Dysfunction of fibroblasts of extrarenal origin underlies renal fibrosis and renal anemia in mice

In chronic kidney disease, fibroblast dysfunction causes renal fibrosis and renal anemia. Renal fibrosis is mediated by the accumulation of myofibroblasts, whereas renal anemia is mediated by the reduced production of fibroblast-derived erythropoietin, a hormone that stimulates erythropoiesis. Despite their importance in chronic kidney disease, the origin and regulatory mechanism of fibroblasts remain unclear. Here, we have demonstrated that the majority of erythropoietin-producing fibroblasts in the healthy kidney originate from myelin protein zero–Cre (P0-Cre) lineage-labeled extrarenal cells, which enter the embryonic kidney at E13.5. In the diseased kidney, P0-Cre lineage-labeled fibroblasts, but not fibroblasts derived from injured tubular epithelial cells through epithelial-mesenchymal transition, transdifferentiated into myofibroblasts and predominantly contributed to fibrosis, with concomitant loss of erythropoietin production. We further demonstrated that attenuated erythropoietin production in transdifferentiated myofibroblasts was restored by the administration of neuroprotective agents, such as dexamethasone and neurotrophins. Moreover, the in vivo administration of tamoxifen, a selective estrogen receptor modulator, restored attenuated erythropoietin production as well as fibrosis in a mouse model of kidney fibrosis. These findings reveal the pathophysiological roles of P0-Cre lineage-labeled fibroblasts in the kidney and clarify the link between renal fibrosis and renal anemia

Bmp7 inhibits the differentiation of cap mesenchyme in kidney explant culture.

<p>Kidney explants were taken from Bmp7^LacZ/fl;Gt(ROSA)26Sor^CreERT2 embryos at E12.5 and cultured for 72 h in the presence or absence of 4-OHT. The expression of Bmp7 mRNA was significantly reduced in Bmp7^LacZ/fl;Gt(ROSA)26Sor^CreERT2 explants treated with 4-OHT compared to the explants from the same embryos treated with a vehicle (n = 3). Data are represented as mean ± SD. Whole explants were costained with pan-cytokeratin (green) to label ureteric buds and Jagged1 (red) to label developing nephrons. The Jagged1-positive red area was measured utilizing Photoshop software. In Bmp7^LacZ/fl;Gt(ROSA)26Sor^CreERT2 explants treated with 4-OHT, Jagged1-positive regions were significantly expanded, indicating accelerated differentiation. Scale bars: 100 µm.</p

Acceleration of nephron maturation and apoptosis of cap mesenchyme at E14.5 in Bmp7 knockout kidneys.

<p>(<b>A</b>) Pregnant mothers bearing Bmp7^LacZ/fl;Gt(ROSA)26Sor^CreERT2 and Bmp7^+/fl;Gt(ROSA)26Sor^CreERT2 embryos were administered tamoxifen at E12.5, and sacrificed at E14.5. (<b>B</b>) The expression of Bmp7 mRNA was significantly reduced at E14.5 in Bmp7^LacZ/fl;Gt(ROSA)26Sor^CreERT2 (Bmp7 knockout kidneys) (n = 4, white column) compared to control kindeys (n = 5, black column). Data are represented as mean ± SD. (<b>C and D</b>) Cap mesenchyme was maintained in both Bmp7 knockout kidneys and control kidneys. (<b>E</b>) Cap mesenchyme positive for WT1 was maintained in both genotypes. (<b>F</b>) The distribution and the number of phospho-Histone H3-positive cells were comparable in both genotypes. pHistone H3 denotes phospho-Histone H3. (<b>G</b>) TUNEL-positive cells were increased in Bmp7 knockout kidneys. (<b>H</b>) The number of TUNEL-positive mesenchymal cells per section was increased in knockout kidneys (white column) compared to control kidneys (black column). Data are represented as mean ± SD. The average numbers of TUNEL-positive mesenchymal cells in three sections were used as the values for each embryo. The mean of the values from three embryos is presented in the graph. (<b>I</b>) Bmp7 knockout kidneys exhibited inappropriately “mature” glomeruli at E14.5. (<b>J</b>) Glomeruli in the mature stages were increased in Bmp7 knockout kidneys (white column) compared to the control kidneys (black column). Criteria for the assessment of glomerular maturity are detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073554#s4" target="_blank">method</a> section. Because C stage is the most common in control kidneys at E14.5, we summed up the glomeruli of stages I, II, and III for comparison of maturation. Glomeruli were counted in 5 slices of each kidney. The mean of the values ± SD is presented in the graphs (n = 7 for control embryos, and 5 for knockout embryos). Scale bars: 100 µm (<b>C, F, G</b>) or 50 µm (<b>D, E, I</b>). n.s.: not significant.</p

Acceleration of nephron maturation and reduction of cap mesenchyme at E18.5 in Bmp7 knockout kidneys.

