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In 2017, the University of Hong Kong and the University of California San Diego co-hosted the first Asian meeting of the recently formed Asia Pacific Research Integrity (APRI) network in Hong Kong. Aligned with planning meetings in 2015 and 2016 funded in part by the US Office of Research Integrity (ORI), the Hong Kong meeting was designed by a multi-national planning committee to address pressing challenges in research integrity: improving multi-national communication; exchanging information on managing misconduct investigations; and sharing best practices to promote research integrity. To create a sustainable, robust international partnership to promote research integrity in the region, the purpose of this 2017 meeting was to foster multi-national awareness, understanding, and opportunities for collaboration. The meeting was defined by four objectives that emerged from the previous meetings: (1) Articulate differences as well as areas of common ground; (2) Identify best or recommended practices; (3) Identify opportunities for research or collaboration; and (4) Set an APRI network agenda for coming years. The key anticipated outcome was to advance the conversation surrounding research integrity among academic institutions and regulators in Asian and Pacific Rim nations. This outcome was evidenced by meeting participation, participant satisfaction, and articulation of next steps for the APRI network.

Recently, two Japanese clinical studies of antihypertensive drugs, supported by pharmaceutical companies and published in the Lancet by groups of Japanese investigators, were later withdrawn (Editors of the Lancet, 2009, 2013a). Four other studies of one of these drugs (Editors of the Lancet, 2013b; Uzu et al., 2008; Muramatsu et al., 2012; Narumi, Hakano, and Shindo, 2011), all funded by the Swiss company Novartis that supported one of the studies, but conducted by different Japanese research groups, were later found to be tainted by misconduct unacceptable by scientific standards (Review Committee for the Recent Cases of Clinical Study on an Antihypertensive Drug, 2013). In this article, we discuss deeply-rooted ties between clinical investigators and pharmaceutical companies that underlie these recently surfaced examples of impropriety in patient-oriented research in Japan, which have been banned in most major U.S. medical centers for some time (Conflict of Interest Policies at Academic Medical Centers, 2014). Typically, companies finance many clinical studies, then pick their preferred research data, and advertise the less-than-scientific conclusions through popular professors.

The renal tissue renin-angiotensin system is activated in chronic kidney diseases. We previously demonstrated that intrarenal ANG II is synthesized primarily from liver-derived angiotensinogen filtered through the glomerulus and that podocyte injury increases the passage of angiotensinogen into the tubular lumen and generation of ANG II. In the present study, we tested the effect of cessation of glomerular filtration by ureteral obstruction on renal ANG II generation in kidneys with podocyte injury under two experimental conditions. Ureteral obstruction is known to activate the renin-angiotensin system in nonproteinuric kidneys. Transgenic mice expressing hCD25 in podocyte (NEP25) were injected with 1.25 or 10 ng/g body wt of LMB2, a hCD25-targeted immunotoxin, subjected to unilateral ureteral ligation on the following day, and euthanized 7 and 4 days later, respectively. In both experiments, compared with the kidney in untreated wild-type mice, renal angiotensinogen protein, as assessed by immunostaining and Western blot analysis, was increased in the contralateral unobstructed kidney. However, it was markedly decreased in the obstructed kidney. Whereas intrarenal ANG II content was increased in the contralateral kidney compared with the untreated kidney (248 ± 83 vs. 106 ± 21 and 298 ± 185 vs. 64.8 ± 20 fmol/g kidney, respectively), this increase was suppressed in the obstructed kidney (161 ± 75 and 113 ± 34 fmol/g kidney, respectively), a pattern opposite to what we expected in obstructed kidneys without podocyte injury. Thus, our study indicates that the major source of increased renal ANG II in podocyte injury is filtered angiotensinogen.

Intrarenal angiotensin II is increased in kidney diseases independently of plasma angiotensin II and is thought to promote progressive deterioration of renal architecture. Here we investigated the mechanism of enhanced renal angiotensin II generation in kidney glomerular diseases. For this, kidney- or liver-specific angiotensinogen gene (Agt) knockout was superimposed on the mouse model of inducible podocyte injury (NEP25). Seven days after induction of podocyte injury, renal angiotensin II was increased ninefold in NEP25 mice with intact Agt, accompanied by increases in urinary albumin and angiotensinogen excretion, renal angiotensinogen protein, and its mRNA. Kidney Agt knockout attenuated renal Agt mRNA but not renal angiotensin II, renal, or urinary angiotensinogen protein. In contrast, liver Agt knockout markedly reduced renal angiotensin II to 18.7% of that of control NEP25 mice, renal and urinary angiotensinogen protein, but not renal Agt mRNA. Renal angiotensin II had no relationship with renal Agt mRNA, or with renal renin mRNA, which was elevated in liver Agt knockouts. Kidney and liver dual Agt knockout mice showed phenotypes comparable to those of liver Agt knockout mice. Thus, increased renal angiotensin II generation upon severe podocyte injury is attributed to increased filtered angiotensinogen of liver origin resulting from loss of macromolecular barrier function of the glomerular capillary wall that occurs upon severe podocyte injury.

Background/Aims:TSPAN8 encoding tetraspanin-8 was identified as a candidate gene for immunoglobulin A nephropathy (IgAN) by a genome-wide association study using microsatellites in the Japanese population. Tetraspanin-8 is a cell surface protein that contributes to the migration and invasion of epithelial cells. Methods: We performed immunohistochemistry for tetraspanin-8 on human renal biopsy specimens associated with IgAN, antineutrophil cytoplasmic antibody-associated nephropathy and interstitial nephritis, as well as normal renal tissue. Furthermore, to study the potential function of tetraspanin-8, we performed cell migration and invasion assays using human renal tubule cells transfected with tetraspanin-8. Results: Tetraspanin-8 was often expressed in vascular smooth muscle cells and occasionally in tubule cells in normal kidney. In the kidneys of all types of nephropathy, tetraspanin-8 staining in the arteries was unaffected, but that in the tubules was enhanced. The degree of tubular staining negatively correlated with the estimated glomerular filtration rate, independently of the type of nephropathy. Tetraspanin-8-expressing tubule cells were found predominantly in distal and collecting tubules, identified by cytokeratin 7 or aquaporin 2 staining. In vitro studies using cultured tubule cells revealed that tetraspanin-8 promoted migration by 2.7-fold without laminin, by 2.8-fold with laminin and invasion into Matrigel by 3.5-fold, suggesting that enhanced tetraspanin-8 may be involved in the repair of tubules. Conclusion: The obtained findings indicate that tetraspanin-8 expression is enhanced in injured distal tubules, which may be involved in the repair of tubules by facilitating migration and invasion.

