Increased renal angiopoietin-1 expression in folic acid-induced nephrotoxicity in mice

Growth factors affect epithelial regeneration after acute renal injury, but less is known regarding the expression of vascular growth factors in this setting. A mouse model of folic acid (FA)-induced nephrotoxicity was used to study the expression of angiopoietins (Ang), factors that bind the Tie-2 receptor and modulate endothelial growth. Tubular damage was detected 1 d after FA administration; in the next 14 d, most tubules regenerated but patchy atrophy, with interstitial fibrosis, was also observed. Ang-1 immunostaining was detected between cortical tubules and in the vasa rectae of vehicle-treated animals. FA-induced nephropathy was associated with the acquisition of Ang-1 immunostaining in renal arterial walls and in a subset of injured cortical tubules that failed to stain with periodic acid-Schiff stain, which indicated that they were distal tubules. Renal Ang-1 protein levels were significantly increased after FA administration, compared with time-matched control values, as assessed by Western blotting. Capillaries between regenerating tubules expressed both Tie-2 and platelet-endothelial cell adhesion molecule. A subset of these endothelia expressed proliferating cell nuclear antigen, whereas capillary proliferation was absent in control samples. Therefore, FA-induced nephropathy is associated with increased Ang-1 protein expression in renal epithelia and arteries. In addition, Tie-2-expressing capillaries near damaged cortical tubules undergo proliferation. Further experiments are required to establish whether these events are functionally related.     

Angiopoietin-2 is a site-specific factor in differentiation of mouse renal vasculature

Angiopoietin-1 (Ang-1) stimulates endothelial and vascular network differentiation through the Tie-2 receptor tyrosine kinase, while Ang-2 modulates this activation in embryo and tumor growth. The nephrogenic pattern of Ang-2 was documented in a mouse strain that expresses the LacZ reporter gene driven by the Ang-2 promoter. Heterozygous animals were healthy with morphologically normal kidneys, and they were examined after X-gal staining. At embryonic days 10.5 (E10.5) and E12.0, transgene expression was absent in the mesonephros and metanephros. At E14.0, expression was noted in the metanephric artery and its major branches. At E19.0 and in neonatal kidneys, expression was maintained in larger renal artery branches, extending to arcuate and smaller cortical vessels. Histologically, transgene expression was located in multiple layers of vessel wall cells, extending further from the endothelium than alpha-smooth muscle actin. The mesangium of immature glomeruli also expressed LacZ. In the first 3 postnatal weeks, a new pattern became evident, with intense X-gal staining in the inner stripe of the outer medulla, where a subset of thin descending limbs of loops of Henle expressed the transgene. This dynamic and developmentally regulated pattern indicates that Ang-2 is an early marker of the renal pericyte and vascular smooth muscle lineage and is also an epithelial-derived growth factor. Because Tie-2 is widely expressed by differentiating renal endothelia, this study is consistent with the hypothesis that Ang-2 has roles in kidney vascular maturation.     

Angiopoietin growth factors and Tie receptor tyrosine kinases in renal vascular development

Angiopoietin-1 (Ang-1) is a secreted growth factor which binds to and activates the Tie-2 receptor tyrosine kinase. The factor enhances endothelial cell survival and capillary morphogenesis, and also limits capillary permeability. Ang-2 binds the same receptor but fails to activate it: hence, it is a natural inhibitor of Ang-1. Ang-2 destabilises capillary integrity, facilitating sprouting when ambient vascular endothelial growth factor (VEGF) levels are high, but causing vessel regression when VEGF levels are low. Tie-1 is a Tie-2 homologue but its ligands are unknown. Angiopoietin and Tie genes are expressed in the mammalian metanephros, the precursor of the adult kidney, where they may play a role in endothelial precursor growth. Tie-1-expressing cells can be detected in the metanephros when it first forms and, based on transplantation experiments, these precursors contribute to the generation of glomerular capillaries. During glomerular maturation, podocyte-derived Ang-1 and mesangial-cell-derived Ang-2 may affect growth of nascent capillaries. After birth, vasa rectae acquire their mature configuration and Ang-2 expressed by descending limbs of loops of Henle would be well placed to affect the growth of this medullary microcirculation. Finally, preliminary data implicate angiopoietins in deregulated vessel growth in Wilms’ kidney tumours and in vascular remodelling after nephrotoxicity. Received: 13 July 2000 / Revised: 6 September 2000 / Accepted: 11 September 2000     

