- [Show abstract] [Hide abstract] ABSTRACT: Background Rapid advancements have been made in alternative treatments for renal diseases. Our goal for renal regeneration is to establish a kidney graft derived from human embryonic tissues. In this study, we investigated the effects of host renal failure on the structure and activity of transplanted embryonic kidney and bladder, and found that diuretics effectively induced urine production in the transplanted kidney. Methods Uremic conditions were reproduced using a 5/6 renal infarction rat model. An embryonic kidney plus bladder (embryonic day 15) was isolated from a pregnant Lewis rat and transplanted into the para-aortic area of a 5/6 renal-infarcted Lewis rat. Following growth, the embryonic bladder was successfully anastomosed to the host ureter. ResultsWe assessed graft function in terms of survival rates and found no differences between normal (n = 5) and renal failure (n = 8) groups (median survival: 70.5 vs 74.5 h; p = 0.331) in terms of survival, indicating that the grafts prolonged rat survival, even under renal failure conditions. Furosemide (n = 9) significantly increased urine volume compared with saline-treated controls (n = 7; p < 0.05), confirming that the grafts were functional. We also demonstrated the possibilities of an in vivo imaging system for determining the viability of transplanted embryonic kidney with bladder. Conclusion The results of this study demonstrate that transplanted embryonic kidney and bladder can grow and function effectively, even under uremic conditions.
- [Show abstract] [Hide abstract] ABSTRACT: There have been several recent attempts to generate, de novo, a functional whole kidney from stem cells using the organogenic niche or blastocyst complementation methods. However, none of these attempts succeeded in constructing a urinary excretion pathway for the stem cell-generated embryonic kidney. First, we transplanted metanephroi from cloned pig fetuses into gilts; the metanephroi grew to about 3 cm and produced urine, although hydronephrosis eventually was observed because of the lack of an excretion pathway. Second, we demonstrated the construction of urine excretion pathways in rats. Rat metanephroi or metanephroi with bladders (developed from cloacas) were transplanted into host rats. Histopathologic analysis showed that tubular lumina dilation and interstitial fibrosis were reduced in kidneys developed from cloacal transplants compared with metanephroi transplantation. Then we connected the host animal's ureter to the cloacal-developed bladder, a technique we called the "stepwise peristaltic ureter" (SWPU) system. The application of the SWPU system avoided hydronephrosis and permitted the cloacas to differentiate well, with cloacal urine being excreted persistently through the recipient ureter. Finally, we demonstrated a viable preclinical application of the SWPU system in cloned pigs. The SWPU system also inhibited hydronephrosis in the pig study. To our knowledge, this is the first report showing that the SWPU system may resolve two important problems in the generation of kidneys from stem cells: construction of a urine excretion pathway and continued growth of the newly generated kidney.
- [Show abstract] [Hide abstract] ABSTRACT: Recent findings have demonstrated that stem cells can differentiate into mature tissue when supplied with a niche containing factors identical to those in the normal developmental program. A niche for the development of an organ can be provided by xenotransplantation of a similar developing organ. However, this process has many technical, safety, and ethical concerns. Here, we established xenotransplantation models that control endogenous mesenchymal stem cell (MSC) differentiation into mature erythropoietin (EPO)-producing tissue in a niche provided by a developing xenometanephros. Transplantation of rat metanephroi into mouse omentum, and similarly pig metanephroi into cat omentum, led to the recruitment of host cells and EPO production. EPO-expressing cells were not differentiated from integrating vessels because they did not coexpress endothelial markers (Tie-2 and VE-cadherin). Instead, EPO-expressing cells were shown to be derived from circulating host cells, as shown by enhanced green fluorescent protein (EGFP) expression in the grown transplants of chimeric mice bearing bone marrow from a transgenic mouse expressing EGFP under the control of the EPO promoter. These results suggest that donor cell recruitment and differentiation in a xenotransplanted developing organ may be consistent between species. The cells responsible for EPO expression were identified as MSCs by injecting human bone marrow-derived MSCs and endothelial progenitor cells into NOD/SCID mice. Furthermore, using metanephroi from transgenic ER-E2F1 suicide-inducible mice, the xenotissue component could be eliminated, leaving autologous EPO-producing tissue. Our findings may alleviate adverse effects due to long-lasting immunosuppression and help mitigate ethical concerns.
