兽研引言
慢性肾病（CKD）是犬和猫常见的发病原因。小动物中报道的患病率差异很大，通常取决于地理区域、年龄和品种。一般来说，估计约1%的成年犬1和猫2患有CKD，而在10岁以上的猫中患病率可达30%。3这些报道的患病率被低估了，因为早期阶段疾病（临床症状出现之前）往往未被发现。这两个物种均有多种病因，疾病进展部分取决于初始损伤。人们普遍认识到疾病进展存在差异，有些动物保持稳定，而另一些则表现出肾功能更快速的恶化。4,5 驱动犬和猫进行性肾损伤的因素尚未明确。可能的因素包括治愈原发性肾脏疾病的能力（或无法治愈）以及叠加的肾脏损伤。在猫中，叠加的损伤包括肾缺血、流出道梗阻和细菌感染。6 在犬中，贫血、低体况评分和高磷血症被确定为增加进行性CKD风险的因素4。
驱动疾病进展的其他因素（主要已在人类和啮齿动物的实验性或自然肾脏疾病中阐明）源于对肾单位丢失的代偿反应（超滤过），导致所谓的“内在性进展”。超滤过导致足细胞肥大以覆盖扩张的肾小球基底膜增加的表面面积。这引起足细胞牵张和剪切应力。7 此外还存在更高的肾小管工作负荷（特别是肾小管对尿滤液中钠的重吸收增加）以及炎症和缺氧损伤通路的激活。7 蛋白尿、高磷血症和贫血与犬和猫CKD的进展相关。4,8–10 间质纤维化和炎症与犬（与氮质血症相关）11和猫（与氮质血症、蛋白尿和高磷血症相关）10–13的进展生物标志物相关。磷酸盐尿14,15和蛋白尿16可触发上皮细胞应激，导致间质区域的纤维化和炎症。如上所述，肾小球超滤过损伤足细胞，这是肾小球硬化的前驱病变。17 间质纤维化和肾小球硬化均为不可逆病变，其严重程度和分布各不相同。在猫18、啮齿动物和人类19中，间质纤维化使肾小管与其管周毛细血管血供分离，加剧与贫血相关的低氧供应。这导致肾小管变性和萎缩。肾小球硬化最终使肾小球毛细血管腔消失，阻碍血流进入管周毛细血管床。20
间质纤维化和肾小球硬化均为胶原为基础的病变，二者均可由转化生长因子（TGF）-β通路启动和维持。该分子通路复杂且具有多向性。它是胚胎发生、出生后生长和伤口愈合过程中间充质组织发育的关键组成部分。TGF-β通路并非CKD中导致胶原产生的唯一机制；WNT、MAPK和RhoGTPase也参与其中。20 然而，TGF-β驱动的肾纤维化被认为是动物CKD发生和进展的主要因素。除促纤维化作用外，TGF-β还具有抗炎作用，这进一步使其在进行性CKD中的作用复杂化。本综述将描述目前关于TGF-β在CKD中作用的认识，重点聚焦于猫和犬，并在适当之处转化至人类和啮齿动物模型。
不同物种间约有30种TGF-β通路基因和蛋白质。各成员通常具有高度同源性，例如TGF-β1氨基酸序列在人、犬、猫和鼠蛋白之间的同一性百分比为89%或更高（计算方式为相同匹配数除以比对位置总数）（图1）。哺乳动物中已知有3种TGF-β亚型（TGF-β1、TGF-β2和TGF-β3），它们由3个独立的基因编码，但氨基酸序列相似。21 TGF-β4是仅在禽类物种中观察到的亚型，而TGF-β5仅存在于两栖动物中。22在各种蛋白质/转录图谱中，尚未证实TGF-β亚型具有细胞或器官特异性。TGF-βs由多种细胞类型合成为前体蛋白，但很快被切割为成熟蛋白和潜伏相关肽（LAP）。成熟部分随后在两个相互强烈结合的相同区域之间被对半切割，形成同源二聚体。同源二聚体然后与其各自的LAP非共价结合，形成小潜伏复合物（SLC），随后分泌到细胞外空间。SLCs然后进一步与潜伏TGF-β结合蛋白（LTBPs）结合，形成大潜伏复合物（LLCs），这些复合物被包埋在细胞外基质（ECM）中。LLCs代表储存供应，以便在需要时释放TGF-β。潜伏TGF-β的释放是通路激活的主要控制点。多种蛋白质可以释放活性形式的TGF-β，包括整合素、血小板反应蛋白、活性氧（ROS）和基质金属蛋白酶（MMPs）。TGF-β1和TGF-β3的结构相似，两者均可由多种细胞产生的整合素释放。然而，TGF-β2缺乏必需的精氨酸-甘氨酸-天冬氨酸（RGD）基序，因此无法被整合素释放。23血小板反应蛋白能够释放所有3种亚型，它们在肾脏疾病中尤为重要。21
[图：TGF-β1氨基酸序列在人、小鼠、猫和犬之间高度保守。（A）人、]
一旦释放，TGF-β1或TGF-β3同源二聚体与跨膜TGF-β受体II（TGFBRII）的单体或二聚体结合。如果细胞因子与TGFBRII的单体结合，则它会吸引另一个单体以形成4蛋白复合物。然后，招募TGF-β受体I（TGFBRI）的二聚体，最终形成6蛋白复合物。细胞因子与TGFBRII之间的亲和力很高，而TGFBRI仅被TGF-β与TGFBRII的复合物吸引。这种逐步结合过程强调了TGFBRII在捕获TGF-β1或3中的重要性，以及TGFBRI启动信号转导所必需的结合。相比之下，TGF-β2不遵循相同的受体结合顺序，因为TGF-β2自身对TGFBRII的亲和力低。体外研究表明，TGF-β2结合TGFBRIII，后者作为共受体增强TGF-β2与TGFBRII的结合亲和力。一旦TGF-β2与TGFBRIII结合，TGFBRI被招募，信号转导以与其他TGF-β亚型相同的方式进行。24 TGF-β受体具有混杂性，可被TGF-β家族中的多种分子激活，包括骨形态发生蛋白（BMPs）、激活素和抑制素。25与TGF-β通路相比，BMPs可产生相反的结果（见下文）。
TGF-β信号传导存在经典和非经典通路。经典通路涉及TGFBRI和TGFBRII二聚体细胞内部分的磷酸化，以招募SMAD2和SMAD3蛋白，这些蛋白也将被磷酸化。磷酸化的SMADs然后与SMAD4和其他辅因子结合，同时从细胞膜转运至细胞核，在那里它们调节基因转录。26存在多种SMADs，其中SMAD2和3是TGF-β通路的一部分，而SMAD1、5和8用于BMP通路。两条通路均使用SMAD4易位至细胞核。SMADs与SMAD结合元件结合，这些元件因细胞和组织类型而异。一些SMAD结合元件涉及表观遗传变化，许多通路需要转录辅因子。抑制性SMADs（通常为SMAD7，程度较轻的SMAD6）可与TGFBRI结合并阻断其与SMAD2和3的相互作用，从而为经典通路创建负反馈环路。与BMP通路竞争SMAD4也可限制TGF-β通路激活。还有许多其他细胞质蛋白可以磷酸化SMADs，以及磷酸酶和蛋白酶使SMADs失活，强调了经典通路的复杂性。非经典TGF-β通路不依赖SMAD，可激活无数细胞功能，包括形态发生、纤维化、免疫和代谢。21经典和非经典通路在纤维化中均发挥重要作用。总体而言，TGF-β通路激活的结果取决于背景，受哪种细胞因子激活受体、受体如何相互作用、激活的是经典还是非经典通路、甚至哪些个体SMADs被招募到受体等因素影响（图2）。
[图：TGF-β通路示意图，该通路在TGF-β2与TGF-β1和3之间存在差异。三者最终均激活SMAD2和SMAD3以]
在肾损伤情况下，TGF-β的3种亚型表达均增加，但TGF-β1的研究最为深入。它可刺激间质成纤维细胞、肾小管上皮细胞、系膜细胞、足细胞和壁上皮细胞产生ECM成分。20 它还导致MMPs下调，减缓新合成胶原的降解。在肝细胞过表达TGF-β1的转基因小鼠中，血浆中TGF-β1水平更高。这通过诱导胶原产生并通过上调基质金属蛋白酶组织抑制剂（TIMPs）抑制其降解，从而启动并维持肾小球硬化。27 尽管该研究证实循环TGF-β可引起肾纤维化，但局部产生的TGF-β也可导致肾瘢痕形成。在自然疾病中，该细胞因子可在血清中检测到，但在自然CKD中，肾外与肾内TGF-β的相对影响尚不清楚。
肾损伤与TGF-β通路的上调相关，通常以旁分泌方式发生。28 受损的肾小管可分泌新的TGF-β，或促进其从ECM中释放。CKD纤维化的一个关键特征是肌成纤维细胞的出现，与典型成纤维细胞相比，肌成纤维细胞具有合成不同类型ECM成分的能力。28 TGF-β是肾脏中该细胞群体出现的主要驱动因素。28 近期证据表明，肌成纤维细胞来源于驻留的间质成纤维细胞和周细胞。某些肾脏肌成纤维细胞谱系可进一步增强TGF-β信号传导，使邻近的成纤维细胞和周细胞转化为更多肌成纤维细胞。29 肾小管上皮细胞向间充质细胞转化（EMT）曾被认为是肌成纤维细胞分化的重要机制，因为TGF-β激活的基因通路与器官发育和癌症中EMT的通路重叠；然而，缺乏真正EMT在肾瘢痕形成中的证据。29,30 肾小球硬化（glomerulosclerosis）是TGF-β在肾脏中的另一个下游效应。本质上，TGF-β通路激活系膜细胞和足细胞，使其合成更多正常类型的胶原。27 然而，这些细胞主要合成非纤维性胶原，而非纤维性胶原。其效应基本相同：ECM不可逆增加，可压迫或闭塞毛细血管腔并阻碍肾小球灌注。实验数据表明，足细胞在TGF-β影响下可发生向间充质细胞表型的部分转化。31 这些间充质细胞合成ECM蛋白（如I型胶原、纤连蛋白和α-平滑肌肌动蛋白），导致肾小球硬化。31 在转基因小鼠中，足细胞响应TGF-β1而发生凋亡。32 相反，BMP在小鼠模型中保护足细胞。33 其他在实验性啮齿动物肾病模型中使用重组BMP7的研究已证明其具有减轻肾小球硬化、肾纤维化和肾小管萎缩的作用。34
随着肾小球硬化的发展，将进一步降低管周毛细血管灌注，促进肾小管萎缩和间质纤维化。管周毛细血管稀疏化是TGF-β通路的另一个不可逆效应。35 间质中可见基质增加，使用Masson三色等突出显示胶原的特殊染色更容易检测。肾小球硬化的特征是大量胶原部分或全局性抹去毛细血管簇（图3）。
[图：（A）基于啮齿动物模型数据和人类自然疾病数据，TGF-β通路在肾脏中的生物学效应]
