Seiji Kishi

Nephron organoids derived from human pluripotent stem cells model kidney development and injury

Kidney cells and tissues derived from human pluripotent stem cells (hPSCs) may enable organ regeneration, disease modeling and drug screening. We report an efficient, chemically defined protocol for differentiating hPSCs into multipotent nephron progenitor cells (NPCs) that can form nephron-like structures. By recapitulating metanephric kidney development in vitro, we generate SIX2+SALL1+WT1+PAX2+ NPCs with 90% efficiency within 9 days of differentiation. The NPCs possess the developmental potential of their in vivo counterparts and form PAX8+LHX1+ renal vesicles that self-organize into nephron structures. In both two- and three-dimensional culture, NPCs form kidney organoids containing epithelial nephron-like structures expressing markers of podocytes, proximal tubules, loops of Henle and distal tubules in an organized, continuous arrangement that resembles the nephron in vivo. We also show that this organoid culture system can be used to study mechanisms of human kidney development and toxicity.

Activation of Bone Morphogenetic Protein 4 Signaling Leads to Glomerulosclerosis That Mimics Diabetic Nephropathy

Diabetic nephropathy (DN) is the most common cause of chronic kidney disease. We have previously reported that Smad1 transcriptionally regulates the expression of extracellular matrix (ECM) proteins in DN. However, little is known about the regulatory mechanisms that induce and activate Smad1. Here, bone morphogenetic protein 4 (Bmp4) was found to up-regulate the expression of Smad1 in mesangial cells and subsequently to phosphorylate Smad1 downstream of the advanced glycation end product-receptor for advanced glycation end product signaling pathway. Moreover, Bmp4 utilized Alk3 and affected the activation of Smad1 and Col4 expressions in mesangial cells. In the diabetic mouse, Bmp4 was remarkably activated in the glomeruli, and the mesangial area was expanded. To elucidate the direct function of Bmp4 action in the kidneys, we generated transgenic mice inducible for the expression of Bmp4. Tamoxifen treatment dramatically induced the expression of Bmp4, especially in the glomeruli of the mice. Notably, in the nondiabetic condition, the mice exhibited not only an expansion of the mesangial area and thickening of the basement membrane but also remarkable albuminuria, which are consistent with the distinct glomerular injuries in DN. ECM protein overexpression and activation of Smad1 in the glomeruli were also observed in the mice. The mesangial expansion in the mice was significantly correlated with albuminuria. Furthermore, the heterozygous Bmp4 knock-out mice inhibited the glomerular injuries compared with wild type mice in diabetic conditions. Here, we show that BMP4 may act as an upstream regulatory molecule for the process of ECM accumulation in DN and thereby reveals a new aspect of the molecular mechanisms involved in DN.

SOX9 protein induces a chondrogenic phenotype of mesangial cells and contributes to advanced diabetic nephropathy.

Diabetic nephropathy (DN) is the most important chronic kidney disease. We previously reported that Smad1 transcriptionally regulates the expression of extracellular matrix in DN. Phenotypic change in mesangial cells (MCs) is a key pathologic event in the progression of DN. The aim of this study is to investigate a novel mechanism underlying chondrogenic phenotypic change in MCs that results in the development of DN. MCs showed chondrogenic potential in a micromass culture, and BMP4 induced the expression of chondrocyte markers (SRY-related HMG Box 9 (SOX9) and type II collagen (COL2)). Advanced glycation end products induced the expression of chondrocyte marker proteins downstream from the BMP4-Smad1 signaling pathway in MCs. In addition, hypoxia also induced the expression of BMP4, hypoxia-inducible factor-1α (HIF-1α), and chondrocyte markers. Overexpression of SOX9 caused ectopic expression of proteoglycans and COL2 in MCs. Furthermore, forced expression of Smad1 induced chondrocyte markers as well. Dorsomorphin inhibited these inductions. Glomerular expressions of HIF-1α, BMP4, and chondrocyte markers were observed in diabetic nephropathy mice. These positive stainings were observed in mesangial sclerotic lesions. SOX9 was partially colocalized with HIF-1α and BMP4 in diabetic glomeruli. BMP4 knock-in transgenic mice showed not only similar pathological lesions to DN, but also the induction of chondrocyte markers in the sclerotic lesions. Here we demonstrate that HIF-1α and BMP4 induce SOX9 expression and subsequent chondrogenic phenotype change in DN. The results suggested that the transdifferentiation of MCs into chondrocyte-like cells in chronic hypoxic stress may result in irreversible structural change in DN.