Podocyte-specific expression of angiopoietin-2 causes proteinuria and apoptosis of glomerular endothelia

Angiopoietin-2 (Ang-2) modulates embryonic vascular differentiation primarily by inhibiting the antiapoptotic effects of Ang-1 on endothelia that express the Tie-2 receptor. Ang-2 is transiently expressed by developing glomeruli but is downregulated with normal maturation. Glomerular Ang-2 expression is, however, markedly upregulated in animal models of diabetic nephropathy and glomerulonephritis, both leading causes of human chronic renal disease, affecting 10% of the world population. It was hypothesized that Ang-2 might have significant roles in the pathobiology of glomerular disease. Mice with inducible podocyte-specific Ang-2 overexpression were generated. When the transgene was induced in adults for up to 10 wk, mice had significant increases in both albuminuria and glomerular endothelial apoptosis, with significant decreases of both vascular endothelial growth factor-A and nephrin proteins, critical for maintenance of glomerular endothelia and filtration barrier functional integrity, respectively. There was, however, no significant change of systemic BP, creatinine clearance, or markers of renal fibrosis, and podocytes appeared structurally intact. In kidneys of young animals in which Ang-2 had been upregulated during organogenesis, increased apoptosis occurred in just-formed glomeruli. In vitro, short-term exposure of isolated wild-type murine glomeruli to exogenous Ang-2 led to decreased levels of vascular endothelial growth factor-A protein. These novel results provide insight into molecular mechanisms underlying proteinuric disorders, highlight potentially complex interactions between subsets of glomerular cells, and emphasize how a vascular growth factor that has critical roles in normal development may be harmful when re-expressed in the context of adult disease.     

High resolution localization of angiotensin II receptors in rat renal medulla.

The cellular localization of angiotensin II (Ang II) receptors in the inner stripe of the outer medulla of the rat kidney was investigated by using high resolution light and electron microscopic autoradiography. Fresh tissue blocks from the inner stripe of the outer medulla were incubated with 125I-[Sar1, Ile8] Ang II and prepared for microscopic autoradiography. At the light microscopic level, 125I-[Sar1, Ile8] Ang II was found to penetrate into the tissue and to bind specifically to sites outlining renal tubules and vasa recta bundles. Electron microscopic autoradiography revealed that silver grains were detected over interstitial cells located between the tubules and components of the vasa recta bundles, but no silver grains were detected overlying the cells of the thin descending or thick ascending limbs of the loop of Henle, the collecting ducts, the vasa recta, or other blood vessels. These interstitial cells contained abundant endoplasmic reticulum, microfilaments, occasional lipid droplets and extensive cytoplasmic processes which closely related to the basement membranes of the vasa recta and loops of Henle. The cells therefore closely resemble type 1 interstitial cells. Since Ang II binding sites are absent in the inner medulla, the cells labelled by this technique must be a subset of type 1 interstitial cells, distinct from the typical lipid-laden interstitial cells most abundant in the inner medulla. These findings demonstrate that type 1 interstitial cells are the primary sites for a high density of Ang II receptors located in the inner stripe of the outer medulla.     

Determinants of intrarenal oxygenation: factors in acute renal failure.

Oxygen tension within the renal parenchyma is influenced by two factors: metabolic demand and oxygen supply. There are three regions within the kidney in which there is an anatomical basis for limited oxygen availability. The first is the inner stripe where oxygen diffusion between arterial and venous vasa recta reduces PO2. The other two are the outer stripe and medullary rays which are fed by O2-poor blood from venous vasa recta. The balance between oxygen demand and supply is most critical in the inner stripe where the PO2 is most influenced by transport activity. In contrast, altering transport activities in the outer stripe will not change the prevalence of hypoxic S3 injury but will alter its type (i.e., cell fragmentation related to high GFR and increased workload versus cell edema related to low GFR and minimal workload). The effect of transport activity on medullary ray PO2 has not been well defined. Using sensitive oxygen microelectrodes, cortical PO2 (52 +/- 2 mm Hg) in the rat was found to be higher than medullary PO2 (21 +/- 2 mm Hg, p less than 0.001). How are these observations reflected in current models of acute renal failure? The ischemia-reflow model affects proximal tubules with a predilection for S3 (located within the outer stripe of medulla) after short-term ischemia. With hyperfiltration (induced by glycine or renal hypertrophy) and the pursuant increase in transport related O2 demand, hypoxic mTAL inner stripe injury becomes prominent. Renal parenchymal hypertrophy exaggerates injury in the contrast nephropathy model, in which mTAL inner stripe injury is a predominant feature and medullary PO2 is very low.(ABSTRACT TRUNCATED AT 250 WORDS)     