TGF-β1不仅具有促纤维化作用，还具有免疫调节作用，能够抑制细胞毒性T细胞、阻断自然杀伤细胞效应功能并诱导调节性T细胞。这说明了TGF-β1可发挥的多样作用，包括在肾炎背景下平息炎症反应的能力。36,37 综上所述，肾脏TGF-β通路涉及多种不同细胞谱系（成纤维细胞、内皮、肾小管上皮、系膜细胞、足细胞、血小板和白细胞38；图3）。人类和啮齿动物研究表明，TGF-β在肾脏疾病中既有正面也有负面效应，这可能反映了通路激活的程度和时机。TGF-β通路的过度激活或慢性上调可能促进CKD进展。
TGF-β通路也与急性肾损伤（AKI）有关。其在近端肾小管上皮中的信号传导迅速上调，主要发挥损伤性效应。仅在肾小管上皮细胞中组成性激活TGFBRI就足以引起AKI，其特征为肾小管细胞凋亡和坏死、氧化应激（通过线粒体来源的ROS）、上皮去分化和间质中巨噬细胞、树突状细胞和T细胞积聚。39 在近端小管中选择性删除TGFBRII可减轻肾损伤并减少毒素诱导AKI模型中的肾小管凋亡，证实了TGF-β信号在急性损伤反应中的直接有害作用。40 在肾小管上皮细胞中条件性敲除SMAD2也通过减轻p53介导的凋亡、RIPK介导的坏死性凋亡和NF-KB驱动的炎症而具有肾脏保护作用。41
CKD中TGF-β通路激活在文献中已被广泛报道，TGF-β亚型转录本丰度升高是其关键特征。尽管基因表达可区分不同TGF-β亚型，但免疫组织化学（IHC）通常特异性较低，且根据抗体识别的表位不同，常可标记一种以上亚型。此外，TGF-β可以潜伏形式存在于肾间质ECM中，因此其在肾脏中的存在并不等同于信号通路处于激活状态。TGF-β与LAP及其他ECM成分的相互作用可能遮蔽表位，降低IHC敏感性。TGF-β存在时间短暂，因此根据评估时间点的不同，在纤维化肾组织中可能无法检出活性形式。通过评估下游促纤维化和免疫调节基因表达（采用bulk转录组学或定量逆转录聚合酶链反应，qRT-PCR）来测定TGF-β通路激活，可提供对通路激活最全面的评估。因此，通路分析是研究各类CKD中TGF-β下游效应（包括间质纤维化、肾小球硬化和炎症）的最佳方法。使用空间定位方法（如原位杂交），可将TGF-β通路转录本定位至特定区域（如肾小球与肾小管间质），从而进一步深入认识发病机制。空间转录组学和单细胞转录组学在阐明TGF-β在人类和啮齿动物CKD中的作用方面具有强大能力。37,42这些方法可展示多种肾脏疾病状态下的TGF-β转录本表达及下游通路激活。由于空间转录组学和单细胞转录组分析本质上是稀疏数据集，研究下游基因特征可提高敏感性，但应注意某些基因集存在非特异性。已对猫43和犬44肾组织进行单细胞转录分析，但每项研究纳入的个体均少于3例，因此仍需更多研究。
猫中的TGF-β
猫CKD的临床表现具有异质性，体现在出现临床症状时所处的分期以及疾病进展速度方面。国际肾脏兴趣学会（IRIS）已提供CKD分期的临床指南。45肾小管间质炎症和纤维化的组织病理学表现较为一致，且与肾功能生化标志物的相关性最佳。12,13具体而言，间质纤维化与氮质血症、高磷血症和贫血的严重程度密切相关，13提示其与CKD进展相关。此外，CKD猫的间质纤维化与胶原交联酶——转谷氨酰胺酶的活性密切相关。46体外研究表明，转谷氨酰胺酶参与将TGF-β结合至LTBPs和ECM，这是TGF-β隔离和最终激活所必需的步骤。47,48
越来越多的证据表明TGF-β参与猫自然发生的肾脏疾病。已在自然发生疾病和实验诱导肾损伤的猫肾脏中评估了TGF-β1基因和蛋白表达。如前所述，由于该细胞因子存在多种潜伏和活性亚型，在病变肾组织中测定和定位TGF-β1蛋白较为困难。比较不同研究结果时，需仔细评估所用IHC抗体的特异性及所采用的验证流程。TGF-β1基因表达的测定问题较少。然而，理想情况下应同时测定转录本和蛋白，以及TGF-β1通路的下游介质。
RNA测序（RNAseq）表征了实验诱导缺血再灌注损伤后6个月肾组织中的基因表达变化。49该团队既往发表的研究已对该模型进行验证，并证明其与自然发生的CKD具有显著的病理相似性。50表达变化是通过与同一只猫的对侧（非缺血）肾脏以及健康对照猫肾脏进行比较而确定的；值得注意的是，健康对照猫的年龄小于接受肾缺血的猫，且未接受假手术。研究采用基因本体分析 approach，与对照猫肾脏相比，既往缺血的肾脏显示促炎和促纤维化通路上调。在“胶原生物合成过程的正调控”分子功能类别（上调2.94倍）中识别出的感兴趣基因包括TGFB1、TGFB3和VIM，分别对应TGF-β1、TGF-β3和波形蛋白。当比较缺血肾脏与非缺血对侧对照肾脏时，差异调节的通路较少，提示即使在损伤后6个月，双肾中促炎和促纤维化通路均被激活。49其原因尚不清楚，但其他缺血再灌注损伤实验模型中也报道了类似的双侧肾脏损伤模式。51
一篇使用相同样本的配套论文采用了更具针对性的方法，通过qRT-PCR检测个体基因表达，包括TGFB1。52 作者显示，与对照猫肾脏中正常化水平相比，缺血肾脏中TGFB1表达显著增加。缺血肾脏与其对侧非缺血对照之间，或对照肾脏与对侧非缺血对照之间未发现显著差异。其他对缺血-再灌注损伤应答上调的靶基因包括MMPs。具体而言，MMP7和MMP9显示出与TGFB1相同的差异表达模式。还评估了基因表达与纤维化组织学严重程度之间的相关性。TGFB1表达与纤维化严重程度之间未发现显著关联；然而，HIF1A（编码缺氧诱导因子-1α）、MMP2、MMP7和TIMP1的表达均与纤维化评分显著且相对较强相关。本研究及其配套研究49均纳入少量动物（6只接受缺血方案的猫和8或9只健康对照），这是局限性之一。
一项随访研究使用了相同的缺血方案，但在缺血后3个月切除对侧肾脏，并在6个月时采集缺血肾脏。评估了肾脏（缺血与非缺血比较）和血清（基线与非终末样本比较）中的微小RNA（miRNA）表达。这项小型研究（4只猫）显示缺血肾脏与对侧肾脏之间有20种miRNA差异表达，其中4种（mir-21、mir-146、mir-199和mir-235）在终末血清样本中也表现出较基线水平升高。这些miRNA预计调控许多与猫CKD相关的基因，包括TGFB1、HIF1A、HGF（编码肝细胞生长因子）、FN1（编码纤连蛋白）和VEGFA（编码血管内皮生长因子A）。53 mir-21在调控TGF-β通路中的作用已在其他物种（包括人类）中进行了研究。54
另一项研究评估了自然发生CKD的猫，靶向与上述相同的基因。55 该研究纳入12只客户拥有的猫，这些猫在年龄、IRIS CKD分期（7只为IRIS 4期CKD）和蛋白尿严重程度方面差异很大。比较组由8只健康实验室猫组成，这些猫年轻（但已成熟），通常未绝育并喂食标准饮食。它们在许多方面与CKD猫不同；因此，它们不是理想的对照组。尽管如此，该研究显示，与对照组相比，自然发生CKD的猫的肾脏组织中TGFB1基因转录更高。这种差异的幅度在CKD个体猫之间高度可变（2至10倍差异），并且同一只猫的左肾和右肾之间存在变异性。其他促纤维化基因（MMP2、7和9、TIMP1和HIF1A）也显示出类似发现，而与对照组相比，CKD肾脏中VEGFA表达显著较低。GAPDH被用作管家基因。与缺血-再灌注研究一样，TGFB1的表达与炎症、纤维化或肾小管萎缩的组织学评分不相关；然而，MMP2、MMP7和TIMP1基因的表达与纤维化呈正相关，VEGFA表达与纤维化呈负相关。相比之下，另一组研究人员的研究报告称，与无病变猫的肾脏（n=6，平均年龄4个月）相比，从6只自然发生CKD的年轻猫（平均年龄2.54岁）安乐死后采集的死后肾脏样本未显示TGFB1基因表达具有统计学显著上调。56 该研究使用β-肌动蛋白作为管家基因。管家基因的差异、CKD猫与其他大多数研究相比年龄较轻，以及对照猫年龄较轻（平均年龄4个月）可能解释该研究56结果不同的原因。
一项涉及51只客户拥有猫的死后肾脏样本的研究初步数据已以摘要形式展示，其中14只为老年对照（肾功能正常），37只患有CKD（IRIS 2-4期）。该研究显示TGFB1表达（通过qRT-PCR）与肾脏纤维化评分之间以及盐皮质激素受体表达与TGFB1表达之间呈弱相关。57 总之，这6项研究表明，猫中TGFB1基因表达上调与诱导性肾损伤和自然发生CKD相关。
有三篇文献使用免疫组化（IHC）对自然发生的猫慢性肾病（CKD）中TGF-β1蛋白的表达进行了定位和半定量分析。研究使用小鼠抗人TGF-β1单克隆抗体标记了17个死后肾脏（7只CKD猫和10只来自其病理档案的猫）。文献未提供验证该抗体与猫TGF-β1交叉反应的方法。平均（±SD）年龄低于许多终末期CKD猫（7.20 ± 3.10岁）。这些猫并非全部完成了完整的IRIS分期，但在已完成分期的猫中，各期均有分布。档案组织并非来自经过IRIS分期的猫，且未提供这些猫的临床数据。研究开发了一种评估IHC染色的客观评分方案，但分析中未纳入已知肾功能正常的猫的肾脏。IHC结果显示TGF-β1在肾脏多个区域表达，但在间质炎症和纤维化区域表达最强（补充表1）。TGF-β1蛋白标记与肾小球硬化和肾小管萎缩强相关。
