gives an overview of the methods used to study the contribution of bone marrow-

derived cells to renal regeneration. To investigate the role of these cells, a method was developed which consisted of a combination of protocols published in the literature during the

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of these cells in recipient mice, but also the induction of AKI are described in detail. Moreover, the different quantification methods to determine the contribution of donor bone marrow cells within the recipient mice and renal function assessments are evaluated. Chapter 2 also discusses critically the advantages and shortcomings of our method and compared it with other methods described.

To study the role of ABC transporters in kidney regeneration, the RNA expression levels of 45 ABC transporters were investigated using quantitative RT-PCR analysis under basal conditions, after induction of ischemia and after regeneration. In chapter 3 we demonstrate that gene expression of ABC transporters is differentially regulated after renal injury; P-

gp/abcb1b, abcb11 and abcc4 expression was increased, and expressions of abca3, abcc2 and

BCRP/abcg2 were decreased. The expression of all transporters returned to baseline after 14 days, except for abcb11, which was still reduced. Using Western blot analysis, we showed that protein expression of P-gp and BCRP was increased after ischemia induction, suggesting that these transporters may have an important function in the renal repair process. Moreover, for the first time expression and subcellular localization of the transporters abca3 and BSEP/abcb11 in mouse kidney were shown.

To investigate the role of P-gp and BCRP in renal regeneration in more detail, knockout mice of each transporter were used. First, renal function of the P-gp- (mdr1a/1b(-/-)) and BCRP (bcrp(-/-))-deficient mice were thoroughly characterized. In chapter 4, we describe the renal phenotype of mice deficient in P-gp expression. After extensive examination of blood and urine samples, it appeared that these mice suffer from proximal tubular dysfunction resembling the human renal Fanconi syndrome. Typical features were increased diuresis, augmented urinary excretion of sodium, amino acids and low molecular weight proteins, and increased lithium clearance. Furthermore, reduced inulin and PAH clearances indicated that P-gp-deficient mice have diminished glomerular filtration rate and renal plasma flow, respectively. Electron microscopy revealed mitochondrial abnormalities in proximal tubular cells and decreased renal cortical ATP levels were found, which can explain the observed tubular dysfunction. Based on the increased expression of P-gp in the kidney after ischemia we expected the P-gp-deficient mice to be more susceptible to acute kidney injury, however,

surprisingly, these mice were resistant to ischemia. This may be the result of ischemic preconditioning due to the hypovolemic condition of the mice. On the other hand, we also found that bone marrow from P-gp-deficient mice show less sensitivity to apoptosis, which suggests that the smaller degree of renal damage may be caused by a decreased number of apoptotic cells causing tubular obstruction. This, evidently, needs to be investigated further. Secondly, BCRP-deficient animals were characterized as well. BCRP transports, like P-gp, a wide variety of chemotherapeutic drugs, but it is also involved in the transport of endogenous steroid hormones, like dehydroepiandrosterone sulfate (DHEAS) and estradiol. The capacity of BCRP to transport estradiol apparently determines the phenotype of the BCRP-deficient mice, which is described in chapter 5. The absence of BCRP leads to a disturbed regulation of sex hormones; of which elevated levels of serum testosterone (10 fold) and luteinizing hormone (LH, 3 fold) seem to be crucial features. The disturbed hormone balance eventually results in a metabolic syndrome-like phenotype, characterized by glucose and insulin intolerance, but also in morphologic abnormalities of the reproductive organs, leading to subfertility. After estradiol administration, serum testosterone, LH and glucose normalized to levels of wild type mice. Adipose tissue is one of the most important sources of peripheral estradiol, which is essential for the negative feedback on the pituitary gland. We found BCRP expression in the adipocyte membrane, suggesting that the transporter may be responsible for controlling peripheral estradiol levels. An absence of BCRP may lead to a disturbed negative feedback on the pituitary gland. These findings imply a new, key role for BCRP in steroid metabolism and glucose homeostasis.

Although expression of BCRP is described in several excretory organs and its mRNA levels were detectable in human kidney tissue, its protein expression and localization in the kidney was still undefined. In chapter 6, we show for the first time expression, localization and transport activity of BCRP in human kidney. In isolated human proximal tubular cells, Hoechst 33342 transport could be inhibited by fumitremorgin C nd nelfinavir indicating BCRP activity. Finally, immunohistochemistry confirmed the localization of BCRP to the proximal tubular apical membrane, the same nephron segment where the ABC transporters, P- gp, Mrp4 and Mrp2, are localized. Furthermore, a clearly higher expression of BCRP was found in rat and mouse kidney as compared to the levels determined in human kidney tissue.

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Nevertheless, the expression of the efflux pump is highly inducible during stress conditions and the transporter may certainly play a role in renal drug excretion.

In chapter 7 we explored the role of P-gp and BCRP in renal regeneration by bone marrow– derived stem cells. Enhanced green fluorescent protein (eGFP)-labeled bone marrow cells isolated from wild type mice, P-gp-deficient mice and BCRP-deficient mice were transplanted into wild type recipient mice. The findings indicate that absence of P-gp or BCRP in the donor bone marrow stimulates renal regeneration in the recipient mice after ischemic reperfusion injury. As a proof of principle that bone marrow cells are important and that the transporters play a crucial role in renal repair, bone marrow from wild type animals was transplanted in BCRP-deficient mice. Without transplantation, these knockout mice are protected against AKI, but interestingly, after transplantation, renal injury and loss of function were observed after ischemic injury. The presence of BCRP and most likely also P-gp, seems to hamper renal (tubular) remodeling by bone marrow cells and the ischemic resistance observed in both knockout mice must originate from (specific populations of) bone marrow cells that promote tissue repair. Analysis of the bone marrow composition revealed that the number of macrophages and granulocytes was increased in P-gp- and BCRP-deficient mice. Moreover, also the amount of endothelial progenitor cells was increased, which possibly fuels the remodeling process.

During the last years, a great deal of attention has been focused on the use of bone marrow- derived stem cells in tissue regeneration, in which the differentiation capacity of bone marrow stem cells into diverse non-hematopoietical cells was shown. Within the bone marrow, a rare cell population, called the multipotent adult progenitor cells (MAPCs), is localized, which has the capacity to differentiate into mesenchymal lineages but also into endothelium, neuroectoderm and endoderm. Their capacity to differentiate towards renal cells was, however, still unknown. In chapter 8, we determined this capacity under influence of basic- Fibroblast Growth Factor, Tumor Growth Factor-α, Leukocyte Inhibitory Factor and Hepatocyte Growth Factor. After initiating differentiation during culturing, morphology of the MAPCs, as well as gene and protein expression of specific renal (tubular) epithelial markers, confirm the differentiation of MAPCs towards renal epithelial cells. These findings were

supported further by an increased alkaline phosphatase and P-gp activities, indicative for renal proximal tubular cells. The ability of MAPCs to differentiate towards renal cells might be a new strategy in the treatment of kidney disease, which needs to be explored further.