Unilateral ureteral obstruction attenuates intrarenal angiotensin II generation induced by podocyte injury.

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.

Genome-wide association study of IgA nephropathy using 23 465 microsatellite markers in a Japanese population.

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, using >20 000 MS markers, provides a new approach regarding susceptible genes for IgAN for investigators seeking new tools for the prevention and treatment of IgAN.

Podocyte injury enhances filtration of liver-derived angiotensinogen and renal angiotensin II generation.

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.

Mice are unable to endogenously regenerate podocytes during the repair of immunotoxin-induced glomerular injury.

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.

Foxc1 gene null mutation causes ectopic budding and kidney hypoplasia but not dysplasia.

BACKGROUND: Mice carrying the null-mutated Foxc1 gene frequently develop an anomalous double collecting system. These mice provide an ideal opportunity to specify the role of ectopic budding in the development of congenital anomalies of the kidney and urinary tract. METHODS: Tissue specimens were collected from Foxc1(ch/ch) mutants at several embryonic stages and at birth. The upper and lower pole kidneys were qualitatively and quantitatively examined by histology, in situ hybridization and immunohistochemistry. RESULTS: Upper pole kidneys of newborn Foxc1(ch/ch) mice were significantly more hypoplastic and contained significantly fewer glomeruli than their lower pole counterparts. On embryonic day 14.5, the stage immediately before the formation of the first urine, the upper pole kidney was already smaller than the lower pole kidney. Neither histology nor immunostaining for kidney markers showed dysplastic regions in either kidney of newborn Foxc1(ch/ch) mice. Of note, expression of Foxc1 was restricted to maturing podocytes and was not detectable in any intermediate structure of nephron development in the nephrogenic zone. CONCLUSION: Ectopic budding alone results only in kidney hypoplasia but not dysplasia. The development of dysplasticity in the maturing kidney involves gene(s) that function beyond the initial budding stage within the metanephros.

Liver angiotensinogen is the primary source of renal angiotensin II.

Angiotensin II content in the kidney is much higher than in the plasma, and it increases more in kidney diseases through an uncertain mechanism. Because the kidney abundantly expresses angiotensinogen mRNA, transcriptional dysregulation of angiotensinogen within the kidney is one potential cause of increased renal angiotensin II in the setting of disease. Here, we observed that kidney-specific angiotensinogen knockout mice had levels of renal angiotensinogen protein and angiotensin II that were similar to those levels of control mice. In contrast, liver-specific knockout of angiotensinogen nearly abolished plasma and renal angiotensinogen protein and renal tissue angiotensin II. Immunohistochemical analysis in mosaic proximal tubules of megalin knockout mice revealed that angiotensinogen protein was incorporated selectively in megalin-intact cells of the proximal tubule, indicating that the proximal tubule reabsorbs filtered angiotensinogen through megalin. Disruption of the filtration barrier in a transgenic mouse model of podocyte-selective injury increased renal angiotensin II content and markedly increased both tubular and urinary angiotensinogen protein without an increase in renal renin activity, supporting the dependency of renal angiotensin II generation on filtered angiotensinogen. Taken together, these data suggest that liver-derived angiotensinogen is the primary source of renal angiotensinogen protein and angiotensin II. Furthermore, an abnormal increase in the permeability of the glomerular capillary wall to angiotensinogen, which characterizes proteinuric kidney diseases, enhances the synthesis of renal angiotensin II.

Maturational regression of glomeruli determines the nephron population in normal mice.

BACKGROUND: Regression is an important process in the normal development of many organs. In this 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, P10, P14, P18, P21, P25, and P28 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% confidence interval: 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 < 0.05 as compared with P7). Thereafter, glomerular number remained constant. Complementary immunohistochemical examination of vascular regression markers showed peak expression of glomerular ANGPT2 and THBS1 at P14. CONCLUSION: Our study reveals that the tissue- and time-saving Weibel-Gomez method commonly used to assess glomerular number is valid only after P18. The data indicate that regulation of glomerular number by regression occurs in normally maturing mouse kidneys. These findings suggest that the process of glomerular regression could be therapeutically targeted to prevent oligonephronia, which otherwise predisposes to chronic kidney disease.

ARB protects podocytes from HIV-1 nephropathy independently of podocyte AT1.