另一项基于IHC的研究从41例生前诊断为CKD的猫死后病例中选取了12个肾脏进行检查。选取这12例是因为它们为稳定性CKD，且无以下任何证据：原发性肾小球疾病、CKD急性失代偿、急性肾损伤（AKI）或多囊肾病。该组包括IRIS 1期猫，其尿比重持续偏低（<1.035）。这些猫的血浆肌酐浓度中位数为2.09（范围0.89-3.1）mg/dL。死亡或安乐死的原因未说明。对照组由4只年轻（23-27月龄）健康实验猫组成。TGF-β1使用兔抗人多克隆抗体标记。对照肾脏间质和基底膜区域可见 faint 标记。相比之下，CKD样本显示肾小管和肾小球系膜有TGF-β1标记。在远端肾单位，顶端细胞表面和管腔染色强烈，而近端肾小管上皮细胞（PTEC）的胞质显示阳性标记。血管内血小板和部分间质白细胞也呈阳性。研究评估了标记强度与血浆肌酐、尿素、肾小球硬化和肾小管间质纤维化之间的相关性。未发现与纤维化的相关性，但远端肾单位管腔内的TGF-β1标记与血浆肌酐和肾小球硬化呈正相关。血浆肌酐与PTEC胞质标记之间以及血浆肌酐与血小板标记之间呈负相关。
最后，在上述一项转录研究中描述了TGF-β IHC。研究纳入6只年轻CKD猫和6只肾功能正常幼猫的肾脏。健康对照肾脏中未发现标记，而CKD猫的肾小管间质区域以及远端小管和集合管胞质内存在标记。同一文献还通过多柔比星处理培养的Crandell-Rees猫肾细胞作为毒性损伤模型，用western blot显示了TGF-β1蛋白表达。这3项IHC研究说明了前述蛋白标记的问题。理想情况下，IHC研究应使用第二种方法（如western blot或ELISA）验证抗体的敏感性。特异性应通过进行同型阴性对照来证明。上述研究中没有数据表明IHC抗体对活性TGF-β1蛋白相对于嵌入ECM中的蛋白具有特异性。这些局限性提示，来自IHC研究的数据应谨慎解读，并应鼓励在未来的发表中对抗体进行充分验证。
TGF-β1是猫肾脏疾病的潜在生物标志物。所测定的异构体（总TGF-β1、潜伏型或活性型）对于理解其生物学意义至关重要。一项研究表明，使用多物种ELISA试剂盒（Biosource International Inc.，Camarillo，CA）测定并以尿肌酐校正的猫尿TGF-β1在CKD猫中显著高于健康对照。他们还测定了血清TGF-β1，循环浓度在组间无差异。该研究的局限性在于，除CKD诊断标准外，对36只猫提供的临床信息很少。60一项检测健康和CKD猫多种尿细胞因子的研究报告了类似结果。61该研究纳入的动物特征明确，为稳定性、非蛋白尿性、非高血压性CKD猫（n=26；中位年龄13.5岁，范围6-19岁），无显著合并症。排除了下尿路疾病作为氮质血症的原因。对照病例为因常规筛查就诊的健康猫（n=18；中位年龄5.8岁，范围8个月至13岁）。使用两种ELISA试剂盒测定尿中TGFβ-1，一种来自R&D Systems，另一种为Invitrogen的多物种检测试剂盒。仅后者能够检测猫TGF-β1，以尿肌酐校正后，CKD猫尿中TGF-β1较对照猫高5至10倍。此外，CKD猫尿TGF-β1与血清肌酐之间存在显著正相关。两项研究均未发表ELISA方法的验证数据，也未报告所测细胞因子为何种形式（总TGF-β1、潜伏型或活性型）。此外，由于研究未获取肾脏组织，无法将尿TGF-β1与组织表达或纤维化程度进行相关性分析。
使用为人开发的ELISA（Biolegend，San Diego，CA）测定猫尿中的活性TGF-β1，该试剂盒在特异性、精密度、重现性和检测限方面对猫进行了部分验证。通过加标回收率评估干扰物质和基质效应。由于当时无重组猫TGF-β1，未评估该方法的准确度。本文呈现了三项回顾性研究，以探讨测定尿中活性TGF-β1的实用性。第一项研究采用横断面设计，检测了肾功能正常（且在12个月内保持正常）的猫、采样时非氮质血症但在12个月内发展为氮质血症性CKD的猫以及氮质血症性CKD猫的尿液。三组之间尿活性TGF-β1：肌酐比值未检测到差异。第二项研究采用纵向设计，涉及3组各6只猫，在1年内采样3次。一组为肾功能在一年内保持正常的健康猫。第二组最初非氮质血症，但在一年内发展为氮质血症性CKD。第三组为氮质血症性CKD猫，其肾功能在一年内恶化。该纵向研究显示个体猫之间差异显著，但时间进程表明，在12个月内发展为氮质血症性CKD的猫在CKD诊断前6个月尿活性TGF-β1：肌酐比值即升高。该升高在12个月时间点持续存在。总体而言，进展性CKD猫尿活性TGF-β1：肌酐比值较高，但差异无统计学意义，可能是因为组样本量小且猫间存在中度变异。该研究的最后一部分检查了在安乐死或死亡前<3个月有库存尿样的猫中尿活性TGF-β1与肾纤维化评分之间的关系。使用多变量分析，仅纤维化评分仍是尿活性TGF-β1：肌酐比值的独立预测因子。这些数据表明，尿活性TGF-β1可作为活动性纤维化的生物标志物，但个体猫之间的变异性可能阻碍其临床应用。62
体外实验研究表明，PTECs原代培养对人重组TGF-β1的刺激有响应。具体而言，对猫PTECs原代培养的上皮细胞标志物表达进行了表征，并与人类肾上皮细胞（HRECs）进行了比较。63 将这些细胞暴露于浓度递增的人重组TGF-β1（0.1-10 nmol/L）中，导致细胞形态改变和基因表达改变，提示细胞表型发生变化及上皮可塑性。这涉及CDH1（E-钙黏蛋白基因）表达缺失以及CDH2（N-钙黏蛋白基因）、COL1A1（胶原1α1基因）和FN1表达增加。此外，猫PTECs对TGFB1基因表达的自身诱导作用明显，结缔组织生长因子（CTGF）的上调也很明显，CTGF是介导TGFβ-1通路促纤维化效应的关键因子。TGFB1表达的自身诱导可能在放大和维持应答中具有重要作用。这些效应均被TGF-β1RI/ALK5拮抗剂SB431542所抑制。猫PTECs中发生Smad2磷酸化。TGF-β1还以浓度依赖性方式抑制猫PTEC增殖并上调凋亡。这些效应被SB431542抑制。在这些研究中，猫PTECs的表现与HERCs相似，支持TGFβ-1在人和猫肾纤维化中具有保守作用。
同一作者发表了姊妹论文，使用来自CKD猫和肾功能正常猫的肾皮质成纤维细胞原代培养。64 通过一系列细胞标志物和特征性细胞形态，这些细胞被鉴定为间充质细胞。TGF-β1以浓度依赖性方式刺激以下基因的表达：COL1A1、FN1、ACTA2（α-平滑肌肌动蛋白基因）和CTGF；它还刺激TGFB1的自身诱导。所有这些效应均被SB431542消除，证明涉及经典TGF-β1通路的激活。一些证据表明，从CKD猫分离的皮质成纤维细胞已被激活，因为它们表达血管细胞黏附分子-1（VCAM-1），而肾功能正常猫的细胞则不表达。TGF-β1诱导成纤维细胞表达α-平滑肌肌动蛋白，提示这些细胞已转化为（或正处于转化为）猫肌成纤维细胞的过程中。细胞在TGF-β1刺激下还发生肥大，这是活化表型的另一特征。综上所述，这些实验表明TGF-β1可在驻留皮质成纤维细胞和PTECs中诱导强烈且持续的促纤维化应答。然而，这些研究并未证明基因表达上调转化为所编码蛋白的产生和分泌。
犬中的TGF-β
与猫相似，犬的进行性CKD与间质性炎症和纤维化的发生相关。很大比例的CKD犬，尤其是老年和老龄犬，有明确的肾性蛋白尿证据。65 如果进行常规尿液分析，蛋白尿通常先于肾小球滤过率下降和不可逆的肾小管间质病变出现。伴有蛋白尿的IRIS 1期犬代表了一个可能从疾病早期治疗性抑制肾纤维化中获益的群体。这与猫形成对比，猫较少出现蛋白尿性CKD。需要明确的是，在群体水平上，尿蛋白是猫进行性CKD的指标，但蛋白尿的程度通常低于犬。此外，猫进行性与稳定性CKD之间尿蛋白与肌酐比值（UPC）的重叠相当大。由于犬中蛋白尿、CKD和TGF-β之间存在多方面关系，蛋白尿性IRIS 1期犬可能代表了一个在肾病进展早期进行干预的有前景的群体。
多项研究支持TGF-β通路在犬肾脏疾病中的作用。较早的体外培养Madin-Darby犬肾（MDCK）细胞文献报道了TGF-β参与EMT。66 然而，如前所述，这种现象在体内肾脏中可能不会发生。最近，在经TGF-β1处理的培养MDCK细胞中观察到了吡非尼酮的通路抑制作用。吡非尼酮的作用机制尚未完全阐明，但有证据表明其部分通过下调Smad 2和3、阻断其磷酸化而发挥作用。在培养MDCK细胞中，它还阻断了ERK1/2磷酸化。67 在其他模型系统中，吡非尼酮似乎通过抑制一种蛋白水解酶——弗林蛋白酶，来抑制活性TGF-β从基质中的释放。68
大多数证明TGF-β在犬CKD中重要性的体内研究涉及患有X连锁遗传性肾病（XLHN）的犬。这些犬存在IV型胶原的突变，IV型胶原是肾小球基底膜的重要组成部分。该犬群接受了系列活检，以及反复的尿液和血液采集，从而能够评估随时间变化的组织基因表达。在一项涉及10只患病犬和5只未患病犬的研究中，在氮质血症和间质纤维化发生之前，观察到肾脏TGF-β（未说明具体亚型）、CTGF和血小板衍生生长因子mRNA表达增加（通过qRT-PCR评估），支持这些基因参与肾小管间质病变的早期/起始事件。69虽然TGF-β mRNA表达在疾病早期肾活检组织中 initially 增加，但在疾病晚期与未患病的雄性同窝犬（对照）相比并未显著过表达；因此，mRNA表达与纤维化程度不相关。此外，使用针对TGF-β所有亚型的抗体进行的IHC显示，患病犬在整个疾病进展过程中以及对照犬的足细胞和一些肾小管中表达相似（补充表1）。据推测，在早期肾小球疾病中，足细胞TGF-β表达导致系膜细胞增殖和基质扩张，这是患病犬最早的 light microscopy 异常。69
