Susceptibility to kidney fibrosis in mice is associated with early growth response-2 protein and tissue inhibitor of metalloproteinase-1 expression
Authors: Gábor Kökény, Ágnes Németh, Jeffrey B. Kopp, Weiping Chen, Andrew J. Oler, Anna Manzéger, László Rosivall, Miklós M. Mózes
Journal: Kidney International (2022)

Patients with chronic kidney disease and experimental animal models of kidney fibrosis manifest diverse progression rates. Genetic susceptibility may contribute to this diversity, but the causes remain largely unknown. We have previously described kidney fibrosis with a mild or severe phenotype in mice expressing transforming growth factor-beta1 (TGF-β1) under the control of a mouse albumin promoter (Alb/TGF-β1), on a mixed genetic background with CBAxC57Bl6 mice. Here, we aimed to examine how genetic background may influence kidney fibrosis in TGF-β1 transgenic mice, and in the unilateral ureteral obstruction (UUO) and subtotal nephrectomy (SNX) mouse models. Congenic C57Bl6(B6)-TGFβ and CBAxB6-TGFβ (F1) transgenic mice were generated and survival, proteinuria, kidney histology, transcriptome and protein expressions were analyzed. We investigated the kidneys of B6 and CBA mice subjected to UUO and SNX, and the effects of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) neutralization on the fibrotic process. CBAxB6-TGFβ mice developed severe kidney fibrosis and premature death, while B6-TGF-β mice had mild fibrosis and prolonged survival. Kidney early growth response factor-2 (EGR2) and TIMP-1 expression were induced only in CBAxB6-TGFβ mice. Similar strain-dependent early changes in EGR2 and TIMP-1 of mice subjected to UUO or SNX were observed. TIMP-1 neutralization in vivo hindered fibrosis both in transgenic mice and the SNX model. EGR2 over-expression in cultured HEK293 cells induced TIMP-1 while EGR2 silencing hindered TGF-β induced TIMP-1 production in HK-2 cells and ureteral obstructed kidneys. Finally, EGR2 and TIMP1 was increased in human kidneys manifesting focal segmental glomerulosclerosis suggesting a correlation between animal studies and patient clinical settings. Thus, our observations demonstrate a strong relationship between genetic background and the progression of kidney fibrosis, which might involve early altered EGR2 and TIMP-1 response, but the relationship to patient genetics remains to be explored. Patients with chronic kidney disease and experimental animal models of kidney fibrosis manifest diverse progression rates. Genetic susceptibility may contribute to this diversity, but the causes remain largely unknown. We have previously described kidney fibrosis with a mild or severe phenotype in mice expressing transforming growth factor-beta1 (TGF-β1) under the control of a mouse albumin promoter (Alb/TGF-β1), on a mixed genetic background with CBAxC57Bl6 mice. Here, we aimed to examine how genetic background may influence kidney fibrosis in TGF-β1 transgenic mice, and in the unilateral ureteral obstruction (UUO) and subtotal nephrectomy (SNX) mouse models. Congenic C57Bl6(B6)-TGFβ and CBAxB6-TGFβ (F1) transgenic mice were generated and survival, proteinuria, kidney histology, transcriptome and protein expressions were analyzed. We investigated the kidneys of B6 and CBA mice subjected to UUO and SNX, and the effects of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) neutralization on the fibrotic process. CBAxB6-TGFβ mice developed severe kidney fibrosis and premature death, while B6-TGF-β mice had mild fibrosis and prolonged survival. Kidney early growth response factor-2 (EGR2) and TIMP-1 expression were induced only in CBAxB6-TGFβ mice. Similar strain-dependent early changes in EGR2 and TIMP-1 of mice subjected to UUO or SNX were observed. TIMP-1 