BACKGROUND: Angiotensin I-converting enzyme inhibitors and angiotensin receptor blockers protect podocytes more effectively than other anti-hypertensive drugs. Transgenic rats overexpressing angiotensin II Type 1 (AT1) receptor selectively in podocytes have been shown to develop glomerulosclerosis. The prevailing hypothesis is that angiotensin II has a capacity of directly acting on the AT1 receptor of podocytes to induce injury. We therefore investigated the mechanism of reno-protective effect of AT1 receptor in a mouse model of HIV-1 nephropathy. METHODS: We generated transgenic mice carrying the HIV-1 gene (control/HIV-1) or both HIV-1 gene and podocyte-selectively nullified AT1 gene (AT1KO/HIV-1). In these mice, we measured urinary protein or albumin excretion and performed histological analysis. RESULTS: At 8 months of age, AT1KO/HIV-1 (n = 13) and control/HIV-1 (n = 15) mice were statistically indistinguishable with respect to urinary albumin/creatinine ratio (median 2.5 versus 9.1 mg/mg), glomerulosclerosis (median 0.63 versus 0.45 on 0-4 scale) and downregulation of nephrin (median 6.90 versus 7.02 on 0-8 scale). In contrast to the observed lack of effect of podocyte-specific AT1KO, systemic AT1 inhibition with AT1 blocker (ARB) significantly attenuated proteinuria and glomerulosclerosis in HIV-1 mice. CONCLUSION: These results indicate that the protective effect of ARB is mediated through its receptors on cells other than podocytes, such as efferent arteriolar smooth muscle cells.

Macrophage polarization by angiotensin II-type 1 receptor aggravates renal injury-acceleration of atherosclerosis.

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<0.05). By contrast, loss of macrophage AT1 which reduced local AT1 expression, prevented any effect of UNx on atherosclerosis (77174±9947 versus 75714±11333 µm(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(+/+). CONCLUSIONS: 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.

Podocyte injury damages other podocytes.

Loss of podocytes promotes glomerulosclerosis, but whether this results from a continued primary insult or a secondary mechanism triggered by the initial loss of podocytes is unknown. We generated chimeric mice in which only a subpopulation of podocytes expressed hCD25, which is the receptor for the immunotoxin LMB2. In addition, genetic labeling of hCD25-negative cells with human placental alkaline phosphatase allowed the study of these two distinct podocyte populations. Administration of LMB2 did not cause podocyte injury in hCD25-negative control mice. In contrast, LMB2 severely damaged or sloughed off the subpopulation of hCD25-positive podocytes within the chimeric glomeruli. Moreover, hCD25-negative podocytes, which were immune to the initial toxin injury, developed injury as early as 4 d after LMB2 injection, evidenced by foot process effacement, upregulation of desmin, and downregulation of nephrin, podocin, and podocalyxin. Furthermore, the magnitude of secondary injury correlated with the magnitude of primary injury, supporting the concept of an amplified cascade of podocyte injury. In conclusion, podocyte damage can propagate injury by triggering secondary damage of "remnant" intact podocytes, even when the primary insult is short-lived. This transmission of podocyte injury may form a vicious cycle leading to accelerated podocyte deterioration and glomerulosclerosis.

Glomerular sclerosis is prevented during urinary tract obstruction due to podocyte protection.

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.

Induction of podocyte-derived VEGF ameliorates podocyte injury and subsequent abnormal glomerular development caused by puromycin aminonucleoside.

Our previous studies using puromycin aminonucleoside (PAN) established that podocyte damage leads to glomerular growth arrest during development and glomerulosclerosis later in life. This study examined the potential benefit of maintaining podocyte-derived VEGF in podocyte defense and survival after 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, antiapoptotic 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. Furthermore, PAN-induced up-regulation of desmin, down-regulation of synaptopodin and nephrin, and disruption of glomerular morphology were significantly attenuated in VEGF-induced transgenic mice. Our data indicate that podocyte-derived VEGF provides self-preservation functions, which can rescue the cell after injury and preempt subsequent deterioration of the glomerulus in developing mice.

Oral activated charcoal adsorbent (AST-120) ameliorates extent and instability of atherosclerosis accelerated by kidney disease in apolipoprotein E-deficient mice.