Angiopoietin correlates with glomerular capillary loss in anti-glomerular basement membrane glomerulonephritis

BACKGROUND: Emerging evidence suggests that endothelial turnover occurs in several glomerular diseases and correlates with resolution or progression of glomerular lesions. We hypothesized that the growth factors modulating embryonic kidney endothelial cell survival and capillary morphogenesis may be implicated in capillary loss that occurs in immune-mediated glomerulonephritis (GN). METHODS: GN was induced in C57BL/6 mice by intravenous administration of sheep anti-mouse glomerular basement membrane (GBM) globulin and assessed with markers of vascularity in glomerular lesions, correlating these with expression of specific vascular growth factors. RESULTS: As assessed by periodic acid Schiff staining, 14 +/- 4% (mean +/- SD) glomeruli were affected by sclerosis at 14 days after globulin administration, and 33 +/- 5% were affected at 21 days. By 21 days, a significant increase of plasma creatinine and urinary protein occurred. P-selectin expression was increased in glomerular capillaries 14 days after disease induction, and capillary loss, as assessed by immunohistochemistry for platelet-endothelial cell adhesion molecule, vascular endothelial growth factor (VEGF) receptor 2 and the angiopoietin (Ang) receptor Tie-2, was recorded at 14 and 21 days in glomeruli affected by proliferative crescents and/or sclerosis. VEGF-A immunostaining, evident in control glomeruli, was qualitatively diminished in glomeruli with lesions. Ang-1 immunostaining was detected in control glomeruli and was diminished at 14 days after administration of anti-mouse GBM globulin; instead, Ang-1 was immunolocalized to distal tubules. In contrast, Ang-2 immunostaining was barely detectable in control glomeruli but was prominent in disease glomeruli. In GN mice, rare apoptotic glomerular endothelia were detected by electron microscopy and in situ end-labeling, but such cells were not seen in controls. CONCLUSIONS: Loss of glomerular capillaries during the course of anti-GBM GN in mice was temporally associated with decreases in endothelial survival molecules VEGF-A and Ang-1, and with up-regulation of Ang-2, an antagonist of Ang-1. A changing balance of these growth factors may contribute to decreased glomerular vascularity in crescentic GN.     

Pericyte regulation of renal medullary blood flow

Pericytes are contractile smooth muscle-like cells that surround descending vasa recta (DVR) and provide their capability for vasomotion. The importance of the medullary pericyte derives from the role of DVR to distribute most or all of the blood flow from juxtamedullary cortex to the renal inner and outer medulla. Physiological processes that are likely to be influenced by pericyte constriction of DVR include the urinary concentrating mechanism and pressure natriuresis. Oxygen tensions in the medulla are low, so that subtle variation of pericyte vasomotion might play a role to abrogate hypoxia and prevent insult to the medullary thick ascending limb of Henle. Known vasoconstrictors of DVR include angiotensin II, endothelins, norepinephrine, acetylcholine, and adenosine. Vasodilators include prostaglandin E2, adenosine, acetylcholine, bradykinin, and nitric oxide.     

