Academic literature on the topic 'Adult mouse kidney'

Abstract. Aquaporin-4 (AQP4) is a member of the aquaporin water-channel family. AQP4 is expressed primarily in the brain, but it is also present in the collecting duct of the kidney, where it is located in the basolateral plasma membrane of principal cells and inner medullary collecting duct (IMCD) cells. Recent studies in the mouse also have reported the presence of AQP4 in the basolateral membrane of the proximal tubule. The purpose of this study was to establish the pattern of AQP4 expression during kidney development and in the adult kidney of both the mouse and the rat. Kidneys of adult and 3-, 7-, and 15-d-old mice and rats were preserved for immunohistochemistry and processed using a peroxidase pre-embedding technique. In both the mouse and the rat, strong basolateral immunostaining was observed in IMCD cells and principal cells in the medullary collecting duct at all ages examined. Labeling was weaker in the cortical collecting duct and the connecting tubule, and there was no labeling of connecting tubule cells in the mouse. In adult mouse kidney, strong AQP4 immunoreactivity was observed in the S3 segment of the proximal tubule. However, there was little or no labeling in the cortex or around the corticomedullary junction in 3- and 7-d-old mice. Between 7 and 15 d of age, distinct AQP4 immunoreactivity appeared in the S3 segment of the mouse proximal tubule concomitant with the differentiation of this segment of the nephron. Labeling of proximal tubules was never observed in the rat kidney. These results suggest that there are differences in transepithelial water transport between mouse and rat or that additional, not yet identified water channels exist in the rat proximal tubule.

Neuropilin-1, a neuronal cell surface semaphorin III receptor protein important for axonal guidance in developing peripheral nervous system efferents, has also been identified as a vascular endothelial growth factor (VEGF) receptor on endothelial cells. To evaluate its expression in kidney, we carried out RT-PCR on newborn and adult total renal RNAs. A 403-bp product, which was predicted to be that from neuropilin-1 mRNA, was found in both samples. Nucleotide sequencing confirmed that these products encoded neuropilin-1. Northern analysis of newborn and adult kidney RNA showed specific hybridization to appropriately sized bands of ∼6 kb. In situ hybridization with a mouse-specific antisense neuropilin-1 35S-cRNA probe showed distinct glomerular localization on sections from both newborns and adults. Similar patterns of hybridization were seen in sections treated with antisense cRNA probes against another VEGF receptor, Flk1, and with VEGF probes. However, the VEGF hybridization signal was markedly less in adult glomeruli than those for neuropilin-1 and Flk1. Because neuropilin-1 specifically binds VEGF165 in humans, we carried out RT-PCR on mouse kidney RNA with primers that amplified the three alternatively spliced isoforms of VEGF mRNA. Our analysis showed that for both newborn and adult kidneys, the relative abundance of VEGF mRNA was VEGF164 ≫ VEGF120 > VEGF188. We conclude that the expression of neuropilin-1, in conjunction with Flk1 and VEGF164, jointly contributes to the development and maintenance of glomerular capillaries.

Renal glomerular capillary tufts have been believed to arise from angiogenic ingrowth of extrinsic vessels. We found, however, that when embryonic day 12 (E12) mouse kidneys were maintained in culture for 6 days and then grafted into anterior eye chambers of adult transgenic ROSA26 host mice (which carry the beta-galactosidase transgene), glomerular endothelial cells within the grafts were predominantly of intrinsic, kidney origin. To identify potential endothelial precursors, we immunolabled kidneys with antibodies against the vascular endothelial growth factor receptor, flk-1. Numerous discrete cells expressing flk-1 were scattered throughout the nephrogenic mesenchyme of both E12 and newborn kidneys, and with development these cells became concentrated in microvessels, glomerular vascular clefts, and glomerular tufts. In adults, flk-1 was weakly expressed in glomeruli but absent elsewhere. To examine abilities of flk-1-positive cells to establish glomeruli, E12 kidneys were grafted into kidney cortices of adult and newborn ROSA26 hosts. Grafts into adults resulted in few glomeruli containing host-derived endothelium, whereas a majority of glomeruli grafted into newborns contained host cells. Cells of graft origin were found in vessels forming in renal cortices of newborn hosts, but not in adults. Our findings indicate that embryonic kidney cells expressing flk-1 are angioblasts that create microvessels and glomeruli by vasculogenesis.