BACKGROUND: Accelerated atherosclerosis and increased cardiovascular events are not only more common in chronic kidney disease (CKD) but are more resistant to therapeutic interventions effective in the general population. The oral charcoal adsorbent, AST-120, currently used to delay start of dialysis, reduces circulating and tissue uremic toxins, which may contribute to vasculopathy, including atherosclerosis. We, therefore, investigated whether AST-120 affects CKD-induced atherosclerosis. METHODS: Apolipoprotein E-deficient mice, a model of atherosclerosis, underwent uninephrectomy, subtotal nephrectomy or sham operation at 8 weeks of age and were treated with AST-120 after renal ablation. Atherosclerosis and its characteristics were assessed at 25 weeks of age. RESULTS: Uninephrectomy and subtotal nephrectomised mice had significantly increased acceleration of atherosclerosis. AST-120 treatment dramatically reduced the atherosclerotic burden in mice with kidney damage, while there was no beneficial effect in sham-operated mice. The benefit was independent of blood pressure, serum total cholesterol or creatinine clearance. AST-120 significantly decreased necrotic areas and lessened aortic deposition of the uremic toxin indoxyl sulfate without affecting lesional macrophage or collagen content. Furthermore, AST-120 lessened aortic expression of monocyte chemoattractant protein-1, tumor necrosis factor-α and interleukin-1ß messenger RNA. CONCLUSIONS: AST-120 lessens the extent of atherosclerosis induced by kidney injury and alters lesion characteristics in apolipoprotein E-deficient mice, resulting in plaques with a more stable phenotype with less necrosis and reduced inflammation.

Genetic podocyte lineage reveals progressive podocytopenia with parietal cell hyperplasia in a murine model of cellular/collapsing focal segmental glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS) is a progressive renal disease, and the glomerular visceral cell hyperplasia typically observed in cellular/collapsing FSGS is an important pathological factor in disease progression. However, the cellular features that promote FSGS currently remain obscure. To determine both the origin and phenotypic alterations in hyperplastic cells in cellular/collapsing FSGS, the present study used a previously described FSGS model in p21-deficient mice with visceral cell hyperplasia and identified the podocyte lineage by genetic tagging. The p21-deficient mice with nephropathy showed significantly higher urinary protein levels, extracapillary hyperplastic indices on day 5, and glomerular sclerosis indices on day 14 than wild-type controls. X-gal staining and immunohistochemistry for podocyte and parietal epithelial cell (PEC) markers revealed progressive podocytopenia with capillary collapse accompanied by PEC hyperplasia leading to FSGS. In our investigation, non-tagged cells expressed neither WT1 nor nestin. Ki-67, a proliferation marker, was rarely associated with podocytes but was expressed at high levels in PECs. Both terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and electron microscopy failed to show evidence of significant podocyte apoptosis on days 5 and 14. These findings suggest that extensive podocyte loss and simultaneous PEC hyperplasia is an actual pathology that may contribute to the progression of cellular/collapsing FSGS in this mouse model. Additionally, this is the first study to demonstrate the regulatory role of p21 in the PEC cell cycle.

Renal dysfunction potentiates foam cell formation by repressing ABCA1.

OBJECTIVE: Patients with chronic kidney disease (CKD) have the highest risk for atherosclerotic cardiovascular disease (CVD). Current interventions have been insufficiently effective in lessening excess incidence and mortality from CVD in CKD patients versus other high-risk groups. The mechanisms underlying the heightened risk remain obscure but may relate to differences in CKD-induced atherogenesis, including perturbation of macrophage cholesterol trafficking. METHODS AND RESULTS: We examined the impact of renal dysfunction on macrophage cholesterol homeostasis in the apoE(-/-) mouse model of atherosclerosis. Renal impairment induced by uninephrectomy dramatically increased macrophage cholesterol content, linked to striking impairment of macrophage cholesterol efflux. This blunted efflux was associated with downregulation of the cholesterol transporter ATP-binding cassette transporter A1 (ABCA1) and activation of the nuclear factor-kappa B (NF-kappaB). Treatment with the angiotensin receptor blocker (ARB) losartan decreased NF-kappaB and restored cholesterol efflux. CONCLUSIONS: Our findings show that mild renal dysfunction perturbs macrophage lipid homeostasis by inhibiting cholesterol efflux, mediated by decreased ABCA1 transporter and activation of NF-kappaB, and that ARB can restore cholesterol efflux.