与未患病同窝犬相比，晚期疾病中TGF-β表达缺乏改变，这与 earlier 一项比较4只XLHN所致终末期疾病犬与4只未患病犬的微阵列研究一致。70在该研究中，两组之间肾脏皮质TGF-β1基因表达无显著差异。70相反，一项更近期的研究基于qRT-PCR证明终末期疾病中TGFB1、TGFB2和TGFBRI的mRNA表达增加，该研究对27只患病犬和5只未患病犬的肾脏皮质尸检样本进行了检测。71此外，在比较11只XLHN犬与10只未患病犬的系列肾活检样本时，miR-21（一种受TGF-β调控的microRNA）的表达在氮质血症发生时首次增加。miR-21的表达还与间质纤维化的严重程度相关。71如上所述，该miR在猫CKD和TGF-β通路中具有研究意义。
另外两项研究使用RNA-seq或small RNA-seq进一步表征XLHN犬的基因表达。在RNA-seq研究中，比较了来自快速进展（n=3）或缓慢进展（n=3）XLHN犬的6份系列肾活检样本，以及来自2只未患病犬的样本。快速进展犬的纤维化评分更高，通路分析在比较快速进展组与缓慢进展组的表达模式时，将TGF-β1确定为主要上游调控因子。然而，TGFB1转录本本身在任何比较中（快速进展 vs 缓慢进展；患病 vs 对照）均未差异表达。72尽管如此，与对照相比，患病犬在疾病中期和晚期TGFB2表达总体增加。在快速进展组中，与对照相比，TGFB3转录本在疾病中期增加。此外，TGFB诱导型转录本（一种TGF-β诱导的转录本）与TGFB2类似地上调。在某些比较中，TGFB1|1（即已知为TGFB1诱导转录本1的转录本）也增加。这些数据支持该疾病过程中TGF-β通路的激活。72
使用small RNAseq比较5只XLHN犬和4只未患病犬的系列肾活检样本，miRNA-21和-146b（两者均与纤维化相关）是在每个时间点患病犬中持续上调的五种miRNA中的两种。73通路分析在最早时间点发现患病犬中“TGFβ受体复合物信号传导”通路富集。这些发现进一步支持TGF-β在早期疾病进展（氮质血症发生之前）中的作用。73
总体而言，犬XLHN研究的结果支持TGF-β通路在CKD进展中的重要性。然而，终末期样本结果的不一致（例如，mRNA存在于一个队列中但不存在于其他队列中，以及疾病各阶段和对照样本中相似的IHC标记模式）凸显了使用基因表达和IHC来确定TGF-β活性的挑战，即使在单一疾病表型内也是如此。
也研究了TGF-β在犬宠物群体肾脏疾病中的作用。值得注意的是，自发性犬CKD的诊断可能基于持续性蛋白尿或氮质血症的存在。或者，它可能在犬从AKI事件向CKD过渡时发生。在一次AKI发作后，犬可能进展为符合CKD的慢性不可逆损伤74。一项研究发现23%的犬在AKI发作后发展为IRIS 2期CKD。75虽然这也可发生在猫中，但被认为较少见。许多细胞因子参与这些过渡，但TGF-β是这些现象中的关键参与者。76,77
全血样本中的TGFB mRNA在尿毒症犬中高于对照组（采用qRT-PCR检测）。该研究纳入46只不同病因的AKI犬、8只CKD犬和10只健康对照犬。有趣的是，AKI犬的全血TGFB表达显著高于对照组，但CKD犬与对照犬之间未检测到差异。78这些发现可能对全血TGFB表达水平作为AKI发作筛查工具具有诊断意义。然而，该细胞因子可受多种全身炎症性疾病调节，这可能限制诊断试验的特异性。进一步研究全血TGFB表达水平与肾纤维化及肾脏基因表达的关系，将是开发此类诊断检测的有价值的下一步。
钩端螺旋体病是犬AKI的常见病因。79–81在钩端螺旋体病流行地区，大多数CKD犬中存在钩端螺旋体无症状排菌，提示其可能在这些犬CKD的发生中起作用。82在人类钩端螺旋体病涉及的众多细胞因子中，TGF-β被认为是感染后肾纤维化最重要的介质之一。83犬中的这种关系尚未充分研究，但可能是未来研究的一个有趣方向。
在流行地区，利什曼病是犬肾小球疾病的常见病因，最常见的是肾小球肾炎。84,85近半数利什曼原虫相关肾小球疾病犬存在肾小球和肾小管间质纤维化。86对自然感染和实验感染犬（使用2种不同寄生虫株）进行了IHC研究。该研究的一个目的是确定2种虫株中是否有任何一种更接近自然利什曼病中所见的肾脏病变。在所有受检犬中，TGF-β标记在肾小球和肾小管基底膜最强。用于实验感染和自然感染的2种虫株之一，与另一实验虫株及未感染对照犬相比，阳性标记区域显著更大。这再次强调了单一疾病实体中IHC标记的变异性。有趣的是，IHC还显示，与对照组相比，同一实验虫株感染犬和自然感染犬中波形蛋白和α-平滑肌肌动蛋白增加。87TGF-β的免疫调节功能在钩端螺旋体病和利什曼病中也可能很重要，因为这两种感染性疾病均引起显著肾炎。
肥胖是人类肾脏病发生的主要危险因素，但其在犬中的相关性知之甚少。实验诱导的肥胖与犬肾灌注过度相关，而减重可逆转这一现象。88这种灌注过度可能导致肾小球高压和随后的肾损伤。实验诱导的肥胖还改变犬肾内细胞因子表达；在肥胖犬的肾髓质中观察到TGFB1增加74%。89TGF-β通路的这种上调可能增加肾纤维化和肾功能丧失的风险。一项研究发现，肥胖犬减重后功能性肾脏变量改善。90这提示对肥胖犬进行早期干预可能有助于通过使肾灌注正常化、或许使肾脏TGF-β1水平正常化来预防相关肾损伤，尽管肥胖与犬CKD风险之间的关联仍需要在可考虑其他协变量的大规模研究中确定。
关于TGF-β在犬肾脏病中作用的数据主要集中于多种原因引起的AKI和两种肾小球驱动的CKD。犬蛋白尿性CKD的许多常见病因尚未评估TGF-β mRNA或蛋白或通路激活情况，包括淀粉样变性和局灶节段性肾小球硬化。后者尤其值得关注，因为已知TGF-β参与人类和啮齿动物的肾小球硬化。在猫和犬中，需要更多研究来表征其在其他类型CKD（如梗阻性肾病和肾盂肾炎）中的作用。
结论
进行性间质纤维化伴肾小管萎缩和炎症是犬和猫进行性肾脏病的标志。如果要发现减缓进行性肾脏病的新疗法，就需要理解其分子发病机制。在伴侣动物物种中，TGF-β通路将影响PTEC的应激因素与间质纤维化联系起来的证据正在积累。TGF-β与肾小球硬化之间的联系也受到怀疑（因为已在实验或人类自然肾脏病中得到证实）。然而，这种关系在小动物中的表征尚不充分。
本综述中呈现的体外与临床数据相结合表明，TGF-β通路是未来治疗干预的合理靶点。近期已有研究采用系统评价和荟萃分析优先报告条目（PRISMA）方法，对人肾脏疾病中特异性抗TGF-β疗法进行了综述。共识别出6篇临床试验相关出版物和14项临床试验，其中仅4项已发表结果（部分在综述时仍处于招募阶段）。已发表的临床试验结果数据存在矛盾，表明TGF-β通路除有害的促纤维化机制外，还具有有益的的抗炎作用。91 当然，人类与小动物对成功肾脏治疗的期望有所不同；在小动物中，维持肾功能一至两年即被视为有益，而在人类中则未必如此，因为移植肾脏可维持数十年。鉴于目前缺乏降低瘢痕化肾脏纤维化的治疗手段，预防或减轻纤维化是猫和犬的治疗目标。
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Introduction
Chronic kidney disease (CKD) is a common cause of morbidity in dogs and cats. The reported prevalences in small animals vary widely and often depend on geography, age, and breed. Generally, it is estimated that approximately 1% of adult dogs1 and cats2 have CKD, whereas the prevalence in cats can reach 30% in individuals over 10 years of age.3 These reported prevalences are underestimates, because early-stage disease (before onset of clinical signs) often goes undetected. There are a variety of causes in both species, and progression partially depends on the initial insult. It is widely recognized that there is variation in disease progression, with some animals remaining stable and others showing more rapid deterioration of renal function.4,5 Factors driving progressive kidney injury in dogs and cats have are not well defined. Possible factors include the ability (or inability) to cure the primary renal disease and superimposed kidney insults. In cats, superimposed insults include renal ischemia, outflow obstruction and bacterial infection.6 In dogs, anemia, low body condition score and hyperphosphatemia are identified as factors that increase risk for progressive CKD4.