- [Show abstract] [Hide abstract] ABSTRACT: The kidney is an important organ for maintaining blood pressure. We have previously reported that transplanted metanephroi can reproduce some kidney functions. The aim of the present study was to determine the metabolic function of transplanted metanephroi with particular reference to maintaining blood pressure. Male Wistar rats were transplanted with metanephroi (transplanted group, n = 28), following unilateral nephrectomy. For comparison, we performed unilateral nephrectomy without transplantation in 32 rats (non-transplanted group, n = 18; haeminephrectomy control group, n = 14). The remaining kidney was removed 2 weeks after the initial operation, while control rats had a sham operation. Hypotension was induced by intravenous infusion of diltiazem hydrochloride or rapid withdrawal of blood. Mean arterial blood pressure (MAP) was invasively monitored and plasma renin activity (PRA) was analysed at multiple time points. Renin expression by metanephroi was evaluated by real-time polymerase chain reaction and immunohistochemistry. Metanephroi in the transplanted group expressed renin messenger RNA. Metanephros transplantation significantly raised PRA and maintained MAP compared with the non-transplanted group. No significant differences between the transplanted and control groups were found with respect to PRA or MAP. PRA was positively correlated with metanephroi weight as well as MAP in the transplanted group. The present study shows that transplantation of metanephroi produces PRA and contributes to raising MAP in a rat model of acute hypotension.
- [Show abstract] [Hide abstract] ABSTRACT: Recent research has shown that transplanted metanephroi form primitive vascularized kidneys with histologically recognizable renal features. The aim of the present study was to determine the metabolic function of transplanted metanephroi in rats with chronic renal failure (CRF), with particular reference to secondary hyperparathyroidism and vascular calcification. CRF was induced in 11-week-old male Wistar rats by maintaining them on a 0.75% adenine diet for 4 weeks, followed by normal diet for an additional 2 weeks. At the end of adenine loading, whole metanephroi from embryonic day 15 rats were transplanted into the omentum and epididymis of the transplantation group. Vascular calcification was evaluated 2 weeks after metanephroi transplantation. Metanephros transplantation significantly reduced vascular calcium and phosphorus content and suppressed the progression of vascular calcification as indicated by von Kossa staining of the media of the thoracic aorta. However, no significant differences between the adenine-fed control and transplantation groups were found regarding the serum levels of 1,25(OH)2D3, calcium or phosphorus or the calcium × phosphorus product. The present study has shown that transplantation of metanephroi suppresses the progression of vascular calcification via a mechanism that is independent of calcium-phosphorus dynamics.
- [Show abstract] [Hide abstract] ABSTRACT: A xenotransplanted metanephros may undergo complete nephrogenesis in the host animal, forming a functional kidney. This suggests that, in future, xenometanephroi could become an unlimited source of material for renal transplantation. Although the omentum (OM) is the primary site for transplantation, we speculated that the growth of the transplant could differ depending on the site of transplantation. Thus, we determined the optimal transplantation site for the metanephros in terms of retaining its ability to produce renin and erythropoietin (EPO). Rat metanephroi were transplanted into the OM, paraaortic area (PA), or both (OM+PA) of unilaterally nephrectomized host rats. After 2-3 weeks, blood was rapidly withdrawn in order to induce production of renin and EPO in the transplants. Histological analysis indicated that transplants in both the PA and OM were well differentiated, demonstrating polarity of the medulla through to the cortex. Plasma renin activity (PRA) increased in response to the induction procedure, but transplants in the PA expressed PRA more effectively than did those in the OM. Real-time polymerase chain reaction (PCR) revealed higher levels of renin mRNA expression in transplants in the PA than in those in the OM. Although EPO production increased 24 hours after the induction procedure, the levels did not differ significantly between OM- and PA-transplanted rats. Compared with transplantation to the OM, transplantation of rat metanephroi to the PA results in better renin production, whereas the transplantation site does not affect EPO production.