neutralization in vivo hindered fibrosis both in transgenic mice and the SNX model. EGR2 over-expression in cultured HEK293 cells induced TIMP-1 while EGR2 silencing hindered TGF-β induced TIMP-1 production in HK-2 cells and ureteral obstructed kidneys. Finally, EGR2 and TIMP1 was increased in human kidneys manifesting focal segmental glomerulosclerosis suggesting a correlation between animal studies and patient clinical settings. Thus, our observations demonstrate a strong relationship between genetic background and the progression of kidney fibrosis, which might involve early altered EGR2 and TIMP-1 response, but the relationship to patient genetics remains to be explored. Translational StatementPatients with chronic kidney disease, regardless of etiology, develop kidney fibrosis, but they manifest varying rates of progressive loss of kidney function. Moreover, ongoing kidney fibrosis is a major therapeutic challenge in nephrology. Differences in genetic susceptibility may contribute to differences in progression rates, driven by as-yet-unknown molecular mechanisms. In the present study, we addressed the question of whether genetic background affects the development and progression of kidney disease in several mouse models of renal fibrosis. We found that kidney fibrosis progression is strongly associated with mouse genetic background. This genetic susceptibility involves very early and marked overproduction of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), which is associated with early growth response factor-2 in vivo and in vitro. Further, we observed similar expression patterns in human renal biopsies manifesting focal segmental glomerulosclerosis. These results may help clarify the initial molecular mechanisms of kidney fibrosis and might lead to identification of early diagnostic markers and the development of pharmacologic inhibitors to slow, halt, or even reverse the fibrotic process. Progressive kidney fibrosis, which often eventuates in end-stage kidney disease, is the final common pathway of chronic renal diseases of different etiologies, and it is one of the major challenges in nephrology. Patients with one particular renal disease, such as diabetic nephropathy or arterionephrosclerosis (often previously misattributed to hypertension in the case of African Americans), show different progression rates, presumably due to genetic susceptibility.1Keith D.S. Nichols G.A. Gullion C.M. et al.Longitudinal follow-up and outcomes among a population with chronic kidney disease in a large managed care organization.Arch Intern Med. 2004; 164: 659-663Crossref PubMed Scopus (1364) Google Scholar Transforming growth factor-beta1 (TGF-β1) is a multifunctional cytokine that regulates the dynamic balance of extracellular matrix components and affects cell growth and differentiation. TGF-β1 plays a pivotal role in not only the pathogenesis of fibrosis, but also development, wound healing, immune processes, and carcinogenesis. Experimental overexpression of TGF-β1 is associated with kidney,2August P. Suthanthiran M. Transforming growth factor beta and progression of renal disease.Kidney Int Suppl. 2003; : S99-S104Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar myocardial,3Khan R. Examining potential therapies targeting myocardial fibrosis through the inhibition of transforming growth factor-beta 1.Cardiology. 2007; 108: 368-380Crossref PubMed Scopus (13) Google Scholar pulmonary,4Murray L.A. Chen Q. Kramer M.S. et al.TGF-beta driven lung fibrosis is macrophage dependent and blocked by serum amyloid P.Int J Biochem Cell Biol. 2011; 43: 154-162Crossref PubMed Scopus (274) Google Scholar and hepatic fibrosis. Several lines of evidence suggest a role for strain-dependent differences in the development and progression of experimental renal diseases. Rat models of puromycin aminonucleoside nephrosis and subtotal nephrectomy5Kökény G. Németh Z. Godó M. Hamar P. The Rowett rat strain is resistant to renal fibrosis.Nephrol Dial Transplant. 