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杨念生

摘  要

单核细胞和巨噬细胞浸润在实验动物模型肾炎及人肾小球肾炎的发生和发展起着重要的作用。一般认为，组织中的巨噬细胞是由血液中单核细胞分化而成。它们一旦在组织中，便失去增生的能力，成为终未期细胞。然而，新近有报导在炎症组织中发现相当数量的巨噬细胞具的增生能力，这对传统观点是一个新的挑战。不久前，澳大利亚Monash Medical Center蓝辉耀博士发现在抗肾小球基底膜肾炎的大鼠肾脏中，存在大量具有增生能力的巨噬细胞，这些增生的巨噬细胞在进行性肾损害中起着重要的作用，巨噬细胞的局部增生是巨噬细胞在炎症局部积聚的重要机制。在大鼠急性肾排斥模型中也有类似的发现。虽然巨噬细胞增生在这些免疫介导的肾损害中起着重要的作用，但是在其他一些“非免疫诱导”的肾脏损害中，如5/6肾切除后的残余肾模型，组织巨噬细胞增生是否也起着重要的作用尚不清楚，而且巨噬细胞局部增生在人肾小球肾炎中的作用，目前尚未见报导。因此，本研究旨在探索下列问题：

1、在大鼠残余肾模型中是否也存在巨噬细胞局部增生。这种现象是否也存在于人肾小球肾炎中？
2、巨噬细胞局部增生在大鼠残余肾模型及人肾小球肾炎的发展中扮演什么角色？
3、抑制炎症局部巨噬细胞增生的意义何在？

一、巨噬细胞局部增生在大鼠残余肾模型中的作用(5/6次全肾切除）
    为探索巨噬细胞局部增生在大鼠残余肾模型中的作用，我们利用蓝辉耀博士新近发明的微波免疫组化双染色技术观察5/6 肾切除后第４、８、２、１６周后大鼠肾脏的改变。增生的巨噬细胞同时表达巨噬细胞标记抗原ＣＤ６８及增生细胞核抗原（ＰＣＮＡ）。小管间质损害在第４周开始出现。这时，肾脏已经有明显的巨噬细胞浸润，并有相当数量的巨噬细胞增生，增生的巨噬细胞占巨噬细胞总数的１９～３４％，随着疾病的进展，增生巨噬细胞数继续增加，在第１２周达到高峰。在假手术组，极少巨噬细胞表达增生细胞核原，和实验组形成鲜明对比。增生的巨噬细胞主要集中于组织损伤处，如肾小球细胞增多、肾小球节段性病变、肾小管损害和间质纤维化处。此外，肾小球及间质中增生巨噬细胞的数量与血压、肾小球硬化、肾小管损害、血清尿素氮和肌酐浓度、尿蛋白总量呈相关关系（Ｐ值均小于0.05）。相反，非增生的巨噬细胞的数量除间质非增生巨噬细胞与肌酐浓度相关外，其余均未见相关关系。

二、巨噬细胞局部增生在人肾小球肾炎中的作用
    我们利用类似上述的方法对８９例人肾小球肾炎的标本进行研究，发现在正常肾脏或微小病变型肾炎的肾脏中，极少有巨噬细胞表达ＰＣＮＡ，而在其他类型的肾小球肾炎中，均存在着不同程度的巨噬细胞增生，其中以新月体肾炎、系膜毛细血管性肾炎和狼疮性肾炎最为显著（占２４～４６％）。增生的巨噬细胞集中在组织损伤严重处，如肾小球细胞增多，肾小球节段性损害、新月体形成、肾小管间质损害和多核巨细胞中，而且肾小球及肾小管间质中增生巨噬细胞的数目和这些组织学改变的指标呈强的相关关系。同时，增生巨噬细胞数目尚和血肌酐浓度（Ｐ＜0.01）及肌酐清除率下降（Ｐ＜0.01）密切相关，但和蛋白尿关系不大，而增生的肾脏固有细胞的数目与这些组织学及肾功能指标的相关要弱得多。另一方面，仅有少数Ｔ细胞呈增生状态。