- [Show abstract] [Hide abstract] ABSTRACT: Significant advances have been made in stem cell research over the past decade. A number of nonhematopoietic sources of stem cells (or progenitor cells) have been identified, including endothelial stem cells and neural stem cells. These discoveries have been a major step toward the use of stem cells for potential clinical applications of organ regeneration. Accordingly, kidney regeneration is currently gaining considerable attention to replace kidney dialysis as the ultimate therapeutic strategy for renal failure. However, due to anatomic complications, the kidney is believed to be the hardest organ to regenerate; it is virtually impossible to imagine such a complicated organ being completely rebuilt from pluripotent stem cells by gene or chemical manipulation. Nevertheless, several groups are taking on this big challenge. In this manuscript, current advances in renal stem cell research are reviewed and their usefulness for kidney regeneration discussed. We also reviewed the current knowledge of the emerging field of renal stem cell biology.
- [Show abstract] [Hide abstract] ABSTRACT: Previously, we reported that human mesenchymal stem cells (hMSCs) that were cultivated in growing embryos differentiated in an appropriate developmental milieu, thereby facilitating the development of a functional renal unit. However, this approach required transfection with an adenovirus that expressed glial cell line-derived neurotrophic factor (GDNF) to enhance the development of hMSC-derived renal tissue, and safety issues restrict the clinical use of such viral vectors. To circumvent this problem, we tested an artificial polymer as a means to diffuse GDNF. This GDNF-polymer, which exists in liquid form at 4 degrees C but becomes a hydrogel upon heating to 37 degrees C, was used as a thermoreversible switch, allowing the injection of hMSCs at low viscosity using a mouth pipette, with subsequent slow diffusion of GDNF as it solidified. The polymer, which was dissolved in a solution of GDNF at 4 degrees C and then maintained at 37 degrees C, acted as a diffuser of GDNF for more than 48 h. LacZ-transfected hMSCs and the GDNF-polymer (at 4 degrees C) were placed in the nephrogenic sites of growing rat embryos that were maintained at 37 degrees C. Forty-eight hours later, the resultant kidney anlagen were dissected out and allowed to continue developing for 6 days in vitro. Whole-organ X-Gal staining and fluorescence activated cell sorter analysis showed that the number of hMSC-derived cells was significantly increased in developed anlagen that have been generated from hMSCs plus GDNF-polymer compared with those from hMSCs plus GDNF-containing medium and was comparable to those from adenovirus-transfected hMSCs. These findings suggest that the GDNF-polymer can be used as a diffuser of GDNF for kidney organogenesis.
- [Show abstract] [Hide abstract] ABSTRACT: Developing animal embryos have been providing human mesenchymal stem cells (hMSCs) with an appropriate environment for their differentiation between species. We previously demonstrated that hMSCs transplanted into the metanephric mesenchyme region of rat embryos differentiate into kidney-specific cells. Here, we assessed whether hMSCs are competent to differentiate into precursors of the collecting duct system when they are transplanted into the ureteric bud progenitor region of chicken embryos that are easier to be manipulated and cultured than mammalian embryos. When chicken Pax2-expressing hMSCs were transplanted into the chicken ureteric bud progenitor region, they migrated caudally with the elongating Wolffian duct and then were integrated into the Wolffian duct epithelia. Also, chicken Pax2-expressing hMSCs started to express human LIM1 after their integration into the Wolffian duct epithelia. These results suggest that chicken Pax2-expressing hMSCs can be competent to differentiate into the Wolffian duct cells by the influence of chicken local signals.
