依那普利在大鼠单侧输尿管梗阻再通模型中的抗纤维化作用

目的:探讨依那普利对大鼠单侧输尿管梗阻再通模型肾脏纤维化的影响.方法:18只SD大鼠随机分为两组:假手术组(6只)以及单侧输尿管梗阻模型组(12只).输尿管梗阻3天后,实施梗阻再通手术,再将大鼠随机分为模型组(6只)以及依那普利组(6只),术后,依那普利组给予依那普利灌胃10mg/kg/d,假手术组以及模型组给予等量0.5%CM-CNa溶液灌胃.用药2周后,取术侧肾组织做HE染色,并采用Raford评分系统对肾间质损伤程度进行评分;用Real-timePCR方法检测Ⅰ、Ⅲ型胶原以及CT-GFmRNA的表达;用Westemblot方法检测CTGF蛋白水平的表达.结果:模型组大鼠肾脏损伤程度,Ⅰ、Ⅲ型胶原mRNA表达水平,以及CTGFmRNA和蛋白表达水平均比假手术组明显上升(P<0.01).经依那普利治疗后,与模型组相比,以上指标均显著下降(P<0.01).结论:依那普利能有效阻止大鼠单侧输尿管梗阻再通后肾脏纤维化的进展.依那普利抗纤维化的作用机制可能与抑制CTGF的表达有关.     

Effects of nitric oxide on renal interstitial fibrosis in rats with unilateral ureteral obstruction

AimsIt is well recognized that microvascular injury is a major determinant of renal fibrosis. Mounting evidence shows that nitric oxide (NO) plays an important role in angiogenesis. Therefore, we investigated to the effects of NO on kidney angiogenesis and renal fibrosis.MethodsIn the present study, a unilateral ureteral obstruction (UUO) model was established with l-arginine (l-Arg, 1 g/dl) and N-nitro-l-arginine methyl ester (L-NAME, 5 mg/dl) serving as interference factors. We investigated the alteration of NO concentration with spectrophotometry, peritubular capillary (PTC) density with aminopeptidase P (JG12) immunohistochemical staining, and the expression of vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), hypoxia inducible factor-1α (HIF-1α) and transforming growth factor-β1 (TGF-β1) with immunohistochemical staining and Western blotting at weeks 2, 3 and 4.Key findingsOur findings showed that the expressions of VEGF, eNOS and PTC density were significantly decreased in rats with UUO, which was accompanied by a progressive increase in HIF-1α, TGF-β1 and an area of renal interstitial fibrosis. The administration of l-Arg promoted the synthesis of NO and significantly elevated the expressions of VEGF, eNOS and PTC density with the conspicuous loss of HIF-1α and TGF-β1 expressions and ultimately ameliorated renal fibrosis, which was markedly aggravated by L-NAME administration.SignificanceThese findings demonstrate that NO appears to play an important role in kidney angiogenesis and in slowing the progression of renal interstitial fibrosis, which suggests that NO may serve as a novel therapeutic strategy for preventing renal fibrosis as well as fibrosis in other organs.     

