缺氧誘導因子對於腎臟水份調節的影響

Abstract

增加缺氧誘導因子(HIF)表現可以通過腎臟血管周細胞產生促紅血球生成素來改善貧血；然而在不同類型的癌症，例如透明細胞腎細胞癌，會看到過度表達。先前研究表明，小鼠腎臟中表達Hoxb7 的腎集尿管上皮細胞若發生von Hippel-Lindau (Vhlh) 基因缺失會出現缺氧誘導因子-1依賴性增生、炎症和纖維化病變。因此研究HIF的調控與釐清其在腎臟病生理的角色至關重要。先前同仁用Tg(Hoxb7-cre);VhlhF/F品系的基因改造小鼠研究透明細胞腎細胞癌時意外發現，Vhlh被剔除的小鼠有多尿等現象。目前已知Vhlh剔除和HIF的大量表達有關。因此，想進一步了解HIF和多尿現象的關係和其他病生理影響。HIF依結構主要可分為 HIF-1和HIF-2兩種蛋白，先前研究指出HIF-1主要和代謝上的調控以及血管新生有關；而HIF-2主要調控紅血球生成素生成，也有許多研究表示其可調節血管生成的多個方面，包括細胞增殖…等。為了解哪一種HIF過度表現造成Vhlh KO小鼠多尿等現象，配種出Tg(Hoxb7-Cre);VhlhF/F;Hif1aF/F (Vhlh;Hif1a DKO和Tg(Hoxb7-Cre);VhlhF/F;Hif2aF/F (Vhlh;Hif2a DKO)，同時剔除集尿管上皮細胞的Vhlh與Hif基因的兩種基因改造小鼠進行後續實驗。先測定24-h給水的情況收集小鼠尿液，觀察到相較同窩對照組的VhlhF/F;Hif2aF/F 和VhlhF/+;Hif2aF/F 小鼠，Vhlh;Hif2a DKO小鼠的尿量仍有較多的現象；然而Vhlh;Hif1a DKO小鼠的多尿現象卻消失了。因臨床上多尿可分為水、溶質利尿兩種，進一步檢測小鼠尿液中離子和滲透克分子，發現對照組小鼠與Vhlh;Hif2a DKO小鼠的24-h尿液離子與滲透克分子排出並無顯著差異，因此推測Vhlh;Hif2a DKO小鼠可能只有水利尿或腎臟水分再吸收能力下降，而無溶質利尿的現象。進一步實驗顯示對照組小鼠與Vhlh;Hif2a DKO小鼠都呈負數的自由水廓清率，表示兩種小鼠腎臟仍都保有水分再吸收的能力，但Vhlh;Hif2a DKO小鼠再吸收水分能力卻不及對照組小鼠。為了鑑別Vhlh;Hif2a DKO小鼠是否因喝多才導致的多尿，接著進行24-h禁水實驗，觀察到Vhlh;Hif2a DKO小鼠的尿量降低和尿液滲透壓升高皆與禁水前有顯著差異，且尿液滲透壓都高於血液滲透壓，但對照組小鼠在禁水前後雖尿液滲透壓有顯著升高，尿量降低並無統計差異。Vhlh;Hif2a DKO小鼠在禁水前後尿量降低與尿液滲透壓升高皆較對照組小鼠變化明顯，顯示Vhlh;Hif2a DKO小鼠保有抗利尿激素-第二型水通道訊息相關的尿液濃縮能力，可在禁水時將水分再吸收回體內，且相關的尿量降低與尿滲透壓升高比對照組更加明顯，暗示多喝水是Vhlh;Hif2a DKO小鼠呈現多尿與尿液滲透壓較低的原因之一。接著測血漿中抗利尿激素(ADH)的濃度與腎髓質集尿管上第二型水通道蛋白(AQP2)的表現量。ADH結果顯示在對照組小鼠和Vhlh;Hif2a DKO小鼠間並無顯著差異，而AQP2的表現在禁水後Vhlh;Hif2a DKO小鼠比對照組小鼠高。這些發現可呼應Vhlh;Hif2a DKO小鼠在禁水前後的尿量降低與尿液滲透壓升高皆較對照組小鼠的變化明顯。然而，因為觀察到禁水後的Vhlh;Hif2a DKO小鼠仍比對照組小鼠有顯著較多的尿量與較低的尿液滲透壓，因此我們懷疑腎臟組織可能影響水分再吸收的變化。經分析腎臟病理組織，發現Vhlh;Hif2a DKO小鼠的腎小管間質組織異常，推測Vhlh;Hif2a DKO小鼠的腎臟可能因損傷而降低間質滲透壓梯度，而導致尿液濃縮上限比對照組小鼠低。然而這推論尚須直接證實其腎臟間質的滲透壓梯度真的較低。相對的，Vhlh;Hif1a DKO小鼠排出的24小時尿量、尿液滲透壓、與尿液總滲透克分子在禁水前都與對照組(VhlhF/F;Hif1aF/F 和VhlhF/+;Hif1aF/F)小鼠相同。進一步實驗顯示對照組小鼠與Vhlh;Hif1a DKO小鼠都呈負數的自由水廓清率，表腎臟仍都有將水分再吸收回來的能力，且Vhlh;Hif1a DKO小鼠的自由水再吸收能力高過對照組小鼠，但滲透克分子廓清率則無差別。接著進行24-h禁水的實驗，可觀察到對照組小鼠的尿量降低，尿液滲透壓升高。雖對照組與Vhlh;Hif1a DKO小鼠在禁水前後的尿液滲透壓都高於血液滲透壓，且對照組與Vhlh;Hif1a DKO小鼠的尿量降低與尿液滲透壓升高的比例皆無差異，但Vhlh;Hif1a DKO小鼠的尿量降低和尿液滲透壓升高都無法像對照組達到統計差異。另外，雖禁水前，24小時尿液總滲透克分子在對照組與Vhlh;Hif1a DKO小鼠無差異，但禁水後卻發現Vhlh;Hif1a DKO小鼠有較高的尿液總滲透克分子排泄。Vhlh;Hif1a DKO小鼠的腎臟也表現出腎小管間質損傷，且血管內皮相關的基因表現增加。另外，從過去的研究中發現Vhlh KO小鼠，體重相較於對照組小鼠輕、且身體較瘦小，但其飲水飲食量都較多，造成Vhlh KO小鼠的多尿現象，推測部分原因可能與能量代謝異常有關。於是同仁有進一步做小鼠養分利用上的實驗，結果顯示Vhlh KO小鼠產生較多熱量、能量利用來源傾向於醣類以及較高的氧氣消耗量，暗示Vhlh KO小鼠可能因為活動力較高而導致易餓所以吃多。後續我們測其自發活動狀態，顯示Vhlh KO小鼠活動量並無較高；而本研究也觀察到Vhlh;Hif2a DKO小鼠的體重相較於對照組輕，進一步檢測其飲水飲食量，結果顯示Vhlh;Hif2a DKO小鼠有喝多但無吃多現象，檢測其新陳代謝率及體組成，並未發現Vhlh;Hif2a DKO小鼠和Vhlh KO 有差異。