Other factors driving progression (which have mostly been elucidated in experimental or natural kidney diseases in humans and rodents) result from adaptive responses to nephron loss (hyperfiltration), leading to so-called “intrinsic progression”. Hyperfiltration results in podocyte hypertrophy to cover the increased surface area of the expanded glomerular basement membrane. This causes podocyte stretching and shear stress.7 There is also higher tubular workload, (specifically higher tubular reabsorption of sodium from the urinary filtrate) and activation of inflammatory and hypoxic injury pathways.7 Proteinuria, hyperphosphatemia and anemia are linked to progression of CKD in dogs and cats.4,8–10 Interstitial fibrosis and inflammation are associated with biomarkers of progression in dogs (correlated to azotemia)11 and cats (correlated to azotemia, proteinuria and hyperphosphatemia).10–13 Phosphaturia14,15 and proteinuria16 can trigger epithelial cell stress, leading to fibrosis and inflammation of the interstitial compartment. As stated above, glomerular hyperfiltration injures podocytes, which is the precursor lesion for glomerulosclerosis.17 Both interstitial fibrosis and glomerulosclerosis are irreversible lesions which can vary in severity and distribution. Interstitial fibrosis separates the tubules from their peritubular capillary blood supply in cats,18 rodents and humans,19 exacerbating the lower oxygen supply associated with anemia. This leads to tubular degeneration and atrophy. Glomerulosclerosis eventually effaces glomerular capillary lumens, impeding blood flow to the peritubular capillary bed.20
Both interstitial fibrosis and glomerulosclerosis are collagen-based lesions, and both can be initiated and perpetuated by the transforming growth factor (TGF)-β pathway. This molecular pathway is complex and multi-directional. It is a key component of mesenchymal tissue development during embryogenesis, post-natal growth, and wound healing. The TGF-β pathway is not the only mechanism that leads to collagen production in CKD; WNT, MAPK and RhoGTPase are also involved.20 Nevertheless, TGF-β-driven renal fibrosis is considered to be a major factor in the development and progression of CKD in animals. In addition to being pro-fibrotic, TGF-β also has anti-inflammatory effects, which further complicates its role in progressive CKD. This review will describe the current knowledge of the role of TGF-β in CKD, focusing on cats and dogs, with translation, where appropriate, to humans and rodent models.
There are approximately 30 TGF-β pathway genes and proteins across species. The members generally share high homology, for example, the TGF-β1 amino acid sequence shares 89% or higher percent identity (calculated as the number of identical matches divided by the total number of aligned positions) between human, canine, feline, and murine proteins (Figure 1). There are 3 known isoforms of TGF-β (TGF-β1, TGF-β2 and TGF-β3) in mammals, which are encoded by 3 separate genes, but which have similar amino acid sequences.21 TGF-β4 is an isoform that has only been observed in avian species, whereas TGF-β5 is only present in amphibians.22 Cell or organ specificity has not been demonstrated for the TGF-β isoforms in various protein/transcriptional atlases. TGF-βs are synthesized by a wide variety of cell types as pro-proteins but are soon cleaved into the mature protein and a latency associated peptide (LAP). The mature portion is subsequently cleaved in half between 2 identical regions that strongly bind to one another, forming a homodimer. The homodimer then binds non-covalently to its respective LAP, forming a small latent complex (SLC) that is then secreted into the extracellular space. The SLCs then further associate with latent TGF-β binding proteins (LTBPs), forming large latent complexes (LLCs) which are enmeshed in the extracellular matrix (ECM). LLCs represent a storage supply, so that TGF-β can be released when needed. Release of latent TGF-beta is a major control point for pathway activation. Multiple proteins can release the TGF-β in its active form, including integrins, thrombospondins, reactive oxygen species (ROS) and matrix metalloproteinases (MMPs). The structures of TGF-β1 and TGF-β3 are similar, and both can be released by integrins that are made by a variety of cells. However, TGF-β2 lacks the requisite arginine-glycine-aspartic acid (RGD) motif, so it is unable to be released by integrins.23 Thrombospondins have the capability to release all 3 isoforms, and they have particular importance in renal diseases.21
[图：TGF-β1 amino acid sequence is highly conserved between human, mice, cats and dogs. (A) Clustal omega alignment of human,]
Once released, a TGF-β1 or TGF-β3 homodimer binds to a monomer or a dimer of transmembrane TGF-β receptor II (TGFBRII). If the cytokine binds to a monomer of TGFBRII, then it will attract another one to create a 4-protein complex. Then, a dimer of TGF-β receptor I (TGFBRI) is recruited, ultimately forming a complex of 6 proteins. The affinity between the cytokine and TGFBRII is high, and TGFBRI is only attracted to complexes of TGF-β and TGFBRII. This stepwise binding process underscores the importance of TGFBRII in the capture of TGF-β1 or 3, and the necessary binding of TGFBRI to initiate signal transduction. In contrast, TGF-β2 does not follow the same sequence of receptor binding because TGF-β2 has a low affinity for TGFBRII on its own. In vitro studies show that TGF-β2 binds TGFBRIII, which acts as a co-receptor to enhance the binding affinity of TGF-β2 to TGFBRII. Once TGF-β2 is bound to TGFBRIII, TGFBRI is recruited, and signal transduction proceeds in the same manner as the other TGF-β isoforms.24 TGF-β receptors are promiscuous and can be activated by a wide variety of molecules in the TGF-β family, including bone morphogenic proteins (BMPs), activins and inhibins.25 BMPs can have opposite results, in comparison to the TGF-β pathway (see below).
There are canonical and non-canonical pathways of TGF-β signaling. The canonical pathway involves phosphorylation of the intracellular portions of the TGFBRI and TGFBRII dimers to recruit SMAD2 and SMAD3 proteins, which will also be phosphorylated. The phosphorylated SMADs then bind to SMAD4 and other co-factors as they are transported from the cell membrane to the nucleus, where they regulate gene transcription.26 There are multiple SMADs, with SMADs 2 and 3 being part of the TGF-β pathway, whereas SMADs 1, 5 and 8 are employed by the BMP pathway. Both pathways use SMAD4 to translocate to the nucleus. SMADs bind to SMAD-binding elements, which vary according to cell and tissue type. Some of the SMAD-binding elements involve epigenetic changes, and many pathways require transcriptional cofactors. Inhibitory SMADs (typically SMAD7, and to a lesser extent SMAD6) can bind to TGFBRI and block its interaction with SMADs 2 and 3, thereby creating a negative feedback loop for the canonical pathway. Competition with the BMP pathway for SMAD4 can also limit TGF-β pathway activation. There are many other cytoplasmic proteins that can phosphorylate SMADs, as well as phosphatases and proteases to inactivate the SMADs, underscoring the complexity of the canonical pathway. Non-canonical TGF-β pathways are SMAD-independent and can activate a myriad of cell functions including morphogenesis, fibrosis, immunity and metabolism.21 The canonical and non-canonical pathways both play important roles in fibrosis. Overall, the outcome of the TGF-β pathway activation is context-dependent, influenced by which cytokine activates the receptors, how the receptors interact with one another, whether the canonical or non-canonical pathway is activated, and even which individual SMADs are recruited to the receptors (Figure 2).
[图：Diagram of the TGF-β pathway which differs between TGF-β2 and TGF-β1 and 3. All 3 ultimately activate SMAD2 and SMAD3 to]
In the setting of renal injury, expression of all 3 isoforms of TGF-β is increased, but TGF-β1 is the best characterized. It can stimulate the production of ECM components by interstitial fibroblasts, tubular epithelial cells, mesangial cells, podocytes, and parietal epithelial cells.20 It also leads to downregulation of MMPs, slowing the breakdown of the newly synthesized collagen. In transgenic mice that overexpress TGF-β1 in hepatocytes, greater levels are found in the plasma. This initiates and perpetuates glomerulosclerosis by inducing production of collagens and inhibiting their breakdown via upregulation of tissue inhibitors of matrix metalloproteinases (TIMPs).27 Although that study proved that circulating TGF-β can cause renal fibrosis, local TGF-β production can also cause renal scarring. In natural disease, the cytokine is detectable in serum, but the relative influence of extra-renal vs intra-renal TGF-β in natural CKD is unknown.
Renal injury is associated with upregulation of the TGF-β pathways, often in a paracrine fashion.28 Injured tubules can secrete new TGF-β or they can facilitate its release from the ECM. A key feature of fibrosis in CKD is the appearance of myofibroblasts, which have the capacity to synthesize different types of ECM components compared to a typical fibroblast.28 TGF-β is a main driver behind the appearance of this population in kidneys.28 Recent evidence has demonstrated that myofibroblasts are derived from resident interstitial fibroblasts and pericytes. Some lineages of renal myofibroblasts can further increase TGF-β signaling to transform adjacent fibroblasts and pericytes into more myofibroblasts.29 Tubular epithelial cell to mesenchymal cell transformation (EMT) was once considered to be an important mechanism for myofibroblast differentiation because gene pathways activated by TGF-β overlap with those of EMT in organ development and cancer; however, evidence for true EMT in renal scarring is lacking.29,30 Scarring of the glomerulus (glomerulosclerosis) is another downstream effect of TGF-β in the kidney. Essentially, TGF-β pathways activate the mesangial cells and podocytes to synthesize increased amounts of their normal types of collagens.27 However, these cells synthesize mostly non-fibrillar collagens, as opposed to fibrillar collagen. The effect is essentially the same: irreversible increase in ECM that can compress or obliterate capillary lumens and impede glomerular perfusion. Experimental data indicate that podocytes can undergo partial transition to a mesenchymal cell phenotype under the influence of TGF-β.31 These mesenchymal cells synthesize ECM proteins (eg, Type I collagen, fibronectin, and α-smooth muscle-actin), resulting in glomerulosclerosis.31 Podocytes undergo apoptosis in response to TGF-β1 in transgenic mice.32 In contrast, BMP protects podocytes in mouse models.33 Other studies using recombinant BMP7 in experimental rodent models of kidney disease have demonstrated a role in attenuating glomerulosclerosis, renal fibrosis, and tubular atrophy.34
As glomerulosclerosis develops, it will further lower peritubular capillary perfusion, contributing to renal tubular atrophy and interstitial fibrosis. Peritubular capillary rarefaction is another irreversible effect of the TGF-β pathway.35 Increased matrix is visible in the interstitium and is easier to detect with special stains like Masson’s trichrome that highlight collagen. Glomerulosclerosis is characterized by abundant collagen that partially or globally effaces the capillary tuft (Figure 3).
