Diabetic nephropathy is characterized by damage to both the glomerulus and tubulointerstitium, but relatively little is known about accompanying cell-specific changes in gene expression. We performed ...unbiased single-nucleus RNA sequencing (snRNA-seq) on cryopreserved human diabetic kidney samples to generate 23,980 single-nucleus transcriptomes from 3 control and 3 early diabetic nephropathy samples. All major cell types of the kidney were represented in the final dataset. Side-by-side comparison demonstrated cell-type–specific changes in gene expression that are important for ion transport, angiogenesis, and immune cell activation. In particular, we show that the diabetic thick ascending limb, late distal convoluted tubule, and principal cells all adopt a gene expression signature consistent with increased potassium secretion, including alterations in Na⁺/K⁺-ATPase, WNK1, mineralocorticoid receptor, and NEDD4L expression, as well as decreased paracellular calcium and magnesium reabsorption. We also identify strong angiogenic signatures in glomerular cell types, proximal convoluted tubule, distal convoluted tubule, and principal cells. Taken together, these results suggest that increased potassium secretion and angiogenic signaling represent early kidney responses in human diabetic nephropathy.
Maximizing the potential of human kidney organoids for drug testing and regenerative medicine and to model development and disease requires addressing cell immaturity, the lack of a mature collecting ...system, and off-target cell types. By independently generating two kidney progenitor cell populations—metanephric mesenchyme and ureteric bud (UB)-like cells—we could generate kidney organoids with a collecting system. We also identify the hormones aldosterone and arginine vasopressin (AVP) as critical to promote differentiation of collecting duct cell types including both principal cells (PCs) and intercalated cells (ICs). The resulting PCs express aquaporin-2 (AQP2) protein, which undergoes translocation to the apical membrane after vasopressin or forskolin stimulation. By single-cell RNA sequencing (scRNA-seq), we demonstrate improved proximal tubule maturation and reduced off-target cell populations. We also show appropriate downregulation of progenitor cell types, improved modeling of tubular injury, the presence of urothelium (Uro), and the ability of Notch pathway modulation to regulate PC:IC ratios during organoid development.
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•Combining differentiated progenitors leads to kidney organoids with collecting duct•Aldosterone and vasopressin drive principal and intercalated cell differentiation•Organoids show improved maturation and model tubular injury•Notch regulates principal:intercalated cell ratios
Uchimura et al. combine independently differentiated metanephric mesenchyme-like and ureteric bud-like progenitors to generate human kidney organoids with a collecting system. Hormones aldosterone and arginine vasopressin drive principal and intercalated cell maturation, and Notch signaling could regulate cell ratios. Organoids also showed improved maturation and injury modeling.
Single-cell genomics techniques are revolutionizing our ability to characterize complex tissues. By contrast, the techniques used to analyze renal biopsy specimens have changed little over several ...decades. We tested the hypothesis that single-cell RNA-sequencing can comprehensively describe cell types and states in a human kidney biopsy specimen.
We generated 8746 single-cell transcriptomes from a healthy adult kidney and a single kidney transplant biopsy core by single-cell RNA-sequencing. Unsupervised clustering analysis of the biopsy specimen was performed to identify 16 distinct cell types, including all of the major immune cell types and most native kidney cell types, in this biopsy specimen, for which the histologic read was mixed rejection.
Monocytes formed two subclusters representing a nonclassical CD16+ group and a classic CD16- group expressing dendritic cell maturation markers. The presence of both monocyte cell subtypes was validated by staining of independent transplant biopsy specimens. Comparison of healthy kidney epithelial transcriptomes with biopsy specimen counterparts identified novel segment-specific proinflammatory responses in rejection. Endothelial cells formed three distinct subclusters: resting cells and two activated endothelial cell groups. One activated endothelial cell group expressed Fc receptor pathway activation and Ig internalization genes, consistent with the pathologic diagnosis of antibody-mediated rejection. We mapped previously defined genes that associate with rejection outcomes to single cell types and generated a searchable online gene expression database.
We present the first step toward incorporation of single-cell transcriptomics into kidney biopsy specimen interpretation, describe a heterogeneous immune response in mixed rejection, and provide a searchable resource for the scientific community.
