臺灣博碩士論文加值系統

腎臟為富含粒線體的器官，負責過濾廢棄物、酸鹼平衡及荷爾蒙分泌。文獻指出失去功能的粒線體與腎臟疾病相關，此外，粒線體內鈣離子的平衡對訊息傳導非常重要且能調節粒線體的功能、細胞功能及存活。然而，粒線體內鈣離子的平衡如何調節腎臟的功能仍然是未知的。粒線體鈣攝取蛋白1 (MICU1) 作為守門員負責避免過多的鈣離子進入粒線體基質中，粒線體鈣單向轉運體 (MCU) 也需要透過它微調粒線體內鈣離子的平衡。但是，仍然沒有文獻報導有關MICU1如何參與腎臟功能的資訊。因此，我們假設MICU1這個作為粒線體內鈣離子的守門員在腎臟功能扮演重要的角色。在我們的實驗結果發現MICU1在腎臟表現量高。另外，免疫螢光染色結果看到了CDH16和MICU1都有表現在野生型小鼠腎臟中，而且MICU1高度表達在集尿管和亨利氏管上升枝的厚段。為了瞭解MICU1在腎臟內的重要性，我們將具有MICU1-floxed (〖Micu1〗^(f/f)) 的小鼠與CDH16-Cre小鼠進行配種而得到在專一性在腎臟缺失MICU1基因的小鼠 (kMICU1 KO) 。藉由mRNA和蛋白質分析確認MICU1基因在kMICU1 KO小鼠已經被成功敲除並伴隨著EMRE表現量的劇烈減少。 我們發現kMICU1 KO小鼠體重較輕之外，在腎臟重量也有下降的趨勢。kMICU1 KO小鼠的尿液中白蛋白與葡萄糖的排泄正常，酸鹼平衡的功能也正常，也沒有看到腎臟纖維化、發炎及近端腎小管的表現。藉由代謝籠的測試， kMICU1 KO小鼠展現多尿且尿液低滲透壓的現象，而且計算每日尿液鈉、鉀、氯的總排泄量沒有差異。kMICU1 KO小鼠的血液電解質及其滲透壓數值表現正常，在鈉轉運蛋白的表現量也無差異。為了探討kMICU1 KO小鼠的多尿現象是否因為腎臟在濃縮尿液的功能損傷，我們將小鼠進行禁水實驗(刺激內源性ADH)及外源性ADH類似物。結果表明kMICU1 KO 小鼠仍然保留對禁水實驗及三種ADH類似物(DDAVP, Pitressin, and Glypressin)的反應去濃縮尿液，然而在整個實驗過程中有發現與野生型小鼠比較有較低的尿液濃縮能力。同樣的，我們發現kMICU1 KO小鼠中的多尿病癥不是因為降低內源性循環ADH水平所引起的。除此之外，kMICU1 KO小鼠改變腎髓質的水通道蛋白的表現量，但是在V1AR與V2R 的相關路徑由西方墨點法的結果表明沒有受到改變。有趣的是我們發現腎臟缺失MICU1後造成表達AQP2的細胞萎縮與集尿管的腎小管擴張的現象發生。我們也發現了針對在腎髓質的PC與IC細胞相關標記的表現量下降。電子顯微鏡的影像結果表明了腎臟缺失MICU1後在PC細胞顯著性誘發粒線體自噬作用，以及伴隨著粒線體及其皺褶數量的大量減少。另外，免疫螢光染色結果展現在PC與IC細胞的LC3的表達量增加。統整先前的結論，腎臟缺失MICU1後造成表達AQP2的PC細胞中粒線體的損傷病及其自噬作用的發生，進一步導致水分再吸收的能力受損及多尿。總體來說，我們研究暗示了MICU1在體內與水的調控平衡扮演重要的角色，並提供了腎臟生理學中粒線體鈣的構想。

Kidney, responsible for filtering waste, water homeostasis, pH balance, and hormone secretion, contains a high density of mitochondria. Studies have shown that mitochondrial dysfunction has been linked to renal diseases. Mitochondrial calcium homeostasis is crucial in signal transduction and regulation of mitochondrial function, cell function and survival. However, how mitochondrial calcium regulates renal function is still unclear. Mitochondrial calcium uptake 1 (MICU1), a gatekeeper which prevents mitochondrial calcium overload and modulates mitochondria calcium uniporter (MCU) complex, is vital for mitochondrial calcium homeostasis. However, how MICU1 regulates renal function remains unclear. Here, we hypothesized that mitochondrial calcium homeostasis regulated by MICU1 plays a key role in renal function. Our results showed that MICU1 was highly expressed in the kidney. Furthermore, we used immunofluorescence staining for MICU1 and CDH16 in the kidney of WT mice, and found that MICU1 was stained the highest in collecting duct (CD) and thick ascending limb (TAL). To investigate the functional significance of MICU1 in kidney, we crossed MICU1-floxed (〖Micu1〗^(f/f)) mice with CDH16-Cre mice to generate renal-specific MICU1 knockout (kMICU1 KO) mice. By determination of mRNA and protein levels, we confirmed the generation of kMICU1 KO mice, which were accompanied by a significant decrease of EMRE. kMICU1 KO mice showed decreases in body and kidney weights. kMICU1 KO mice exhibited normal urinary excretion of albumin and glucose and regulation of acid–base balance, as well as normal expression of markers for renal fibrosis, inflammation and proximal tubular injury. By using metabolic cages, kMICU1 KO mice showed increased urine output and decreased urine osmolality, without a significant change in daily excretion of urine sodium, potassium, and chloride. Normal plasma electrolytes and osmolality, as well as normal expression of sodium transporters, were found in kMICU1 KO mice. To explore whether polyuria in kMICU1 KO mice was caused by impaired renal ability to concentrate urine, we challenged the mice with water deprivation (stimulation by endogenous antidiuretic hormone (ADH)) and exogenous ADH analogues. kMICU1 KO mice preserved the response to water deprivation and three ADH analogues (DDAVP, Pitressin, and Glypressin) for concentrating urine, but exhibited a decreased urine concentrating ability throughout the whole experiment procedures. Consistently, we found that polyuria in kMICU1 KO mice was not caused by a reduction in endogenous circulating ADH level. Furthermore, while kMICU1 KO mice altered expression of aquaporins in medulla, V1AR and V2R pathways, revealed by immunoblotting, were not altered in kMICU1 KO mice. Interestingly, we found that loss of MICU1 in kidney resulted in shrinkage of AQP2-expressing cells and tubular dilatation of CD. We also found downregulation of principal cell (PC) and intercalated cell (IC) related markers, particularly in the medulla. Electron microscopy showed that loss of MICU1 in kidney significantly induced mitophagy with decreases in mitochondria and cristae numbers in PCs. Moreover, immunofluorescence staining showed upregulation of LC3 in the PCs and ICs of kMICU1 KO mice. In conclusion, lack of MICU1 in kidney damages mitochondria and causes mitophagy in AQP2-expressing PC cells, resulting in impaired water absorption and polyuria. Together, our study implicates MICU1 in body-water homeostasis and provides a concept of mitochondrial calcium in renal physiology.