Roles of angiopoietins in kidney development and disease

Angiopoietins are a family of growth factors, the best studied being angiopoietin 1 (Ang-1), which binds to and tyrosine-phosphorylates endothelial Tie-2, causing enhanced survival and cell-cell stabilization. Ang-2 and Tie-1 downregulate Ang-1-induced Tie-2 signaling, and angiopoietin actions are further modified by vascular endothelial growth factor A and integrins. Metanephric capillaries express Tie genes, whereas metanephric mesenchyme, maturing tubules, and mature podocytes express Ang-1. Ang-1 null embryos begin to form blood vessels, but subsequent vascular remodeling fails, and analyses of chimeric wild-type/Tie null mutant embryos show that Tie genes are needed for renal endothelial survival. Ang-2 is transiently expressed in renal arterial smooth muscle and mesangial cells, and tubules around adult vasa rectae express Ang-2. Ang-2 null mice have increased pericytes around kidney cortical peritubular capillaries, perhaps an indirect consequence of upregulated Tie-2 signaling. Ang-1 therapies attenuate peritubular capillary loss in adult models of tubulointerstitial disease, although, in one study, this was accompanied by enhanced inflammation and fibrosis. Podocyte-directed Ang-2 transgenic overexpression causes glomerular endothelial apoptosis, downregulated nephrin expression, and increased albuminuria, and glomerular Ang-2 is upregulated in hyperglycemic and immune-mediated glomerulopathies. Thus, angiopoietins affect podocyte as well as glomerular endothelial biology, and imbalanced angiopoietin signaling contributes to glomerular pathobiology.     

The medullary microcirculation

Like other regional circulations, the medullary circulation supplies oxygen and other primary substrates to the medulla and removes carbon dioxide and other waste metabolites. It also acts as a countercurrent exchanger and simultaneously removes water reabsorbed from the renal tubule to preserve mass balance. Our present understanding of how the medulla serves both these functions at the same time is illustrated in Figure 3. Blood leaves the efferent arteriole with an elevated plasma protein concentration as a consequence of glomerular filtration, and flows down descending vasa recta within a vascular bundle. The increased interstitial osmotic-concentration coupled with a finite capillary reflection coefficient for small solutes causes additional water to be extracted so that at the termination of descending vasa recta, the plasma protein concentration exceeds that in the systemic circulation by approximately twofold. Solute, urea more than sodium chloride, also enters descending vasa recta. As blood flows through the interconnecting capillary plexus and up ascending vasa recta, transcapillary oncotic and osmotic pressure differences combine to cause capillary uptake of fluid. There is also simultaneous loss of urea such that the medullary trapping of urea is very effective. Countercurrent exchange of sodium chloride, however, appears to be less efficient and as a consequence, not only water but sodium chloride is removed from the medulla. Antidiuretic hormone reduces medullary blood flow, both directly by its vasoconstrictor (V1-receptor mediated) effect and indirectly by its antidiuretic (V2-receptor mediated) effects. Prostaglandins are able to enhance medullary blood flow by counteracting vasoconstrictive influences.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cycloxygenase-2 is expressed in vasculature of normal and ischemic adult human kidney and is colocalized with vascular prostaglandin E2 EP4 receptors

The study was performed to elucidate the distribution and cellular localization of cyclooxygenase (COX)-2 in human kidney and to address localization of downstream targets for COX-derived prostanoids. Cortex and outer and inner medulla tissue were obtained from control kidneys (cancer specimens), kidneys with arterial stenosis, and kidneys of patients who received angiotensin II inhibition or acetylsalicylic acid. Ribonuclease protection assay and Western blot test revealed that COX-1 and -2 mRNA and protein were expressed in all regions of human kidney (mRNA ratio, cortex:outer medulla:inner medulla COX-1 1:3:20 and COX-2 1:1:3). In adult kidney, immunohistochemical labeling for COX-2 was associated with smooth muscle cells in pre- and postglomerular vessels and with endothelium, particularly in vasa recta and medullary capillaries. Western blot test confirmed COX-2 expression in renal artery. COX-2 had a similar localization in fetal kidney and was additionally observed in Henle's loop and macula densa. Human tissue arrays displayed COX-2 labeling of vascular smooth muscle in multiple extrarenal tissues. Vascular COX-2 expression was significantly increased in kidneys with arterial stenosis. COX-1 was colocalized with microsomal prostaglandin E(2) synthase (PGES) in collecting ducts, and PGES was also detected in macula densa cells. Vascular COX-2 was colocalized with prostaglandin E(2) EP4 receptors but not with EP2 receptors. Thus, renovascular COX-2 expression was a constitutive feature encountered in human kidneys at all ages, whereas COX-2 was seen in macula densa only in fetal kidney. Vascular COX-2 activity in human kidney and extrarenal tissues may support blood flow and affect vascular wall-blood interaction.     

