Hydralazine, enalapril, and losartan treatment did not changethe number of Ki-67–expressing cells in TSLPtg and WTmice (Ki-67–expressing cells/50 glomeruli: females at8 wk: TSLPtg untreated 26.6 ± 6.6, hydralazine 28.3 ±3.1, enalapril 26.2 ± 1.8, losartan 27.6 ± 1.5,WT untreated 18.8 ± 1.4; males at 24 wk: TSLPtg untreated31.2 ± 2.8, enalapril 31.8 ± 3.6, losartan 30.2± 2.6, WT untreated 22.8 ± 3.1). As assessed byterminal deoxynucleotidyl transferase-mediated dUTP nick-endlabeling (TUNEL) staining, TSLPtg mice had few apoptotic cellsin glomeruli (two to three positive cells in 50 glomeruli) withonly rare scattered TUNEL-positive cells in the interstitium.Hydralazine, enalapril, and losartan treatment did not changethe number of apoptotic cells in glomeruli and interstitium(data not shown).
Proteinuria Was Markedly Reduced by Enalapril and Losartan but not by Hydralazine Treatment
Urine albumin excretion increased dramatically in untreatedTSLPtg mice (Figure 7). Hydralazine-treated TSLPtg mice showeda similar level of massive proteinuria. Both enalapril and losartanmarkedly reduced albumin excretion in TSLPtg mice (urine albumin-creatinineratio [µg/mg]: females at 8 wk: TSLPtg untreated 156.8± 42.4, hydralazine 161.6 ± 30.8, enalapril 66.9± 17.4, losartan 56.8 ± 12.0, WT untreated 27.2± 4.9; males at 24 wk: TSLPtg untreated 107.3 ±13.4, enalapril 66.9 ± 7.5, losartan 60.0 ± 12.0,WT untreated 30.0 ± 5.9; P < 0.05 versus matched untreatedand hydralazine treated TSLPtg mice). There was no differencein serum creatinine levels among the various study groups (datanot shown).
Figure 7. (A and B) Proteinuria of 4- and 8-wk females of untreated WT and TSLPtg mice and hydralazine-, enalapril-, or losartan-treated TSLPtg mice (A), and 24-wk males of untreated WT and TSLPtg mice and enalapril- or losartan-treated TSLPtg mice (B). Proteinuria was measured as urine albumin (µg)-creatinine (mg) ratio. Untreated TSLPtg mice have marked increased urine albumin excretion compared with WT mice. The proteinuria level of hydralazine-treated TSLPtg mice is similar to that of untreated TSLPtg mice. Proteinuria was significantly decreased in enalapril- or losartan-treated TSLPtg mice. Data are means ± SEM (n = 4 to 6 per group). *P < 0.05 versus matched untreated TSLPtg control; ++P < 0.01 versus WT control.
BP was Effectively Decreased by Enalapril, Losartan, and Hydralazine
The systolic BP increased progressively from 4 to 8 wk and from4 to 24 wk in female and male TSLPtg mice, respectively, ascompared with age-matched WT controls. Treatment with hydralazine,enalapril, and losartan reduced BP in TSLPtg mice and WT miceat every time point, but the decrease of BP in WT mice is lessthan in TSLPtg mice (Figure 8). Enalapril and losartan had similarantihypertensive effects in males, but in the 4- and 8-wk femaleTSLPtg mice, systolic BP of the enalapril group was lower thanthat of the losartan group but was similar to that of the hydralazinegroup (BP of TSLPtg 4 wk [mmHg]: untreated 123 ± 4, hydralazine88 ± 6, enalapril 82 ± 5; losartan 104 ±4; TSLPtg 8 wk: untreated 131 ± 4, hydralazine 94 ±5, enalapril 90 ± 2, losartan 110 ± 4; P <0.05 losartan groups versus matched enalapril or hydralazinegroups).
Figure 8. Time course of systolic BP in untreated and hydralazine-, enalapril- or losartan-treated wild type and TSLP transgenic mice. BP of each mouse at each time point is the mean of 6 to 10 measurements. Every group had 4 to 6 mice. Data are mean ± SEM (mmHg). ***P < 0.001; **P < 0.01; *P < 0.05 hydralazine-, enalapril- or losartan-treated TSLPtg mice versus matched untreated TSLPtg control.