Background Regression is an important process in normal development of many organs. In the present study, we investigated whether glomerular regression occurs after normal glomerulogenesis and determined the time course for this process. Methods Glomerular number was analyzed in normal mouse kidneys at postnatal day (P) 7, 10, 14, 18, 21, 25 and 28 by the gold-standard fractionator/dissector method, which involves exhausting the kidney tissue. Vascular regression markers, angiopoietin 2 (ANGPT2) and thrombospondin 1 (THBS1), were examined by immunohistochemistry. Results The maximum glomerular number was reached at P7 with 14,051 glomeruli per kidney (95% CI: 12,084-16,018). This peak was followed by a progressive reduction with a nadir of 11,060 (10,393-11,727) occurring at P18 (p

Background: We previously showed that angiotensin type 1 receptor (AT1) blocker (ARB) attenuates glomerular injury in Nphs1-hCD25 (NEP25) transgenic mice, a model of selective podocyte injury. However, subsequent studies in NEP25 mice with podocyte-specific deficiency of AT1 revealed that the protective effects of ARB are not through the podocyte AT1, thereby raising the possibility that the protective effects of ARB involve mineralocorticoids. Methods: NEP25 mice were treated with the mineralocorticoid receptor blocker (MRB) spironolactone (25 mg/kg/day, n = 10), the ARB losartan (250 mg/kg/day, n = 11), both (ARB+MRB, n = 8) or vehicle (Vehicle, n = 9) from day –7 to day 9 of induction of podocyte injury. Results: Although MRB did not reduce systolic blood pressure or proteinuria, addition of MRB to ARB significantly attenuated glomerulosclerosis (glomerulosclerosis index: ARB+MRB 1.67 ± 0.19 vs. MRB 2.01 ± 0.29, ARB 2.35 ± 0.19, and Vehicle 2.25 ± 0.26, p < 0.05) and preserved the number of WT1-positive podocytes (ARB+MRB 152.5 ± 9.7 vs. MRB 117.2 ± 9.0 or ARB 113.6 ± 7.4, and ARB+MRB vs. Vehicle 97.5 ± 4.0 per glomerulus; p < 0.05). Conclusion: These data suggest that, while MRB does not attenuate proteinuria caused by podocyte-specific injury, it provides protective effects against glomerulosclerosis that is independent of systemic blood pressure.

Objective— Angiotensin II is a major determinant of atherosclerosis. Although macrophages are the most abundant cells in atherosclerotic plaques and express angiotensin II type 1 receptor (AT1), the pathophysiologic role of macrophage AT1 in atherogenesis remains uncertain. We examined the contribution of macrophage AT1 to accelerated atherosclerosis in an angiotensin II-responsive setting induced by uninephrectomy (UNx). Methods and Results— AT1 −/− or AT1 +/+ marrow from apolipoprotein E deficient (apoE −/− ) mice was transplanted into recipient apoE −/− mice with subsequent UNx or sham operation: apoE −/− /AT1 +/+ →apoE −/− +sham; apoE −/− /AT1 +/+ →apoE −/− +UNx; apoE −/− /AT1 −/− →apoE −/− +sham; apoE −/− /AT1 −/− →apoE −/− +UNx. No differences in body weight, blood pressure, lipid profile, and serum creatinine were observed between the 2 UNx groups. ApoE −/− /AT1 +/+ →apoE −/− +UNx had significantly more atherosclerosis (16907±21473 versus 116071±8180 μm 2 , P 2 , P =NS). Although UNx did not affect total macrophage content in the atheroma, lesions in apoE −/− /AT1 −/− →apoE −/− +UNx had fewer classically activated macrophage phenotype (M1) and more alternatively activated phenotype (M2). Further, UNx did not affect plaque necrosis or apoptosis in apoE −/− /AT1 −/− →apoE −/− whereas it significantly increased both (by 2- and 6-fold, respectively) in apoE −/− /AT1 +/+ →apoE −/− mice. Instead, apoE −/− /AT1 −/− →apoE −/− had 5-fold–increase in macrophage-associated apoptotic bodies, indicating enhanced efferocytosis. In vitro studies confirmed blunted susceptibility to apoptosis, especially in M2 macrophages, and a more efficient phagocytic function of AT1 −/− macrophages versus AT1 +/+ . Conclusion— AT1 receptor of bone marrow-derived macrophages worsens the extent and complexity of renal injury-induced atherosclerosis by shifting the macrophage phenotype to more M1 and less M2 through mechanisms that include increased apoptosis and impaired efferocytosis.

Our previous studies using puromycin aminonucleoside (PAN) established that podocyte damage leads to glomerular growth arrest during development and glomerulosclerosis later in life. The present study examined the potential benefit of maintaining podocyte-derived vascular endothelial growth factor (VEGF) in podocyte defense and survival following PAN injury using conditional transgenic podocytes and mice, in which human VEGF-A (hVEGF) transgene expression is controlled by tetracycline responsive element (TRE) promoter and reverse tetracycline transactivator (rtTA) in podocytes. In vitro experiments used primary cultured podocytes harvested from mice carrying podocin-rtTA and TRE-hVEGF transgenes, in which hVEGF can be induced selectively. Induction of VEGF in PAN-exposed podocytes resulted in preservation of intrinsic VEGF, α-actinin-4 and synaptopodin, anti-apoptotic marker Bcl-xL/Bax, as well as attenuation in apoptotic marker cleaved/total caspase-3. In vivo, compared with genotype controls, PAN-sensitive neonatal mice with physiologically relevant levels of podocyte-derived VEGF showed significantly larger glomeruli. Further, PAN-induced up-regulation of desmin, down-regulation of synaptopodin and nephrin, and disruption of glomerular morphology was significantly attenuated in VEGF-induced transgenic mice. Our data indicate that podocyte-derived VEGF provides self-preservation functions, which can rescue the cell following injury and preempt subsequent deterioration of the glomerulus in developing mice.

