Pathogenesis of Idiopathic Interstitial Pulmonary Fibrosis

The histopathology and ultrastructure of idiopathic interstitial pulmonary fibrosis is described and illustrated. There is evidence that proliferating type II pneumocytes in regenerating alveolar epithelium are implicated in the pathogenesis of this disorder.     

肾间质纤维化中MicroRNA表达研究进展

MicroRNA (miRNA)是一种高度保守、内源性的非编码小分子RNA,主要参与生物体中转录后水平基因表达的调控.miRNAs在各种肾脏疾病的发病机制中起重要作用.肾间质纤维化是各种慢性肾脏病进展至终末期,最终导致器官功能丢失的共同的病理过程和特征.转化生长因子β(TGF-β)在肾纤维化过程中是公认的一个重要的介质,它能刺激细胞外基质(ECM)的积聚并损害肾功能.近年研究发现,在肾脏组织中存在的一些miRNAs对肾脏纤维化的发生与发展有着重要的作用,深入地了解它们之间的关系可以为临床肾脏纤维化的治疗提供新的治疗靶点.     

Ultrastructure of the Kidney in Acute Interstitial Nephritis

Fifteen percutaneous renal biopsies from patients with acute renal failure due to acute interstitial nephritis (AIN), in almost all cases due to drugs, were studied by electron microscopy. Differential counting of interstitial cells showed an average of 69% lymphocytes (small and large) and 11 % macrophages. Plasma cells and eosinophils were comparatively rare. The infiltrate resembled that of acute rejection, suggesting a cellular hypersensitivity reaction. Proximal and distal tubules were severely affected focally. Migration of lymphocytes through the tubular basement membrane of otherwise well-preserved tubules was considered to be the first phase. Other tubules showed extreme thinning of the tubular basement membrane, with still intact cellular walls. Rupture of the tubular basement membrane and necrotic disintegration of tubular epithelial cells are probably late phenomena. The non-necrotic tubules displayed severe reduction of proximal brush border and proximal as well as distal tubular basolateral infoldings. Focal tubular disintegration leading to tubular block and/or backleak as well as decrease of proximal tubular sodium resorption leading to a decreased glomerular filtration (a mechanism probably also acting in ischemic acute renal failure) may all be factors responsible for the acute renal failure in AIN.     

肾移植患者肾组织和尿中巨细胞病毒DNA的动态观察及其临床意义

肾移植病人巨细胞病毒（ＣＭＶ）感染对排异反应的影响认识不一，国内目前无资料可考。我们采用原位杂交及聚合酶链反应技术，动态观察了８例肾移植患者肾活检当日尿标本中ＣＭＶ－ＤＮＡ的检出状况，结合肾组织病理形态学分析，发现正常肾组织即有少数ＣＭＶ感染；肾组织ＣＭＶ感染率随移植时间延长而增加；ＣＭＶ感染与移植肾慢性排异有较为密切的关系，可能是造成慢性排异反应发生的因素之一。     

博莱霉素所致大鼠肺间质纤维化中内皮素作用的研究

以博莱霉素Ａ５造成大鼠肺间质纤维化动物模型，测定实验组和对照组鼠血浆及肺组织匀浆的内皮素（ＥＴ）含量。实验组大鼠血浆ＥＴ含量２１１．６７±１８．３７ｎｇ／Ｌ，显著高于对照组的（１８３．９１±２３．８６ｎｇ／Ｌ，Ｐ＜０．０５），而其肺组织匀浆的ＥＴ含量却显著低于对照组的（２０８．０３±８５．９８ｎｇ／Ｌｖｓ２８１．３７±６５．３０ｎｇ／Ｌ，Ｐ＜０．０５）。同时检测两组动物的动脉血血气分析及肺组织的病理观察。提示内皮素在肺间质纤维化形成中有一定作用。     