2010; 25: 1458-1462Crossref PubMed Scopus (5) Google Scholar, 6Grond J. Beukers J.Y. Schilthuis M.S. et al.Analysis of renal structural and functional features in two rat strains with a different susceptibility to glomerular sclerosis.Lab Invest. 1986; 54: 77-83PubMed Google Scholar, 7Grond J. Muller E.W. van Goor H. et al.Differences in puromycin aminonucleoside nephrosis in two rat strains.Kidney Int. 1988; 33: 524-529Abstract Full Text PDF PubMed Scopus (16) Google Scholar have demonstrated the importance of strain differences. Studies involving albumin overload,8Chen J.S. Chen A. Chang L.C. et al.Mouse model of membranous nephropathy induced by cationic bovine serum albumin: antigen dose-response relations and strain differences.Nephrol Dial Transplant. 2004; 19: 2721-2728Crossref PubMed Scopus (61) Google Scholar,9Ishola Jr., D.A. van der Giezen D.M. Hahnel B. et al.In mice, proteinuria and renal inflammatory responses to albumin overload are strain-dependent.Nephrol Dial Transplant. 2006; 21: 591-597Crossref PubMed Scopus (57) Google Scholar subtotal nephrectomy,10Ma L.J. Fogo A.B. Model of robust induction of glomerulosclerosis in mice: importance of genetic background.Kidney Int. 2003; 64: 350-355Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar,11Salzler H.R. Griffiths R. Ruiz P. et al.Hypertension and albuminuria in chronic kidney disease mapped to a mouse chromosome 11 locus.Kidney Int. 2007; 72: 1226-1232Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar and diabetic nephropathy12Gurley S.B. Clare S.E. Snow K.P. et al.Impact of genetic background on nephropathy in diabetic mice.Am J Physiol Renal Physiol. 2006; 290: F214-F222Crossref PubMed Scopus (207) Google Scholar mouse models show that the C57Bl6/J (B6) strain is more resistant to kidney fibrosis, compared to other mouse strains, including 129Sv, Balb/C, and DBA/2. Furthermore, despite early renal TGF-β1 overexpression after injury, B6 mice are resistant to fibrosis induced by renal ablation.10Ma L.J. Fogo A.B. Model of robust induction of glomerulosclerosis in mice: importance of genetic background.Kidney Int. 2003; 64: 350-355Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar,13Rumberger B. Vonend O. Kreutz C. et al.cDNA microarray analysis of adaptive changes after renal ablation in a sclerosis-resistant mouse strain.Kidney Blood Press Res. 2007; 30: 377-387Crossref PubMed Scopus (8) Google Scholar Important to note is that the molecular mechanisms underlying strain-dependent renal fibrosis remain largely unknown. We examined the role of genetic background on the progression of TGF-β1–induced renal fibrosis in transgenic mice overexpressing TGF-β1.14Sanderson N. Factor V. Nagy P. et al.Hepatic expression of mature transforming growth factor beta 1 in transgenic mice results in multiple tissue lesions.Proc Natl Acad Sci U S A. 1995; 92: 2572-2576Crossref PubMed Scopus (601) Google Scholar Originally, TGF-β1 transgenic mice maintained on mixed (CBAxB6) genetic background developed progressive renal fibrosis with phenotypic variability, characterized as either mild or severe.15Mozes M.M. Bottinger E.P. Jacot T.A. et al.Renal expression of fibrotic matrix proteins and of transforming growth factor-beta (TGF-beta) isoforms in TGF-beta transgenic mice.J Am Soc Nephrol. 