Urine outflow obstruction activates a variety of profibrotic factors, including the intrarenal renin-angiotensin system. However, the obstruction also nullifies the transmural hydraulic pressure difference across the glomerular capillary wall, an established inducer of glomerulosclerosis. In the present study, we investigated whether, and by what mechanism, urine outflow obstruction affects the process of progressive glomerulosclerosis. For this purpose, we tested the effect of unilateral ureteral obstruction (UUO) of 7 days duration in two distinct mouse models of glomerulosclerosis. In the human immunodeficiency virus (HIV) nephropathy model, where HIV-1 genes are selectively expressed in podocytes and develop progressive podocyte damage and glomerulosclerosis, UUO protected against sclerosis with preservation of podocytes morphologically and immunohistochemically. In contrast, the nonobstructed contralateral kidneys of these mice, as well as sham-operated HIV-1 mouse kidneys, developed severe podocyte injury and glomerulosclerosis. The protection against glomerulosclerosis imparted by ureteral obstruction was also documented in the NEP25 model of podocyte injury, in which a single injection of immunotoxin, LMB2, triggers selective podocyte injury followed by glomerulosclerosis, both of which were protected by UUO. Notably, intervention with an angiotensin II type 1 receptor antagonist provided only a partial protective effect in each of the models. These results demonstrate that urine outflow obstruction protects the glomerulus from progressive sclerosis. The results further reveal that this protection occurs at a very early stage of the pathologic process, namely, damage of podocytes.

In the present study, we tested the hypothesis that the renoprotective effect of an angiotensin receptor blocker depends on the angiotensin II type 1 (AT 1 ) receptor on podocytes. For this purpose, we generated podocyte-specific knockout mice for the AT 1 gene ( Agtr1a ) and crossed with NEP25, in which selective podocyte injury can be induced by immunotoxin, anti-Tac(Fv)-PE38. Four weeks after the addition of anti-Tac(Fv)-PE38, urinary albumin:creatinine ratio was not attenuated in Agtr1a knockout / NEP25 mice (n=18) compared with that in control NEP25 mice (n=13; 8.08±2.41 in knockout versus 4.84±0.73 in control). Both strains of mice showed similar degrees of sclerosis (0.66±0.17 versus 0.82±0.27 on a 0 to 4 scale) and downregulation of nephrin (5.78±0.45 versus 5.65±0.58 on a 0 to 8 scale). In contrast, AT 1 antagonist or an angiotensin I–converting enzyme inhibitor, but not hydralazine, remarkably attenuated proteinuria and sclerosis in NEP25 mice. Moreover, continuous angiotensin II infusion induced microalbuminuria similarly in both Agtr1a knockout and wild-type mice. Thus, angiotensin inhibition can protect podocytes and prevent the development of glomerulosclerosis independent of podocyte AT 1 . Possible mechanisms include inhibitory effects on AT 1 of other cells or through mechanisms independent of AT 1 . Our study further demonstrates that measures that directly affect only nonpodocyte cells can have beneficial effects even when sclerosis is triggered by podocyte-specific injury.

Background: Composition of nonselective proteinuria includes several endogenous ligands of Toll-like receptors (TLRs) not normally present in Bowman’s space, thus raising the possibility that TLRs are involved in proteinuria-mediated podocyte injury. Methods: Kidneys of NEP25 mice, a model of glomerular sclerosis induced by podocyte-specific injury, were immunohistochemically evaluated for the presence of fibrin/fibrinogen, which are potent ligands for TLRs. A podocyte cell line was treated with fibrinogen or lipopolysaccharides and examined for expression of cytokines. siRNAs were used to knockdown components of TLR signaling. Results: We found deposits of fibrin/fibrinogen only in the damaged podocytes of proteinuric kidneys, indicating that podocytes are exposed to these potent TLR ligands in proteinuric state. In cultured podocytes, we confirmed mRNA expressions of TLR2, TLR4, as well as their major TLR signal transducer, MyD88. Fibrinogen and lipopolysaccharides dose-dependently upregulated mRNA expressions of MCP-1, TNF-α and TLR2 in podocytes as well as increased the MCP-1 protein in the medium. Knockdown of TLR2 and TLR4 inhibited the fibrinogen-induced MCP-1 mRNA upregulation. Knockdown of MyD88 also inhibited the upregulation. Conclusion: These results suggest that plasma macromolecules that appear in Bowman’s space in proteinuric conditions have the capacity to induce podocyte cytokines through TLRs, and thereby accelerate podocyte injury.

糸球体疾患において，高分子蛋白尿の量が多いほど末期腎不全へと移行するリスクが高く，蛋白尿は腎疾患の指標であるのみでなく，さらなる腎傷害の原因となる可能性がある。 糸球体から漏出した様々な蛋白質は，主にmegalinというレセプターを介したエンドサイトーシスにより管腔側から近位尿細管細胞へと取り込まれ，ライソソームへと運ばれて分解されるか，そのまま基底膜側へと運ばれる。それにともなって，MCP-1やRANTESなどの炎症に関わるメディエーターや，TGF-βなどの線維化に関わるメディエーターが尿細管間質に放出される。また，近位尿細管のアポトーシスも惹き起こされる。このように，漏出した蛋白質が直接的な尿細管傷害の起因となることによって悪循環を形成し，慢性腎不全を進行させている可能性がある。

Acute thyroparathyroidectomy (TPTX) in rats results in a marked increase in the glomerular capillary ultrafiltration coefficient, Kf. Infusion of a submaximal dose of bovine PTH (1 U/kg/min) returns Kf to non-TPTX levels. Infusion of 10 U/kg/min of bovine PTH further reduces Kf and leads to a net reduction in single nephron GFR. These findings suggest that PTH may play an important role in modulating Kf and, consequently, GFR.

This study aimed to identify the causative gene for HIV-1 associated nephropathy, a paradigmatic podocytopathy. A previous study demonstrated that transgenic expression of nonstructural HIV-1 genes selectively in podocytes in mice with FVB/N genetic background resulted in podocyte injury and glomerulosclerosis. In this study, transgenic mice that expressed individual HIV-1 genes in podocytes were generated. Five of six transgenic mice that expressed vpr developed podocyte damage and glomerulosclerosis. Analysis of an established vpr transgenic line revealed that transgenic mice on FVB/N but not on C57BL/6 genetic background developed podocyte injury by 8 wk of age, with later glomerulosclerosis. Four of 11 transgenic mice that expressed nef also developed podocyte injury. One transgenic line was established from the nef founder mouse with the mildest phenotype. Transgenic mice in this line developed mesangial expansion at 3 wk of age and mild focal podocyte damage at 10 wk of age. Mating with FVB/N mice did not augment nephropathy. None of the transgenic mice that expressed vif, tat, rev, or vpu in podocytes, even with the FVB/N genetic background, developed podocyte injury. For testing effects of simultaneous expression of vpr and nef, these two lines were mated. All nef:vpr double-transgenic mice showed severe podocyte injury and glomerulosclerosis by 4 wk of age. In contrast, all vpr or nef single-transgenic mice in the same litter uniformly showed no or much milder podocyte injury. These findings indicate that vpr and nef each can induce podocyte injury with a prominent synergistic interaction.