Fibrinogen that appears in Bowman's space of proteinuric kidneys in vivo activates podocyte Toll-like receptors 2 and 4 in vitro.

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-alpha 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.

Toward global standardization of conducting fair investigations of allegations of research misconduct.

In the United States, through nation-wide discussions, the procedures for handling allegations of research misconduct are now well established. Procedures are geared toward carefully treating both complainants and respondents fairly in accordance with the US framework. Other countries, which have their own cultural and legal framework, also need fair and legally compatible procedures for conducting investigations of allegations of research misconduct. Given the rapid growth of international collaboration in research, it is desirable to have a global standard, or common ground, for misconduct investigations. Institutions need clear guidance on important subjects such as what information should be included in the investigation reports, how the investigation committee should be organized once research misconduct allegation has been received, how to conduct the investigation, how the data and information obtained should be taken as evidence for vs. against misconduct, and what policies the investigation committee should follow. We explore these issues from the viewpoint of members of committees investigating accusations of research misconduct (hereafter referred to as "investigation committees") as well as persons overseeing the committees in Japan. We hope to engender productive discussions among experts in misconduct investigations, leading to a formulation of international standards for such investigation.

First meeting in Asia of the Asia Pacific Research Integrity network.

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.

Bmp in podocytes is essential for normal glomerular capillary formation.

Bone morphogenetic protein (BMP) 4 exerts multiple biological effects on kidney and ureter development. To examine the role of BMP4 in glomerular morphogenesis, we generated transgenic mice with altered BMP4 function in podocytes by conferring tissue-specificity with the nephrin (Nphs1) promoter. At birth, Tg(Nphs1-Nog) mice, which had loss of BMP4 function in podocytes, were found to have glomerular microaneurysms, collapsed glomerular capillary tufts, enlarged Bowman's capsules, and fewer normal proximal tubules. Conversely, Tg(Nphs1-Bmp4) mice, which had increased BMP4 function in podocytes, demonstrated defects in glomerular capillary formation, but podocytes were not appreciably affected. The Tg(Nphs1-Nog) and Tg(Nphs1-Bmp4) mice shared morphological characteristics with the previously reported podocyte-specific Vegf-A over-expressing and knockout mice, respectively. Consistent with the morphological similarity, in situ hybridization revealed an intense signal for podocyte expression of Vegf in Tg(Nphs1-Nog) mice, whereas the signal was markedly suppressed in Tg(Nphs1-Bmp4) mice. However, in vitro studies with metanephroi failed to demonstrate a direct interaction between BMP4 or Noggin and VEGF in podocytes. Instead, immunostaining showed that phosphorylated Smads, the mediators of BMP signaling, are present in endothelial and/or mesangial cells, but not in podocytes, within the developing glomeruli. Therefore, this study suggests that podocyte-derived BMP plays an important role in glomerular capillary formation, perhaps by acting on non-podocyte glomerular cells in a paracrine fashion.

Megalin contributes to the early injury of proximal tubule cells during nonselective proteinuria.

Megalin, a member of the LDL receptor family, is expressed on the apical membrane of proximal tubules and serves as an endocytic scavenger of filtered proteins and hence might contribute to the tubule injury as a consequence of glomerular disease. To study its role, we crossed megalin knockout mosaic mice (lacking megalin expression in 60% of proximal tubule cells) with NEP25 mice (a transgenic line expressing human CD25 in the podocyte). Treatment of this transgenic mouse with the immunotoxin causes nephrotic syndrome, focal segmental glomerulosclerosis and tubule-interstitial injury. Following this treatment, the double transgenic mice had massive non-selective proteinuria and mild glomerular and tubular injury. Comparison of megalin-containing to megalin-deficient proximal tubule cells within each kidney showed that albumin, immunoglobulin light chain, IgA and IgG were preferentially accumulated in proximal tubule cells expressing megalin. Tubule injury markers such as heme-oxygenase-1, monocyte chemoattractant protein-1 and cellular apoptosis were also preferentially found in these megalin-expressing cells. These results show that megalin plays a pivotal role in the reabsorption of small to large molecular size proteins and provides direct in vivo evidence that reabsorption of filtered proteins triggers events leading to tubule injury.