Vhlh;Hif1a DKO小鼠的體重則與對照組無差別。總結，相較於腎臟集尿管Vhlh KO的小鼠，Vhlh;Hif2a DKO的小鼠並未表現溶質利尿現象，但持續表現多尿和體重降低；Vhlh;Hif2a DKO小鼠的腎小管間質組織異常可能會降低間質滲透壓梯度，而導致尿液濃縮上限比對照組小鼠低，而引起多尿。但禁水實驗發現Vhlh;Hif2a DKO小鼠能比對照組小鼠更高比例降低尿量與升高尿液滲透壓，可能暗示多喝水是造成其多尿的重要原因之一。養分利用的分析顯示Vhlh;Hif2a DKO小鼠並未像Vhlh KO小鼠會產生較多熱量、能量利用來源傾向於醣類以及較高氧氣消耗量，且多吃現象也消失。目前仍無法確定Vhlh;Hif2a DKO小鼠為何仍比對照組的體重輕，只能推測是多尿引起。而Vhlh;Hif1a DKO的小鼠並未表現多尿和體重降低的情況，但Vhlh;Hif1a DKO的小鼠仍出現腎小管間質組織的異常，為何不如Vhlh;Hif2a DKO小鼠引起多尿則需更多研究加以釐清。

Hypoxia-inducible factor (HIF) is one of important cellular responders when facing decreased oxygen tension or under hypoxia. HIF stabilization can improve anemia through erythropoietin production from kidneys; however, its overexpression could be seen in different cancer types, such as clear cell renal cell carcinoma (ccRCC). Previous studies have shown wide-spread HIF-1-dependent hyperplastic, inflammatory and fibrotic lesions in the kidney of mice with von Hippel-Lindau gene (Vhlh) deletion in Hoxb7-expressing renal collecting duct (CD) epithelia. Therefore, it is important to investigate the regulation of HIF and its mechanism. Previously, the colleagues of lab used the of the Tg(Hoxb7-cre);VhlhF/F (Vhlh KO) to investigate ccRCC and unexpectedly found that Vhlh KO mice exhibited higher food intake and osmotic diuresis, but decreased body weight. It has been well-established that Vhlh KO is related to the stable or massive expression of HIF. Therefore, we want to further understand the relationship between HIF and polyuria, as well as other pathophysiological influences. Hifs can be divided into HIF-1 and HIF-2. HIF-1 is primarily associated with metabolic pathway and angiogenesis; while HIF-2 has also been proved to regulate multiple aspects of angiogenesis, including cell proliferation…etc. To further investigate, We bred two strains of mice, Tg(Hoxb7-Cre);VhlhF/F;Hif1aF/F(Vhlh;Hif1aDKO) and Tg(Hoxb7-Cre);VhlhF/F;Hif2aF/F(Vhlh;Hif2a DKO) mice. First, mice were put in metabolism cage with 24 hr water ad libitum for collecting the urine. Compared to littermate control VhlhF/F;Hif2aF/F and VhlhF/+;Hif2aF/F mice, we found that Vhlh;Hif2a DKO mice had more urine excretion, while Vhlh;Hif1a DKO had no more polyuria. There are two kinds of polyuria in clinical, water and osmotic polyuria, respectively. We measured ions and osmoles in urine. The results showed that Vhlh;Hif2a DKO mice still showed polyuria but without