Purpose of review Recognition of the dramatically expanded functional repertoire of the renin–angiotensin system now includes a role in morphogenesis of the kidney and urinary tract. On the basis of published data, the article presents formulations of mechanisms through which the system operates. Recent findings Studies in humans and animals carrying defective angiotensin-related genes have provided unequivocal evidence that the renin–angiotensin system is involved in the normal development of both the kidney and the urinary tract. Angiotensin exerts its function through at least two different types of receptors, AT1 and AT2. AT1 mediates establishment of the ureteral peristaltic machinery, while AT2 mediates the early kidney and urinary tract morphogenesis. Disruption in receptor functions promotes development of congenital anomalies of the kidney and urinary tract. Summary Angiotensin is involved in multiple steps of normal development of the kidney and urinary tract through two types of receptors. This takes place in concert with other functionally overlapping genes.

Experimentally sustained increase in angiotensin II (AngII) promotes tissue destruction in various cardiovascular disorders. We examined whether transiently heightened AngII affects subsequent atherosclerosis and aneurysm formation. AngII or saline was administered for 2 weeks to apolipoprotein E (apoE)-deficient mice. Mice were sacrificed at the end of the 2-week infusion or 6- or 14 weeks later. Short-term AngII did not affect atherosclerosis immediately following the infusion or 6 weeks later. By contrast, 14 weeks after infusion there was remarkably more atherosclerosis in previously AngII-exposed mice. Preceding the build up of atherosclerotic lesions, AngII-exposure increased mRNA expression and immunostaining of monocyte chemoattractant protein-1 (MCP-1) and its receptor, CCR2. This was followed by greater macrophage-positivity in AngII-exposed aortae. In contrast to the delayed effects on atherosclerosis, 20% of mice were found to have abdominal aneurysms at the end of AngII-exposure. This effect was not contingent on blood pressure. Moreover, despite amplification in atherosclerosis following AngII, no aneurysms were found 14 weeks later. Our studies reveal that even transient exposure to AngII primes the vessel for subsequent amplification of atherosclerosis which involves activation of MCP-1/CCR2 and influx of macrophages into the nascent atherosclerotic plaque. By contrast, transient AngII-exposure causes prompt aneurysm formation that does not parallel atherosclerosis and disappears even in the face of progressively greater atherosclerotic lesions.

Immunoglobulin A nephropathy (IgAN) is the most common form of primary glomerulonephritis in many parts of the world. Although previous genome-wide association studies (GWAS) identified the major susceptibility loci for IgAN, the causal genes currently remain unknown. We performed a GWAS using 23 465 microsatellite (MS) markers to identify genes related to IgAN in a Japanese population. A pooled sample analysis was conducted in three-stage screenings of three independent case-control populations, and after the final step of individual typing, 11 markers survived. Of these, we focused on two regions on 6p21 and 12q21 because they (i) showed the strongest relationship with IgAN, and (ii) appeared to be highly relevant to IgAN in view of several previous studies. These regions contained the HLA, TSPAN8 and PTPRR genes. This study on GWAS, using20 000 MS markers, provides a new approach regarding susceptible genes for IgAN for investigators seeking new tools for the prevention and treatment of IgAN.

PURPOSE OF REVIEW For some time, the so-called vicious cycle has been believed to underlie progression of glomerular sclerosis. This mechanism describes a circumstance when loss of some glomeruli imposes injurious stress on the remnant glomeruli. Evidence from recent genetic approaches, however, has prompted revision of this classical view and now points toward a new direction of investigations. RECENT FINDINGS Whereas experimental maneuvers that selectively injure mesangial cells have failed to induce glomerular sclerosis, genetic approaches that target visceral epithelial cells, or podocytes, in embryos and adult animals regularly produce glomerular sclerosis. Association between podocyte damage and glomerular sclerosis observed in many human diseases and animal models have identified podocyte injury as a common, if not universal, trigger leading to glomerular sclerosis. The process from podocyte injury to sclerosis is remarkably rapid, and the rate of progression depends upon the degree of initial podocyte injury. A single brief injurious stimulus on a podocyte activates a 'domino effect', whereby progressive damage of the initially hit podocyte spreads to involve cells that escaped the initial insult. SUMMARY The mouse, a species highly useful for studying the function of specific gene products, is notoriously resistant to development of glomerular sclerosis in adulthood. However, recent genetic engineering in this species has overcome this disadvantage and brought about a new dimension to our understanding of the mechanisms involved in progressive glomerular sclerosis.

Evidence that bone morphogenetic protein 4 has multiple biological functions during kidney and urinary tract development. Background We have suggested that bone morphogenetic protein 4 (BMP4), acting on the Wolffian duct and ureter epithelium, determines the budding site of the ureter by locally antagonizing ubiquitous inductive signal(s) from the metanephric mesenchyme. In the present study, we examine the effect of BMP4 on the development of metanephric and periureteral mesenchymal cells, which express the BMP type I receptor gene, Bmpr1a ( Alk3 ). Methods Urogenital tissues obtained from Bmp4 heterozygous null mutant ( Bmp4 +/-) embryos at different stages, and metanephric and ureteral tissue explants cultured in the presence of recombinant BMP4 were subjected to morphologic, immunohistochemical and in situ hybridization analyses. To examine the chemotactic activity of BMP4 for periureteral mesenchymal cells, a modified Boyden chamber assay was performed. Results Many of the kidneys of newborn Bmp4 +/- mice contained multicystic dysplastic regions. This morphology was preceded by abnormally high apoptotic activity in the metanephric mesenchyme of mutant embryos at E14.5. In whole metanephric explants, BMP4 uniformly promoted the expansion of the Pax2 -negative and weakly Foxd1 (previously Bf2 ) -positive peripheral stromal compartment of metanephric mesenchyme in the presence of fibroblast growth factor 2 (FGF2). In addition, in isolated metanephric mesenchyme, BMP4-loaded beads prevented apoptosis locally. Thus, BMP4 prevents cell death and promotes the growth of the metanephric mesenchyme. The effect of BMP4 on periureteral mesenchyme is different from its effect on metanephric mesenchyme. In utero, periureteral mesenchymal cells condense around the ureter epithelium, followed by differentiation into smooth muscle cells at a site where Bmp4 is intensely expressed. Analysis of Bmp4 +/- ureters at E15.5 reveals that the α-smooth muscle actin (α-SMA)–positive cells are low in number. In vitro, BMP4-loaded beads promote the accumulation of periureteral mesenchymal cells to form several cell layers surrounding the beads. In addition, in a Boyden chamber assay, BMP4 increases the migration of periureteral mesenchymal cells through the filter. Thus, BMP4 can serve as a chemoattractant for periureteral mesenchymal cells and induce locally the smooth muscle layer of the ureter at Bmp4 -expressing sites. Conclusion Depending on local context, BMP4 has several biological actions on the morphogenesis of different portions of the excretory system, namely, the development of the ureterovesical junction, the ureter, and the kidney.