大黄蟅虫超微粉剂对大鼠肾间质纤维化的影响

目的比较大黄蟅虫传统丸剂与超微粉剂对肾间质纤维化大鼠模型的药效作用。方法实验动物分为假手术组、模型组、传统丸剂组（0．54g／kg／d）、超微粉剂组（0．27g／kg／d）4组。采用单侧输尿管结扎的方法复制大鼠肾间质纤维化模型,用大黄蟅虫传统丸剂与超微粉剂进行干预,观察不同剂型对肾间质纤维化大鼠肾功能指标、血清纤维化指标、病理组织变化的影响。结果与假手术组相比,模型组血清肌酐（Cr）、尿素氮（Urea）、透明质酸（HA）、层黏蛋白（LN）、Ⅳ型胶原（IV—C）、Ⅲ型前胶原（PCⅢ）显著升高（P〈0．01）,肾间质纤维化明显。与模型组相比,大黄蟅虫传统丸剂组与超微粉剂组血清Cr、Urea、HA、LN、PCⅢ、Ⅳ—C降低（P〈0．01）,肾小管损害和肾间质纤维化程度减轻。与传统丸剂组相比,超微粉剂组血清Cr、Urea较低（P〈0．05）,其余指标差异无统计学意义。结论大黄蟅虫传统丸剂与超微粉剂对实验性肾间质纤维化都有较好的防治作用,能降低肾间质纤维化程度。改善肾功能;在改善肾功能方面,超微粉剂型优于传统丸剂型。复方“大黄蟅虫”药材超微粉碎后减少药量的同时有助于提高药效,有利于剂型现代化。     

Two Series of Familial Cases With Unclassified Interstitial Pneumonia With Fibrosis

Several children presenting with mild symptoms of respiratory tract infection were diagnosed with unclassified interstitial pneumonia with fibrosis. Their clinical and radiological findings were similar to those of acute interstitial pneumonia, but there were some differences in the pathological findings. Unclassified interstitial pneumonia with fibrosis is characterized by histological findings of centrilobular distribution of alveolar damage and bronchiolar destruction with bronchiolar obliteration. This report describes two different series of familial cases of unclassified interstitial pneumonia with fibrosis, which developed almost simultaneously in the spring. Some of the individual cases showed rapidly progressive respiratory failure of unknown cause, with comparable clinical courses and similar radiological and pathological features, including lung fibrosis. Each family member was affected almost simultaneously in the spring, different kinds of viruses were detected in two patients, and all members were negative for bacterial infection, environmental and occupational agents, drugs, and radiation. These findings implicate a viral infection and/or processes related to a viral infection, such as an exaggerated or altered immune response, or an unknown inhaled environmental agent in the pathogenesis of unclassified interstitial pneumonia with fibrosis.     

人间质胶原酶基因高效表达对体外肝纤维化的影响

目的:观察人间质胶原酶(MMP1)基因高效表达对体外肝纤维化的影响。方法:利用DNA重组技术构建了pcDNA3-MMP1真核表达重组质粒。经脂质体包裹后,并将其转染张氏传代人肝细胞,Western blot检测MMP1蛋白表达量。在体外乙醇诱导纤维化培养条件下,LSAB免疫组织化学法检测肝细胞周围Ⅰ、Ⅲ型胶原生成状况,经图象分析仪半定量分析胶原纤维的相对含量(relative content of collagen,RCC)。结果:成功构建了真核表达质粒pcD-NA3-MMP1。RCC结果显示未转染组为132.6±5.7,转染组为118.8±4.8;未转染组明显高于转染组(P<0.01)。从而证明重组质粒pcDNA3-MMP1能在体外致纤维化培养条件下拮抗细胞间质过多胶原产生。结论:本研究结果表明通过MMP1基因的高效表达,加速Ⅰ、Ⅲ型胶原降解,在体外表现一定抗肝纤维化作用,从而为以后肝纤维化基因导入治疗打下基础。     