- [Show abstract] [Hide abstract] ABSTRACT: Significant advances have been made in stem cell research over the past decade. A number of non-hematopoietic sources of stem cells (or progenitor cells) have been identified including endothelial stem cells and neural stem cells. These discoveries have been a major step towards the potential regeneration of organs for clinical applications using stem cells. The worldwide shortage of donor kidneys means that this approach has garnered significant attention in the field of nephrology. Here, we review recent findings on renal stem cells and their possible therapeutic application for renal diseases. KeywordsKidney regeneration–Mesenchymal stem cell–Renal failure–Xenobiology–Metanephros–Embryo
- [Show abstract] [Hide abstract] ABSTRACT: Differentiation of autologous stem cells into functional transplantable tissue for organ regeneration is a promising regenerative therapeutic approach for cancer, diabetes, and many human diseases. Yet to be established, however, is differentiation into tissue capable of producing erythropoietin (EPO), which has a critical function in anemia. We report a novel EPO-producing organ-like structure (organoid) derived from human mesenchymal stem cells. Using our previously established relay culture system, a human mesenchymal stem cell-derived, human EPO-competent organoid was established in rat omentum. The organoid-derived levels of human EPO increased in response to anemia induced by rapid blood withdrawal. In addition, the presence of an organoid in rats suppressed for native (rat) EPO production enhanced recovery from anemia when compared with control animals lacking the organoid. Together these results confirmed the generation of a stem cell-derived organoid that is capable of producing EPO and sensitive to physiological regulation.
- [Show abstract] [Hide abstract] ABSTRACT: Establishment of a functional whole kidney de novo has not received much attention because of the formidable challenges and the slow pace of advances in this field of research. This situation has changed recently with publication of data revealing the catastrophic nature of Medicaid costs for dialysis-related diseases. An innovative approach is needed in our search for therapies for kidney diseases and to provide a substitute for dialysis as soon as possible. Regenerative medicine offers great hope for realizing this goal. We established a system by which human mesenchymal stem cells can differentiate into a functional renal unit using a program of nephrogenesis in a developing xeno-embryo. In this article, recent research in the field of developing whole kidneys is reviewed, and possible therapeutic applications for kidney diseases are proposed in combination with our knowledge of the emerging field of kidney stem cell biology.
Article: Stem cells and kidney organogenesis[Show abstract] [Hide abstract] ABSTRACT: The discovery of tissue stem cells has launched the current boom in the field of regenerative research, which is tremendously exciting and holds enormous therapeutic potential. Despite such optimism, recent findings have tempered the potential for medical practice. Anatomically complicated organs, such as the kidney, have proved refractory to stem cell-based regenerative techniques. As the kidney has the capacity to regenerate after renal injury, investigations into the mechanisms underlying kidney organogenesis may provide the clues to solving the puzzle of complex organ regeneration. This article reviews the current understanding of kidney organogenesis and kidney stem cells, and discusses the potential of kidney organogenesis as a therapeutic strategy for renal failure.
- [Show abstract] [Hide abstract] ABSTRACT: Correction of anemia by erythropoietin (EPO) is often associated with a rise in blood pressure (BP; EPO-induced hypertension). Most studies regarding EPO-induced hypertension have involved evaluation using office/clinic BP (OBP). However, recent investigations suggest that BP measured at home (HBP) may be of more importance for clinical practice in hypertension. In this context, the present study addressed whether or not HBP measured in the morning could be useful to predict EPO-induced hypertension. The study involved patients with mild to moderate renal impairment who had renal anemia requiring EPO treatment. BP control was evaluated based on the relationship between OBP and HBP in the morning. The BP categories used were well-controlled BP, poorly controlled BP, hypertension with a white-coat effect (white-coat hypertension), and masked hypertension. Comparison was made of the BP categories before and after EPO treatment. Before EPO treatment, 38% of patients had well-controlled BP, 30% had poorly controlled BP, 20% had masked hypertension, and 12% had white-coat hypertension, revealing a predominance of morning hypertension (poorly controlled BP plus masked hypertension). Following EPO treatment, the prevalence of morning hypertension in patients with masked hypertension and poorly controlled BP increased significantly, by 5% (HBP in those with masked hypertension increased from 152 +/- 18 mmHg to 162 +/- 25 mmHg, and HBP in those with poorly controlled BP increased from 157 +/- 18 mmHg to 168 +/- 25 mmHg; P < 0.05 by paired t-test). And there was a significant decrease in the prevalence of the well-controlled category, by 8%, with an increased level of morning HBP (from 128 +/- 14 mmHg to 137 +/- 16 mmHg; P < 0.05 by paired t-test). In contrast, OBP remained unchanged in all groups. The development of EPO-induced hypertension was effectively predicted by HBP in the morning (from 62% to 72% before and after EPO treatment; P = 0.0031 by Wilcoxon's analysis), but not by OBP (from 42% to 47% before and after treatment; P = 0.1399). The present study indicates that, despite receiving concurrent antihypertensive therapy, the majority of patients with renal disease had morning hypertension. Furthermore, HBP in the morning can be more useful than OBP to predict the development of EPO-induced hypertension in patients with renal anemia.