We examined the in vivo function of the angiotensin II type 1 receptor (Agtr1) on macrophages in renal fibrosis. Fourteen days after the induction of unilateral ureteral obstruction (UUO), wild-type mice reconstituted with marrow lacking the Agtr1 gene (Agtr1(-/-)) developed more severe interstitial fibrosis with fewer interstitial macrophages than those in mice reconstituted with Agtr1(+/+) marrow. These differences were not observed at day 5 of UUO. The expression of profibrotic genes - including TGF-beta1, alpha1(I) collagen, and alpha1(III) collagen - was substantially higher in the obstructed kidneys of mice with Agtr1(-/-) marrow than in those with Agtr1(+/+) marrow at day 14 but not at day 5 of UUO. Mice with Agtr1(-/-) marrow were characterized by reduced numbers of peripheral-blood monocytes and macrophage progenitors in bone marrow. In vivo assays revealed a significantly impaired phagocytic capability in Agtr1(-/-) macrophages. In vivo treatment of Agtr1(+/+) mice with losartan reduced phagocytic capability of Agtr1(+/+) macrophages to a level comparable to that of Agtr1(-/-) macrophages. Thus, during urinary tract obstruction, the Agtr1 on bone marrow-derived macrophages functions to preserve the renal parenchymal architecture, and this function depends in part on its modulatory effect on phagocytosis.

▪ Abstract When angiotensin II or AT1 receptor is experimentally inhibited during the perinatal period, either by pharmacological intervention or genetic manipulation, the kidney develops with profound structural abnormalities. Most prominent are hypertrophy of arterial vasculatures and atrophy of the papilla. Although the mechanism by which the vascular hypertrophy occurs remains unknown, study of the atrophic papilla gives us a new clue for understanding the physiological role of angiotensin. Mutant mice completely devoid of AT1 receptor fail to develop the renal pelvis and the ureteral peristaltic movement. Normally, angiotensin and AT1 receptor are transiently up-regulated around the renal outlet at birth. Thus angiotensin II induces the peristaltic machinery during the perinatal period in a timely fashion to accommodate the dramatic increase in urine production that occurs during the transition from intra- to extra-uterine life. Further studies revealed that in adult animals angiotensin augments the peristaltic movement when the urinary tract is partially obstructed, thereby protecting the kidney from hydronephrosis. This newly discovered function of angiotensin to protect kidney architecture at the time of urine outflow obstruction is reminiscent of its similar kidney structure-protecting function that is active during arterial blood flow obstruction.

Paradigm shift from classic antatomic theories to contemporary cell biological views of CAKUT. Ectopic budding of the ureter from the Wolffian duct is the first ontogenic misstep that leads to many—if not all—congenital anomalies of the kidney and urinary tract (CAKUT). The ectopia results in hypoplastic kidney, ectopia of ureterovesical orifice, urinary outflow obstruction and/or reflux. Studies in several mutant mouse models have verified that ectopic ureteric budding indeed precedes formation of CAKUT. Often, the genes involved in navigating ureteric budding to the correct site also regulate later ontogenic events of the kidney and urinary tract. The wide spectrum of CAKUT, for example, multicystic dysplastic kidney, megaureter and atretic ureter, portray the additional important functions of these same genes that are activated at multiple sites and stages during the normal morphogenesis of the kidney and urinary tract

The adrenal cortex consists of three zones producing different hormones: zona glomerulosa (zG), zona fasciculata (zF) and zona reticularis (zR). Accumulated knowledge has revealed the contribution of transcriptional factors to the functional development of the adrenal cortex, however, the regulatory mechanisms have not yet been fully understood. We studied the expression and localization of transcriptional factors, SF-1, GATA4 and GATA6 by immunohisto-chemistry in the developmental process, establishing three zones of the rat adrenal cortex. The development of the adrenal cortex was monitored by the expression of cortical enzymes, P45011β, P450aldo, and P450scc on gestational days 15, 16, 18, 19, 20 (G15, G16, G18, G19, G20) and postnatal days 1, 2, 20 (P1, P2, P20) in Spraque-Dawley rats. GATA4 expression was observed in a limited number of cortical cells from early gestation (G15) to the neonatal period (P20) before complete establishment of the cortical zones. Nearing the final phase of zG establishment (P20), GATA4 changed its intracellular localization from the nuclei to the perinuclear region. Another noteworthy finding is that GATA6 translocated from the cytoplasm into the nuclei on G19, followed by a leaning distribution of SF-1 in zG (P1) and changing the distribution of SF-1 from zG to zF parallel to the establishment of zG (P20). In conclusion, we report that GATA4 was detected in the immature phase of the adrenal cortex in limited expression but diminished in the mature adrenal cortex. GATA4 may participate in the gene transcription required for the development of the adrenal cortex.

