Calreticulin in kidney function and disease: chronic low level of calreticulin impairs Ca2+ homeostasis leading to mitochondrial dysfunction and chronic renal injury
Bedeutung der Calreticulin in Nierenfunktion und -Erkrankung: chronisch niedrige Calreticulin-Konzentration beeinträchtigt die Ca2+-Homöostase und führt zu mitochondrialer Dysfunktion und chronischer Nierenschädigung
by Asima Bibi
Date of Examination:2012-10-11
Date of issue:2012-11-28
Advisor:Prof. Dr. Hassan Dihazi
Referee:Prof. Dr. Sigrid Hoyer-Fender
Referee:Prof. Dr. Jürgen Brockmöller
Persistent Address:
http://dx.doi.org/10.53846/goediss-1451
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Abstract
English
Chronic kidney disease (CKD) is becoming a major public health problem worldwide. The persistent progression of CKD is postulated to result from a self-perpetuating vicious cycle of events activated after initial injury. Being a major excretory and homeostatic organ of the body, kidney is continuously exposed to toxic wastes, excess of water and ions. In an attempt to understand the molecular mechanisms, which lead a normal functioning kidney towards disease state, proteomic screening of renal cells under various physiological conditions such as osmotic stress, oxidative stress and cytokines were performed. The data highlighted the expression regulation of an endoplasmic reticulum resident Ca2+ binding protein, calreticulin. Within endoplasmic reticulum (ER), calreticulin plays important function as a chaperon directing proper conformation of proteins, as well as a major ER Ca2+ binding protein, which controls cytosolic and ER Ca2+ levels. The purpose of this study was to investigate the potential role of calreticulin and mechanisms connecting this protein in regulating the renal cells function and progression of renal injury. In vitro investigations described in Chapter 2 using two-dimensional fluorescence difference gel electrophoresis combined with mass spectrometry analysis revealed an expression alteration of calreticulin in renal cells under osmotic stress conditions. It was also found that downregulation of calreticulin is combined with continuous change in the level of free intracellular Ca2+. On the other hand, inhibition of the Ca2+ release, through IP3R antagonist, prevented calreticulin expression alteration under hyperosmotic stress, whereas the cell viability was significantly impaired. An increase in ER Ca2+ storage with decreased cell viability was observed in cells overexpressing wild type calreticulin compared to no significant change in Ca2+ level and viability in cells overexpressing mutant calreticulin, lacking the Ca2+ binding domain. Furthermore, free Ca2+ level and cell survival were significantly improved under osmotic stress conditions by silencing calreticulin with siRNA. Taken together, our data clearly highlight the crucial role of calreticulin in renal cells functioning and survival through modulating Ca2+ homeostasis under osmotic stress conditions. The work presented in Chapter 3 was performed with adult heterozygote Calr+/- mice having chronic low level of calreticulin to further investigate the in vivo impact of downregulation of calreticulin on kidney structure and function. A progression of renal injury evidenced by development of glomerulosclerosis and tubulointerstitial damage was observed in histological analysis of Calr+/- mice kidneys from different age groups. The significant overexpression of cytosolic Ca2+ binding proteins with an insignificant alteration of ER stress proteins, suggested the role of intracellular Ca2+ homeostasis disturbance in renal impairments in Calr+/- mice. It was also found that endoplasmic reticulum stress protein markers are not significantly induced. Proteomic analysis further highlighted the role of oxidative stress and mitochondrial dysfunction in renal injury in Calr+/- mice kidneys. Especially, the reactive oxidative species scavenging enzyme, Sod1 expression was not only significantly downregulated but also showed irregular aggregates with immunohistochemical staining. Ultrastructural analysis further indicated significantly impaired mitochondrial morphology characterized by enlarged, swollen mitochondria with disturbed membranous structures in Calr+/- mice. These morphological changes were accompanied by biochemical abnormalities with specific decreases in the activity of cytochrome c oxidase of the mitochondrial electron transfer chain. Consequently, the oxidative stress together with mitochondrial damage and energy imbalance resulted in kidney injury in Calr+/- mice. In conclusion, the work presented in this thesis, revealed for the first time, the role of calreticulin in renal cells function and in the progression of chronic kidney injury. The study also points out that low level of calreticulin mediated Ca2+ homeostasis disturbances impacts the mitochondrial morphology, function and expression of Sod1. It will be interesting to investigate the exact mechanism by which calreticulin modulates Sod1 downregulation, at the molecular level. This should provide more concentrated foci for future experimentation. However, our findings highlighted a new potential mechanism of the progression of CKD and encourage new directions in CKD research, which in turn should have impact on treatment approach, diagnosis and prevention of CKD.
Keywords: Calreticulin; Kidney; CKD; chronic kidney disease
Other Languages
Chronische Nierenerkrankung (CKD, chronic
kidney disease) ist weltweit zu einem großen Gesundheitsproblem
geworden. Das kontinuierliche Fortschreiten der CKD scheint das
Ergebnis der Selbsterhaltung der Mechanismen zu sein, die einer
anfänglichen Schädigung zugrunde liegen. Die Niere, als das
Blutfiltrationsorgan, ist stets toxische Metabolismus-Abfälle,
Wasserüberschuss und Ionen ausgesetzt. Mit dem Ziel, die
molekularen Mechanismen zu verstehen, die eine normal
funktionierende Niere zu einer kranken Niere überführen, wurden
proteomische Analyse von Nierenzellen unter verschiedenen
physiologischen Bedingungen wie osmotischer Stress, oxidativer
Stress und Zytokinbehandlung durchgeführt. Interessanteweise haben
die Daten gezeigt, dass Calreticulin, eine Ca2+-bindendes
Endoplasmatische Reticulum (ER)-Protein, eine starke
Expressionsregulierung aufweist. Innerhalb des ERs, spielt die
Calreticulin eine wichtige Rolle als Chaperon, die zur richtigen
Proteinfaltung beiträgt. Des Weitern kommt der Calreticulin als
Ca2+-bindendes ER Protein, eine wesentliche Rolle bei der
Regulierung der cytososlischen Calciumkonzentration zu. Der Ziel
dieser Studie war es, die mögliche Rolle von Calreticulin in der
Nierenerkrankungen zu studieren wie auch die Mechanismen zu
untersuchen, die dieses Protein mit der Regulierung der Funktion
der Nierenzellen und das Fortschreiten der Nierenschädigung
verbinden. Zur Verwirklichung der Ziele wurden sowohl in-vitro als
auch in vivo Studien durchgeführt. Kapitel 2 der Dissertation
beschäftigt sich mit der in-vitro-Untersuchungen: Kombination der
zweidimensionalen Gelelektrophorese (mit Fluoreszenz gefärbten
Proteine) und Massenspektrometrie, zeigt eine starke
Runterregulierung der Calreticulin-Expression in Nierenzellen unter
osmotischen Stress. Die Expressionsänderung der Calreticulin
korrelierte mit einer kontinuierlichen Steigerung der Konzentration
des freien intrazellulären Ca2+. Andererseits, die Hemmung der
Ca2+-Freisetzung, durch IP3R Antagonisten, verhinderte die
Expressionsänderung der Calreticulin unter Stressbedingungen. Dies
resultierte in einer erheblichen Beeinträchtigung des
Zellüberlebens.
Schlagwörter: Calreticulin; Niere; CKD; chronischer Nierenschädigung