依帕司他对单侧输尿管梗阻大鼠肾间质纤维化的干预作用及其机制

目的:观察依帕司他(EPS)对单侧输尿管梗阻(UUO)大鼠间质纤维化的保护作用及其机制。方法:实验设假手术组(Sham)组、UUO、UUO+EPS(50 mg/kg)及UUO+EPS(100 mg/kg)剂量组,每组n=8。左侧输尿管结扎制备UUO大鼠模型。造模后连续灌胃给药3周,sham和UUO组给予等体积的羟甲基纤维素钠。HE和Masson染色观察肾组织病理变化及胶原沉积情况。免疫组化法观察肾组织醛糖还原酶(AR)表达情况,分别采用real-time PCR和(或) Western blot检测肾脏I型胶原(collagen I)、III型胶原(collagen III)、α-平滑肌肌动蛋白(α-SMA)、成纤维细胞特异蛋白-1(FSP-1)、纤连蛋白(FN)、E-钙粘蛋白(E-cadherin)、转化生成因子-β1(TGF-β1)和AR mRNA及蛋白表达。结果:与Sham组相比,UUO组大鼠小管上皮细胞萎缩、空泡样变性,肾间质成纤维细胞及肌成纤维细胞大量增殖并伴大量炎症细胞浸润,胶原沉积明显增加,collagen I、collagen III、TGF-β1和AR mRNA及蛋白表达水平明显升高(P<0.01),同时EMT标志性蛋白α-SMA、FSP-1、FN mRNA及蛋白表达水平明显升高(P<0.01),而E-cadherin mRNA及蛋白表达水平明显降低。与UUO组相比,经EPS治疗3周后,肾间质纤维化程度明显减轻,胶原沉积明显减少,collagen I、collagen III、TGF-β1和AR mRNA及蛋白表达水平明显降低(P<0.01或P<0.05),另外α-SMA、FSP-1、FN mRNA及蛋白表达水平明显降低(P<0.01或P<0.05),而E-cadherin mRNA及蛋白表达水平明显升高(P<0.01或P<0.05),而且100 mg/kg剂量组上述指标的改变均好于低剂量组(P<0.05,P<0.01)。结论:依帕司他对肾间质纤维化具有一定的改善作用,其机制可能与其抑制TGF-β1介导的AR表达、进而抑制大鼠肾小管上皮细胞EMT有关。     

Persistent activation of autophagy in kidney tubular cells promotes renal interstitial fibrosis during unilateral ureteral obstruction

Renal fibrosis is the final, common pathway of end-stage renal disease. Whether and how autophagy contributes to renal fibrosis remains unclear. Here we first detected persistent autophagy in kidney proximal tubules in the renal fibrosis model of unilateral ureteral obstruction (UUO) in mice. UUO-associated fibrosis was suppressed by pharmacological inhibitors of autophagy and also by kidney proximal tubule-specific knockout of autophagy-related 7 (PT-Atg7 KO). Consistently, proliferation and activation of fibroblasts, as indicated by the expression of ACTA2/α-smooth muscle actin and VIM (vimentin), was inhibited in PT-Atg7 KO mice, so was the accumulation of extracellular matrix components including FN1 (fibronectin 1) and collagen fibrils. Tubular atrophy, apoptosis, nephron loss, and interstitial macrophage infiltration were all inhibited in these mice. Moreover, these mice showed a specific suppression of the expression of a profibrotic factor FGF2 (fibroblast growth factor 2). In vitro, TGFB1 (transforming growth factor β 1) induced autophagy, apoptosis, and FN1 accumulation in primary proximal tubular cells. Inhibition of autophagy suppressed FN1 accumulation and apoptosis, while enhancement of autophagy increased TGFB1-induced-cell death. These results suggest that persistent activation of autophagy in kidney proximal tubules promotes renal interstitial fibrosis during UUO. The profibrotic function of autophagy is related to the regulation on tubular cell death, interstitial inflammation, and the production of profibrotic factors.     

Transplantation of endothelial progenitor cells alleviates renal interstitial fibrosis in a mouse model of unilateral ureteral obstruction

AimsThe present study investigated whether transplantation of bone marrow-derived endothelial progenitor cells (BM-EPCs) in renal capillary network improves renal interstitial fibrosis in unilateral ureteral obstruction (UUO) model in mice.Main methodsEx vivo generated, characterized, and cultivated mice BM-EPCs were identified by their vasculogenic properties in vitro. BM-EPCs were labelled with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) before transplantation. The animal models of UUO were used. Histological changes in renal tubular interstitium were observed with HE and Masson staining. The protein levels of vascular endothelial growth factor(VEGF), hypoxia inducible factor-1α (HIF-1α) and connective tissue growth factor (CTGF) were analyzed by western blotting and immunohistochemistry. Transforming growth factor-β1 (TGF-β1) was detected by immunohistochemistry. Peritubular capillary (PTC) density was determined by CD31 immunostaining.Key findingsTransplanted BM-EPCs were successfully incorporated into the capillary network in the obstructed kidney in vivo. UUO induced a significant decrease in VEGF levels and PTC density in the kidney tissue, which was accompanied by a significant increase in HIF-1α, CTGF and TGF-β1. Transplantation of BM-EPCs increased PTC density, VEGF expression and alleviated the development of renal interstitial fibrosis in UUO mice. No significant pathological changes were found in control mice.SignificanceThe reduction of PTC density and up-regulation of HIF-1α are the important mechanisms of interstitial fibrosis in UUO mice. BM-EPCs transplantation may increase the number of capillary density and alleviate the development of renal fibrosis in obstructive nephropathy in mice.     