螺内酯对马兜铃酸所致大鼠肾间质纤维化防治作用的研究

目的探讨螺内酯对马兜铃酸(Aristolochic Acid,AA)所致大鼠肾间质纤维化的防治.方法大白鼠随机分成3组,正常对照组、木通组、木通螺内酯组.在实验前及实验的第2、4周,分别从各组大鼠眼内眦静脉采血,测定血肌酐(SCr)、血尿素氮(BUN)、血钾、血钠等生化指标以及Ⅲ型前胶原(PC Ⅲ)、血清透明质酸(HA)、层粘连蛋白(LN)等放免指标,42d时从心脏直接采血复查上述各指标,而后处死动物取肾脏进行组织形态学及特殊染色检查.结果1、木通螺内酯组和木通组用药前后自身比较血生化、放免指标有统计学意义.2、木通螺内酯组和木通组用药后组间比较血生化、放免指标有统计学意义.3、木通螺内酯组和木通组用药后组间比较免疫组化指标有统计学意义.结论1、醛固酮抑制对AA所致的慢性肾间质纤维化有一定的防治作用.2、在AA所致的慢性肾间质损伤时血电解质紊乱以钾、钠表现为明显,血氯和血钙变化不明显.3、血放免指标PC Ⅲ、HA、LN可以作为早期慢性肾间质纤维化的重要临床观察指标.     

miR‐128 Regulates Genes Associated with Inflammation and Fibrosis of Rat Kidney Cells In Vitro

microRNAs (miRNAs) regulate diverse cellular functions and signaling pathways via inhibiting the expression of their target genes. Given that miR‐128 mediates mitogen‐activated protein kinase signaling and production of reactive oxygen species and pro‐inflammatory chemokines in various types of cells and tissues, and that miR‐128 is differentially expressed in aged and diseased kidneys, we hypothesized that miR‐128 may play key roles in kidney inflammation. Hence, in this study, we evaluated the biological effects of miR‐128 in normal rat kidney (NRK) cells in vitro. Our results revealed that overexpression of miR‐128 enhanced expression of genes associated with inflammation, pro‐inflammatory cytokines and fibrosis in NRK cells. The recent reports showing that expression of miR‐128 is increased in liver and lung fibrosis, together with the findings in this study, suggest that miR‐128 may be a pro‐fibrotic miRNA that regulates fibrosis in various tissues. Anat Rec, 301:913–921, 2018. © 2017 Wiley Periodicals, Inc.     

Endothelial-Myofibroblast Transition Contributes to the Early Development of Diabetic Renal Interstitial Fibrosis in Streptozotocin-Induced Diabetic Mice