Cisplatin is a well-known chemotherapy medication used to treat numerous cancers. However, treatment with cisplatin in cancer therapy has major side effects, such as nephrotoxic acute kidney injury. Adult vertebrate kidneys are commonly used as models of cisplatin-induced nephrotoxic acute kidney injury. Embryonic zebrafish kidney is more simplified and is composed simply of two nephrons and thus is an excellent model for the investigation of cisplatin nephrotoxicity. Here, we developed a novel model to induce cisplatin nephrotoxicity in adult zebrafish and demonstrated that intraperitoneal injection of cisplatin caused a decline in kidney proximal tubular function based on fluorescein-labeled dextran uptake and alkaline phosphatase staining. We also showed that cisplatin induced histological injury of the kidney tubules, quantified by tubular injury scores on the periodic acid-Schiff-stained kidney sections. As shown in a mouse model of cisplatin-induced nephrotoxicity, the activation of poly(ADP-ribose) polymerase (PARP), an enzyme implicated in cisplatin-induced cell death, was markedly increased after cisplatin injection in adult zebrafish. Furthermore, pharmacological inhibition of PARP using a specific PARP inhibitor PJ 34 hydrochloride (PJ34) or 3-aminobenzamide ameliorated kidney proximal tubular functional and histological damages in cisplatin-injected adult zebrafish kidneys. Administration of a combination of PARP inhibitors PJ34 and 3-aminobenzamide additively protected renal function and histology in zebrafish and mouse models of cisplatin nephrotoxicity. In conclusion, these data suggest that adult zebrafish are not only suitable for drug screening and genetic manipulation but also useful as a simplified but powerful model to study the pathophysiology of cisplatin nephrotoxicity and establish new therapies for treating human kidney diseases.

It has been postulated that developmental pathways are reutilized during repair and regeneration after injury, but functional analysis of many genes required for kidney formation has not been performed in the adult organ. Mutations in SALL1 cause Townes-Brocks syndrome (TBS) and nonsyndromic congenital anomalies of the kidney and urinary tract, both of which lead to childhood kidney failure. Sall1 is a transcriptional regulator that is expressed in renal progenitor cells and developing nephrons in the embryo. However, its role in the adult kidney has not been investigated. Using a mouse model of TBS ( Sall1 TBS), we investigated the role of Sall1 in response to acute kidney injury. Our studies revealed that Sall1 is expressed in terminally differentiated renal epithelia, including the S3 segment of the proximal tubule, in the mature kidney. Sall1 TBS mice exhibited significant protection from ischemia-reperfusion injury and aristolochic acid-induced nephrotoxicity. This protection from acute injury is seen despite the presence of slowly progressive chronic kidney disease in Sall1 TBS mice. Mice containing null alleles of Sall1 are not protected from acute kidney injury, indicating that expression of a truncated mutant protein from the Sall1 TBS allele, while causative of congenital anomalies, protects the adult kidney from injury. Our studies further revealed that basal levels of the preconditioning factor heme oxygenase-1 are elevated in Sall1 TBS kidneys, suggesting a mechanism for the relative resistance to injury in this model. Together, these studies establish a functional role for Sall1 in the response of the adult kidney to acute injury.