1999; 10: 271-280Crossref PubMed Google Scholar Therefore, we generated congenic B6-TGFβ transgenic mice that manifested only a mild renal phenotype, and CBAxB6-TGFβ F1 mice that had severe proteinuria and glomerulosclerosis, in order to analyze the expression of various fibrosis-related molecules in these transgenic strains. Further, we made similar inter-strain comparisons in the unilateral ureter obstruction (UUO) and subtotal renal ablation (SNX) models, complemented with cell culture studies and analysis of human focal segmental glomerulosclerosis (FSGS) kidney biopsies. Our experimental data show that genetic background and progression of TGF-β1–induced renal fibrosis strongly correlate with altered tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) expression, which is associated with early response of early growth response factor-2 (EGR2). We suggest that the underlying mechanism of resistance to renal fibrosis in B6 mice might involve the lack of early EGR2 and TIMP-1 response. Concise methods are provided in the Supplementary Methods. In order to confirm the role of genetic background on kidney fibrosis progression in this model, male CBA.B6-Alb/TGF-β1 transgenic mice were backcrossed to both C57Bl6 (B6) and CBA inbred strains, as a CBAxB6 F1 hybrid mouse was the founder of this transgenic line. The backcrossing of Alb/TGF-β1 transgenic mice to the CBA strain failed, as 70% of the CBAxB6-TGFβ F1 males died before reaching 6 weeks of age (Figure 1a; Supplementary Figure S1A). In contrast, backcrossing of Alb/TGF-β1 transgenic mice for 22 generations to the B6 strain greatly increased survival, compared with that in the original transgenic strain, which exhibited 100% mortality by age 52 weeks. We found that 72% of B6-TGFβ mice survived to age 15 weeks (Figure 1a), and at 52 weeks, 39% were alive, compared to 100% of B6 wild type controls (n = 33, log-rank test, P < 0.001). At the age of 4 and 9 months, plasma TGF-β1 levels were 2-fold higher in B6-TGFβ transgenic mice, compared with levels in age-matched B6 controls (Supplementary Table S1). Body weights of both B6 and B6-TGFβ mice increased comparably with age (Supplementary Table S1). No differences were seen in kidney weights normalized to body weight, or in urinary protein-to-creatinine ratios among the groups. We observed mild but significant glomerulosclerosis in B6-TGFβ kidneys at ages 4 and 9 months, compared with age-matched B6 controls (Supplementary Table S1; Supplementary Figure S1B). We did not observe tubulointerstitial damage in B6–TGF-β1 mice at any of the ages investigated. Plasma levels of TGF-β1 at age 14 days in B6-TGFβ and CBAxB6-TGFβ F1 mice were similar, and they were 11–14-fold higher than those in controls (Table 1). Body weights of transgenic mice were similar to their wild-type control (Table 1). In contrast to B6-TGFβ mice, the survival time of CBAxB6-TGFβ F1 mice was dramatically shorter, as only 60% of these F1 mice survived to age 2 weeks, and all mice died by age 12 weeks (Figure 1a). Despite comparable plasma TGF-β1 levels in B6-TGFβ and CBAxB6-TGFβ strains, only CBAxB6-TGFβ mice had significantly elevated urine protein-to-creatinine ratio (Figure 1b). Similarly, kidney weights and levels of serum urea were significantly higher only in CBAxB6-TGFβ F1 mice (Figure 1c). Due to the early uremic death of most CBAxB6-TGFβ F1 mice, we were not able to continue the backcross to the CBA strain, and all experimental samples were obtained at age 14 days.Table 1Pathology and laboratory data of transgenic B6-TGFβ and CBAxB6-TGFβ F1 mice and wild-type controls, at age 14 daysMouse strainBody weight, gKidney weight/body weight, mg/gPlasma TGF-β1, ng/mlUrea nitrogen, mg/dlProteinuria, mg protein/mg creatinineB6 (n = 10)7.25 ± 0.627.60 ± 1.034.04 ± 2.0136.5 ± 10.65.03 ± 1.18CBAxB6 F1 (n = 8)8.45 ± 0.95aP < 0.05 vs. B6.7.40 ± 0.793.10 ± 1.7634.8 ± 6.45.41 ± 1.11B6-TGFβ (n = 14)7.21 ± 0.87cP < 0.05 vs. CBAxB6 