Diabetic nephropathy is the leading cause of chronic renal failure. Myofibroblasts play a major role in the synthesis and secretion of extracellular matrix in diabetic renal fibrosis. Increasing evidence suggests that endothelial cells may undergo endothelial-myofibroblast transition under physiological and pathophysiological circumstances. Therefore, this study investigates whether endothelial-myofibroblast transition occurs and contributes to the development of diabetic renal interstitial fibrosis. Diabetes was induced by administration of streptozotocin to Tie2-Cre;LoxP-EGFP mice, an endothelial lineage-traceable mouse line generated by crossbreeding B6.Cg-Tg(Tek-cre)12F1v/J mice with B6.Cg-Tg(ACTB-Bgeo/GFP)21Lbe/J mice. The endothelial-myofibroblast transition was also studied in MMECs (a mouse pancreatic microvascular endothelial cell line) and primary cultures of CD31⁺/EYFP⁻ (enhanced yellow fluorescent protein) endothelial cells isolated from adult normal α-smooth muscle actin promoter-driven-EYFP (α-SMA/EYFP) mouse kidneys. Confocal microscopy demonstrated that 10.4 ± 4.2 and 23.5 ± 7.4% of renal interstitial myofibroblasts (α-SMA⁺) in 1- and 6-month streptozotocin-induced diabetic kidneys were of endothelial origin (EGFP⁺/α-SMA⁺ cells), compared with just 0.2 ± 0.1% of myofibroblasts in vehicle-treated Tie2-Cre;LoxP-EGFP mice (P < 0.01). Confocal microscopy and real-time PCR showed that transforming growth factor (TGF)-β1 induced de novo expression of α-SMA and loss of expression of VE-cadherin and CD31 in MMECs and primary cultures of renal endothelial cells in a time- and dose-dependent fashion. These findings demonstrate that the endothelial-myofibroblast transition occurs and contributes to the early development and progression of diabetic renal interstitial fibrosis and suggest that the endothelial-myofibroblast transition may be a therapeutic target.Diabetic nephropathy (DN) is the leading cause of end-stage renal disease in the Western world. Approximately 25 to 35% of patients with type 1 diabetes¹ and 5 to 10% of patients with type II diabetes² develop DN. Glomerulosclerosis and interstitial fibrosis are the key morphological features of DN, and both correlate well with the development and progression of renal disease.³ Myofibroblasts play a major role in the synthesis and secretion of extracellular matrix in the development and progression of renal fibrosis. In DN, cells expressing α-smooth muscle actin (α-SMA), the putative marker of myofibroblasts, are located primarily in the renal interstitium and to a lesser extent in glomeruli in association with mesangial proliferation.⁴ The number of myofibroblasts is inversely correlated with renal function in DN.⁵Importantly however, the origin of myofibroblasts in DN remains unclear. It is generally believed that myofibroblasts may be derived from resident fibroblasts, epithelial cells through the epithelial-myofibroblast transition, mesangial cells, or bone marrow-derived cells. Interestingly, increasing evidence suggests that endothelial cells may undergo endothelial-myofibroblast transition (EndoMT) under physiological and pathophysiological circumstances^6,7 and thereby give rise to myofibroblasts. Arciniegas et al⁸ demonstrated that transforming growth factor (TGF)-β1 can induce aortic endothelial cells to differentiate into α-SMA⁺ cells in vitro, suggesting a novel role for TGF-β1 in atherogenesis. Moreover, embryonic endothelial cells have been shown to transdifferentiate into mesenchymal cells expressing α-SMA in vitro and in vivo,⁹ and vascular endothelium-derived cells contain α-SMA in restenosis,¹⁰ inflammation, and hypertension,¹¹ suggesting that myofibroblasts may be of endothelial origin.The involvement of TGF-β1 in renal fibrosis, including DN, has been the subject of extensive investigation.¹² TGF-β1 exerts its biological effects by signaling through TGF-β type II and type I receptors,¹³ and their downstream effectors, R-Smads (Smad2 and Smad3). TGF-β/Smad2/3 signaling pathways are activated in human DN¹⁴ and diabetic mouse kidneys.^15,16 Smad3-null mice are resistant to streptozocin (STZ)-induced DN.¹⁷ It remains largely unknown whether TGF-β1 can induce EndoMT in microvascular endothelial cells in DN, one of the major microvascular complications of diabetes and whether Smad3 plays a pivotal role in the process of TGF-β1-induced EndoMT.In this study we investigated whether EndoMT occurs and contributes to the development of renal interstitial fibrosis in STZ-induced DN in an endothelial lineage-traceable mouse line, the Tie2-Cre;LoxP-EGFP mouse. We also assess whether a specific inhibitor for Smad3 (SIS3)¹⁸ can inhibit TGF-β-induced EndoMT in a mouse microvascular endothelial cell line (MMECs).     

Hypoalbuminaemia – A Marker of Cardiovascular Disease in Patients with Chronic Kidney Disease Stages II - IV

Cardiovascular disease (CVD) is a major cause of morbidity and mortality in patients with chronic kidney disease (CKD) patients. Serum albumin, a negative acute-phase reactant and marker for underlying inflammation and/or malnutrition, is an independent predictor of CVD and mortality in CKD VI patients. Such an association in patients with less severe CKD is not well established.     