Prostasin (PRSS8) is a serine protease that metabolizes and moderates the effect of specific substrates. Epidermal growth factor receptor (EGFR), which modulates insulin secretion and pancreatic ...β-cell proliferation, is regulated via proteolytic shedding by PRSS8. We first detected PRSS8 expression in β-cells of pancreatic islets of mice. To better understand the molecular processes involved in PRSS8-associated insulin secretion, pancreatic β-cell-specific PRSS8 knockout (βKO) and PRSS8-overexpressing (βTG) male mice were generated. We found that glucose intolerance and reduction in glucose-stimulated insulin secretion developed in βKO mice compared with the control subjects. A higher response to glucose was noted in islets retrieved from βTG mice. Erlotinib, a specific blocker of EGFR, blocks EGF- and glucose-stimulated secretion of insulin among MIN6 cells, and glucose improves EGF release from β-cells. After silencing PRSS8 in MIN6 cells, glucose-stimulated insulin secretion decreased, and EGFR signaling was impaired. Conversely, overexpression of PRSS8 in MIN6 cells induced higher concentrations of both basal and glucose-stimulated insulin secretion and increased phospho-EGFR concentrations. Furthermore, short-term exposure to glucose improved the concentration of endogenous PRSS8 in MIN6 cells through inhibition of intracellular degradation. These findings suggest that PRSS8 is involved in glucose-dependent physiological regulation of insulin secretion via the EGF-EGFR signaling pathway in pancreatic β-cells.
Hypoxia occurs in the kidneys of chronic kidney disease (CKD) patients, inducing interstitial fibrosis and tubule cell death. Renal tubule cell death is an important determinant of mortality in CKD. ...We focused on the regulation of cell-cycle-mediated protein expression to prevent cell death under chronic hypoxia in the kidneys of CKD patients. Paraffin-embedded kidney sections from patients with CKD (diabetes nephropathy, nephrosclerosis, or IgA nephropathy) were analyzed for the expression of hypoxia-inducible factor (HIF), thyroid hormone receptor (TR) β, or p21 and levels of interstitial fibrosis. Human renal proximal tubule cells were exposed to hypoxia and analyzed for the expression of HIF, TRβ, or p21 and the cell-cycle stage. TRβ expression was enhanced early on when fibrosis was not fully developed in the tubule cells of CKD patients. HIF1α bound to the TRβ promoter and directly induced its transcription. Further, HIF1α expression induced the expression of TRβ and inhibited cell-cycle progression. In the early stage of kidney injury, TRβ might act as a guardian to prepare and organize cell-cycle proliferation and prevent cell death. While the molecular mechanism that regulates the expression of cell-cycle regulators in renal tubule cells remains controversial, TRβ has strong potential as a new therapeutic target.
Thyroid hormones are critical regulators of vertebrate development and metabolism. Under hyperthyroid conditions, excess thyroid hormones induce expression of several enzymes and activities via ...activation of ligand-bound thyroid hormone receptors (TRs). Arginase (ARG) is downstream of a ligand-bound TR and overexpression of ARG2 induces the production of reactive oxygen species and subsequent exacerbation of kidney ischemia/reperfusion (I/R) injury. To clarify the association between I/R-induced kidney injury and hyperthyroidism, mice were pretreated with L-thyroxine (LT4) or vehicle alone, then subjected to I/R. Proximal tubular cell-specific conditional knockout of thyroid hormone receptor β (TRβcKO) mice was generated and the effects of I/R were analyzed. Hyperthyroidism enhanced tubular damage and fibrosis in the kidneys of mice after I/R. Hyperthyroidism induced tubular cell necroptosis following inflammatory cell accumulation in the kidney after I/R. ARG2 expressions and reactive oxygen species accumulated in the kidneys of hyperthyroid mice after I/R, but these changes were ameliorated in the kidneys of TRβcKO mice. Hyperthyroidism-enhanced kidney injury was ameliorated in the kidney of TRβcKO mice after I/R. These results suggest that excess thyroid hormones are disadvantageous for the kidney under ischemic stress. Overt hypothyroidism represents a severe thyroid hormone deficiency disease that requires LT4 treatment, while overreplacement or iatrogenic thyrotoxicosis might cause kidney injury.
The liver has a most indispensable role in glucose and lipid metabolism where we see some of the most serious worldwide health problems. The serine protease prostasin (PRSS8) cleaves toll-like ...receptor 4 (TLR4) and regulates hepatic insulin sensitivity under PRSS8 knockout condition. However, liver substrate proteins of PRSS8 other than TLR4 and the effect to glucose and lipid metabolism remain unclarified with hepatic elevation of PRSS8 expression. Here we show that high-fat-diet-fed liver-specific PRSS8 transgenic mice improved glucose tolerance and hepatic steatosis independent of body weight. PRSS8 amplified extracellular signal-regulated kinase phosphorylation associated with matrix metalloproteinase 14 activation in vivo and in vitro. Moreover, in humans, serum PRSS8 levels reduced more in type 2 diabetes mellitus (T2DM) patients than healthy controls and were lower in T2DM patients with increased maximum carotid artery intima media thickness (>1.1 mm). These results identify the regulatory mechanisms of PRSS8 overexpression over glucose and lipid metabolism, as well as excessive hepatic fat storage.