The acyl-CoA synthetase medium-chain family member 2 ( Acsm2) gene was first identified and cloned by our group as a kidney-specific “ KS” gene. However, its expression pattern and function remain to be clarified. In the present study, we found that the Acsm2 gene was expressed specifically and at a high level in normal adult kidneys. Expression of Acsm2 in kidneys followed a maturational pattern: it was low in newborn mice and increased with kidney development and maturation. In situ hybridization and immunohistochemistry revealed that Acsm2 was expressed specifically in proximal tubular cells of adult kidneys. Data from the Encyclopedia of DNA Elements database revealed that the Acsm2 gene locus in the mouse has specific histone modifications related to the active transcription of the gene exclusively in kidney cells. Following acute kidney injury, partial unilateral ureteral obstruction, and chronic kidney diseases, expression of Acsm2 in the proximal tubules was significantly decreased. In human samples, the expression pattern of ACSM2A, a homolog of mouse Acsm2, was similar to that in mice, and its expression decreased with several types of renal injuries. These results indicate that the expression of Acsm2 parallels the structural and functional maturation of proximal tubular cells. Downregulation of its expression in several models of kidney disease suggests that Acms2 may serve as a novel marker of proximal tubular injury and/or dysfunction.

The adult proximal tubule is a low-resistance epithelium where there are high rates of both active transcellular and passive paracellular NaCl transport. We have previously demonstrated that the neonatal rabbit and rat proximal tubule have substantively different passive paracellular transport properties than the adult proximal tubule, which results in a maturational change in the paracellular passive flux of ions. Neonatal proximal tubules have a higher PNa/PCl ratio and lower chloride and bicarbonate permeabilities than adult proximal tubules. Claudins are a large family of proteins which are the gate keepers of the paracellular pathway, and claudin isoform expression determines the permeability characteristics of the paracellular pathway. Previous studies have shown that claudins 1, 2, 3, 4, 5, 7, 8, 10, 11, 12, 15, and 16 are expressed in the adult mouse kidney. To determine whether there are developmental claudin isoforms, we compared the claudin isoforms present in the neonatal and adult kidney using RT-PCR to detect mRNA of claudin isoforms. Claudin 6, claudin 9, and claudin 13 were either not expressed or barely detectable in the adult mouse kidney using traditional PCR, but were expressed in the neonatal mouse kidney. Using real-time RT-PCR, we were able to detect a low level of claudin 6 mRNA expression in the adult kidney compared with the neonate, but claudin 9 and claudin 13 were only detected in the neonatal kidney. There was the same maturational decrease in these claudin proteins with Western blot analysis. Immunohistochemistry showed high levels of expression of claudin 6 in neonatal proximal tubules, thick ascending limb, distal convoluted tubules, and collecting ducts in a paracellular distribution but there was no expression of claudin 6 in the adult kidney. Using real-time RT-PCR claudin 6 and 9 mRNA were present in 1-day-old proximal convoluted tubules and were virtually undetectable in proximal convoluted tubules from adults. Claudin 13 was not detectable in neonatal or adult proximal convoluted tubules. In summary, we have identified developmentally expressed claudin isoforms, claudin 6, claudin 9, and claudin 13. These paracellular proteins may play a role in the maturational changes in paracellular permeability.

Dystroglycan is a cell surface protein which, in muscle, links the extracellular matrix protein laminin-2 to the intracellular cytoskeleton. Dystroglycan also binds laminin-1 and the binding occurs via the E3 fragment of laminin-1. Recently, it was found that dystroglycan is expressed in developing epithelial cells of the kidney. Moreover, antibodies against dystroglycan can perturb epithelial development in kidney organ culture. Therefore, dystroglycan may be an important receptor for cell–matrix interactions in non-muscle tissues. However, information about the tissue distribution of dystroglycan is limited, especially in adult tissues. Here we show that dystroglycan is present in epithelial cells in several non-muscle organs of adult mice. Dystroglycan is enriched towards the basal side of the epithelial cells that are in close contact with basement membranes. We suggest that dystroglycan is involved in linking basement membranes to epithelial and muscle cells. Dystroglycan may be important for the maintenance of tissue integrity.