[图：(A) Biologic effects of the TGF-β pathways in the kidney, based on data from rodent models and natural disease in humans]
TGF-β1 is not only pro-fibrotic, but it is also immunomodulatory, with the ability to suppress cytotoxic T cells, block natural killer cell effector functions, and induce T-regulatory cells. This illustrates the diverse roles that TGF-β1 can play, with the ability to quell inflammatory responses in the context of nephritis.36,37 Taken together, the renal TGF-β pathway involves multiple different cell lineages (fibroblast, endothelium, renal tubular epithelium, mesangial cells, podocytes, platelets, and leukocytes38; Figure 3). Studies in humans and rodents show both positive and negative effects of TGF-β in kidney disease, and it likely reflects degree and timing of pathway of activation. Overactivation or chronic upregulation of the TGF-β pathway are scenarios that might promote CKD progression.
The TGF-β pathway is also implicated in acute kidney injury (AKI). Its signaling in the proximal tubular epithelium is rapidly upregulated and exerts predominantly injurious effects. Constitutive activation of TGFBRI in tubular epithelial cells alone is sufficient to cause AKI, characterized by tubular cell apoptosis and necrosis, oxidative stress (via mitochondrial-derived ROS), epithelial dedifferentiation and interstitial accumulation of macrophages, dendritic cells and T cells.39 Selective deletion of TGFBRII in proximal tubules attenuates renal injury and reduces tubular apoptosis in toxin-induced AKI models, confirming a direct detrimental role of TGF-β signaling in acute injury response.40 Conditional knockout of SMAD2 in tubular epithelial cells also has renoprotective effects by alleviating p53-mediated apoptosis, RIPK-mediated necroptosis, and NF-KB-driven inflammation.41
TGF-β pathway activation in CKD is widely reported in the literature, with higher transcript abundance of TGF-β isoform(s) being a key feature. Although gene expression can distinguish among the TGF-β isoforms, immunohistochemistry (IHC) is generally less specific and often labels more than 1 isoform depending on the epitope recognized by the antibody. Moreover, TGF-β can remain latent in the renal interstitial ECM, so its presence in the kidney does not equate to active signaling. The interaction with the LAP and other ECM components has the potential to mask epitopes, decreasing IHC sensitivity. TGF-β is ephemeral such that identification of the active form could be missed in fibrotic kidney tissue, depending on the timepoint of evaluation. Measuring TGF-β pathway activation through evaluation of downstream pro-fibrotic and immunomodulatory gene expression (through bulk transcriptomics or quantitative reverse transcriptase polymerase chain reaction, qRT-PCR) provides the most comprehensive assessment of pathway activation. As such, pathway analysis is an optimal method to investigate which downstream effects of TGF-β including interstitial fibrosis, glomerulosclerosis, inflammation are expected in various categories of CKD. Using spatially aware approaches, such as in situ hybridization, TGF-β pathway transcripts can be localized to specific regions (eg, glomerulus vs tubulointerstitium), enabling further insights to pathogenesis. Spatial and single cell transcriptomics are powerful in illustrating the role of TGF-β in human and rodent CKD.37,42 These methods demonstrate TGF-β transcript expression and downstream pathway activation in various renal disease states. With spatial and single cell transcriptomic analyses being inherently sparse datasets, investigation of downstream gene signatures can enhance sensitivity. Although caution should be used due to the non-specificity of some gene sets. Single cell transcription analysis of feline43 and canine44 renal tissue has been performed, but it has been limited to <3 individuals in each study, so more research is needed.
TGF-β in cats
The clinical presentation of CKD in cats is heterogeneous, in terms of stage at which clinical signs are noted and the rate of progression. Clinical guidelines for staging CKD have been provided by the International Renal Interest Society (IRIS).45 The histopathological findings of tubulointerstitial inflammation and fibrosis are consistent and correlate best with biochemical markers of kidney function.12,13 Specifically, interstitial fibrosis correlates strongly with severity of azotemia, hyperphosphatemia and anemia,13 suggesting that it is associated with CKD progression. Furthermore, interstitial fibrosis in cats with CKD is strongly associated with the activity of a collagen cross-linking enzyme, transglutaminase.46 In vitro studies show that transglutaminase is involved in binding TGF-β to LTBPs and ECM, a step required for both sequestration and eventual activation.47,48
The evidence that TGF-β is involved in naturally occurring kidney disease in cats is accumulating. TGF-β1 gene and protein expression have been assessed in the kidneys of cats with naturally occurring disease and experimentally induced kidney injury. As discussed previously, measurement and localization of TGF-β1 protein within diseased kidney tissue is difficult because of the existence of multiple latent and active isoforms of this cytokine. Comparison of results between studies needs to carefully assess the specificity of the IHC antibody used and the validation process applied. Measurement of TGF-β1 gene expression is less problematic. However, ideally one would measure both transcript(s) and protein(s), as well as downstream mediators in the TGF-β1 pathway.
RNA sequencing (RNAseq) characterized gene expression changes in renal tissue 6 months after experimentally induced ischemia and reperfusion injury.49 Previous publications from this group had validated the model and demonstrated remarkable pathological similarities to naturally occurring CKD.50 The expression changes were established through comparisons to the contralateral (non-ischemic) kidney of the same cat and to healthy control feline kidneys; of note, healthy control cats were younger than the cats undergoing renal ischemia and had not been subjected to sham operation. A gene ontology analysis approach was taken, and in comparison to control cat kidneys, the previously ischemic kidneys showed upregulation of pro-inflammatory and profibrotic pathways. Genes of interest identified within the molecular function category of “positive regulation of collagen biosynthetic process” (which was upregulated 2.94-fold), included TGFB1, TGFB3, and VIM. These are the genes for TGF-β1, TGF-β3, and vimentin proteins, respectively. Fewer pathways were differentially regulated when comparisons were made between the ischemic and non-ischemic contralateral control kidney, suggesting proinflammatory and pro-fibrotic pathways were activated in both kidneys, even 6 months after injury.49 The reason for this was not clear, but similar bilateral patterns of renal injury have been reported in other experimental models of ischemia-reperfusion injury.51
A companion paper that used the same samples took a more targeted approach to examine individual gene expression by qRT-PCR, including TGFB1.52 The authors showed that TGFB1 expression was significantly increased in the ischemic kidney compared to normalized levels found in control cat kidneys. No significant difference was found between the ischemic kidney and its contralateral non-ischemic control, or between the control kidneys and the contralateral non-ischemic controls. Other targeted genes that were upregulated in response to the ischemia–reperfusion injury included MMPs. Specifically, MMP7 and MMP9 showed the same pattern of differential expression as TGFB1. Correlations between gene expression and histological severity of fibrosis were also assessed. No significant association was found between TGFB1 expression and severity of fibrosis; however, expression of HIF1A (which encodes hypoxia inducible factor-1α), MMP2, MMP7 and TIMP1 were all significantly and relatively strongly correlated with the fibrosis score. Both this study and its companion study49 included a small number of animals (6 cats with the ischemic protocol and 8 or 9 healthy controls), which is one of the limitations.
A follow-up study used the same ischemia protocol, but the contralateral kidney was removed 3 months after ischemia, and the ischemic kidney was harvested at 6 months. Micro(mi)-RNA expression was evaluated in the kidneys (ischemic compared to non-ischemic) and serum (baseline compared to terminal samples). The small study (4 cats) demonstrated differential expression of 20 miRNAs between ischemic and contralateral kidneys, 4 of which (mir-21, mir-146, mir-199, and mir-235) also exhibited increased levels in terminal serum samples compared to the baseline. These miRNAs are predicted to regulate many genes associated with CKD in cats, including TGFB1, HIF1A, HGF (which encodes hepatocyte growth factor), FN1 (which encodes fibronectin), and VEGFA (which encodes vascular endothelial growth factor A).53 The role of mir-21 in regulating the TGF-β pathway has been studied in other species, including humans.54
An additional study evaluated cats with naturally occurring CKD, targeting the same genes as detailed above.55 This study included 12 client-owned cats which varied greatly in terms of age, IRIS stage of CKD (7 were IRIS Stage 4 CKD) and severity of proteinuria. The comparator group consisted of 8 healthy laboratory cats which were young (but mature), often not neutered and fed a standard diet. They differed in many respects to the CKD cats; as such, they were not an ideal control group. Even so, this study demonstrated that kidney tissue from cats with naturally occurring CKD showed higher transcription of the TGFB1 gene when compared to the control group. The magnitude of this difference was highly variable (2- to 10-fold difference) between individual cats with CKD, and there was variability between left and right kidneys from the same cat. Similar findings were shown for other profibrotic genes (MMPs 2,7 & 9, TIMP1 and HIF1A), whereas VEGFA expression was significantly lower in CKD kidneys compared to controls. GAPDH was used as the house-keeping gene. Like the ischemia-reperfusion study, expression of TGFB1 did not correlate with histological scores for inflammation, fibrosis, or tubular atrophy; however, expression of MMP2, MMP7, and TIMP1 genes was positively correlated, and VEGFA expression was negatively correlated with fibrosis. By contrast, a study by different investigators reported that postmortem kidney samples collected after euthanasia from 6 young cats (mean age 2.54-years) with naturally occurring CKD did not show a statistically significant upregulation in TGFB1 gene expression compared to kidneys from cats with no lesions (n = 6, mean age 4 months).56 This study used β-actin as the housekeeping gene. The difference in house-keeping genes, young age of the CKD cats compared with most other studies, and the young age of the control cats (mean age 4-months) might account for the disparate results from this study.56
Preliminary data from a study involving post-mortem kidney samples from 51 client-owned cats, 14 of which were geriatric controls (with normal kidney function) and 37 of which had CKD (IRIS stages 2-4) have been presented as an abstract. This study showed weak correlations between TGFB1 expression (via qRT-PCR) and kidney fibrosis score and also between mineralocorticoid receptor expression and TGFB1 expression.57 Together, these 6 studies suggest that upregulation of TGFB1 gene expression occurs in the cat in association with induced kidney injury and naturally occurring CKD.