- [Show abstract] [Hide abstract] ABSTRACT: A 77-year-old man with a history of hypertension and hyperuricemia was admitted to our hospital complaining of limb weakness, persistent constipation, and worsening hypertension. He had been taking a Chinese herbal remedy for allergic rhinitis for the past 10 years, together with an angiotensin-converting enzyme inhibitor (ACE-I; enalapril, 20 mg daily). After the dosage of enalapril had been reduced to 10 mg daily about 1(1/2) years before the current admission, he had developed persistent constipation. Therefore, he had started taking another traditional Chinese herbal remedy, a laxative, for the constipation, about 4 months prior to this hospitalization. Laboratory data on admission demonstrated marked metabolic alkalosis with severe hypokalemia associated with urinary wasting of potassium and chloride. A diagnosis of pseudoaldosteronism was made based upon his past history of exposure to various traditional Chinese medicines containing glycyrrhizin. Discontinuation of the Chinese remedies and supplementation of potassium successfully normalized the electrolyte imbalance and relieved all symptoms within a short time. The present case describes the occurrence of pseudoaldosteronism induced by a patient taking two traditional Chinese herbs, both containing glycyrrhizin, resulting in an overdose of this causative chemical agent. The development of pseudoaldosteronism appeared to be of particular interest with regard to the interaction of the renin-angiotensin-aldosterone (RAA) system with glycyrrhizin, in which an ACE-I retarded the development of pseudoaldosteronism.
- [Show abstract] [Hide abstract] ABSTRACT: Blood pressure (BP) measured at home early in the morning (HBP) has been recognized as a useful predictor for organ damage and has been viewed as an important therapeutic target in patients with hypertension. The present study was aimed to determine whether this notion holds true in patients with progressive renal disease. The study enrolled patients with mild to moderate renal impairment. They were all directed to record self-measured HBP to evaluate the adequacy of BP control. In addition to the conventional antihypertensive therapy, intensive treatment to more efficiently reduce elevated morning HBP was applied, especially in patients with diabetic nephropathy. The results were as follows: 1) The status of BP control assessed using HBP and office/clinic BP (OBP) shows predominance of morning hypertension. The prevalence of patients with well-controlled systolic HBP was 38%, those with poorly-controlled HBP 30%, masked hypertension 20% and white coat hypertension 12%. 2) Early morning systolic HBP in diabetics was significantly higher than that in non-diabetics. However, when evaluated on systolic OBP, both groups were comparable.3)Logistic regression analysis showed that the predictive variables to explain morning hypertension (more than 130 mmHg and increased systolic HBP) were age, amount of daily urinary protein excretion and left ventricular mass index (LVMI).4)Following conventional therapy, intensive antihypertensive therapy consisting of calcium channel blockers (CCB) and/or diuretics given in the morning, and angiotensin receptor blockers (ARB) given in the evening, together with alpha1-blockers given at bedtime, efficaciously reduced elevated HBP in the morning. This result was associated with significant reduction in daily urinary protein excretion and in serum plasminogen-activator inhibitor (PAI-1) concentration. The present study indicates that, regardless of ongoing conventional antihypertensive therapy, the majority of patients with renal disease had morning hypertension, suggesting that these patients are at a higher risk for cardiovascular disease. For the purpose of improving morning hypertension, intensive treatments with combined CCB, ARB and alpha1-blockers could have substantial benefit on the morbidity and prognosis in patients with diabetic nephropathy.