Loss of poly(ADP-ribose) polymerase 1 attenuates renal fibrosis and inflammation during unilateral ureteral obstruction

Poly(ADP-ribose) polymerase 1 (PARP1) contributes to necrotic cell death and inflammation in several disease models; however, the role of PARP1 in fibrogenesis remains to be defined. Here, we tested whether PARP1 was involved in the pathogenesis of renal fibrosis using the unilateral ureteral obstruction (UUO) mouse model. UUO was performed by ligation of the left ureter near the renal pelvis in Parp1-knockout (KO) and wild-type (WT) male mice. After 10 days of UUO, renal PARP1 expression and activation were strongly increased by 6- and 13-fold, respectively. Interstitial fibrosis induced by UUO was significantly attenuated in Parp1-KO kidneys compared with that in WT kidneys at 10 days, but not at 3 days, based on collagen deposition, α-smooth muscle actin (α-SMA), and fibronectin expression. Intriguingly, the UUO kidneys in Parp1-KO mice showed a dramatic decrease in infiltration of neutrophil and reduction in expression of proinflammatory proteins including intercellular adhesion molecule-1, tumor necrosis factor-α, inducible nitric oxide synthase, and toll-like receptor 4 as well as phosphorylation of nuclear factor-κB p65, but not transforming growth factor-β1 (TGF-β1) at both 3 and 10 days. Pharmacological inhibition of PARP1 in rat renal interstitial fibroblast (NRK-49F) cell line or genetic ablation in primary mouse embryonic fibroblast cells did not affect TGF-β1-induced de novo α-SMA expression. Parp1 deficiency significantly attenuated UUO-induced histological damage in the kidney tubular cells, but not apoptosis. These data suggest that PARP1 induces necrotic cell death and contributes to inflammatory signaling pathways that trigger fibrogenesis in obstructive nephropathy.     

Mast cells are required for the development of renal fibrosis in the rodent unilateral ureteral obstruction model

Mast cells are associated with inflammation and fibrosis. Whether they protect against or contribute to renal fibrosis is unclear. Based on our previous findings that mast cells can express and secrete active renin, and that angiotensin (ANG II) is profibrotic, we hypothesized that mast cells play a critical role in tubulointerstitial fibrosis. We tested this hypothesis in the 14-day unilateral ureteral obstruction (UUO) model in rats and mast cell-deficient (MCD) mice (WBB6F1-W/Wv) and their congenic controls (CC). In the 14-day UUO rat kidney, mast cell number is increased and they express active renin. Stabilizing mast cells in vivo with administration of cromolyn sodium attenuated the development of tubulointerstitial fibrosis, which was confirmed by measuring newly synthesized pepsin-soluble collagen and blind scoring of fixed trichrome-stained kidney sections accompanied by spectral analysis. Fibrosis was absent in UUO kidneys from MCD mice unlike that observed in the CC mice. Losartan treatment reduced the fibrosis in the CC UUO kidneys. The effects of mast cell degranulation and renin release were tested in the isolated, perfused kidney preparation. Mast cell degranulation led to renin-dependent protracted flow recovery. This demonstrates that mast cell renin is active in situ and the ensuing ANG II can modulate intrarenal vascular resistance in the UUO kidney. Collectively, the data demonstrate that mast cells are critical to the development of renal fibrosis in the 14-day UUO kidney. Since renin is present in human kidney mast cells, our work identifies potential targets in the treatment of renal fibrosis.     