Enalapril and Losartan Increased the Long-Term Survival of TSLPtg Mice
Observation of the 24-wk groups of male TSLPtg and WT mice demonstratedthat untreated male TSLPtg mice had a high mortality rate (58%at 24 wk), similar to previous studies.21 In contrast, enalapril-and losartan-treated male TSLPtg mice had much lower mortalityrates (8% in enalapril group and 17% in losartan group at 24wk of treatment; Figure 9). Treatment of WT mice had no effecton survival.
Figure 9. Survival curves of 24-wk male TSLPtg and WT mouse groups. At the start point, all groups had 12 male mice. Treatment groups started enalapril or losartan treatment at 3 wk of age and continued for 24 wk. Seven of 12 untreated TSLPtg mice died in this duration, whereas one of 12 enalapril-treated TSLPtg mice and two of 12 losartan-treated TSLPtg mice died in this duration, suggesting enalapril and losartan treatment markedly improved survival in TSLPtg mice. They did not show an impact on survival of WT mice in this time frame.
Systemic Effects of Enalapril and Losartan
All TSLPtg mice had circulating cryoglobulins, whereas WT micedid not. Treatment with enalapril and losartan did not changecirculating cryoglobulin levels (data not shown).
In TSLPtg mice, manifestations of systemic cryoglobulinemiaand B cell defects included marked increase of the weight ofspleen and lung as a result of increased cellularity or inflammation.Hydralazine, enalapril, and losartan treatment did not changespleen weights. Lung weights tended to decrease with enalapriland losartan treatment, although only the decrease of lung weightsin the 12-wk losartan group and the 24-wk enalapril group reachedstatistical significance (Supplemental Figure 4). In contrast,hydralazine-treated TSLPtg mice had similar lung weights asuntreated TSLPtg control. The weights of kidney and liver werenot different among groups (data not shown).
Histologic evaluation of the liver showed portal leukocyte infiltrationas described previously,21,23 which was unchanged by treatmentwith hydralazine, enalapril, or losartan (Table 1). The lunglesion in TSLPtg mice is characterized by marked perivascularand peribronchiolar leukocyte infiltration (Figure 10). Hydralazine-treatedTSLPtg mice showed a similar level of lung inflammation. Bothenalapril and losartan treatment resulted in decreased leukocyteinfiltration in the lung with more open bronchioli and alveoli(Figure 10, Table 1).
Figure 10. Representative lung sections with hematoxylin and eosin staining of 8-wk female groups of untreated WT and TSLPtg mice, and hydralazine-, enalapril-, or losartan-treated TSLPtg mice. Magnification, x100.
The value of inhibition of the RAS, whether by ACEI or by ARB,has been established in clinical trials of patients with mostforms of CKD, including diabetic nephropathy and hypertensivekidney disease. In this study, we demonstrated that these agentsalso provide a clear and continuous benefit in amelioratingthe glomerular injury consequent to persistent, ongoing immunecomplex deposition. These benefits are both structural (diminishedextracellular matrix [ECM] accumulation and reduced activationof mesangial cells) and functional (decreased proteinuria).The renal and systemic benefits confer reduced mortality incryoglobulinemic mice. In contrast, despite a comparable loweringof BP, hydralazine was not effective in ameliorating glomerularinjury and reducing proteinuria. These findings, in aggregate,suggest that RAS blockade can be effective as a treatment foractive glomerulonephritis in humans, in addition to their establishedbenefits in CKD.
The findings underscore the specific renoprotective effectsof RAS blockade that are independent of any benefit conferredby reduction in BP. Although hydralazine, enalapril, and losartanshowed marked lowering of BP in the TSLPtg mice throughout thetreatment course, only enalapril and losartan achieved significantstructural and functional improvement of renal lesions. Treatmentdid not significantly affect the levels of circulating cryoglobulinsor the extensive immune complex deposition or complement fixationin glomeruli, all components of the process by which the glomerularinjury was initiated. Our studies do not allow us to concludethat the survival benefit in the ACEI- and ARB-treated micewas due to a specific effect of RAS blockade independent ofeffects on systemic BP lowering, because a needed control groupof hydralazine-treated mice with lowered BP was not done forthe survival study component of this experiment.