F1.7.14 ± 0.5557.61 ± 16.77aP < 0.05 vs. B6.,cP < 0.05 vs. CBAxB6 F1.58.5 ± 11.9aP < 0.05 vs. B6.,cP < 0.05 vs. CBAxB6 F1.5.58 ± 0.97CBAxB6-TGFβ F1 (n = 15)8.54 ± 1.06aP < 0.05 vs. B6.,bP < 0.05 vs. B6-TGFβ.9.94 ± 1.03aP < 0.05 vs. B6.,bP < 0.05 vs. B6-TGFβ.,cP < 0.05 vs. CBAxB6 F1.44.49 ± 19.15aP < 0.05 vs. B6.,cP < 0.05 vs. CBAxB6 F1.152.0 ± 31.8aP < 0.05 vs. B6.,bP < 0.05 vs. B6-TGFβ.,cP < 0.05 vs. CBAxB6 F1.15.20 ± 6.66aP < 0.05 vs. B6.,bP < 0.05 vs. B6-TGFβ.,cP < 0.05 vs. CBAxB6 F1.ANOVA/Kruskal-WallisP < 0.0001P < 0.0001P < 0.0001P < 0.0001P < 0.005ANOVA, analysis of variance; TGFβ, transforming growth factor-beta.Data from male TGF-β1 transgenic mice and male wild-type controls at the age of 14 days are shown. Despite having plasma TGFβ1 levels similar to those of B6-TGFβ mice, CBAxB6-TGFβ mice had significant renal hypertrophy, accompanied by elevated serum urea nitrogen levels and proteinuria. Values are expressed as mean ± SD. ANOVA or Kruskal-Wallis test.a P < 0.05 vs. B6.b P < 0.05 vs. B6-TGFβ.c P < 0.05 vs. CBAxB6 F1. Open table in a new tab ANOVA, analysis of variance; TGFβ, transforming growth factor-beta. Data from male TGF-β1 transgenic mice and male wild-type controls at the age of 14 days are shown. Despite having plasma TGFβ1 levels similar to those of B6-TGFβ mice, CBAxB6-TGFβ mice had significant renal hypertrophy, accompanied by elevated serum urea nitrogen levels and proteinuria. Values are expressed as mean ± SD. ANOVA or Kruskal-Wallis test. At 14 days of age, B6-TGFβ mice exhibited kidney histology similar to wild-type controls. However, CBAxB6-TGFβ mice manifested glomerular hypertrophy with mesangial expansion and capillary obliteration affecting 60% of glomeruli, together with mild tubulointerstitial fibrosis and tubular hyaline deposits (Figure 1d–f [right panel of f]). Significant glomerular and tubulointerstitial collagen-4 (Figure 2a and b) and fibronectin accumulation were present (Figure 2c and d). Glomerular expression of TGFB1 protein was significant in only CBAxB6-TGFβ kidneys (Figure 2e and f), whereas mild tubular expression was present in B6-TGFβ as well. The evaluation of renal cortical gene expression at age 14 days showed that collagen-1 (Cola1) and collagen-3 (Col3a1) mRNA expression in B6-TGFβ kidneys were mildly elevated, compared with that in controls, and were significantly increased (5- and 4-fold, respectively) in kidneys of CBAxB6-TGFβ mice (Figure 3a and b). This increase was accompanied by renal Lcn2 (Figure 3c), Tgfb1, and connective tissue growth factor (Ctgf) mRNA overexpression in CBAxB6-TGFβ mice (Table 2), in parallel with increased expression of the TGF-β1 inhibitors biglycan (Bgn) and decorin (Dcn; Table 2). At the age of 14 days, expression of matrix metalloproteinease-2 (Mmp2) was similar in wild-type and transgenic kidneys. No significant alteration occurred in the expression of TGF-β1 receptor-II (Tgfbr2) or any of the Smad mRNAs (Smad2, 3, 4, 6, 7) or SMAD3 phosphorylation (Supplementary Figure S2A). Interestingly, Mmp9 levels in B6 and B6-TGF-β kidneys were comparable, whereas CBAxB6 and CBAxB6-TGF-β kidneys had 3- to 4-fold increases in Mmp9 mRNA expression, respectively. Expression of Timp2 mRNA was comparable in wild-type and B6-TGFβ kidneys, but it was elevated in CBAxB6-TGF-β mice (Table 2).Table 2Renal expression of fibrosis pathway genes in transgenic B6-TGFβ and CBAxB6-TGFβ F1 mice at age 14 daysGene symbolB6 (n = 5)CBAxB6 F1 (n = 5)B6-TGFβ (n = 7)CBAxB6-TGFβ F1 (n = 7)Kruskal-Wallis testTgfb11.00 ± 0.181.05 ± 0.120.80 ± 0.171.61 ± 0.42aP < 0.05 vs. B6.,bP < 0.05 vs. B6-TGFβ.,cP < 0.05 vs. CBAxB6 F1.P < 0.0001Ctgf1.00 ± 0.190.69 ± 0.130.29 ± 0.061.88 ± 0.56aP < 0.05 vs. B6.,bP < 0.05 vs. B6-TGFβ.,cP < 0.05 vs. CBAxB6 