血管生成素及其受体、血管内皮生长因子与糖尿病肾脏微血管病变的关系

目的 探讨血管生成素(Ang)及其受体(Tie-2)和血管内皮生长因子(VEGF)在糖尿病肾脏中的表达变化规律，研究其与肾脏微血管结构变化的关系。 方法 将雄性成年SD大鼠分成糖尿病组和对照组，糖尿病组采用链脲佐菌素(STZ)腹腔注射造模。采用RT-PCR以及免疫组化技术，连续多时点观察肾脏Ang-1、Ang-2、Tie-2、VEGF以及血栓调节蛋白(TM-1) mRNA和蛋白表达变化规律，并分析其相关性。 结果 (1)糖尿病组Ang-1 mRNA于第4、8周时显著上调(吸光度A，83.58±10.23、88.59±6.97)， 第24周时低于对照组 (47.13±8.02比64.53±8.77，P < 0.05)。免疫组化显示Ang-1突出表达于肾小球，糖尿病组第4周后肾小球阳性染色明显强于对照组(A对数值，4周1.64±0.12比1.08±0.16，24周1.24±0.11比1.11±0.17)。(2) 糖尿病组Tie-2 mRNA在4~16周显著高于对照组(A，4周87.31±11.69比63.62±5.61，16周81.75±8.58比60.15±2.66)。免疫组化显示Tie-2突出表达于肾小球，糖尿病组各时点均显著高于对照组，高峰在4~8周。(3)糖尿病组仅在第16和20周时检测到明显的Ang-2 mRNA表达。免疫组化显示12周后仅在皮质区肾小管周围有Ang-2染色的微血管，而在第16周时最明显。(4) 糖尿病组2~20周时肾小球TM-1染色显著高于对照组(A对数值，2周0.99±0.15比0.68±0.17，20周1.03±0.17比0.74±0.13)， 第24周时低于对照组，但差异无统计学意义。(5)糖尿病组VEGF mRNA和蛋白表达均较对照组明显升高。(6) 糖尿病组Ang-1、Tie-2、VEGF和TM-1相互间均呈正相关，它们与尿蛋白、肾重/体重、肾小球体积、肾小球面积也均呈正相关。结论 (1)糖尿病肾脏存在VEGF、Ang及Tie-2表达的改变，早期Ang-1、Tie-2表达上调， 后期下调并伴Ang-2表达上调。(2)Ang、Tie及VEGF的改变与糖尿病肾脏新生血管生成有关，其Ang-1下调和VEGF、Ang-2上调起重要作用。(3)糖尿病肾脏中后期皮质区肾小管周围已有Ang-2染色的新生微血管生成。     

反转录-聚合酶链式反应检测促血管生成素-1和促血管生成素-2及Tie-2受体的mRNA在血管瘤组织中的表达及其意义

目的研究血管瘤组织中促血管生成素-1（Ang-1）、促血管生成素-2（Ang-2）及Tie-2受体的mRNA表达水平,探讨其在血管瘤血管生成中的作用与意义。方法用反转录-聚合酶链式反应（RT—PCR）的方法分别检测30例血管瘤组织（增生期血管瘤17例,消退期血管瘤13例）以及10例小儿正常皮肤组织中Ang-1mRNA、Ang-2mRNA及Tie-2mRNA的表达水平。用免疫组织化学方法检测血管瘤组织Ki-67蛋白的表达。结果增生期血管瘤组织中Ang-2mRNA及Tie-2mRNA表达水平高于消退期血管瘤组织（P〈0．01）。血管瘤组织中Ang-1mRNA表达为阴性。结论Ang—Tie-2体系表达失衡在血管瘤的发生发展过程中可能起重要的作用。     

P-selectin-dependent macrophage migration into the tubulointerstitium in unilateral ureteral obstruction