Changing patterns in collaborative research networks reflect a new geography of science that is increasingly shaped by interactions between established and emerging scientific powers, whose cultural and political diversity are now reflected in perhaps never before thought of interactions. The various partners in these international collaborations are likely to produce research results that should gradually lead to stronger diversity in research output, including an increase in international co-authorship. When it comes to multinational co-authored publications, among sensitive questions that may complicate these relationships is the way different research systems deal with research integrity. It is well worth examining how to accommodate different cultural traditions and normative assumptions embedded in academic practices for collaborative research networks. Given the increasingly international and interdisciplinary nature of science and engineering, any guidelines for authorship practices in international collaborations must be clear about the variations that exist across disciplines and cultures, variations which will have an impact on accepted practices and expectations for collaboration. Collaborative endeavors in research will succeed only if a broader understanding of potential obstacles to these collaborations is reached.

In wild-type mice, 2-wk administration of losartan, an angiotensin (Ang) II type 1 (AT1) receptor antagonist, along with dietary sodium restriction, resulted in an elevation of plasma aldosterone greater than that seen with sodium restriction alone (2.75 +/- 0.35 vs. 1.38 +/- 0.16 ng/ml, P < 0.01). Plasma potassium increased in sodium-restricted, losartan-treated mice (6.0 +/- 0.2 mEq/liter), while potassium remained unchanged in mice with sodium restriction alone. To study the effect of Ang II on glomerulosa cells that may operate independently of plasma potassium in situ, we used chimeric mice made of cells with or without the intact AT1A gene (Agtr1a). When animals were fed a normal diet or chronically infused with Ang II, the aldosterone synthase mRNA was detectable only in Agtr1a+/+ but not Agtr1a-/- zona glomerulosa cells. After 2 wk of sodium restriction, plasma aldosterone increased (1.51 +/- 0.27 ng/ml) and potassium remained on average at 4.5 +/- 0.2 mEq/liter, with aldosterone synthase mRNA expressed intensively in Agtr1a+/+, but not detectable in Agtr1a-/- cells. Simultaneous sodium restriction and losartan treatment caused increases in plasma potassium (5.5 +/- 0.1 mEq/liter) and aldosterone (1.84 +/- 0.38 ng/ml), with both Agtr1a-/- and Agtr1a+/+ cells intensively expressing aldosterone synthase mRNA. Thus, aldosterone production is regulated by Ang II in the adrenal gland during chronic alterations in extracellular fluid volume when plasma potassium is maintained within the normal range. In the light of a previous observation that dietary potassium restriction superimposed on sodium restriction abolished secondary hyperaldosteronism in angiotensinogen null-mutant mice, the present findings demonstrate that when the renin-Ang system is compromised, plasma potassium acts as an effective alternative mechanism for the volume homeostasis through its capacity to induce hyperaldosteronism.

Purpose: Deletion of the angiotensin type II receptor gene (Agtr2) in mice results in a spectrum of urinary tract anomalies similar to that in humans. The mechanism behind this anomalous development is poorly understood. We evaluated Agtr2 expression as it relates to normal and abnormal ureteral budding.Materials and Methods: A total of 400 wild type mice were inspected at birth for gross evidence of a urinary tract anomaly. In addition, the urinary tracts of 30 wild type embryos were evaluated at 11.0/11.5 and 13.5 weeks of gestation. These embryos were examined for ureteral budding site via section and whole mount in situ hybridization with c-ret probe and Agtr2 expression via in situ hybridization with Agtr2 riboprobe. There were 740 newborn mice homozygous for the null mutation of Agtr2 also evaluated along with 55 angiotensin type II knockout embryos at the aforementioned gestational ages.Results: All wild type newborn animals were grossly normal. Of the angiotensin type II knockout newborns 23 (3.1%...

Recent studies of the human genome and genetic engineering experiments in mice revealed that congenital anomalies of the kidney and urinary tract commonly seen in newborns in various anatomical forms are polygenic disease, that is a disease caused by simultaneous defects in multiple genes. We discuss some possible genetic explanations of the classic theories of the formation of congenital kidney and urinary tract anomalies.We reviewed classic and current theories regarding urinary tract development. Included in our review are recent results from our laboratory evaluating the genetic role of normal and abnormal urinary tract development.We observed a genetic abnormality that may explain many classic anatomical theories of congenital kidney and urinary tract anomalies. One of the genes involved in urinary tract ontogenesis is the angiotensin type 2 receptor gene, which is the "other" angiotensin receptor. While the type 1 receptor mediates essentially all known actions of angiotensin, including its hypertensive effect, relatively little is known about the angiotensin type 2 receptor. Careful dissection studies in mutant mouse embryos selectively lacking the angiotensin type 2 receptor gene revealed that this gene is pleiotropic, that is its defect causes not only ectopic ureteral budding from the wolffian duct, but also disturbance in other subsequent ontogenic events that are critical for the normal growth of the kidney and urinary tract.Many congenital anomalies of the kidney and urinary tract appear to share a common genetic cause. While these anomalies are caused by various genetic hits, abnormalities in the angiotensin type 2 receptor gene are often involved in this anomalous development. This review article offers a better understanding of the genetics involved in urinary tract development and ties some of the newly emerging genetic theories with classic anatomical theories.

Background Recent studies have reported that podocytes are postnatally generated from progenitor cells localized in Bowman's capsule or in the bone marrow. In the present study, we investigated whether or not podocyte regeneration is important in the repair of injured glomeruli after mild podocyte injury in mice. Methods Mild podocyte injury was induced in NEP25 mice (n = 8) by injecting an immunotoxin, LMB2 (0.625 ng/g body weight). Control mice, not injured by LMB2 injection (n = 7) was used as a comparison. Proliferating cells were labeled by continuous infusion of bromodeoxyuridine (BrdU). Podocytes, identified by nephrin, WT1 or podocin staining, that had incorporated BrdU were enumerated 4 weeks later. Results A total of 742 corpuscles were inspected in serial sections stained for BrdU and nephrin; 19% showed sclerosis. BrdU(+) cells were observed in both the glomeruli and Bowman's capsules, averaging 2.5 ± 3.1 in non-sclerotic corpuscles and 7.0 ± 5.8 in sclerotic corpuscles. Only one BrdU(+) cell was also positive for nephrin. Another cell, localized at a position consistent with its potential identification as a podocyte, was nephrin negative but had incorporated BrdU. WT1 staining similarly revealed that only two nuclei were doubly positive for BrdU and WT1. Additional 1676 corpuscles were inspected by double staining for BrdU and podocin; none were doubly positive. Conclusions Podocytes are not replenished by proliferation of endogenous progenitor cells in mice with glomerular injury.