osmotic diuresis. From the result of free water clearance, suggesting that Vhlh;Hif2a DKO mice kidney had the ability of water reabsorption, but worse than control mice. To identify the reason whether Vhlh;Hif2a DKO drink more water to cause polyuria, next we conducted the experiment of 24 hr water deprivation.We found that Vhlh;Hif2a DKO mice had lower urine amount and higher urine osmolality compared with the condition of water ad libitum . The change of urine osmolality in Vhlh;Hif2a DKO mice is higher than control mice with or without water deprivation, suggesting that both control and Vhlh;Hif2a DKO mice have urine concentration ability related to vasopressin-AQP2 axis. To figure out this problem, we measured the concentration of ADH in plasma and the expression of AQP2 on the collecting duct of renal medulla. The results showed ADH was no difference but the expression of AQP2 was higher in Vhlh;Hif2a DKO mice after water deprivation. Due to these results, We proposed that Vhlh;Hif2a DKO mice kidney may have other damage and lead to poor water absorption. In contrast, Vhlh;Hif1a DKO mice 24-h urine amount, osmolality, and daily urine osmole were the same as control mice without water deprivation. In addition, it has been found that Vhlh;Hif1a DKO mice had higher daily urine osmole after water deprivation. Besides, Vhlh;Hif1a DKO mice also exhibited tubulointerstitial damage and increased expression of vascular endothelium-related genes. It has been found that Vhlh KO mice were thinner, but they drank and ate a lot. We proposed that there may be a problem with energy metabolism, and results showed that Vhlh KO mice produced more heat, but the locomotor activity is no difference than control mice. We found that Vhlh;Hif2a DKO mice was lighter than control mice, and they drank but not ate a lot. The metabolic rate and body composition showed there was no difference between Vhlh;Hif2a DKO mice and control mice. The body weight of Vhlh;Hif1a DKO mice was no different than control mice. To sum up, Compared to Vhlh KO mice, KO Vhlh and Hif2a simultaneously, osmotic diuresis disappeared but still showed polyuria and lower body weight. Abnormal tubulointerstitial organization in Vhlh;Hif2a DKO mice may reduce the interstitial osmotic pressure gradient, resulting in polyuria. While KO Vhlh and Hif1a simultaneously, polyuria disappeared. However, the reasons need to be clarified by more research.