Scleraxis modulates bone morphogenetic protein 4 (BMP4)-Smad1 protein-smooth muscle α-actin (SMA) signal transduction in diabetic nephropathy.

Background: Activated mesangial cells exhibit SMA and contribute to the progression of diabetic nephropathy. Results: Scleraxis negatively regulated the AGE-induced expression and secretion of BMP4. Conclusion: Scleraxis and Id1 are involved in the BMP4-SMA pathway and modulate phenotypic changes. Significance: Deeper insight into the impact of regulatory mechanism of scleraxis-BMP4-Smad1 signal activation might help to prevent diabetic glomerular damage. Activation of mesangial cells (MCs), which is characterized by induction of smooth muscle α-actin (SMA) expression, contributes to a key event in various renal diseases; however, the mechanisms controlling MC differentiation are still largely undefined. Activated Smad1 induced SMA in a dose-dependent manner in MCs. As a direct regulating molecule for SMA, we identified and characterized scleraxis (Scx) as a new phenotype modulator in advanced glycation end product (AGE)-exposed MCs. Scx physically associated with E12 and bound the E-box in the promoter of SMA and negatively regulated the AGE-induced SMA expression. Scx induced expression and secretion of bone morphogenetic protein 4 (BMP4), thereby controlling the Smad1 activation in AGE-treated MCs. In diabetic mice, Scx was concomitantly expressed with SMA in the glomeruli. Inhibitor of differentiation 1 (Id1) was further induced by extended treatment with AGE, thereby dislodging Scx from the SMA promoter. These data suggest that Scx and Id1 are involved in the BMP4-Smad1-SMA signal transduction pathway besides the TGFβ1-Smad1-SMA signaling pathway and modulate phenotypic changes in MCs in diabetic nephropathy.

Bone morphogenetic protein 4 and Smad1 mediate extracellular matrix production in the development of diabetic nephropathy

Diabetic nephropathy is the leading cause of end-stage renal disease. It is pathologically characterized by the accumulation of extracellular matrix in the mesangium, of which the main component is α1/α2 type IV collagen (Col4a1/a2). Recently, we identified Smad1 as a direct regulator of Col4a1/a2 under diabetic conditions in vitro. Here, we demonstrate that Smad1 plays a key role in diabetic nephropathy through bone morphogenetic protein 4 (BMP4) in vivo. Smad1-overexpressing mice (Smad1-Tg) were established, and diabetes was induced by streptozotocin. Nondiabetic Smad1-Tg did not exhibit histological changes in the kidney; however, the induction of diabetes resulted in an ∼1.5-fold greater mesangial expansion, consistent with an increase in glomerular phosphorylated Smad1. To address regulatory factors of Smad1, we determined that BMP4 and its receptor are increased in diabetic glomeruli and that diabetic Smad1-Tg and wild-type mice treated with a BMP4-neutralizing antibody exhibit decreased Smad1 phosphorylation and ∼40% less mesangial expansion than those treated with control IgG. Furthermore, heterozygous Smad1 knockout mice exhibit attenuated mesangial expansion in the diabetic condition. The data indicate that BMP4/Smad1 signaling is a critical cascade for the progression of mesangial expansion and that blocking this signal could be a novel therapeutic strategy for diabetic nephropathy.