Three publications have used IHC to localize and semi-quantify the expression of TGF-β1 protein in naturally occurring feline CKD. A monoclonal mouse anti-human TGF-β1 antibody was used to label 17 postmortem kidneys (7 cats with CKD and 10 cats from their pathology archives).58 The method of validation showing that this antibody cross-reacts with feline TGF-β1 was not provided. The mean (±SD) age was lower than many cats with end-stage CKD (7.20 ± 3.10 years). The cats were not all completely IRIS-staged, but for those that were, the stages were variable. The archived tissues were not from IRIS-staged cats, and clinical data were not provided for them. An objective scoring scheme to assess IHC staining was developed, but kidneys from cats with known normal kidney function were not included in the analysis. The IHC results showed TGFβ-1 expression in many areas of the kidney, but expression was strongest in areas of interstitial inflammation and fibrosis (Supplementary Table 1). TGF-β1 protein labeling was strongly correlated with glomerulosclerosis and tubular atrophy.
Another IHC-based study examined 12 kidneys selected from 41 postmortem cases of cats with antemortem diagnoses of CKD. The 12 cases were selected because they had stable CKD with no evidence of any of the following: primary glomerular disease, acute decompensation of CKD, AKI, or polycystic kidney disease. This group included IRIS stage 1 cats that had persistently low urine specific gravity measurements (<1.035). The median plasma creatinine concentration of these cats was 2.09 (range 0.89-3.1) mg/dL. The reasons for death or euthanasia were not stated. A comparator group consisted of 4 young (23-27 months) healthy laboratory cats. TGF-β1 was labeled with a polyclonal rabbit anti-human antibody. Faint labeling was found in the control kidneys, within the interstitium and the basement membrane regions. By contrast, CKD samples showed TGF-β1 labeling of tubules and glomerular mesangium. In the distal nephron, apical cell surfaces and lumens stained strongly, whereas the cytoplasm of the proximal tubule epithelial cells (PTECs) showed positive labeling. Platelets inside blood vessels and some of the interstitial leukocytes were also positive. Correlations between the intensity of labeling, plasma creatinine and urea, glomerulosclerosis, and tubulointerstitial fibrosis were assessed. No correlations were found with fibrosis, but TGF-β1 labeling in the lumen of the distal nephron correlated positively with plasma creatinine and glomerulosclerosis. Negative correlations were found between plasma creatinine and labeling of PTEC cytoplasm, and also between plasma creatinine and labeling of platelets.59
Lastly, TGF-β IHC was described in one of the transcriptional investigations discussed above.56 Kidneys from 6 young CKD cats and from 6 kittens with normal kidney function were included. No labeling was found in the healthy control kidneys, whereas labeling was present in tubulointerstitial areas and within the cytoplasm of distal tubules and collecting ducts of CKD cats. TGF-β1 protein expression was shown by western blot of cultured Crandell-Rees Feline Kidney cells treated with doxorubicin as a model of toxic injury, in the same publication.56 These 3 IHC studies illustrate the previously discussed issues of protein labeling. Ideally, an IHC study would validate the antibody’s sensitivity using a second modality (eg, western blot or ELISA). Specificity should be demonstrated by performing isotype negative controls. There are no data in the above studies to indicate that the IHC antibodies were specific for the active TGF-β1 protein vs the protein embedded in the ECM. These limitations suggest that data reported from IHC studies should be interpreted with caution, and full validation of antibodies should be encouraged for future publications.
TGF-β1 is a potential biomarker of kidney disease in cats. The isoform measured (total, latent, or active) is important to understand its biological significance. One study showed that urinary TGF-β1 measured by a multispecies ELISA kit (Biosource International Inc., Camarillo, CA) normalized to urine creatinine was significantly higher in CKD cats compared to healthy controls. They also measured serum TGF-β1 and circulating concentrations did not differ between groups. A limitation of this study was that little clinical information was provided for the 36 cats, other than the diagnostic criteria for CKD.60 Similar results were reported as part of a study examining multiple urinary cytokines obtained from healthy and CKD cats.61 The animals included in this study were well characterized as stable, non-proteinuric, non-hypertensive CKD cats (n = 26; median age 13.5 years, range 6-19 years) without significant comorbidities. Lower urinary tract disorders as causes of azotemia were excluded. Control cases were healthy cats (n = 18; median age 5.8 years, range 8 months-13 years) presented for routine screening. Two ELISA kits were used to measure TGFβ-1 in urine, one from R&D Systems and one multispecies assay from Invitrogen. Only the latter assay was able to detect feline TGF-β1, which was 5 to 10-fold higher in urine from CKD cats compared to control cats when normalized to urine creatinine. In addition, a significant positive correlation was found between urinary TGF-β1 and serum creatinine in CKD cats. Neither study published a validation of the ELISA assay, nor did they report what form of the cytokine (total, latent, or active) was measured. Furthermore, because their studies did not have kidney tissue, they could not correlate urinary TGF-β1 with tissue expression or the degree of fibrosis.
Active TGF-β1 was measured in cat urine with an ELISA developed for humans (Biolegend, San Diego, CA), which was partially validated for the cat in terms of specificity, precision, reproducibility, and limit of detection. Interfering substances and matrix effects were assessed by determining spike recovery. The accuracy of the assay was not assessed because recombinant feline TGF-β1 was not available at the time. This paper presented three retrospective studies to explore the utility of measuring active TGF-β1 in urine. The first looked cross-sectionally at urine from cats that had normal kidney function (and remained normal over 12 months), cats that were non-azotemic at the time of sampling but developed azotemic CKD within 12 months, and cats with azotemic CKD. No differences in urinary active TGF-β1: creatinine ratio were detected among 3 three groups. The second study had a longitudinal design and involved 3 groups of 6 cats sampled 3 times over the course of 1 year. One group consisted of normal healthy cats whose kidney function remained normal during the year. The second group was non-azotemic initially but developed azotemic CKD over the course of a year. The third group had azotemic CKD, and their kidney function deteriorated during the year. This longitudinal study showed marked variability between individual cats, but the time course indicated that cats that developed azotemic CKD during the 12 months had elevated active urinary TGF-β1: creatinine ratios 6 months before CKD diagnosis. This elevation was sustained at the 12-month time point. Generally, progressive CKD cats had high urinary active TGF-β1: creatinine ratios, but the differences were not statistically significant, possibly because group sizes were small and there was moderate inter-cat variability. The final aspect of this study examined the relationship between urinary active TGF-β1 and renal fibrosis scores in cats that had a banked urine sample <3 months before euthanasia or death. Using multivariate analysis, only the fibrosis score remained an independent predictor of urinary active TGF-β1: creatinine ratio. These data suggest that urinary active TGF-β1 can be used as a biomarker of active fibrosis but that variability between individual cats might hamper its clinical utility.62
Experimental in vitro work shows that primary cultures of PTECs respond to stimulation by human recombinant TGF-β1. Specifically, the expression of epithelial cell markers by primary cultures of feline PTECs was characterized and compared to human renal epithelial cells (HRECs).63 Exposing these cells to increasing concentrations of human recombinant TGF-β1 (0.1-10 nmol/L) resulted in changes to cell morphology and altered gene expression, suggesting a change in phenotype of the cells and epithelial plasticity. This involved loss of expression of CDH1 (gene for E-cadherin) and increased expression of CDH2 (gene for N-cadherin), COL1A1 (gene for collagen 1α1), and FN1. Furthermore, auto-induction of TGFB1 gene expression by feline PTECs was evident, as was upregulation of connective tissue growth factor (CTGF), which is a key factor mediating the pro-fibrotic effects of the TGFβ-1 pathway. Auto-induction of TGFB1 expression is likely important in amplifying and sustaining the response. These effects were all inhibited by the TGF-β1RI/ALK5 antagonist, SB431542. Phosphorylation of Smad2 occurs in the feline PTECs. TGF-β1 also inhibited feline PTEC proliferation and upregulated apoptosis in a concentration-dependent manner. This was inhibited by SB431542. Feline PTECs behaved in a similar way to HERCs in these studies, supporting a conserved role for TGFβ-1in renal fibrosis in humans and cats.
The same authors published a companion paper using primary cultures of renal cortical fibroblasts from cats with CKD and from cats with normal kidney function.64 The cells were characterized as mesenchymal cells by use of a range of cell markers and characteristic cell morphology. TGF-β1 stimulated concentration-dependent expression of the following genes: COL1A1, FN1, ACTA2 (the gene for α-smooth muscle actin) and CTGF; it also stimulated auto-induction of TGFB1. All these effects were abrogated by SB431542, demonstrating that activation of the canonical TGF-β1 pathway was involved. Some evidence indicated that the cortical fibroblasts isolated from CKD cats were activated because they expressed vascular cell adhesion molecule-1 (VCAM-1), whereas those from cats with normal kidney function did not. TGF-β1 induction of fibroblasts to express α-smooth-muscle actin suggests that these cells had been transformed (or were in the process of transforming) into feline myofibroblasts. Cells also underwent hypertrophy in response to TGF-β1 stimulation, another feature of the activated phenotype. Taken together, these experiments demonstrate that TGF-β1 can induce a strong and sustainable profibrotic response in resident cortical fibroblasts and PTECs. However, these studies did not prove that upregulation of gene expression translated to production and secretion of the encoded proteins.
TGF-β in dogs
Similar to cats, progressive CKD in dogs is correlated with development of interstitial inflammation and fibrosis. A large proportion of CKD dogs, particularly senior and geriatric dogs, have evidence of overt renal proteinuria.65 If routine urinalyses are performed, proteinuria often precedes declining glomerular filtration rate and irreversible tubulointerstitial lesions. IRIS stage 1 dogs with proteinuria represent a cohort that could potentially benefit from therapeutic inhibition of renal fibrosis early in its development. This is in contrast to cats, which less frequently have proteinuric CKD. To be clear, urine protein is an indicator of progressive CKD in the cat at the population level, but the magnitude of the proteinuria is often lower than in dogs. Moreover, the overlap of urine protein to creatinine ratio (UPC) between progressive and stable CKD in cats is considerable. Because of the multifaceted relationship of proteinuria, CKD, and TGF-β in dogs, proteinuric IRIS stage 1 dogs might represent a promising cohort for early intervention in renal disease progression.