F1.P < 0.0001Bgn (biglycan)1.00 ± 0.390.37 ± 0.061.01 ± 0.231.78 ± 0.46aP < 0.05 vs. B6.,bP < 0.05 vs. B6-TGFβ.,cP < 0.05 vs. CBAxB6 F1.P < 0.0001Dcn (decorin)1.00 ± 0.280.43 ± 0.131.09 ± 0.27cP < 0.05 vs. CBAxB6 F1.1.84 ± 0.43aP < 0.05 vs. B6.,bP < 0.05 vs. B6-TGFβ.,cP < 0.05 vs. CBAxB6 F1.P < 0.0001Mmp21.00 ± 0.290.40 ± 0.210.97 ± 0.190.91 ± 0.38NSMmp91.00 ± 0.333.24 ± 0.57aP < 0.05 vs. B6.0.88 ± 0.30cP < 0.05 vs. CBAxB6 F1.4.06 ± 1.04aP < 0.05 vs. B6.,bP < 0.05 vs. B6-TGFβ.P < 0.0001Timp11.00 ± 0.530.52 ± 0.281.39 ± 0.45108.48 ± 28.57aP < 0.05 vs. B6.,bP < 0.05 vs. B6-TGFβ.,cP < 0.05 vs. CBAxB6 F1.P < 0.0001Timp21.00 ± 0.200.61 ± 0.080.98 ± 0.091.53 ± 0.11cP < 0.05 vs. CBAxB6 F1.P < 0.05Timp31.00 ± 0.211.31 ± 0.241.16 ± 0.071.35 ± 0.39NSTgbr21.00 ± 0.130.91 ± 0.151.03 ± 0.341.15 ± 0.16NSSmad21.00 ± 0.340.72 ± 0.180.86 ± 0.140.95 ± 0.39NSSmad31.00 ± 0.080.81 ± 0.190.89 ± 0.121.03 ± 0.23NSSmad41.00 ± 0.280.85 ± 0.220.97 ± 0.120.91 ± 0.24NSSmad61.00 ± 0.470.95 ± 0.391.12 ± 0.191.10 ± 0.60NSSmad71.00 ± 0.100.70 ± 0.170.86 ± 0.111.07 ± 0.24NSTGFβ, transforming growth factor-beta; NS, not significant.Renal mRNA expression values of male TGF-β1 transgenic mice and male wild-type controls at age 14 days. Expression of each gene was normalized to 18S rRNA (Rn18s) using the 2-ΔΔCt formula. Data are presented as fold expression to a pooled control sample (mean ± SD).a P < 0.05 vs. B6.b P < 0.05 vs. B6-TGFβ.c P < 0.05 vs. CBAxB6 F1. Open table in a new tab TGFβ, transforming growth factor-beta; NS, not significant. Renal mRNA expression values of male TGF-β1 transgenic mice and male wild-type controls at age 14 days. Expression of each gene was normalized to 18S rRNA (Rn18s) using the 2-ΔΔCt formula. Data are presented as fold expression to a pooled control sample (mean ± SD). Strikingly, a 100-fold increase occurred in Timp1 mRNA expression in CBAxB6-TGFβ kidneys, compared to that in B6-TGF-β and wild-type mice (Figure 3d). Immunoblot analysis revealed a 10-fold increase in TIMP1 protein expression (Figure 3e), mainly localized to tubules in CBAxB6-TGFβ kidneys (Figure 3g). This TIMP1 overexpression in CBAxB6-TGFβ kidneys resulted in reduced renal matrix metalloproteinase-9 (MMP9) activity, as shown by gelatin zymography (Figure 3f). We wished to elucidate whether either the hepatic expression of the TGF-β transgene exerts local fibrotic effects in the liver or the elevated plasma TGF-β1 levels distantly affect the heart in ways that could substantially contribute to the short survival time of CBAxB6-TGFβ mice. However, the extent of fibrosis in the liver and the myocardial expression of Col1a1 mRNA were similar in the 2 transgenic strains (Supplementary Figure S2B and C). To investigate whether alterations in other kidney disease–related genes could be responsible for this phenotype, we performed cDNA microarray analysis of B6-TGFβ and CBAxB6-TGFβ kidneys. The microarray showed 311 significant gene alterations (Supplementary Figures S3 and S4; Supplementary Tables S4 and S5) and confirmed that Timp1 is the most upregulated gene related to matrix remodeling. Significant changes in RNA expression were seen for 27 transcription factors (Supplementary Table S6). Among them, Egr2 was the most strikingly induced in CBAxB6-TGFβ kidneys (Figure 4a–c). EGR2 drew our attention, as it has been implicated in the pathogenesis of skin and lung fibrosis.16Fang F. Ooka K. Bhattacharyya S. et al.The early growth response gene Egr2 (Alias Krox20) is a novel transcriptional target of transforming growth factor-beta that is up-regulated in systemic sclerosis and mediates profibrotic responses.Am J Pathol. 