Organoide aus adulten Mäusen sind vielversprechende Modelle für die Nierenforschung. Ihre Charakterisierung wurde jedoch nicht auf ein zufriedenstellendes Niveau gebracht. Hier habe ich ein langfristiges 3D-Maus-Organoid (Tubuloid)-Modell etabliert und charakterisiert, das die Erneuerung und die Reparatur sowie die Architektur und die Funktionalität der adulten tubulären Epithelien rekapituliert. In der Zukunft wird das Modell detaillierte Untersuchungen der Trajektorien selbsterneuernder Zellen sowohl zur teilweisen Wiederherstellung der Niere als auch zur malignen Transformation der Niere ermöglichen. Das klarzellige Nierenzellkarzinom (ccRCC) ist der häufigste und aggressivste Nierenkrebs. Die Inaktivierung des Tumorsuppressorgens Von Hippel-Lindau (VHL) ist der Haupttreiber des ccRCCs. Zuvor hatten wir die Hochregulation der Wnt- und Notch-Signalübertragung in den CXCR4+MET+CD44+-Krebsstammzellen (CSC) aus primären humanen ccRCC-Tumoren identifiziert. Das Blockieren von Wnt und Notch in von Patienten stammenden Xenotransplantaten, Organoiden und nicht-anhaftenden Sphären unter Verwendung von niedermolekularen Inhibitoren beeinträchtigte die Selbsterneuerung der CSC und das Tumorwachstum. Um CSC-gesteuertes humanes ccRCC in genetischen Mausmodellen nachzuahmen, begann ich mit der Erzeugung von zwei Doppelmausmutanten; β-Catenin-GOF; Notch-GOF und Vhl-LOF; β-Catenin-GOF. Sowohl die β-Catenin-GOF; Notch-GOF Mausmutante als auch die Vhl-LOF; β-Catenin-GOF Mausmutante entwickelten innerhalb einiger Monate schwere Krankheitssymptome. Überraschenderweise beobachtete ich weder Tumore oder Tumorvorläuferläsionen noch höhere Zellproliferationsraten in den mutierten Nieren. Weitere Analysen ergaben, dass die Mausmutanten Merkmale chronischer Nierenerkrankung (CKD) aufwiesen.
Adult mouse organoids are promising models for kidney research. However, their characterization has not been pushed forward to a satisfying level. Here, I have generated and characterized a long-term 3D mouse organoid (tubuloid) model, which recapitulates renewal and repair, and the architecture and functionality of the adult tubular epithelia. In the future, the model will allow detailed investigations of trajectories of self-renewing cells towards both the partial recreation and malignant transformation of the kidney. Clear cell renal cell carcinoma (ccRCC) is the most common and aggressive kidney cancer. Inactivation of the Von Hippel-Lindau (VHL) tumor suppressor gene is the major driver of ccRCC. Earlier, we identified the upregulation of Wnt and Notch signaling in CXCR4+MET+CD44+ cancer stem cells (CSCs) from primary human ccRCCs. Blocking Wnt and Notch in patient-derived xenografts, organoids and non-adherent spheres using small-molecule inhibitors impaired self-renewal of CSCs and tumor growth. To mimic CSC-governed human ccRCC in genetic mouse models, I started from the generation of two double mouse mutants; β-catenin-GOF; Notch-GOF and Vhl-LOF; β-catenin-GOF. Surprizingly, I observed neither tumors or tumor precursor lesions nor higher cell proliferation rates in the mutant kidneys. Further analyses revealed that the mutant mice displayed features of chronic kidney disease (CKD). Thus, β-catenin-GOF; Notch-GOF and Vhl-LOF; β-catenin-GOF mouse mutants did not develop kidney tumors under the given experimental conditions.