<p>(<b>A</b>) Pregnant mothers bearing Bmp7^LacZ/fl;Gt(ROSA)26Sor^CreERT2 and Bmp7^+/fl;Gt(ROSA)26Sor^CreERT2 embryos were administered tamoxifen at E12.5, and sacrificed at E18.5. (<b>B</b>) The expression of Bmp7 mRNA was significantly reduced in knockout kidneys (n = 4, white column) compared to control kidneys (n = 3, black column). Data are represented as mean ± SD. (<b>C and D</b>) Bmp7 knockout embryos at E18.5 exhibited small kidneys. (<b>E and F</b>) The number of cap mesenchymal cells, as shown by the immunostaining of WT1, was decreased in Bmp7 knockout kidneys. (<b>G</b>) The number of phospho-Histone H3-positive cells was reduced in Bmp7 knockout kidneys. pHistone H3 denotes phospho-Histone H3. (<b>H</b>) TUNEL-positive mesenchymal cells were increased in Bmp7 knockout kidneys. (<b>I</b>) The number of TUNEL-positive mesenchymal cells per section was increased in knockout kidneys (white column) compared to control kidneys (black column). Data are represented as mean ± SD. The average numbers of TUNEL-positive mesenchymal cells in three sections were used as the values for each embryo. The mean of the values from three embryos is presented in the graph. (<b>J</b>) Inappropriately “mature” glomeruli were observed in Bmp7 knockout kidneys. (<b>K</b>) Glomeruli in the mature stages were increased in Bmp7 knockout kidneys (white column) compared to the control kidneys (black column). Criteria for the assessment of glomerular maturity are detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073554#s4" target="_blank">method</a> section. Because stage I is the most common in control kidneys at E18.5, we summed up the glomeruli of stages II and III for comparison of maturation. Glomeruli were counted in 3 slices of each kidney. The mean of the values ± SD is presented in the graphs (n = 4 for control embryos, and 6 for knockout embryos). (<b>L</b>) Kidneys were stained with LTA (green) and WT1 (red) to label proximal tubules and glomeruli, respectively. The volume of LTA-positive proximal tubule sections in knockout kidneys was comparable to the control kidneys, whereas the number of glomeruli was significantly reduced. (<b>M</b>) The number of LTA⁺ proximal tubule cross sections normalized by the number of WT1⁺ glomeruli was increased in knockout kidneys (white column) compared to control kidneys (black column). Data are represented as mean ± SD. At least three sections were stained for each kidney. The sum of the number of LTA-positive proximal tubule cross sections was divided by the sum of the number of WT1-positive glomeruli. The mean of the values from four embryos is presented in the graph. Scale bars: 100 µm (<b>D, G, L</b>) or 50 µm (<b>E, F, H, J</b>).</p

Bmp7 inhibits the epithelialization of cap mesenchyme in colony-forming assay.

<p>(<b>A</b>) Administration of Bmp7 dose-dependently inhibited the formation of sheetlike colonies of cap mesenchyme (n = 3). Data are represented as mean ± SD. Scale bars: 100 µm. The expression of E-cadherin, but not vimentin, in these colonies was confirmed by immunostaining. Because the colonies were formed on the feeder cells, some DAPI-positive nuclei were observed outside of the colony. (<b>B</b>) Simultaneous administration of Noggin (300 ng/ml) reversed the inhibitory effect of Bmp7 (10 ng/ml) on colony formation (n = 3). Data are represented as mean ± SD. *: P<0.001. Scale bars: 100 µm.</p

ADAMTS-13: A Prognostic Biomarker for Portal Vein Thrombosis in Japanese Patients with Liver Cirrhosis