Abstract i
Abstract in Chinese iii
誌謝 v
Contents vi
Introduction 1
Kidney biology 1
Urine formation 2
Water homeostasis 2
AQP2 3
Acid-base homeostasis in ICs 4
Renal function related to mitochondria 4
Mitochondrial dysfunction in kidney diseases 5
Mitochondrial homeostasis 6
MCU complex 6
Polyuria related to intracellular calcium homeostasis 7
Hypothesis and Significance 7
Material and methods 9
Animals and treatment 9
Metabolic cage studies 9
Urine and blood measurements 10
Storage of tissue samples 10
Protein extraction 10
Western blotting 10
Tissue RNA extraction 11
Real-time PCR 12
Opal Multiplexed IHC 12
Data analysis 13
Results 14
Expression of MICU1 in mice 14
Generation of renal-specific MICU1 knockout (kMICU1 KO) mice 14
The basic profile of renal function is not altered between WT and kMICU1 KO mice 15
Loss of MICU1 in kidney mice does not induce kidney fibrosis 16
Loss of MICU1 in kidney causes polyuria 16
Urinary electrolytes are diluted but daily excretion is the same 17
Normal plasma electrolytes and osmolality and expression of sodium transporters in kMICU1 KO mice 17
Loss of MICU1 in kidney preserves the response to water deprivation 18
Loss of MICU1 in kidney retains the response to ADH analogue 19
Loss of MICU1 in kidney does not alter endogenous ADH in circulation 20
Loss of MICU1 in kidney does not has a significant impact on V1R and V2R pathways 21
Loss of MICU1 in kidney causes shrinkage of AQP2 positive cells 22
Loss of MICU1 in kidney results in tubular dilatation of CD 23
Loss of MICU1 in kidney downregulates PC and IC related markers, particularly in the medulla 23
Loss of MICU1 in kidney significantly decreases mitochondria number and cristae number, and increases mitophagy in PCs 24
Loss of MICU1 in kidney upregulates LC3 in PCs and ICs 25
Discussion 26
Differential expression of MICU1 in renal tubule 26
MCU complex composition in kidney 27
Mitophagy in kMICU1 KO kidney 28
Other mechanisms involved in regulation of urine concentrating in kMICU1 KO mice 29
Conclusion 31
Reference 32
Tables 38
Table 1. Antibody list 38
Table 2. Primer list 40
Figures 42
Figure 1. Expression and location of MICU1 in WT mice kidney 42
Figure 2. The renal tubule-specific MICU1 knockout mice were generated 43
Figure 3. Kidney function in WT and kMICU1 KO mice 45
Figure 4. Renal fibrosis, inflammation and renal proximal tubule injury markers related mRNA expression in WT and kMICU1 mice 47
Figure 5. Metabolic cage study and urine osmolality in WT and kMICU1 KO mice 48
Figure 6. Urinary electrolytes in WT and kMICU1 KO mice 49
Figure 7. Plasma electrolytes, plasma osmolality and expression of sodium transporters in WT and kMICU1 KO mice 50
Figure 8. Changes in body weight, osmolality and electrolytes of WT and kMICU1 KO mice during 48-hour water deprivation 51
Figure 9. Changes in osmolality and electrolytes of WT and kMICU1 KO mice responds to ADH analogues 52
Figure 10. Endogenous ADH and V1AR/V2R mRNA expression in WT and kMICU1 KO mice 54
Figure 11. Expression of V1AR and V2R and downstream proteins in WT and kMICU1 KO mice 55
57
Figure 12. AQP2 and V-ATPase expression in kidney of WT and kMICU1 KO mice 58
Figure 13. Histology (H＆E staining) in WT and kMICU1 KO mice 60
Figure 14. Expression of PC and IC related markers in WT and kMICU1 KO mice 61
Figure 15. Electron microscopy of CD in WT and kMICU1 KO mice 63
Figure 16. Electron microscopy of mitochondria in WT and kMICU1 KO mice 64
Figure 17. LC3 expression in kidney of WT and kMICU1 KO mice 66
Figure 18. Postulated mechanism of polyuria in kMICU1 KO mice 67

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