博士
國立成功大學
臨床醫學研究所
98
In Taiwan, the incidence of end-stage renal disease ranked first and the prevalence ranked second in the world. Patients with end-stage renal disease need hemodialysis, peritoneal dialysis or kidney transplant to maintain their life. For the shortage of available organ, most of the patients are under regular dialysis. To accelerate renal repair or even make a functioning kidney is the emergent issue to be solved and interesting topic for research. Following the discovery of tissue-specific progenitor cells in other organs and their ability to improve regeneration after injury, progenitor cell-based therapy is a new strategy in the treatment of acute kidney injury and has potentially more value than single-agent drug therapy due to the highly versatile response of cells to their environment. These cells may not only secrete cytokines within the injured kidney, but also participate in tubular cell proliferation or angiogenesis to facilitate renal regeneration. In rodents, increasing evidence suggests that the therapeutic potential of mesenchymal stem cells derived from bone marrow could be beneficial in the kidney injury. Thereby, we hypothesize that kidney progenitor cells may accelerate renal regeneration after injury. We first observed the regenerative process of acute tubular necrosis in rodents. In the normal kidney, only interstitial cells but not tubular cells expressed vimentin. Following acute renal failure, vimentin-positive renal interstitial cells proliferated and surrounded the damaged renal tubules as early as 12 hours after injury. Within the regenerating tubules, vimentin staining was found intensely two days after injury, and disappeared after full recovery of tubular epithelial cells. By known interstitial cell markers, only few vimentin-positive renal interstitial cells were characterized as endothelial cells or fibroblasts one day after acute renal failure. Most of the other proliferating cells were not specified and we hypothesize that kidney progenitor cells could reside in these areas. Using bromodeoxyuridine (BrdU) as a marker of proliferating cells, we monitor the distribution of the interstitial cells by immunohistochemistry during acute renal failure. Following one injection of BrdU, eighty five percent of BrdU labeling cells located in the interstitium 12 hours after acute renal failure and the count decreased to 25% at the 4th day. Interestingly, BrdU labeling cells redistributed to the regenerating tubules at the 1st and 4th day. Seventy-five percentage of BrdU labeling cells located in the tubules at the 4th day. As assessed by ELISA, the uptake of BrdU in the kidney peaked at the 1st day, decreased to constant level after 3 days, and maintained till 7 days following one injection of BrdU before acute renal failure. These results indicate that interstitial cells might be engaged in the process of tubular regeneration after acute renal failure. We test the hypothesis that renal progenitor cells isolated from adult mouse kidney accelerate renal regeneration via participation in the repair process. A unique population of cells exhibiting characteristics consistent with renal progenitor cells, mouse kidney progenitor cells (MKPC), was isolated from Myh9 targeted mutant mice. Features of these cells include: (1) spindle-shaped morphology, (2) self-renewal of more than 100 passages without evidence of senescence, (3) expression of Oct-4, Pax-2, Wnt-4, WT-1, vimentin, alpha-smooth muscle actin, CD29 and S100A4 but no SSEA-1, c-kit, or other markers of more differentiated cells. MKPC exhibit plasticity as demonstrated by the ability to differentiate into endothelial cells and osteoblasts in vitro and endothelial cells and tubular epithelial cells in vivo. The origin of the isolated MKPC was from the interstitium of medulla and papilla. Importantly, intra-renal injection of MKPC in mice with ischemic injury rescued renal damage, as manifested by decreases in peak serum urea nitrogen, the infarct zone and the necrotic injury. Seven days after the injury, some MKPC formed vessels with red blood cells inside and some incorporated into renal tubules. In addition, MKPC treatment reduces the mortality in mice after ischemic injury. Our results indicate that MKPC represent a multipotent adult progenitor cell population, which may contribute to the renal repair and prolong survival after ischemic injury. The PhD study not only raised a novel method to treat acute renal failure but also open a new window to elucidate the relationship between kidney progenitor cells and tubular regeneration. Based on these, we will be able to unveil the mechanism of how tissue-specific progenitor cells involve in the process of tissue regeneration.