Sustained hyperosmolarity increses TGF-ß1 and Egr-1 expression in the rat renal medulla

Abstract

Background: Although TGF-ß and the transcription factor Egr-1 play an important role in both kidney fibrosis and in response to acute changes of renal medullary osmolarity, their role under sustained hypo- or hyperosmolar conditions has not been elucidated. We investigated the effects of chronic hypertonicity and hypotonicity on the renal medullary TGF-ß and Egr-1 expression.

Methods: Male adult Sprague Dawley rats (n = 6/group) were treated with 15 mg/day furosemide, or the rats were water restricted to 15 ml/200 g body weight per day. Control rats had free access to water and rodent chow. Kidneys were harvested after 5 days of treament. In cultured inner medullary collecting duct (IMCD) cells, osmolarity was increased from 330 mOsm to 900 mOsm over 6 days. Analyses were performed at 330, 600 and 900 mOsm.

Results: Urine osmolarity has not changed due to furosemide treatment but increased 2-fold after water restriction (p < 0.05). Gene expression of TGF-ß and Egr-1 increased by 1.9-fold and 7-fold in the hypertonic medulla, respectively (p < 0.05), accompanied by 6-fold and 2-fold increased c-Fos and TIMP-1 expression, respectively (p < 0.05) and positive immunostaining for TGF-ß and Egr-1 (p < 0.05). Similarly, hyperosmolarity led to overexpression of TGF-ß and Egr-1 mRNA in IMCD cells (2.5-fold and 3.5-fold increase from 330 to 900 mOsm, respectively (p < 0.05)) accompanied by significant c-Fos and c-Jun overexpressions (p < 0.01), and increased Col3a1 and Col4a1 mRNA expression.

Conclusion: We conclude that both TGF-ß and Egr-1 are upregulated by sustained hyperosmolarity in the rat renal medulla, and it favors the expression of extracellular matrix components.

Keywords: Egr-1; Fibrosis; Osmotic stress; Sodium chloride; TGF-ß; Urea.

Fig. 1

Effect of increased or decreased medullary solute concentration in rats. a Compared to the initial body weights and to controls, both water restricted and furosemide treated rats had significantly lower body weights at harvest. The urine volume dropped by 50% due to water restriction, but increased almost 2-fold in furosemide treated rats, accompanied by significantly decreased or increased excretion of solutes, respectively. b Water restriction increased mRNA expression of TGF-ß by 2-fold in the medulla, accompanied by marked TGF-ß immunostaining c, d, as compared to control and furosemide treated rats. d The cytoplasm of outer medullary tubular epithelial cells were negative in controls and only weakly stained in furosemide treated rats, but no interstitial staining was observed. However, water restriction increased the cytoplasmic TGF-ß staining intensity in several tubular cells in the inner stripe of the outer medulla (red cytoplasm, see arrows). In contrast, TGF-ß immunostaining showed mostly interstitial staining in the inner medulla of controls and furosemide treated rats, but also tubular cytoplasmic staining in water restricted rats (see insets, arrows pointing on positive cells). Primary antibody was omitted as negative control for immunostaining. Data are presented as mean ± SEM, n = 6/group. * p < 0.05, ** p < 0.01, *** p < 0.001 (one-way ANOVA with Tukey’s post-hoc test or Kruskal-Wallis test with Dunn’s post-hoc test)

Fig. 2

Expression of profibrotic transcription factors due to chronic increased or decreased medullary solute concentration in rats. a Compared to control rats, water restriction increased the medullary expression of Egr-1 mRNA and protein, while furosemide treated rats had similar expression levels to controls. b The Egr-1 overexpression was confirmed by immunohistochemistry, depicting a significant amount of Egr-1 positive tubular cell nuclei in water restricted rats (arrows), as compared to both control and furosemide treated rats. c Interestingly, apart of the tubular staining (arrows), we also found Egr-1 positive interstitial cells only in the medulla of water restricted rats (asterisk). d Water restriction significantly increased the mRNA expression of the AP-1 component cFos. The expression of the AP-1 component cJun did not change due to increased medullary solute concentration, but decreased by 50% after furosemide treatment. e Increased medullary osmolarity had no effect on the mRNA expression of collagen-III, but it was reduced by furosemide treatment. As compared to controls, the medullary TIMP-1 expression was almost 2-fold elevated after chronic water restriction, and furosemide did not alter TIMP-1. Data are presented as mean ± SEM, n = 6/group. * p < 0.05, ** p < 0.01, *** p < 0.001 (Kruskal-Wallis test with Dunn’s post-hoc test)

Fig. 3

Effect of chronic increase in medium osmolarity on viability and TGF-ß expression of IMCD cells. a Chronic hyperosmolarity significantly reduced the viability of IMCD cells (n = 8/group). b The expression of TGF-ß, however, increased dramatically both at mRNA and protein level (n = 4/group). c Accordingly, immunocytochemistry depicted significant TGF-ß staining at 600 mOsm and even stronger staining at 900 mOsm (n = 3/group). Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 (one-way ANOVA with Tukey’s post-hoc test)

Fig. 4

Effect of chronic hyperosmolarity on the expression of profibrotic genes and transcription factors in IMCD cells. a The progressive increase of medium osmolarity induced overexpression of the Egr-1 mRNA. Mild type III collagen upregulation was induced only by 900 mOsm, dramatic (6-fold) type IV collagen overexpression was observed at medium osmolarity of 600 mOsm. b Egr-1 and TGF-ß mRNA expressions showed the strongest correlation. Both type III and type IV collagen expressions correlated well to the expression of the profibrotic Egr-1. c Components of the transcription factor AP-1, cFos and cJun, were strongly overexpressed only at high osmolar concentration of 900 mOsm. Data are presented as mean ± SEM, n = 4/group. * p < 0.05, ** p < 0.01 (Kruskal-Wallis test with Dunn’s post-hoc test)

Fig. 5

Effect of TGF-ß administration on IMCD cells under normal osmotic concentration. TGF-ß administration for 48 h significantly upregulated Egr-1 mRNA expression by 2-fold (a) but had no effect on type III collagen expression (b). As compared to the effects of increased osmotic concentration, TGF-ß administration induced only a mild type IV collagen overexpression (c). Data are presented as mean ± SEM, n = 3/group. * p < 0.05 (Mann-Whitney test)

Fig. 6

Proposed molecular effect of chronic osmotic stress on collagen production. Osmotic stress induces the expression of the profibrotic early growth response factor-1 (Egr-1), which can directly induce the transcription of collagen-III and collagen-IV. Hyperosmolarity also induces profibrotic TGF-ß expression, which can lead to AP-1 activation through cFos and cJun, therefore directly upregulate collagen expression. Egr-1 might also directly activate cJun and promote further AP-1 activation. Moreover, we postulate that TGF-ß and Egr-1 might induce each other in the renal medulla (dotted arrows), enhancing their profibrotic effects