Salutary role for angiotensin in partial urinary tract obstruction. Background In the neonatal period, angiotensin II (Ang II) is up-regulated and induces a timely development of the renal pelvis and ureteral peristalsis, thereby protecting the kidney from hydronephrosis. We tested the possibility that in adulthood, Ang II may act salutarily on the kidney structure during partial urinary tract obstruction by inducing adaptive changes in the peristaltic machinery. Methods Adult male Sprague-Dawley rats were subjected to partial unilateral ureteral obstruction (UUO) and divided into two groups, that is, those treated with (group L, N = 21) and those without (group C, N = 21) an angiotensin type 1 (AT1) receptor antagonist (losartan). Control animals were sham operated (N = 10). Rats were sacrificed either at day 7 or day 14. Results The degree of hydronephrosis determined morphometrically was significantly more severe in group L than group C at both day 7 and day 14, indicating that Ang II inhibition accentuated hydronephrosis. The measurement of upstream pressure within the partially ligated ureter in vivo revealed that losartan significantly attenuates the frequency of ureteral peristaltic activities. In in vitro studies using ureteral strips harvested from normal adult Sprague-Dawley rats (N = 10), Ang II (10-8 mol/L) was shown to augment contraction, which was completely inhibited by losartan (10-6 mol/L). Conclusions Ang II has a salutary effect of protecting kidneys from hydronephrosis during partial ureteral obstruction through its ability to augment ureteral peristalsis.

As the only ex utero mechanism for the removal of nitrogenous waste, the mammalian kidney achieves some 50-fold increase in urine production during the perinatal period when the placental circulation becomes no longer available as a functional dialyzer. This urine is efficiently removed from the kidney by the renal pelvis, a smooth muscle structure unique to mammals, which develops during the perinatal period. We found that mutant mice completely devoid of angiotensinogen or its type 1 receptor, as well as wild-type neonates given an ACE inhibitor, fail to develop a renal pelvis or a ureteral peristaltic movement. These structural and functional defects in the urinary tract are followed by severe obstructive injury of the renal parenchyma. The ability of angiotensin to directly induce the pelvis is demonstrated in an organ culture system, in which treatment with angiotensin induces the characteristic smooth muscle layer in the wild type, but not in homozygous null mutants. Upregulation of both renal angiotensin content and type 1 receptor at the renal hilum are also demonstrated in the wild type during the transition from intra- to extra-uterine life. By inducing the timely development of the renal pelvis, angiotensin thus facilitates the removal from the renal parenchyma of the urine that promptly increases at birth, thereby effectively preventing a buildup of intrarenal pressure and a consequent development of dysmorphic kidney.

Accumulating data show that excess of angiotensin II (Ang II) is involved in cardiac fibrosis. Many experimental studies suggested that Ang II induces cardiac fibrosis not by its blood pressure-raising action, but rather by a direct action on the heart. However, it has been difficult to distinguish the local and systemic actions in vivo. Recent genetic technology sheds new light on this problem. This review focuses on the recent advances and newly arising issues regarding the mechanism of Ang II-induced cardiac fibrosis.

CAKUT are problems that often require surgical intervention or, in the worst case, lead to renal failure and the need for dialysis and/or renal transplantation. It is believed that these anomalies share a common genetic cause and to date there has been no good animal model with which to study these abnormalities. Although the abnormal interaction between the ureteral bud and metanephric blastema leads to renal hypodysplasia, vesicoureteral reflux, and ectopic ureters to name a few, the genetic and biochemical modulation of urinary tract development is not understood. Studies using the mouse strain mutant for angiotensin type 2 (AT2) receptors have given new insight into this mystery. The animals show defective apoptosis of undifferentiated mesenchymal cells in the area surrounding the developing kidney and urinary tract. This abnormal apoptosis may well interfere with the normal interaction between the ureteral bud and metanephric blastema resulting in CAKUT. This abnormal interaction would theoretically lead to preexisting intrinsic abnormalities of the kidney, which are programmed and take effect early in embryonic development. In the worst cases, the renal abnormalities would lead to progressive deterioration of renal function. Undoubtedly, there are more genes and biochemical modulators involved in this process other than the RAS and AT2 receptors. Our current animal model gives new and unique possibilities with which to study development of the kidney and urinary tract and ultimately seek ways of preventing an often debilitating disease process.

Potent antihypertrophic effect of the bradykinin B2 receptor system on the renal vasculature. Background Angiotensin type 1 (AT1) receptor-deficient mice ( Agtr1 -/-), which selectively lack both AT1A and AT1B receptor genes, are characterized by marked intrarenal vascular thickening. In the present study, we explored the possible involvement of the kinin-kallikrein system in the development of this renal vascular hypertrophy. Methods Wild-type and Agtr1 -/- mice were examined for the developmental regulation pattern of the kinin-kallikrein system and treated with aprotinin (a kallikrein inhibitor), AcLys [D-b Nal 7 , Ile 8 ] des-Arg 9 -bradykinin (a bradykinin B1 receptor antagonist), or Hoe-140 (a bradykinin B2 receptor antagonist) from 3 to 14 days of age. Results The normal postnatal up-regulation of kininase II was organ-specifically suppressed in Agtr1 -/- kidneys at 2 and 3 weeks of age. Immunohistochemical staining in Agtr1 -/- mice revealed tissue kallikrein staining along the nephron from connecting tubules to cortical collecting tubules in proximity to the hypertrophic vasculature, whereas tissue kallikrein staining was confined to connecting tubules in wild-type mice. Aprotinin and Hoe-140 accelerated the vascular hypertrophy significantly as determined by wall thickness ratio, whereas B1 receptor antagonism had no effect. Conclusion The kinin-kallikrein system in the Agtr1 -/- mouse kidney is functionally activated by local suppression of kininase II and extensive redistribution of kallikrein to perivascular areas. This activation, specific to the kidney, serves to dampen a development of the marked vascular hypertrophy. These results demonstrate, to our knowledge for the first time, the antihypertrophic effect of the bradykinin B2 receptor system on the renal vasculature in vivo.