Portal vein thrombosis (PVT), one of the most prevalent hepatic vascular conditions in patients with liver cirrhosis (LC), is associated with high mortality rates. An imbalance between a disintegrin-like metalloproteinase with thrombospondin type-1 motifs 13 (ADAMTS-13) enzyme and von Willebrand factor (VWF) is responsible for hypercoagulability, including spontaneous thrombus formation in blood vessels. Herein, we aimed to identify potential prognostic and diagnostic biomarkers in Japanese patients with LC and PVT. In total, 345 patients were divided into two groups: 40 patients who developed PVT (PVT group) and 305 who did not develop PVT (NPVT group). Among the 345 patients with LC, 81% (279/345) were deemed ineligible due to the presence of preventive comorbidities, active or recent malignancies, and organ dysfunction. The remaining 66 patients were divided into two groups: the PVT group (n = 33) and the NPVT group (n = 33). Plasma ADAMTS-13 activity (ADAMTS-13:AC) and the vWF antigen (VWF:Ag) were measured using enzyme-linked immunosorbent assays. Contrast-enhanced, three-dimensional helical computed tomography (CT) was used to detect and characterize PVT. ADAMTS-13:AC was significantly lower in the PVT group than in the NPVT group. No significant differences in plasma vWF:Ag or liver stiffness were observed between the two groups. ADAMTS-13:AC of p < 0.002). The receiver operating characteristic analysis of ADAMTS-13:AC revealed an area under the curve of 0.913 in PVT detection. Patients with PVT having ADAMTS-13:AC ≥18.8 (n = 17) had higher albumin levels and better prognoses than those with ADAMTS-13:AC <18.8 (n = 16). No significant correlations of ADAMTS-13:AC levels with either fibrin degradation product or D-dimer levels were observed. ADAMTS-13:AC levels could be potential diagnostic and prognostic biomarkers for PVT in Japanese patients with LC

Loss of the BMP antagonist USAG-1 ameliorates disease in a mouse model of the progressive hereditary kidney disease Alport syndrome

The glomerular basement membrane (GBM) is a key component of the filtering unit in the kidney. Mutations involving any of the collagen IV genes (COL4A3, COL4A4, and COL4A5) affect GBM assembly and cause Alport syndrome, a progressive hereditary kidney disease with no definitive therapy. Previously, we have demonstrated that the bone morphogenetic protein (BMP) antagonist uterine sensitization–associated gene-1 (USAG-1) negatively regulates the renoprotective action of BMP-7 in a mouse model of tubular injury during acute renal failure. Here, we investigated the role of USAG-1 in renal function in Col4a3–/– mice, which model Alport syndrome. Ablation of Usag1 in Col4a3–/– mice led to substantial attenuation of disease progression, normalization of GBM ultrastructure, preservation of renal function, and extension of life span. Immunohistochemical analysis revealed that USAG-1 and BMP-7 colocalized in the macula densa in the distal tubules, lying in direct contact with glomerular mesangial cells. Furthermore, in cultured mesangial cells, BMP-7 attenuated and USAG-1 enhanced the expression of MMP-12, a protease that may contribute to GBM degradation. These data suggest that the pathogenetic role of USAG-1 in Col4a3–/– mice might involve crosstalk between kidney tubules and the glomerulus and that inhibition of USAG-1 may be a promising therapeutic approach for the treatment of Alport syndrome

Twisted Gastrulation, a BMP Antagonist, Exacerbates Podocyte Injury

<div><p>Podocyte injury is the first step in the progression of glomerulosclerosis. Previous studies have demonstrated the beneficial effect of bone morphogenetic protein 7 (Bmp7) in podocyte injury and the existence of native Bmp signaling in podocytes. Local activity of Bmp7 is controlled by cell-type specific Bmp antagonists, which inhibit the binding of Bmp7 to its receptors. Here we show that the product of <i>Twisted gastrulation</i> (Twsg1), a Bmp antagonist, is the central negative regulator of Bmp function in podocytes and that <i>Twsg1</i> null mice are resistant to podocyte injury. Twsg1 was the most abundant Bmp antagonist in murine cultured podocytes. The administration of Bmp induced podocyte differentiation through Smad signaling, whereas the simultaneous administration of Twsg1 antagonized the effect. The administration of Bmp also inhibited podocyte proliferation, whereas simultaneous administration of Twsg1 antagonized the effect. Twsg1 was expressed in the glomerular parietal cells (PECs) and distal nephron of the healthy kidney, and additionally in damaged glomerular cells in a murine model of podocyte injury. <i>Twsg1</i> null mice exhibited milder hypoalbuminemia and hyperlipidemia, and milder histological changes while maintaining the expression of podocyte markers during podocyte injury model. Taken together, our results show that Twsg1 plays a critical role in the modulation of protective action of Bmp7 on podocytes, and that inhibition of Twsg1 is a promising means of development of novel treatment for podocyte injury.</p></div