三、Deoxyspergualin治疗抑制大鼠新月体肾炎巨噬细胞局部增生。
    上面的实验进一步证明巨噬细胞增生在肾炎的进展中起着重要的作用，那么抑制巨噬细胞的增生是否能抑制疾病的进展呢？免疫抑制剂Deoxyspergualin在体外能抑制单核细胞株的增生，因此，下面的实验探索该药是否能抑制大鼠新月体肾炎时巨噬细胞局部增生。首先建立起八组（各５只动物）大鼠抗基底膜肾炎模型，各四组分别给予Deoxyspergualin（５ mg/kg，每天腹腔注射一次）治疗或生理盐水作为对照。治疗从注射肾毒血清时开始，直至第１、７、１４、２１天动物被杀。生理盐水对照组大鼠的肾脏有大量的巨噬细胞浸润，其中４２～６０％的巨噬细胞表达ＰＣＮＡ。而在 Deoxyspergualin治疗组，肾小球及肾间质的增生巨噬数量明显减少，分别减少７２～７８％（Ｐ＜0.01）和８２～９７％（Ｐ＜0.01）。组织巨噬细胞浸润也相应减少，组织学损害减轻，肾功能改善。Ｔ细胞在大鼠抗肾小球基底膜肾炎中也有局部增生，但其程度远不如巨噬细胞， DSP治疗同时抑制Ｔ细胞增生。

综上所述，这些结果证明下列结论：

1、巨噬细胞局部增生是大鼠实验性肾炎，包括新月体肾炎和残余肾模型的共同特征。它是巨噬细胞在肾组织积聚的重要机制之一。
2、巨噬细胞局部增生也是人肾小球肾炎的常见特征，增生的程度和疾病的严重性成正比，它很可能是巨噬细胞在肾组织积聚的重要机制之一。
3、巨噬细胞局部增生对大鼠5/6肾切除后残余肾脏纤维化及肾功能衰竭的进展起着重要作用。
4、巨噬细胞局部增生在介导人肾小球肾炎，特别是严重类型的肾炎的进展中起重要作用。此外，间质增生巨噬细胞的数目可用于作为预测肾功能恶化的指标。
5、该研究提示抑制巨噬细胞增生可能是Deoxyspergualin抑制大鼠新月体肾炎的新的重要机制。
6、该研究提示抑制人体肾组织中的巨噬细胞增生可能有利于治疗人肾小球肾炎，特别是严重类型的肾小球肾类。

关键词；巨噬细胞 增生 肾小球肾炎 T－细胞 炎症 残余肾 Deoxyspergualin

Abstract

The accumulation of monocytes/macrophages has been considered to be of central importance for the development of renal injury in both experimental and human glomerulonephritis. The conventional view of macrophage accumulation in glomerulonephritis is of recruitment of blood monocytes into the kidney with no significant local macrophage proliferation. However, this conventional view has been questioned by a few recent studies reporting significant numbers of proliferating macrophages within inflammatory lesions. Previously, we had demonstrated local proliferation of macrophages is an important mechanism in both macrophage accumulation and mediation of progressive renal injury in a number of immunologically-induced renal diseases, including rat anti-GBM glomerulonephritis and rat renal allograft rejection. However, whether there is similar local macrophage proliferation in the non-immune mediated rat remnant kidney model after 5/6 subtotal nephrectomy is still undetermined. Furthermore, few studies had been conducted in human GN. Therefore, the aim of the current study is to determine:

1. Whether there is similar local macrophage proliferation in rat remnant kidney and also in human GN.

2. The potential role of local macrophage proliferation in the progression of rat remnant kidney and human glomerulonephritis.

3. What is the possible therapeutic significance of targeting this local macrophage proliferation?

Local Macrophage Proliferation in rat remnant kidney model

To examine local macrophage proliferation in a rat remnant kidney model, groups of 5 rats were euthanised 4,8,12 and 16 weeks after 5/6 subtotal nephrectomy. Proliferating macrophages were identified by co-expression of CD68 and PCNA using double immunohistochemistry labeling. Macrophage proliferation was evident at week 4 when tubulointerstitial injury became apparent, accounting for 19-34% of the total infiltrated macrophages. The number of proliferating macrophages continued to rise as the disease progressed, reaching its peak at week 12. In contrast, local macrophage proliferation is very rare in sham group. Proliferating macrophages were almost restricted to areas of tissue damage such as glomerular hypercellularity, focal segmental lesions, and tubular lesions. Furthermore, the number of proliferating macrophage in glomeruli and interstitium is closely correlated to blood pressure, glomerulosclerosis, tubular lesions, serum urea, serum creatinine, and proteinuria(all P<0.05). In contrast, there is no significant correlation between the numbers of non-proliferating macrophages in either glomerulus or interstitium and histology or renal function, except for the correlation between the numbers of non-proliferating macrophages in the interstitium and serum creatinine. In conclusion, this study has demonstrated that local macrophage proliferation is the major mechanism of macrophage accumulation contributing to glomerulosclerosis and interstitial fibrosis and development of renal failure following subtotal nephrectomy in rat.

Local Macrophage Proliferation in Human Glomerulonephritis .