To characterize the mode of action of angiotensin II (Ang II) in cardiac remodeling, we generated chimeric mice that are made of both homozygous Ang II receptor type 1A gene (Agtr1a) null mutant cells and Agtr1a intact cells expressing the lacZ gene (ROSA26). Both Agtr1a null and intact myocytes and interstitial cells independently form areas that are randomly distributed throughout the heart. The distribution of ROSA26 cardiomyocytes overlaps completely with that of Ang II binding, indicating that the majority of Ang II receptors reside on cardiomyocytes. When Ang II (1 ng/g body weight/min) was infused for 2 weeks, mice developed mild to moderate hypertension. The proliferating cardiac fibroblasts identified by bromodeoxyuridine staining were present predominantly in the areas surrounded by Agtr1a intact cardiomyocytes. When control chimeric mice made of wild-type cells and ROSA26 cells (i.e., both carrying intact Agtr1a) were infused with Ang II, fibroblast proliferation was found equally in these cardiomyocyte types. When compared with Agtr1a null mutant chimeras, the control chimeras had more extensive cardiac fibrosis, most prominently in perivascular regions. Therefore, in response to Ang II, cardiac fibroblasts proliferate through both the local and systemic action of Ang II. Importantly, the former is determined by the Ang II receptor of neighboring cardiomyocytes, indicating that a communication between myocytes and fibroblasts plays an important role during Ang II-dependent cardiac remodeling.

Over the last decade, gene targeting technologies have provided investigators with powerful new tools to study the physiology and pathophysiology of the kidney. In that, the renin-angiotensin system (RAS) has been a subject of intense investigation. Detailed analyses of mutant mice have not only confirmed notions already suggested by other studies, but also shed a new light on previously unrecognized functions of RAS. In this review, we will focus on what we have learned from these gene targeted animals in particular relevance to nephrology.

Angiotensin type 2 receptor gene null mutant mice display congenital anomalies of the kidney and urinary tract (CAKUT). Various features of mouse CAKUT impressively mimic human CAKUT. Studies of the human type 2 receptor (AGTR2) gene in two independent cohorts found that a significant association exists between CAKUT and a nucleotide transition within the lariat branchpoint motif of intron 1, which perturbs AGTR2 mRNA splicing efficiency. AGTR2, therefore, has a significant ontogenic role for the kidney and urinary tract system. Studies revealed that the establishment of CAKUT is preceded by delayed apoptosis of undifferentiated mesenchymal cells surrounding the urinary tract during key ontogenic events, from the ureteral budding to the expansive growth of the kidney and ureter.

The embryonic development of mammalian kidneys is completed during the perinatal period with a dramatic increase in urine production, as the burden of eliminating nitrogenous metabolic waste shifts from the placenta to the kidney. This urine is normally removed by peristaltic contraction of the renal pelvis, a smooth muscle structure unique to placental mammals. Mutant mice completely lacking angiotensin type 1 receptor genes do not develop a renal pelvis, resulting in the buildup of urine and progressive kidney damage. In mutants the ureteral smooth muscle layer is hypoplastic and lacks peristaltic movements. We show that angiotensin can induce the ureteral smooth muscles in organ cultures of wild-type, but not mutant, ureteral tissues and that, in wild-type mice, expression of both renal angiotensin and the receptor are transiently upregulated at the renal outlet at birth. These results reveal a new role for angiotensin in the unique cellular adaptations of the mammalian kidney to the physiological stresses of postnatal life.

To examine whether angiotensin II (Ang II) can trigger apoptosis via Ang II type 2 (AT2) receptor, two genotypes of skin fibroblasts cultured from the AT2 receptor gene targeting homozygous (AT2−/−) and wild-type (AT2+/+) mouse embryos, respectively, were studied when exposed to Ang II. In the AT2+/+ fibroblasts, mRNA expression of the AT2 receptor was modulated by Ang II in a dose-dependent manner and apoptosis appeared with the convincing features of internucleosomal DNA fragmentation and DNA content decrease after stimulation with Ang II, whereas Ang II had no significant impact on the AT2−/− fibroblasts due to the AT2 receptor gene deletion. This is the first report using a gene targeting study to demonstrate that Ang II induces apoptosis through the AT2 receptor in the fibroblasts of the mouse embryo.

Plasma volume, nitrogenous wastes in the plasma, and plasma osmolality profoundly affect the function of various organs in the body, particularly the central nervous system and the heart. Some parameters are more critical for these organs than others, so that, when homeostatic mechanisms come into play, the body often defends the parameter of greatest importance at the expense of others. The hyponatremia and azotemia that develop in dehydrated infants exemplify this prioritization of the body. The primary goal of therapeutic intervention in such circumstances is, therefore, geared toward a correction of dehydration. While alterations in plasma and urinary electrolyte patterns commonly reflect adjustments made by the body's machinery, it is essential at the bedside to identify factitiously abnormal laboratory results produced by laboratory analytical machines. They measure not only biologically active but also insignificant components of plasma electrolytes and osmolality.

Angiotensinogen gene null-mutant mice lack homeostatic regulation of glomerular filtration and tubular reabsorption. Chronic volume depletion by dietary salt restriction causes marked decrease in glomerular filtration rate (GFR) with little increase in urine osmolality in angiotensinogen gene null mutant (Agt−/−) mice. Moreover, urine osmolality is insensitive to both water and vasopressin challenge. In contrast, in normal wild-type (Agt+/+) mice, GFR remains remarkably constant and urine osmolality is adjusted promptly. Changes in volume status also cause striking divergence in renal structure between Agt−/− and Agt+/+ mice. Thus, in contrast to the remarkably stable glomerular size of Agt+/+ mice, glomeruli of Agt−/− mice are atrophied during a low salt and hypertrophied during a high salt diet. Moreover, the renal papilla, a structure unique to mammals and essential for urine diluting and concentrating mechanisms, is hypoplastic in Agt−/− mice. Thus, angiotensin is essential for the two fundamental homeostatic functions of the mammalian kidney, namely stable GFR and high urine diluting and concentrating capacity during alteration in extracellular fluid (ECF) volume. This is not only accompanied by angiotensin’s tonic effects on renal vasomotor tone and tubule transporters, but also accomplished through its capacity to affect the structure of both the glomerulus and the papilla directly or indirectly.

Summary: Clinical studies revealed that angiotensin converting enzyme (ACE) inhibitor reduces proteinuria and attenuates progressive decline in renal function in IgA nephropathy. Recent studies by us and others have demonstrated that the homozygote of the D allele (DD) of the ACE insertion/deletion (I/D) polymorphism is a potential risk factor for poor prognosis in IgA nephropathy, and that this deletion polymorphism predicts the therapeutic efficacy of ACE inhibition on proteinuria and, potentially, on progressive deterioration of renal function in patients with the nephropathy.