Fate Tracing Reveals the Pericyte and Not Epithelial Origin of Myofibroblasts in Kidney Fibrosis

Understanding the origin of myofibroblasts in kidney is of great interest because these cells are responsible for scar formation in fibrotic kidney disease. Recent studies suggest epithelial cells are an important source of myofibroblasts through a process described as the epithelial-to-mesenchymal transition; however, confirmatory studies in vivo are lacking. To quantitatively assess the contribution of renal epithelial cells to myofibroblasts, we used Cre/Lox techniques to genetically label and fate map renal epithelia in models of kidney fibrosis. Genetically labeled primary proximal epithelial cells cultured in vitro from these mice readily induce markers of myofibroblasts after transforming growth factor β₁ treatment. However, using either red fluorescent protein or β-galactosidase as fate markers, we found no evidence that epithelial cells migrate outside of the tubular basement membrane and differentiate into interstitial myofibroblasts in vivo. Thus, although renal epithelial cells can acquire mesenchymal markers in vitro, they do not directly contribute to interstitial myofibroblast cells in vivo. Lineage analysis shows that during nephrogenesis, FoxD1-positive(⁺) mesenchymal cells give rise to adult CD73⁺, platelet derived growth factor receptor β⁺, smooth muscle actin-negative interstitial pericytes, and these FoxD1-derivative interstitial cells expand and differentiate into smooth muscle actin⁺ myofibroblasts during fibrosis, accounting for a large majority of myofibroblasts. These data indicate that therapeutic strategies directly targeting pericyte differentiation in vivo may productively impact fibrotic kidney disease.Understanding the origin and differentiation pathways of myofibroblasts in vivo is critical for identifying new therapeutic strategies for fibrosing disease. Myofibroblasts, contractile cells that deposit pathological extracellular matrix, were first believed to derive from a specialized perivascular cell known as the hepatic stellate cell when studied in the liver. In health these cells store retinoic acid in intracellular vesicles and cultured stellate cells possess all of the hallmarks of myofibroblasts in vitro.¹ In other organ systems, similar perivascular cells have been postulated to be the source of myofibroblasts, but have been hard to define.^2,3 Mesoderm-derived cells, when cultured in vitro, differentiate into cells with hallmarks of myofibroblasts, including most notably mesenchymal stem cells from bone marrow, as well as mesangial cells of the kidney, and cultured monocyte-derived macrophages.^4–6 Whether mesoderm-derived pericytes (also called perivascular fibroblasts) give rise to kidney myofibroblasts remains controversial, partly because primary epithelial cells when cultured in vitro can be induced to express some genes that are also expressed in myofibroblasts.^7–9 During carcinogenesis phenotypic alterations termed epithelial-to-mesenchymal transition (EMT) have been well characterized and promote cell migration, invasion, and metastasis.¹⁰ Further, a recent report suggests that other terminally differentiated cells such as endothelial cells can develop a myofibroblast phenotype in vitro and in vivo.^11,12It has been postulated that during kidney injury in vivo, epithelial cells undergo a phenotypic transition or can transdifferentiate into interstitial myofibroblasts by this same process of EMT.^13,14 Subsequent studies both in vivo and in vitro support this hypothesis.^15,16 The implication from these observations is that if the molecular mechanisms by which epithelial cells traverse the basement membrane and differentiate into myofibroblasts can be understood, novel antifibrotic strategies will be identified.Epithelial cells are known to respond to injury in several ways. They undergo morphological changes, lose polarity, acquire stress fibers, and migrate along the basement membrane.¹⁷ They up-regulate inflammatory genes and genes that enhance their ability to survive in a hostile environment.^18,19 In addition, they express some genes shared by embryonic mesenchymal cells transitioning to epithelium during nephrogenesis.^20–22 Thus it has been suggested that in response to injury epithelial cells undergo EMT, recapitulating primitive mesenchymal cells of the intermediate mesoderm.