Because the improvement of MPGN was similar in mice that weretreated either with an ACEI or a blocker targeting the AT1R,the benefit is not due to interruption of activities of angiotensinII consequent to engagement of other receptors (e.g., AT2R)or blockade of other activities attributable to ACE such asdegradation of bradykinin. It is well established that angiotensinII acting via AT1R triggers vasoconstriction and aldosteronerelease and mediates proinflammatory and profibrogenic effectsthat perpetuate progressive kidney injury.24,25 AngiotensinII activates mesangial cells, induces the expression of TGF-β1and PAI-1, and thus promotes ECM accumulation.25 Our study demonstratesthat treatment of TSLPtg mice with enalapril or losartan, possiblyacting through each of these mechanisms, effectively reducesglomerular ECM expansion and mesangial cell activation, largelyabolishing the amplification phase of MPGN after injury wasinitiated.
Decreased degradation of ECM is also an important factor inthe expansion of mesangial matrix and glomerulosclerosis. PAI-1is an important factor in the maintenance of the delicate balancebetween ECM synthesis and degradation; its activities on plasminand other proteases limit matrix degradation. Angiotensin inducesPAI-1, and, conversely, angiotensin blockade decreases PAI-1expression and decreases sclerosis.9 Both enalapril and losartandecreased renal mRNA and protein expression of PAI-1 in TSLPtgmice, likely contributing to the reduction in matrix accumulation.
There is increasing evidence that angiotensin II has immunomodulatoryeffects.26,27 Some immunomodulatory effects of RAS blockadelikely contributed to the improvement of the systemic well-beingof TSLP mice. Enalapril- and losartan-treated TSLPtg mice hadlower mortality, which we attribute in part to reduction ofrenal injury and part to the reduction of the severe lung inflammationthat occurs in these mice. In contrast, hydralazine-treatedTSLPtg mice showed a similar degree of lung inflammation asuntreated TSLPtg mice. This immunomodulatory effect of RAS blockade,not yet identified more specifically, may be organ specificbecause improvement in the concurrent hepatitis that occursin the TSLPtg model was not observed using any of the treatmentmodalities.28
In summary, we demonstrated that treatment with enalapril andlosartan resulted in a dramatic attenuation of MPGN and hada beneficial effect on long-term survival in mice with cryoglobulinemia-associatedglomerulonephritis. The long-term effects of RAS blockade havebeen well established in a variety of chronic injury models;what is surprising is the speed and efficacy of this effectin glomerulonephritis, where there is active, ongoing depositionof immune reactants. The beneficial effects included reducedaccumulation of glomerular ECM, decreased mesangial cell activation,decreased expression of PAI-1, reduced proteinuria, and decreasedmortality. We conclude that RAS blockade has the potential tobe a useful therapeutic strategy in the treatment of at leastsome types of immune complex–mediated glomerulonephritisin humans.
Animals and Experimental Design
The experimental protocol was approved by the Animal Care Committeeof the University of Washington. TSLPtg mice on a C57BL/6 backgroundhave been characterized previously.21,29,30
Pups weaned at age 3 wk were randomly assigned to the followingtreatment groups: (1) Enalapril treatment groups: enalapril(Sigma-Aldrich, St. Louis, MO) 125 mg/L in drinking water, adosage equal to 30 mg/kg body wt per d; (2) losartan treatmentgroups: losartan (Merck, Whitehouse Station, NJ) 100 mg/L indrinking water, equal to 25 mg/kg body wt per d; (3) hydralazinetreatment groups: hydralazine (Sigma-Aldrich) 200 mg/L in drinkingwater, equal to 50 mg/kg body wt per d; (4) control groups:normal drinking water. Each group had four to eight TSLPtg andWT littermates each except for the 24-wk groups, which had 12mice of each genotype for assessment of the long-term survivaleffect of ACEI and ARB treatment. Hydralazine treatment continuedfor 4 and 8 wk in females. Enalapril and losartan treatmentcontinued for 4 or 8 wk in females and for 12 or 24 wk in malesuntil they were killed. Different time frames for treatmentof females and males were based on previous observations thatalthough male and female mice develop an identical pattern ofglomerular injury in this model, the effects of gender are manifestedby a greater rapidity in onset and progression of disease infemales as compared with males, with females typically havingovert disease by age 30 d and developing the full manifestationsof disease by day 50. In contrast, the onset of disease is slowerin males, typically evident by day 50, and may take upwardsof 120 d to be fully manifested. At the time the disease fullymanifests, the renal and systemic manifestations of the cryoglobulinemicprocess are indistinguishable in males and females.