Proliferating macrophages in 89 human kidney biopsies were identified by co-expression of CD68 and PCNA using double immunohistochemistry labeling. Local macrophage proliferation is absent or mild in normal kidneys and in minimal change lesion. In contrast, varying percentage of macrophages expressed PCNA in a variety forms of GN, being most prominent in crescentic GN, mesangiocapillary GN and lupus nephritis(27.4-42.4%). Proliferating macrophages were almost restricted to areas of tissue damage such as glomerular hypercellularity, focal segmental lesions, crescents, and tubulointerstitial lesions and in multinucleated macrophage giant cells within granulomatous lesions. Furthermore, the number of proliferating macrophages showed a strong correlation with these histological parameters. In addition, the number of proliferating macrophages in both glomeruli and interstitium is closely correlated with serum creatinine(P<0.01)and fall in creatinine clearance(P<0.01), but not with proteinuria. In contrast, correlation between the number of non-proliferating macrophages and histology or renal function is less prominent. On the other hand, only relatively small portion of T cells showed proliferative activity. In conclusion, this study, for the first time, demonstrated that local macrophage proliferation is one of major mechanisms contributing to macrophage accumulation in human glomerulonephritis, and proliferating macrophages may play an important role in mediation of progressive renal injuries.

Deoxyspergualin Suppresses Local Macrophage Proliferation in Rat Crescentic Glomerulonephritis

Local proliferation is a major mechanism whereby macrophage accumulation within the kidney during rat anti-glomerular basement membrane (GBM) glomerulonephritis, and this local proliferation is associated with severe renal injury. It is tempting that inhibition of local macrophage proliferation could suppress the progression of kidney diseases. The immunosuppressive drug deoxyspergualin, has recently been shown to inhibit the proliferation of monocyte cell lines in vitro. Thus, the ability of deoxyspergualin to inhibit local macrophage proliferation was investigated in rat crescentic anti-GBM glomerulonephritis. Groups of 5 primed rats were treated with deoxyspergualin (5mg/kg i.p. per day) or vehicle control (saline) from the time of nephrotoxic serum administration until being killed on day 1, 7, 14 or 21. Double immunohistochemistry staining of tissue sections was used to detect ED1+ macrophages and the subpopulation of proliferating ED1+ macrophages expressing proliferating cell nuclear antigen (PCNA). In saline treated animals, there was prominent macrophage accumulation within the glomerulus and interstitium over the disease course which was associated with a high level of local macrophage proliferation, accounting for 42-60% of the total macrophage population. In contrast, deoxyspergualin treatment inhibited macrophage accumulation in association with a marked reduction in local macrophage proliferation (ED1+PCNA+ cells) in both the glomerulus (?72-78%, p<0.01) and interstitium (?82-97%, p<0.01). The dramatic suppression of local macrophage proliferation correlated with DSP-mediated inhibition of glomerular and tubulointerstitial histological damage and improvement in renal function. Whilst local T-cell proliferation in this disease model is not as pronounced as macrophage proliferation, this too was inhibited by deoxyspergualin treatment (?76-93%; p<0.01). In conclusion, this study suggests that suppression of local macrophage proliferation as a novel and important mechanism by which deoxyspergualin inhibits rat crescentic glomerulonephritis.

In summary, these results demonstrated that

1. Local macrophage proliferation is a common feature and is one of major mechanisms contributing to macrophage accumulation in experimental glomerulonephritis including rat crescentic glomerulonephritis, in rat renal allograft rejection, and in remnant kidney after 5/6 subtotal nephrectomy.

2. Local macrophage proliferation is also a common feature contributing to macrophage accumulation in a variety of human glomerulonephritis, being most prominent in severe forms of glomerulonephritis including crescentic GN, mesangiocapillary GN and lupus.

3. Local macrophage proliferation plays an important role in the progression of renal fibrosis and development of chronic renal failure following subtotal nephrectomy in rat.

4. Local macrophage proliferation plays an important role in mediation of progressive renal injuries in human glomerulonephritis, particularly in severe forms of glomerulonephritis. In addition, the number of interstitial proliferating macrophages may be used as a better prognostic parameter predicting progression to end-stage renal failure.

5. This study suggests suppression of local macrophage proliferation as a novel and important mechanism by which deoxyspergualin inhibits rat crescentic glomerulonephritis.

6. This study suggests that Targeting local macrophage proliferation in human glomerulonephritis, particularly in the severe form of glomerulonephritis, may also be therapeutically beneficial.

KEY WORDS：Macrophages, Proliferation, Glomerulonephritis, T-cells, Inflammation, remnant kidney, Deoxyspergualin