1H NMR-based metabonomics study on serum of renal interstitial fibrosis rats induced by unilateral ureteral obstruction

Renal fibrosis is the common pathway of progressive renal disease with complex pathogenesis. Investigating the metabolic changes in the evaluation process of renal fibrosis may enhance the understanding of its pathogenesis. In this study, (1)H nuclear magnetic resonance ((1)H NMR) measurements combined with multivariate statistical techniques were performed to study the metabolic changes in serum samples of renal interstitial fibrosis (RIF) rats, induced by unilateral ureteral obstruction (UUO). Partial least squares-discriminant analysis (PLS-DA) showed satisfactory clustering between UUO and sham operation (SO) rats, suggesting that the metabolic profiles of the RIF groups are markedly different from those of the controls. Alterations in the levels of some metabolites such as valine, isoleucine, lactate, 3-hydroxybutyrate, alanine, acetate, acetoacetate, pyruvate, and glutamate, with time dependence in UUO rats, were observed in PLS-DA loading plots. These changed metabolites represent potential metabolic biomarkers and provide clues that can elucidate the mechanisms underlying the generation and development of RIF. Enhanced metabolic pathways of lipid and ketone body synthesis were predominant in RIF rats. Energy metabolism seemed to be impaired at the early stage of fibrosis but enhanced at a late stage. Our results suggest that (1)H NMR-based metabonomics can provide novel insights into the pathogenesis of RIF.     

Ablation of Gadd45β ameliorates the inflammation and renal fibrosis caused by unilateral ureteral obstruction

The growth arrest and DNA damage‐inducible beta (Gadd45β) protein have been associated with various cellular functions, but its role in progressive renal disease is currently unknown. Here, we examined the effect of Gadd45β deletion on cell proliferation and apoptosis, inflammation, and renal fibrosis in an early chronic kidney disease (CKD) mouse model following unilateral ureteral obstruction (UUO). Wild‐type (WT) and Gadd45β‐knockout (KO) mice underwent either a sham operation or UUO and the kidneys were sampled eight days later. A histological assay revealed that ablation of Gadd45β ameliorated UUO‐induced renal injury. Cell proliferation was higher in Gadd45β KO mouse kidneys, but apoptosis was similar in both genotypes after UUO. Expression of pro‐inflammatory cytokines after UUO was down‐regulated in the kidneys from Gadd45β KO mice, whereas UUO‐mediated immune cell infiltration remained unchanged. The expression of pro‐inflammatory cytokines in response to LPS stimulation decreased in bone marrow‐derived macrophages from Gadd45β KO mice compared with that in WT mice. Importantly, UUO‐induced renal fibrosis was ameliorated in Gadd45β KO mice unlike in WT mice. Gadd45β was involved in TGF‐β signalling pathway regulation in kidney fibroblasts. Our findings demonstrate that Gadd45β plays a crucial role in renal injury and may be a therapeutic target for the treatment of CKD.     

Liproxstatin-1 attenuates unilateral ureteral obstruction-induced renal fibrosis by inhibiting renal tubular epithelial cells ferroptosis

Renal fibrosis is a common pathological process that occurs with diverse etiologies in chronic kidney disease. However, its regulatory mechanisms have not yet been fully elucidated. Ferroptosis is a form of non-apoptotic regulated cell death driven by iron-dependent lipid peroxidation. It is currently unknown whether ferroptosis is initiated during unilateral ureteral obstruction (UUO)-induced renal fibrosis and its role has not been determined. In this study, we demonstrated that ureteral obstruction induced ferroptosis in renal tubular epithelial cells (TECs) in vivo. The ferroptosis inhibitor liproxstatin-1 (Lip-1) reduced iron deposition, cell death, lipid peroxidation, and inhibited the downregulation of GPX4 expression induced by UUO, ultimately inhibiting ferroptosis in TECs. We found that Lip-1 significantly attenuated UUO-induced morphological and pathological changes and collagen deposition of renal fibrosis in mice. In addition, Lip-1 attenuated the expression of profibrotic factors in the UUO model. In vitro, we used RSL3 treatment and knocked down of GPX4 level by RNAi in HK2 cells to induce ferroptosis. Our results indicated HK2 cells secreted various profibrotic factors during ferroptosis. Lip-1 was able to inhibit ferroptosis and thereby inhibit the secretion of the profibrotic factors during the process. Incubation of kidney fibroblasts with culture medium from RSL3-induced HK2 cells promoted fibroblast proliferation and activation, whereas Lip-1 impeded the profibrotic effects. Our study found that Lip-1 may relieve renal fibrosis by inhibiting ferroptosis in TECs. Mechanistically, Lip-1 could reduce the activation of surrounding fibroblasts by inhibiting the paracrine of profibrotic factors in HK2 cells. Lip-1 may potentially be used as a therapeutic approach for the treatment of UUO-induced renal fibrosis.Subject terms: Cell death, RNAi, Urinary tract obstruction     