2011; 178: 2077-2090Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar Of note, renal expression of Egr2 mRNA was similar in B6-TGFβ and wild-type mice (Figure 4a–c). We observed nuclear EGR2 protein expression mostly in some interstitial cells in control kidneys and B6-TGFβ mice; this increased significantly in CBAxB6-TGFβ kidneys, accompanied by tubular overproduction that coexpressed with TIMP1 (Figure 4c). As histology revealed nearly end-stage glomerulosclerosis in CBAxB6-TGFβ (F1) kidneys at age 14 days, we investigated the initiation and early development of kidney fibrosis in TGF-β1–transgenic mice at birth and at 5 days. Glomerular size and mesangial matrix content increased significantly in CBAxB6-TGFβ mice at 5 days (glomerulosclerosis index [GSI] B6-TGFβ: 0.05 ± 0.01 vs. CBAxB6-TGFβ: 0.45 ± 0.21, P < 0.05; Figure 1f, left). Renal Tgfb1 mRNA expression in 5-day-old CBAxB6-TGFβ mice was also significantly higher compared to that in B6-TGF-β or controls (Table 3), although expression of Ctgf, and type-I (Col1a1) and type III (Col3a1) collagens, was similar in all groups at this age. However, Timp1 mRNA expression at this early age was 50% higher in CBAxB6-TGFβ kidneys, accompanied by elevated Egr2 expression (Table 3). In B6-TGFβ kidneys, EGR2 immunostaining was observed in interstitial cells, similar to that in controls, despite a mild tubular TIMP1 immunostaining (Supplementary Figure S5). However, EGR2 and TIMP1 were already coexpressed in tubules of CBAxB6-TGFβ mice at this early age, although to a lesser extent than in 14-day-old mice (Figure 4c). In contrast, kidneys of newborn B6-TGFβ and CBAxB6-TGFβ mice depicted no renal histologic alterations (data not shown) and had comparable Timp1 and Egr2 mRNA expression levels. The kinetics of Egr2 and Timp1 mRNA expression from 0 to 14 days of age, while only correlative, could indicate a mechanistic role for EGR2 and TIMP1 in the pathophysiological process of kidney fibrosis in this model (Figure 4d and e).Table 3Renal expression of fibrosis-related genes in transgenic B6-TGFβ and CBAxB6-TGFβ F1 mice at age 5 daysGene symbolB6 (n = 4)CBAxB6 F1 (n = 4)B6-TGFβ (n = 6)CBAxB6-TGFβ F1 (n = 6)Kruskal-Wallis testTgfb11.00 ± 0.251.09 ± 0.060.83 ± 0.151.34 ± 0.32aP < 0.05 vs. B6.,bP < 0.05 vs. B6-TGFβ.,cP < 0.05 vs. CBAxB6 F1.P < 0.05Ctgf1.00 ± 0.061.14 ± 0.231.01 ± 0.321.18 ± 0.07NSCol1a1.00 ± 0.360.85 ± 0.141.08 ± 0.301.01 ± 0.37NSCol3a11.00 ± 0.250.78 ± 0.141.07 ± 0.451.13 ± 0.29NSMmp21.00 ± 0.110.89 ± 0.140.90 ± 0.101.75 ± 0.90aP < 0.05 vs. B6.,bP < 0.05 vs. B6-TGFβ.,cP < 0.05 vs. CBAxB6 F1.P < 0.05Mmp91.00 ± 0.350.92 ± 0.211.06 ± 0.691.27 ± 0.51NSTimp11.00 ± 0.531.25 ± 0.501.02 ± 0.441.94 ± 0.62aP < 0.05 vs. B6.,bP < 0.05 vs. B6-TGFβ.,cP < 0.05 vs. CBAxB6 F1.P < 0.05Egr21.00 ± 0.260.94 ± 0.310.99 ± 0.272.20 ± 0.42aP < 0.05 vs. B6.,bP < 0.05 vs. B6-TGFβ.,cP < 0.05 vs. CBAxB6 F1.P < 0.001TGFβ, transforming growth factor-beta; NS, not significant.Timp1 and Egr2 expression were already elevated in CBAxB6-TGFβ mice at this very early age. Gene expression was normalized to 18S rRNA (Rn18s) using the 2-ΔΔCt formula. Data are presented as fold expression to a pooled control sample (mean ± SD).a P < 0.05 vs. B6.b P < 0.05 vs. B6-TGFβ.c P < 0.05 vs. CBAxB6 F1. Open table in a new tab TGFβ, transforming growth factor-beta; NS, not significant. Timp1 and Egr2 expression were already elevated in CBAxB6-TGFβ mice at this very early age. Gene expression was normalized to 18S rRNA (Rn18s) using the 2-ΔΔCt formula. Data are presented as fold expression to a pooled control sample (mean ± SD). As these data suggested the central role of TIMP1 in TGF-β1–induced kidney fibrosis, we injected male CBAxB6-TGFβ mice i.p. with anti-TIMP1 neutralizing antibody or isotype IgG for 5 consecutive days, beginning at age 8 days, and the animals were euthanized at age 14 days. The