BP Measurement
BP was measured by CODA6 noninvasive tail-cuff system (KentScientific, Torrington, CT). Mice were kept in a quiet and warmenvironment in this procedure. The first five measuring cycleswere acclimation cycles, and these measurements were not recorded.BP of each mouse at each time point is the mean of 6 to 10 subsequentmeasurements. For female mice, BP was measured at 4 and 8 wkof treatment. For males, BP of losartan groups was measuredat 4, 8, 12, 16, 20, and 24 wk of treatment and of enalaprilgroups at 12 and 24 wk of treatment.
Analysis of Kidney Function
Before the mice were killed, spot urine was collected from eachmouse. Blood was collected by retro-orbital bleeding after anesthetization.Urine albumin was measured by ELISA using the Albuwell kit (Exocell,Philadelphia, PA), and urine creatinine was measured using theCreatinine Companion kit (Exocell). Proteinuria was calculatedas the urine albumin-creatinine ratio. Serum creatinine wasmeasured using HPLC.
Serum Cryoglobulin Evaluation
Serum cryocrit was measured by aliquotting 100 µl of seruminto Natelson blood collecting glass tubes (Chase ScientificGlass, Rockwood, TN). After 1 wk at 4°C, the heights ofcryoprecipitates were measured and expressed as a percentageof total serum heights.
Tissue Collection and Histologic Study
Kidney, spleen, liver, and lung were collected when the micewere killed. Tissues were fixed, embedded, and stained withhematoxylin and eosin and methenamine silver as described previously.21Portions of kidney were snap-frozen and stored at –80°Cfor RNA extraction and for immunofluorescence studies. Tissuefor electron microscopy was fixed in half-strength Karnovsky'ssolution and processed as described previously.21,31
Immunofluorescence
Acetone-fixed frozen kidney sections were incubated with fluorescein-conjugatedantibodies against mouse IgG, IgM, IgA, and complement C3 (allfrom Cappel Pharmaceuticals, Aurora, OH) as described previously.21,30In a blinded manner, the glomerular fluorescence intensity wasscored semiquantitatively (0, negative; 1, weak; 2, moderate;3, strong). For every sample, 15 glomeruli were counted anda mean score was calculated.
Immunohistochemistry
The immunohistochemistry protocols for type IV collagen, -SMA,macrophage marker Mac-2, and PAI-1 have been described previously.21,29Cellular proliferation was assessed with a rat monoclonal anti-Ki-67antibody (Dako, Carpinteria, CA). Apoptosis was evaluated byTUNEL using Apoptag apoptosis kit (Chemicon, Temecula, CA) asdescribed previously.9
Quantitative Real-Time PCR
Total kidney RNA was extracted from frozen kidney tissue usingTRIzol reagent (Invitrogen, Carlsbad, CA). Real-time PCR wasperformed using Taqman gene expression assay kits for the primersand probes of mouse PAI-1 and glyceraldehyde-3-phosphate dehydrogenasegene (Applied Biosystems, Foster City, CA). The fold changeof the expression level of PAI-1 gene in TSLPtg mice comparedwith WT mice was normalized to the endogenous housekeeping geneglyceraldehyde-3-phosphate dehydrogenase.
Analytical Methods and Statistical Analysis
Morphometry study for GTA and the glomerular area occupied bysilver staining or stained by antibodies to type IV collagen,-SMA, Mac-2, and PAI-1 were quantified as described previously.29,30Glomerular cell proliferation and apoptosis were assessed bycounting the number of Ki-67–and TUNEL-positive cellsin 50 random glomeruli. Liver and lung tissue sections stainedwith hematoxylin and eosin were assessed for evidence of inflammation.Liver and lung inflammation was scored semiquantitatively ona scale of 0 to 3: 0, no inflammation; 1, mild; 2, moderate;3, severe with regard to the extent and density of leukocyticinfiltration.
All data are expressed as mean ± SEM. Statistical analysisof the data for multiple groups was performed by ANOVA withTukey-Kramer multiple comparisons test using the InStat program(version 3.0; Intuitive Software for Science, San Diego, CA).P < 0.05 was considered significant.
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This article has been cited by other articles:
S. Guo, A. S. Muhlfeld, T. A. Wietecha, C. J. Peutz-Kootstra, J. Kowalewska, K. Yi, M. Spencer, W. Pichaiwong, F. Nimmerjahn, K. L. Hudkins, et al. Deletion of Activating Fc{gamma} Receptors Does Not Confer Protection in Murine Cryoglobulinemia-Associated Membranoproliferative Glomerulonephritis
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Abstract
Introduction
-smooth muscle actin (