Meclizine Preconditioning Protects the Kidney Against Ischemia-Reperfusion Injury.

Global or local ischemia contributes to the pathogenesis of acute kidney injury (AKI). Currently there are no specific therapies to prevent AKI. Potentiation of glycolytic metabolism and attenuation of mitochondrial respiration may decrease cell injury and reduce reactive oxygen species generation from the mitochondria. Meclizine, an over-the-counter anti-nausea and -dizziness drug, was identified in a ‘nutrient-sensitized’ chemical screen. Pretreatment with 100 mg/kg of meclizine, 17 h prior to ischemia protected mice from IRI. Serum creatinine levels at 24 h after IRI were 0.13 ± 0.06 mg/dl (sham, n = 3), 1.59 ± 0.10 mg/dl (vehicle, n = 8) and 0.89 ± 0.11 mg/dl (meclizine, n = 8). Kidney injury was significantly decreased in meclizine treated mice compared with vehicle group (p < 0.001). Protection was also seen when meclizine was administered 24 h prior to ischemia. Meclizine reduced inflammation, mitochondrial oxygen consumption, oxidative stress, mitochondrial fragmentation, and tubular injury. Meclizine preconditioned kidney tubular epithelial cells, exposed to blockade of glycolytic and oxidative metabolism with 2-deoxyglucose and NaCN, had reduced LDH and cytochrome c release. Meclizine upregulated glycolysis in glucose-containing media and reduced cellular ATP levels in galactose-containing media. Meclizine inhibited the Kennedy pathway and caused rapid accumulation of phosphoethanolamine. Phosphoethanolamine recapitulated meclizine-induced protection both in vitro and in vivo.

Dual involvement of growth arrest-specific gene 6 in the early phase of human IgA nephropathy.

Background Gas6 is a growth factor that causes proliferation of mesangial cells in the development of glomerulonephritis. Gas6 can bind to three kinds of receptors; Axl, Dtk, and Mer. However, their expression and functions are not entirely clear in the different glomerular cell types. Meanwhile, representative cell cycle regulatory protein p27 has been reported to be expressed in podocytes in normal glomeruli with decreased expression in proliferating glomeruli, which inversely correlated with mesangial proliferation in human IgA nephropathy (IgAN). Methods The aim of this study is to clarify Gas6 involvement in the progression of IgAN. Expression of Gas6/Axl/Dtk was examined in 31 biopsy proven IgAN cases. We compared the expression levels with histological severity or clinical data. Moreover, we investigated the expression of Gas6 and its receptors in cultured podocytes. Results In 28 of 31 cases, Gas6 was upregulated mainly in podocytes. In the other 3 cases, Gas6 expression was induced in endothelial and mesangial cells, which was similar to animal nephritis models. Among 28 podocyte type cases, the expression level of Gas6 correlated with the mesangial hypercellularity score of IgAN Oxford classification and urine protein excretion. It also inversely correlated with p27 expression in glomeruli. As for the receptors, Axl was mainly expressed in endothelial and mesangial cells, while Dtk was expressed in podocytes. In vitro, Dtk was expressed in cultured murine podocytes, and the expression of p27 was decreased by Gas6 stimulation. Conclusions Gas6 was uniquely upregulated in either endothelial/mesangial cells or podocytes in IgAN. The expression pattern can be used as a marker to classify IgAN. Gas6 has a possibility to be involved in not only mesangial proliferation via Axl, but also podocyte injury via Dtk in IgAN.