BACKGROUND: Interstitial infiltration of macrophages (M?) is one of the main causal factors for the tubulointerstitial injury. However, precise mechanisms of M? infiltration into tubulointerstitium have not been fully explored. The purposes of this study were to assess the role of selectins in the acute infiltration of M? in rats with unilateral ureteral obstruction (UUO) and to evaluate the role of vasa recta, that is, whether they facilitate massive influx of M? into the interstitium by functioning as specialized vessels. METHODS: To evaluate the role of selectins in M? infiltration into tubulointerstitium, the expression of selectins and L-selectin ligands was examined by immunohistochemistry and immunoelectron microscopy. The functional role of P-selectin in vasa recta was studied by Stamper-Woodruff assay, in vivo p-M? migration assay and in vivo blocking experiments with the monoclonal antibody (mAb) ARP2-4. RESULTS: Selective expression of P-selectin was detected in vasa recta as early as one hour after UUO, and the expression increased thereafter for 96 hours. In contrast, endothelial expression of L-selectin ligands and E-selectin were not detectable. In the Stamper-Woodruff assay on kidney sections of rats with UUO, the adhesion of isolated rat peritoneal M? (p-M?) to vasa recta was significantly inhibited by the mAb ARP2-4 (P-selectin blocker; P < 0.01), but not by mAb ARE-5 (E-selectin blocker) or rLECIg (rat L-selectin chimeric protein). In the in vivo transfer experiments with fluorescein-labeled p-M? into rats 48 hours after UUO, labeled p-M? had accumulated around vasa recta at three minutes and had infiltrated predominantly into the outer medulla at 180 minutes. The number of labeled p-M? was reduced when the rats were pretreated with ARP2-4 (P < 0.01). Finally, ARP2-4 (10 mg/kg), injected 15 minutes before UUO, reduced the number of infiltrated M? (P < 0.01). CONCLUSION: The results suggest that vasa recta, which express P-selectin, contribute to massive infiltration of M? into the interstitium by functioning as specialized post-capillary venules.     

Urea and renal function in the 21st century: insights from knockout mice

Since the turn of the 21st century, gene knockout mice have been created for all major urea transporters that are expressed in the kidney: the collecting duct urea transporters UT-A1 and UT-A3, the descending thin limb isoform UT-A2, and the descending vasa recta isoform UT-B. This article discusses the new insights that the results from studies in these mice have produced in the understanding of the role of urea in the urinary concentrating mechanism and kidney function. Following is a summary of the major findings: (1) Urea accumulation in the inner medullary interstitium depends on rapid transport of urea from the inner medullary collecting duct (IMCD) lumen via UT-A1 and/or UT-A3; (2) as proposed by Robert Berliner and colleagues in the 1950s, the role of IMCD urea transporters in water conservation is to prevent a urea-induced osmotic diuresis; (3) the absence of IMCD urea transport does not prevent the concentration of NaCl in the inner medulla, contrary to what would be predicted from the passive countercurrent multiplier mechanism in the form proposed by Kokko and Rector and Stephenson; (4) deletion of UT-B (vasa recta isoform) has a much greater effect on urinary concentration than deletion of UT-A2 (descending limb isoform), suggesting that the recycling of urea between the vasa recta and the renal tubules quantitatively is less important than classic countercurrent exchange; and (5) urea reabsorption from the IMCD and the process of urea recycling are not important elements of the mechanism of protein-induced increases in GFR. In addition, the clinical relevance of these studies is discussed, and it is suggested that inhibitors that specifically target collecting duct urea transporters have the potential for clinical use as potassium-sparing diuretics that function by creation of urea-dependent osmotic diuresis.     

Urea transporters and renal function: lessons from knockout mice

PURPOSE OF REVIEW: Gene knockout mice have been created for the collecting duct urea transporters UT-A1 and UT-A3, the descending thin-limb urea transporter UT-A2 and the descending vasa recta isoform, UT-B. In this brief review, the new insights in our understanding of the role of urea in the urinary concentrating mechanism and kidney function resulting from studies in these mice are discussed. RECENT FINDINGS: The major findings in studies on urea transporter knockout mice are as follows: rapid transport of urea from the inner medulla collecting duct lumen via UT-A1 or UT-A3 is essential for urea accumulation in the inner medullary interstitium; inner medulla collecting duct urea transporters are essential in water conservation by preventing urea-induced osmotic diuresis; an absence of inner medulla collecting duct urea transport does not prevent the concentration of sodium chloride in the inner medulla interstitium; deletion of the vasa recta isoform UT-B has a much greater effect on urinary concentration than deleting the descending limb isoform UT-A2. SUMMARY: Multiple urea transport mechanisms within the kidney are essential for producing maximally concentrated urine.