Toll-like receptor downstream signaling

The family of Toll-like receptors (TLRs) senses conserved structures found in a broad range of pathogens, causing innate immune responses that include the production of inflammatory cytokines, chemokines and interferons. The signal transduction is initiated from the Toll/interleukin-1 receptor (TIR) domain of TLRs after pathogen recognition. Almost all TLRs use a TIR-containing adapter MyD88 to activate a common signaling pathway that results in the activation of NF-κB to express cytokine genes relevant to inflammation. Recently, three further TIR-containing adapters have been identified and shown to selectively interact with several TLRs. In particular, activation of the TRIF-dependent pathway confers antiviral responses by inducing anti-viral genes including that encoding interferon-β. Taken together, these results indicate that the interaction between individual TLRs and the different combinations of adapters directs appropriate responses against distinct pathogens.     

Toll-like receptor downstream signaling

The family of Toll-like receptors (TLRs) senses conserved structures found in a broad range of pathogens, causing innate immune responses that include the production of inflammatory cytokines, chemokines and interferons. The signal transduction is initiated from the Toll/interleukin-1 receptor (TIR) domain of TLRs after pathogen recognition. Almost all TLRs use a TIR-containing adapter MyD88 to activate a common signaling pathway that results in the activation of NF-kappaB to express cytokine genes relevant to inflammation. Recently, three further TIR-containing adapters have been identified and shown to selectively interact with several TLRs. In particular, activation of the TRIF-dependent pathway confers antiviral responses by inducing anti-viral genes including that encoding interferon-beta. Taken together, these results indicate that the interaction between individual TLRs and the different combinations of adapters directs appropriate responses against distinct pathogens.     

The effect of unilateral ureteral obstruction on renal function in pigs measured by diffusion-weighted MRI

The objective of this study was to examine the effect of unilateral ureteral obstruction on the apparent diffusion coefficient (ADC) in pig kidney. Changes in ADC is suggested to reflect changes in the ratio of extracellular to intracellular volume. Thirteen pigs were allocated into three groups: 1) pigs subjected to acute unilateral ureteral obstruction (AUO) (n = 3), 2) pigs subjected to chronic partial unilateral obstruction (CPUO) (n = 3), and 3) control pigs (n = 7). The extra- to intracellular volume ratio was indirectly measured in both the ipsilateral obstructed kidney and contralateral non-obstructed kidney by the ADC of the renal tissue using diffusion-weighted echo-planar magnetic resonance imaging. ADC was 2.07 +/- 0.27 x 10(-3) mm2/s in the cortex and 2.10 +/- 0.24 x 10(-3) mm2/s in the medulla of normal control kidneys. In the obstructed kidney from the AUO group the ADC of the medulla was significantly reduced 24 hours after occlusion of the ureter (1.65 +/- 0.05 x 10(-3) mm2/s vs 2.10 +/- 0.24 x 10(-3) mm2/s; p < 0.05). Similarly ADC decreased slightly in the cortex of the ipsilateral kidney. In contrast, ADC of the ipsilateral kidney of CPUO pigs was increased both in the renal medulla (3.13 +/- 0.21 x 10(-3) mm2/s vs. 2.10 +/- 0.24 x 10(-3) mm2/s; p < 0.05) and cortex (3.09 +/- 0.14 x 10(-3) mm2/s vs. 2.07 x 10(-3) mm2/s, p < 0.05). In conclusion, the results of the present study suggest that diffusion weighted imaging (ADC) may be a useful parameter to incorporate when identifying whether a ureteric obstruction is acute or chronic.     