Sirtuins (SIRT1-7) have attracted considerable attention as regulators of metabolism over the past decade. However, the physiological functions and molecular mechanisms of SIRT7 are poorly ...understood. Here we demonstrate that Sirt7 knockout mice were resistant to high-fat diet-induced fatty liver, obesity, and glucose intolerance, and that hepatic triglyceride accumulation was also attenuated in liver-specific Sirt7 knockout mice. Hepatic SIRT7 positively regulated the protein level of TR4/TAK1, a nuclear receptor involved in lipid metabolism, and as a consequence activated TR4 target genes to increase fatty acid uptake and triglyceride synthesis/storage. Biochemical studies revealed that the DDB1-CUL4-associated factor 1 (DCAF1)/damage-specific DNA binding protein 1 (DDB1)/cullin 4B (CUL4B) E3 ubiquitin ligase complex interacted with TR4, leading to its degradation, while binding of SIRT7 to the DCAF1/DDB1/CUL4B complex inhibited the degradation of TR4. In conclusion, we propose that hepatic SIRT7 controls lipid metabolism in liver by regulating the ubiquitin-proteasome pathway.
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•Sirt7 knockout mice are resistant to high-fat diet-induced hepatic steatosis•SIRT7 inhibits degradation of nuclear receptor TR4/TAK1 involved in lipid metabolism•SIRT7 binds to the DCAF1/DDB1/CUL4B ubiquitin ligase complex•SIRT7 controls hepatic lipid metabolism by regulating the ubiquitin pathway
Yoshizawa et al. show that loss of Sirt7 is protective against high-fat diet-induced hepatic steatosis. Mechanistically, SIRT7 binds the DCAF1/DDB1/CUL4B ubiquitin ligase complex and prevents the degradation of the nuclear receptor TR4/TAK1, which is involved in lipid metabolism.
Metabolic syndrome (MetS) is associated with chronic kidney disease and proteinuria. Previously, we reported that a synthetic serine protease inhibitor, camostat mesilate (CM), mitigated hypertension ...and proteinuria in rodent disease models. The present study evaluated the anti-hypertensive and anti-proteinuric effects of CM in MetS model rats (SHR/ND mcr-cp). Rats were divided into normal salt-fed (NS), high salt-fed (HS), HS and CM-treated (CM), and HS and hydralazine-treated (Hyd) groups. Rats were sacrificed after four weeks of treatment. Severe hypertension and proteinuria were observed in the HS group. Although CM and Hyd equally alleviated hypertension, CM suppressed proteinuria and glomerular sclerosis more efficiently than Hyd. The HS group revealed a decrease in podocyte number and podocyte-specific molecules, together with an increase in glomerular apoptotic cells and apoptosis-related proteins in the kidney. These changes were significantly attenuated by CM, but not by Hyd. Furthermore, CM ameliorated the apoptotic signals in murine cultured podocytes stimulated with the high glucose and aldosterone medium. In conclusion, CM could exert renoprotective effects in MetS model rats, together with the inhibition of podocyte apoptosis. Our study suggests that serine protease inhibition may become a new therapeutic strategy against MetS-related hypertension and renal injuries.
For chronic kidney disease, regeneration of lost nephrons with human kidney organoids derived from induced pluripotent stem (iPS) cells is proposed to be an attractive potential therapeutic option. ...It remains unclear, however, whether organoids transplanted into kidneys in vivo would be safe or functional. Here, we purified kidney organoids and transplanted them beneath the kidney capsules of immunodeficient mice to test their safety and maturity. Kidney organoid grafts survived for months after transplantation and became vascularized from host mouse endothelial cells. Nephron-like structures in grafts appeared more mature than kidney organoids in vitro, but remained immature compared with the neighboring mouse kidney tissue. Ultrastructural analysis revealed filtration barrier-like structures, capillary lumens, and tubules with brush border in the transplanted kidney organoids, which were more mature than those of the kidney organoids in vitro but not as organized as adult mammalian kidneys. Immaturity was a common feature of three separate differentiation protocols by immunofluorescence analysis and single cell RNA sequencing. Stroma of transplanted kidney organoid grafts were filled with vimentin-positive mesenchymal cells, and chondrogenesis, cystogenesis, and stromal expansion were observed in the long term. Transcription profiles showed that long-term maintenance after kidney organoid transplantation induced transcriptomic reprogramming with prominent suppression of cell-cycle-related genes and upregulation of extracellular matrix organization. Our data suggest that kidney organoids derived from iPS cells may be transplantable but strategies to improve nephron differentiation and purity are required before they can be applied in humans as a therapeutic option.
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