Multiple studies support the role of the TGF-β pathway in kidney disease in dogs. Older literature of in vitro cultured Madin-Darby canine kidney (MDCK) cells reported the involvement of TGF-β in EMT.66 However, as stated earlier, this phenomenon likely does not occur in the kidney in vivo. More recently, pathway inhibition by pirfenidone was seen in cultured MDCK cells that were treated with TGF-β1. The mechanism of pirfenidone action is not fully understood, but there is evidence that it acts in part through downregulation of Smads 2 and 3, by blocking their phosphorylation. In cultured MDCK cells, it also blocked ERK1/2 phosphorylation.67 In other model systems, pirfenidone appears to inhibit release of active TGF-β from the matrix by inhibiting one of the proteolytic enzymes, furin.68
The majority of in vivo studies demonstrating the importance of TGF-β in canine CKD involve dogs with X-linked hereditary nephropathy (XLHN). These dogs have a mutation in Type IV collagen, which is an integral component of the glomerular basement membrane. A colony of these dogs underwent serial biopsies, in addition to repeated urine and blood collections, allowing assessment of tissue gene expression over time. In one study of 10 affected and 5 unaffected dogs, increased renal expression of TGF-β (specific isoform not stated), CTGF, and platelet-derived growth factor mRNAs (assessed with qRT-PCR) was observed before development of azotemia and interstitial fibrosis, supporting that these genes are involved in the early/initiating events of tubulointerstitial lesions.69 While TGF-β mRNA expression initially increased in renal biopsy tissue early in the disease, it was not significantly overexpressed compared with unaffected male littermates (controls) at late-stage disease; therefore, mRNA expression did not correlate with degree of fibrosis. Additionally, IHC using an antibody directed against all isoforms of TGF-β showed similar expression in podocytes and some tubules in both affected dogs throughout their disease progression as well as controls (Supplementary Table 1). It was hypothesized that, in early glomerular disease, podocyte TGF-β expression results in mesangial cell proliferation and matrix expansion, which are the earliest light microscopy abnormalities in affected dogs.69
The lack of altered TGF-β expression at late-stage disease compared with unaffected littermates is consistent with an earlier microarray study comparing 4 dogs with end-stage disease due to XLHN to 4 unaffected dogs.70 In that study, cortical renal gene expression of TGF-β1 was not significantly different between the 2 groups.70 In contrast, a more recent study demonstrated increased mRNA expression of TGFB1, TGFB2, and TGFBRI at end-stage disease based on qRT-PCR, performed on autopsy samples of renal cortex from 27 affected and 5 unaffected dogs.71 Additionally, expression of miR-21, a microRNA regulated by TGF-β, was first increased at the onset of azotemia when comparing serial renal biopsy samples from 11 XLHN dogs to 10 unaffected dogs. The expression of miR-21 also correlated with the severity of interstitial fibrosis.71 This miR is of interest in cat CKD and TGF-β pathways, as noted above.
Two additional studies used RNA-seq or small RNA-seq to further characterize gene expression in XLHN dogs. In the RNA-seq study, 6 serial renal biopsies from XLHN dogs with either rapid (n = 3) or slow (n = 3) progression were compared, along with those from 2 unaffected dogs. Fibrosis scores were higher in dogs with rapid progression, and pathway analysis identified TGF-β1 as the primary upstream regulator when comparing expression patterns between the rapid vs slow groups. However, the TGFB1 transcript itself was not differentially expressed in any comparison (rapid vs slow; affected vs control).72 Despite this, TGFB2 expression was generally increased in affected dogs at mid- and late-stage disease compared to controls. The TGFB3 transcript was increased at mid-stage disease in the rapidly progressive group compared to controls. Additionally, TGFB inducible (a TGF-β-induced transcript) was upregulated, similar to TGFB2. In some comparisons, TGFB1|1 (which is the transcript known as TGFB1-induced transcript 1) was also increased. These data support activation of the TGF-β pathway in this disease process.72
Using small RNAseq to compare serial renal biopsies from 5 XLHN dogs and 4 unaffected dogs, miRNA-21 and -146b, both of which are associated with fibrosis, were two of the five miRNAs consistently upregulated in affected dogs at each timepoint.73 Pathway analysis identified enrichment of “signaling by TGFβ receptor complex” pathway in affected dogs at the earliest timepoint. These findings further support the role of TGF-β in early disease progression (before onset of azotemia).73
Overall, the results of the canine XHLN studies support the importance of the TGF-β pathway in CKD progression. However, discrepancies in the results from end stage samples (eg, mRNA present in one cohort but not others and similar IHC labeling patterns across disease stages and control samples) highlight the challenges of using gene expression and IHC to determine TGF-β activity, even within one disease phenotype.
The role of TGF-β in renal disease of the canine pet population has also been examined. Notably, diagnosis of spontaneous canine CKD could be based on the presence of persistent proteinuria or azotemia. Alternatively, it might occur when dogs are transitioning from an AKI event to CKD. After an AKI episode, a dog might progress to develop chronic irreversible damage consistent with CKD74. One study found 23% of dogs developed IRIS stage 2 CKD after an AKI episode.75 Although this can occur in cats, it is thought to be less common. Numerous cytokines are involved in these transitions, but TGF-β is a key player in these phenomena.76,77
TGFB mRNA from whole blood samples was higher in uremic dogs compared to controls (using qRT-PCR). This study included 46 dogs with AKI of various etiologies, 8 dogs with CKD, and 10 healthy control dogs. Interestingly, whole blood TGFB expression was significantly higher in AKI dogs compared to controls, but there was no detectable difference between CKD and control dogs.78 These findings might have diagnostic implications for whole blood TGFB expression levels as a screening tool for AKI episodes. However, modulation of the cytokine occurs with multiple systemic inflammatory conditions, and that could limit the specificity of a diagnostic test. Further investigations of whole blood TGFB expression levels with renal fibrosis and renal gene expression would be a valuable next step in developing this type of diagnostic assay.
Leptospirosis is a common cause of AKI in dogs.79–81 Asymptomatic shedding of leptospires is found in most dogs with CKD living in leptospirosis endemic regions, suggesting a possible role in the development of CKD in these dogs.82 Of the numerous cytokines involved in leptospirosis in humans, TGF-β is considered one of the most important mediators of post-infection renal fibrosis.83 The relationship in dogs is not well-studied but might be an interesting avenue for future investigation.
In endemic regions, leishmaniosis is a common cause of glomerular disease in dogs, most commonly glomerulonephritis.84,85 Glomerular and tubulointerstitial fibrosis was present in nearly half of dogs with leishmania-associated glomerular disease.86 An IHC study was performed in naturally and experimentally infected dogs (using 2 different parasite strains). One aim of the study was to determine if either of the 2 strains more closely mimicked renal lesions seen in natural leishmaniosis. TGF-β labeling was greatest in glomeruli and tubular basement membranes in all examined dogs. One of the 2 strains used for experimental infections and natural infections resulted in significantly larger regions of positive labeling compared to the other experimental strain and uninfected control dogs. Again, this underscores the variability of IHC labeling within a single disease entity. Interestingly, IHC also showed that vimentin and α-smooth muscle actin were increased by infections with the same experimental strain and in naturally infected dogs compared to controls.87 The immunomodulatory functions of TGF-β could also be important in both leptospirosis and leishmaniosis, because both infectious diseases incite significant nephritis.
Obesity is a major risk factor for the development of kidney disease in people, but less is known about its relevance in dogs. Experimentally-induced obesity is associated with renal hyperperfusion in dogs, which can be reversed with weight loss.88 This hyperperfusion might result in glomerular hypertension and subsequent renal injury. Experimentally induced obesity also alters intrarenal cytokine expression in dogs; a 74% increase in TGFB1 was observed in the medulla of obese dogs.89 This upregulation of the TGF-β pathway might increase the risk of renal fibrosis and the loss of renal function. One study identified improvement in functional kidney variables after weight loss in obese dogs.90 This suggests that early intervention in obese dogs might help prevent associated renal injury, through normalization of renal perfusion and perhaps normalization of renal TGF-β1 levels, although the association between obesity and risk of CKD in dogs remains to be determined in any large-scale study where other co-variates can be taken into account.
The data regarding the role of TGF-β in canine kidney disease has focused on AKI from a wide variety of causes and two types of glomerular-driven CKD. Many common causes of proteinuric CKD in dogs have not been evaluated for TGF-β mRNA or proteins, or pathway activation, including amyloidosis and focal segmental glomerulosclerosis. The latter disease would be of special interest, given that TGF-β is known to be involved in human and rodent glomerulosclerosis. In both cats and dogs, more research is needed to characterize its role in other types of CKD, such as obstructive nephropathy and pyelonephritis.
Conclusions
Progressive interstitial fibrosis with tubular atrophy and inflammation are the hallmarks of progressive kidney diseases in dogs and cats. Understanding the molecular pathogenesis is required if novel therapies of slowing progressive kidney disease are to be identified. The evidence that the TGF-β pathway links the stressors affecting PTECs to interstitial fibrosis is building for companion animal species. Links between TGF-β and glomerulosclerosis are also suspected (because they have been shown experimentally or in natural kidney disease in humans). However, this relationship is not as well characterized in small animals.
The combination of in vitro and clinical data presented in this review suggests the TGF-β pathway is a logical target for future therapeutic interventions. Specific anti-TGF-β therapies for renal disease in humans were recently reviewed, using a Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) approach. Six publications on clinical trials and 14 clinical trials were identified, only 4 of which had published results (some were still in recruitment phase at the time of the review). Conflicting data from published clinical trial results indicated that there are beneficial anti-inflammatory roles of the TGF-β pathways in addition to its harmful pro-fibrotic mechanisms.91 Certainly, expectations for a successful renal therapy differ between humans and small animals; preserving renal function for one to two years would be considered beneficial in small animals, whereas it might not be in humans, because a transplanted kidney can last for decades. Given the lack of treatment modalities to lower fibrosis in scarred kidneys, prevention or mitigation is a therapeutic goal in cats and dogs.
Supplementary Material
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