Toll-like receptor 2 is required for LPS-induced Toll-like receptor 4 signaling and inhibition of ion transport in renal thick ascending limb

Previously we demonstrated that basolateral LPS inhibits HCO(3)(-) absorption in the renal medullary thick ascending limb (MTAL) through TLR4-dependent ERK activation. Here we report that the response of the MTAL to basolateral LPS requires TLR2 in addition to TLR4. The basolateral addition of LPS (ultrapure Escherichia coli K12) decreased HCO(3)(-) absorption in isolated, perfused MTALs from wild-type mice but had no effect in MTALs from TLR2(-/-) mice. In contrast, inhibition of HCO(3)(-) absorption by lumen LPS was preserved in TLR2(-/-) MTALs, indicating that TLR2 is involved specifically in mediating the basolateral LPS response. LPS also did not increase ERK phosphorylation in MTALs from TLR2(-/-) mice. TLR2 deficiency had no effect on expression of TLR4, MD-2, or MyD88. However, LPS-induced recruitment of MyD88 to the basolateral membrane was impaired in TLR2(-/-) MTALs. Inhibition of HCO(3)(-) absorption by LPS did not require CD14. Co-immunoprecipitation studies demonstrated an association between TLR4 and TLR2. Inhibition of HCO(3)(-) absorption by TLR2-specific ligands was preserved in MTALs from TLR4(-/-) mice. These results indicate that the effect of basolateral LPS to inhibit HCO(3)(-) absorption in the MTAL through MyD88-dependent ERK activation depends on a novel interaction between TLR4 and TLR2. TLR2 plays a dual role in the induction of intracellular signals that impair MTAL function, both through cooperation with TLR4 to mediate ERK signaling by LPS and through a TLR4-independent signaling pathway activated by Gram-positive bacterial ligands. Regulation of TLR2 expression and its interaction with TLR4 may provide new mechanisms for controlling and therapeutic targeting of TLR4-mediated LPS responses.     

Tyrosine phosphorylation in Toll-like receptor signaling

There is a wealth of knowledge about how different Ser/Thr protein kinases participate in Toll-like receptor (TLR) signaling. In many cases, we know the identities of the Ser/Thr residues of various components of the TLR-signaling pathways that are phosphorylated, the functional consequences of the phosphorylation and the responsible protein kinases. In contrast, the analysis of Tyr-phosphorylation of TLRs and their signaling proteins is currently incomplete, because several existing analyses are not systematic or they do not rely on robust experimental data. Nevertheless, it is clear that many TLRs require, for signaling, ligand-dependent phosphorylation of specific Tyr residues in their cytoplasmic domains; the list includes TLR2, TLR3, TLR4, TLR5, TLR8 and TLR9. In this article, we discuss the current status of knowledge of the effect of Tyr-phosphorylation of TLRs and their signaling proteins on their biochemical and biological functions, the possible identities of the relevant protein tyrosine kinases (PTKs) and the nature of regulations of PTK-mediated activation of TLR signaling pathways.     

Effect of acute and chronic renal denervation on renal function after release of unilateral ureteral obstruction in the rat.

The role of the renal nerves in determining renal function after relief of 24-h unilateral ureteral obstruction (UUO) was studied using clearance techniques in anaesthetized rats. Acute renal denervation during the first 1--2 h after relief of UUO resulted in a significant increase in glomerular filtration rate (GFR), renal plasma flow (RPF), urine flow, and sodium and potassium excretion, changes which were not seen in the sham-denervated postobstructive kidney. Acute denervation of sham-operated normal kidneys caused a similar natriuresis and diuresis but with no change in GFR or RPF. Chronic renal denervation 4--5 days before UUO denervated postobstructive controls, while chronic denervation alone was associated with a significantly higher urine flow and sodium excretion rate from the denervated kidney. The effectiveness of renal denervation was confirmed by demonstrating marked depletion of tissue catecholamines in the denervated kidney. It was concluded that renal nerve activity plays a significant but not a major role in the functional changes present after relief of UUO. Chronic renal denervation did not protect against the functional effects of unilateral ureteral obstruction.     

Renal haemodynamics in chronic unilateral ureteral obstruction in the dog

Renal blood flow and its intrarenal distribution have been investigated after 7 and 14 days of unilateral ureteral ligation (UUL) in both the ligated and the undisturbed kidney by the radioactive microsphere technique without opening the abdomen and the obstructed ureter. It has been found that renal blood flow (RBF) decreased to about 26% after 7 days and to about 10% after 14 days of normal controls in the obstructed kidney. There was a pronounced inward shifting of the much reduced RBF. The slight increase in RBF of the undisturbed kidney was not significant and did not compensate the pronounced decrease of RBF in the obstructed one.