⁹ This, however, is misleading since intermediate mesoderm cells do not express inflammatory and cell-survival genes that injured adult epithelial cells up-regulate, and expression of a limited number of genes shared by embryonic mesenchyme such as α smooth muscle actin (SMA), by itself, does not define injured epithelial cells as mesenchymal.^23–25 Neoplastic epithelial cells have the capacity to metastasize, share some characteristics with myofibroblasts, and express or down-regulate key regulators of metastasis such as mts1 (S100A4 or FSP-1), Twist, Snail, and β-catenin, genes whose expression can also be activated in cultured epithelial cells.^26–28 Proponents of the hypothesis that myofibroblasts in inflammation and scarring derive from epithelial cells have drawn on these observations to extend the term EMT to mean epithelial-to-myofibroblast transition.Interstitial myofibroblasts are the principle source of interstitial collagens, including fibrillar collagens I and III. They are widely held to be the primary cell in the injured kidney that lays down the interstitial matrix that becomes fibrotic (For review see²⁹). Many myofibroblasts express the actin fiber, αSMA that correlates with contractile and activated morphology, and recent studies confirmed that in the fibrotic kidney more than 80% of these produce fibrillary collagen.³⁰ Although this is not specific to interstitial myofibroblasts (αSMA is also expressed by vascular smooth muscle cells), αSMA has long been used as a marker of myofibroblasts.Although it is widely accepted that primary epithelial cells cultured in vitro up-regulate genes that result in a myofibroblast phenotype,^9,25 and generate fibrillar collagens, the evidence that this occurs in vivo is less well-established. There are some published examples of epithelial cells transgressing intact or disrupted basement membrane or cells co-expressing established epithelial and fibroblast markers in vivo,^31–33 but histological snapshots do not prove a lineage relationship, and cells may express a variety of antigens during injury. In our own extensive studies of injured epithelial cells in kidney repair, we concluded that non-epithelial cells do not migrate from interstitium into the tubule.³⁴ Similarly, we have never observed a cell outside of the confines of the epithelial basement membrane that was positive for markers of epithelial injury. Explanations for a failure to make these observations in fixed tissues include the hypothesis that a cell exiting the confines of the basement membrane rapidly loses epithelial markers and only subsequently gains myofibroblast markers.³⁵ However, in vitro, epithelial cells can express both fibroblast markers and epithelial markers simultaneously.³⁶Because efforts to design new antifibrotic therapies require a rigorous understanding of the cellular origin of myofibroblasts in vivo, we have performed lineage analysis of both renal epithelial cells and interstitial stromal cells during fibrosis in vivo. Transgenic or knock-in mice with lineage-restricted expression of bacterial Cre recombinase were used for genetic tracking of three cell populations. The HoxB7-Cre driver is expressed exclusively in the mesonephric duct and its derivatives, resulting in labeling of collecting duct epithelium and ureteral epithelium of adult kidney.³⁷ In the Six2-Cre transgenic mouse, expression of Cre occurs in cap-mesenchyme and labels all non-ureteric, bud-derived, nephron epithelia, including podocytes, proximal tubule, loop of Henle, and connecting segment, but it does not label any interstitial cell population.^34,38 FoxD1 is a well characterized marker of renal stromal cells, but not epithelia, during development, and we used FoxD1-Cre knock-in mice to genetically label renal stroma.³⁹ We crossed these three Cre drivers against two different reporter lines to permanently and heritably label all epithelial cells of the entire nephron in adult mouse kidney or all stromal cells.^34,38 We demonstrate that, contrary to the prevailing model, kidney epithelial cells do not become myofibroblasts in vivo during fibrotic disease. Rather, we show by genetic tracing that myofibroblasts derive from interstitial pericytes/perivascular fibroblasts.     

周细胞在慢性肾脏病中缺氧与纤维化相互关系中的作用

肾间质纤维化、肾小管损伤和炎性细胞浸润是慢性肾脏病的重要标志.组织缺氧是肾间质损伤发生的重要机制之一.肾周毛细血管损伤导致的间质血流减少为肾间质纤维化的重要病因.纤维化时,周细胞从肾小管周围毛细血管分离并生成ECM,毛细血管稀疏介导了肾小管间质损伤、缺氧和纤维化间错综复杂的相互关系.理清上述三者之间的关系,以及周细胞在其中所起到的作用,有助于肾纤维化的治疗,延缓慢性肾脏病的进展.