early systemic TIMP1 inhibition was associated with a 20% reduction in kidney weight in fibrosis-prone CBAxB6-TGFβ mice, compared to that in isotype IgG-treated transgenic littermates (Figure 5a), accompanied by a 50% reduction in proteinuria (Figure 5b) and serum urea levels (Figure 5c), and by less glomerular and tubulointerstitial damage than in IgG-treated littermates (Figure 5d–f). In order to test whether strain-dependent early onset kidney fibrosis is associated with EGR2 or TIMP1 protein overexpression in other fibrosis models, we investigated B6 and CBA wild-type mice after UUO and SNX. Overt kidney fibrosis usually develops 3–5 days after UUO.17Klahr S. Morrissey J. Obstructive nephropathy and renal fibrosis.Am J Physiol Renal Physiol. 2002; 283: F861-F875Crossref PubMed Scopus (488) Google Scholar We first examined young wild-type B6 and CBA male mouse kidneys 24 hours after UUO. Kidney histology at this early stage showed only a few dilated tubules, otherwise normal structure, and no signs of extracellular matrix accumulation (Figure 6a). Renal mRNA expression of Lcn2, as a marker of tubular damage (Figure 6b) and Tgfb1 (Figure 6c), increased comparably in both B6 and CBA UUO. By contrast, Col1a1 mRNA expression was 2-fold higher in CBA UUO, compared with that in B6 UUO kidneys (Figure 6d), and was accompanied by significant overexpression of Egr2 and Timp1 mRNA (Figure 6e and f) and EGR2 protein (Supplementary Figure S6A and B). The higher TIMP1 expression resulted in lower renal MMP9 activity in CBA UUO kidneys (Figure 6g and h). We also analyzed kidneys at 7 days after UUO, at which point significant interstitial fibrosis had developed in B6, but slightly more-severe fibrosis had developed in CBA kidneys, accompanied by higher Timp1 mRNA expression and reduced renal MMP9 activity, but only Egr2 mRNA expression tended to be higher in CBA at this late time point (Supplementary Figure S7A–D). In the second set of experiments, we observed marked interstitial fibrosis and glomerulosclerosis in male CBA mice at 6 weeks after SNX, compared with B6 mice (Figure 7a and b). The significant renal fibrosis of CBA SNX mice was associated with an elevated urinary protein-to-creatinine ratio (Figure 7c), although Tgfb1 mRNA was comparable in B6 and CBA SNX mice (Figure 7d). However, CBA SNX kidneys overexpressed Col1a1 and Lcn2 mRNA (Figure 7e and f), as well as TIMP1 (Figure 7g and h), EGR2 mRNA, and protein (Figure 7i; Supplementary Figure S8), the latter of which co-localized with TIMP1, as shown by immunostaining in most of the CBA SNX tubules (Figure 7h). In order to confirm the central role of TIMP1 in the initiation and progression of kidney fibrosis, we compared TIMP1-deficient (Timp1-null) male mice on a fibrosis-prone CBAxB6 background, to wild-type CBAxB6 F1 male mice (Timp1+) subjected to SNX. Six weeks after nephrectomy, Timp1+ SNX kidneys manifested glomerulosclerosis, tubular atrophy, and interstitial matrix accumulation, whereas Timp1-null SNX kidneys exhibited only mild, nonsignificant glomerulosclerosis and interstitial fibrosis (Figure 8a and b). This difference was reflected by a marked difference in proteinuria (Figure 8c). Of note, expression of type I and type III collagen mRNA was slightly but significantly elevated in Timp1-null SNX kidneys (Figure 8d and e), even though their histology was similar to that of control kidneys. Notably, Timp1+ SNX kidneys had 10-fold increased expression of Col1a1 and Col3a1, accompanied by a 2-fold increase in Tgfb1 mRNA (Figure 8f). In contrast, Timp1-null SNX kidneys had Tgfb1 expression similar to that in controls. The lack of TIMP1 resulted in higher MMP9 gelatinase activity in Timp1-null SNX kidneys (Figure 8g). For in vitro ev