Romanov V, Noiri E, Czerwinski G, Finsinger D, Kessler H, Goligorsky M S
State University of New York, Stony Brook, New York, USA.
Kidney Int. 1997 Jul;52(1):93-102. doi: 10.1038/ki.1997.308.
We have previously demonstrated that RGD peptides prevent tubular obstruction in ischemic acute renal failure (ARF) suggested that exposed unoccupied integrin receptors represent the target for such therapy. The present study investigated the topography of RGD binding sites and integrin receptors in ischemic rat kidneys. Two RGD peptides were synthesized: a cyclic biotinylated (Bt) RGD peptide and a linear RGD peptide (GRGDSP) labeled with rhodamine green (RhoG). Rats were subjected to 45 minutes of renal artery occlusion kidneys were harvested at different times post-ischemia, and stained with RGD peptides and a panel of antibodies to integrins. In control, Bt-RGD staining was undetectable in alkaline phosphatase histochemistry, whereas immunofluorescence detection with Rho-streptavidin conjugate as well as RhoG-GRGDSP staining faintly decorated the basolateral aspect of the proximal tubular cells in a punctate fashion. In contrast, ischemic kidneys showed binding to the basolateral and apical aspects of proximal tubules, peritubular capillaries, and desquamated cells within tubular lumen. The most conspicuous staining of ischemic kidneys was obtained with antibodies to the beta 1 (labeling of the apical aspect of proximal and distal tubules, as well as desquamated cells obstructing tubular lumen) and the alpha V (glomeruli, tubular epithelia, intima of blood vessels stained faintly, while the obstructing cellular conglomerates showed intense staining) subunits. Double staining with Bt-RGD and antibodies against the beta 1 and alpha V beta 3 integrins showed co-localization of staining within the tubules and vasculature, respectively. In vitro attachment of HL-60 leukocytes to the endothelial cells was inhibited by the cyclic RGD peptide. In conclusion, expression of RGD binding sites and beta 1 integrin subunits along the apical aspect of tubular epithelia and on the surface of desquamated cells is in concert with the hypothesis on the pathogenetic role of RGD-recognizing integrins in tubular obstruction. The expression of RGD binding sites along the intimal surface of blood vessels in ischemic kidneys suggests an additional target for RGD peptides in vascular endothelial cells.
我们之前已经证明，RGD肽可预防缺血性急性肾衰竭（ARF）中的肾小管阻塞，这表明暴露的未占据整合素受体是此类治疗的靶点。本研究调查了缺血大鼠肾脏中RGD结合位点和整合素受体的拓扑结构。合成了两种RGD肽：一种环化生物素化（Bt）RGD肽和一种用罗丹明绿（RhoG）标记的线性RGD肽（GRGDSP）。对大鼠进行45分钟的肾动脉闭塞，在缺血后不同时间采集肾脏，并用RGD肽和一组整合素抗体进行染色。在对照中，碱性磷酸酶组织化学检测不到Bt-RGD染色，而用Rho-链霉亲和素偶联物进行的免疫荧光检测以及RhoG-GRGDSP染色以点状方式微弱地修饰了近端肾小管细胞的基底外侧。相比之下，缺血性肾脏显示与近端小管的基底外侧和顶端、肾小管周围毛细血管以及管腔内的脱落细胞有结合。缺血性肾脏最明显的染色是用针对β1（标记近端和远端小管的顶端以及阻塞管腔的脱落细胞）和αV（肾小球、肾小管上皮、血管内膜微弱染色，但阻塞性细胞团显示强烈染色）亚基的抗体获得的。用Bt-RGD与针对β1和αVβ3整合素的抗体进行双重染色，分别显示在肾小管和脉管系统内染色共定位。环化RGD肽可抑制HL-60白细胞在体外与内皮细胞的黏附。总之，沿着肾小管上皮顶端以及脱落细胞表面的RGD结合位点和β1整合素亚基的表达与关于识别RGD的整合素在肾小管阻塞中的致病作用的假说一致。缺血性肾脏血管内膜表面RGD结合位点的表达表明RGD肽在血管内皮细胞中还有一个额外的靶点。