An Autopsy Case of Mitochondrial Myopathy, Encephalopathy, Lactic Acidosis, and Stroke-Like Episodes (MELAS) with Intestinal Bleeding in Chronic Renal Failure

Abstract A 50-year-old man who underwent hemodialysis (HD) at local outpatient HD center due to end-stage renal disease (ESRD) was transferred to our hospital because of pneumonia. He had severe emaciation and past history of congestive heart failure. Presenting symptoms almost consistently involved difficulty in hearing and recurrent attacks of migraine-like headaches. He was diagnosed with dilated cardiomyopathy, showing diastolic mechanical dyssynchrony by tissue Doppler echocardiography. On the day of death, he had hematemesis and hemorrhagic shock. Autopsy revealed perforation of duodenum, and genetic analysis using mitochondrial DNA from cardiac muscle and iliopsoas muscle revealed a 3243A > G mutation in the mitochondrial tRNALeu(UUR) gene, which is related to mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). Multiple organ failure due to the mutation of mitochondrial DNA with gastrointestinal bleeding is not a common.

Transcription factor 7-like 2 (TCF7L2) regulates activin receptor-like kinase 1 (ALK1)/Smad1 pathway for development of diabetic nephropathy.

Smad1 has previously been shown to play a key role in the development of diabetic nephropathy (DN), by increasing synthesis of extracellular matrix. However, the regulatory mechanism of Smad1 in DN is still unclear. This study aims to elucidate molecular interactions between activin receptor-like kinase 1 (ALK1)/Smad1 signaling pathway and transcription factor 7-like 2 (TCF7L2) in the progression of DN in vitro and in vivo. The expressions of TCF7L2 and ALK1 were induced by advanced glycation end products (AGEs) in parallel with Smad1, phosphorylated Smad1 (pSmad1), and alpha-smooth muscle actin (α-SMA) through TGF-β1 in cultured mesangial cells. Constitutively active ALK1 increased pSmad1 and α-SMA expressions. The binding of TCF7L2 to ALK1 promoter was confirmed by chromatin immunoprecipitation assay. Furthermore, TCF7L2 induced promoter activity of ALK1. AGEs and TGF-β1 induced a marked increase in TCF7L2 expression in parallel with ALK1. Overexpression of TCF7L2 increased the expressions of ALK1 and Smad1. Inversely, TCF7L2 knockdown by siRNA suppressed α-SMA expression as well as ALK1 and Smad1. The iNOS transgenic mice (iNOS-Tgm), which developed diabetic glomerulosclerosis resembling human diabetic nephropathy, exhibited markedly increased expressions of ALK1, TCF7L2, Smad1, pSmad1, and α-SMA in glomeruli in association with mesangial matrix expansion. These results provide a new evidence that the TCF7L2/ALK1/Smad1 pathway plays a key role in the development of DN.

Dietary iron restriction alleviates renal tubulointerstitial injury induced by protein overload in mice

Increased proteinuria causes tubulointerstitial injury due to inflammation in chronic kidney disease (CKD). Iron restriction exhibits protective effects against renal dysfunction; however, its effects against protein overload-induced tubulointerstitial damage remain unclear. Here, we investigated dietary iron restriction effect on tubulointerstitial damage in mice with protein-overload tubulointerstitial injury. Renal tubulointerstitial injury in animal model was induced by intraperitoneal injection of an overdose of bovine serum albumin (BSA). We divided mice into three groups: normal saline + normal diet (ND), BSA + ND, and BSA + iron-restricted diet (IRD). BSA overload induced renal tubulointerstitial injury in the ND mice, which was ameliorated in the IRD mice. Inflammatory cytokines and extracellular matrix mRNA expression was upregulated in BSA + ND mice kidneys and was inhibited by IRD. BSA-induced increase in renal superoxide production, NADPH oxidase activity, and p22phox expression was diminished in the IRD mice. IRD suppression increased BSA-induced renal macrophage infiltration. Moreover, BSA mice exhibited nucleotide-binding oligomerisation domain-like receptor pyrin domain-containing protein (NLRP) inflammasome activation, which was inhibited by IRD. Ferrous iron increased in kidneys with BSA overload and was inhibited by IRD. Thus, iron restriction inhibited oxidative stress and inflammatory changes, contributing to the protective effect against BSA overload-induced tubulointerstitial injury.