Direct evidence of proximal tubular proliferation in early diabetic nephropathy.

Kidney hypertrophy is a common clinical feature in patients with diabetes and is associated with poor renal outcomes. Initial cell proliferation followed by cellular hypertrophy are considered the responsible mechanisms for diabetic kidney hypertrophy. However, whether similar responses against hyperglycemia continue in the chronic phase in diabetes is unclear. We performed lineage tracing analysis of proximal tubular epithelia using novel type 2 diabetic mice with a tamoxifen-inducible proximal tubule-specific fluorescent reporter. Clonal analysis of proximal tubular epithelia demonstrated that the labeled epithelia proliferated in type 2 diabetic mice. Based on the histological analysis and protein/DNA ratio of sorted labeled tubular epithelia, there was no evidence of cellular hypertrophy in type 2 diabetic mice. Lineage tracing and histological analyses of streptozocin-induced type 1 diabetes also revealed that cellular proliferation occurs in the chronic phase of type 1 diabetes induction. According to our study, epithelial proliferation accompanied by SGLT2 upregulation, rather than cellular hypertrophy, predominantly occurs in the hypertrophic kidney in both type 1 and type 2 diabetes. An increased number of SGLT2+ tubular epithelia may be an adaptive response against hyperglycemia, and linked to the hyper-reabsorption of sodium and glucose observed in type 2 diabetes patients.

Kidney vascular congestion exacerbates acute kidney injury in mice.

Heart failure is frequently accompanied by kidney failure and co-incidence of these organ failures worsens the mortality in patients with heart failure. Recent clinical observations revealed that increased kidney venous pressure, rather than decreased cardiac output, causes the deterioration of kidney function in patients with heart failure. However, the underlying pathophysiology is unknown. Here, we found that decreased blood flow velocity in peritubular capillaries by kidney congestion and upregulation of endothelial nuclear factor-κB (NF-κB) signaling synergistically exacerbate kidney injury. We generated a novel mouse model with unilateral kidney congestion by constriction of the inferior vena cava between kidney veins. Intravital imaging highlighted the notable dilatation of peritubular capillaries and decreased kidney blood flow velocity in the congestive kidney. Damage after ischemia reperfusion injury was exacerbated in the congestive kidney and accumulation of polymorphonuclear leukocytes within peritubular capillaries was noted at the acute phase after injury. Similar results were obtained in vitro, in which polymorphonuclear leukocytes adhesion on activated endothelial cells was decreased in flow velocity-dependent manner but cancelled by inhibition of NF-κB signaling. Pharmacological inhibition of NF-κB for the mice subjected by both kidney congestion and ischemia reperfusion injury ameliorated the accumulation of polymorphonuclear leukocytes and subsequent exacerbation of kidney injury. Thus, our study demonstrates the importance of decreased blood flow velocity accompanying activated NF-κB signaling in aggravation of kidney injury. Hence, inhibition of NF-κB signaling may be a therapeutic candidate for the vicious cycle between heart and kidney failure with increased kidney venous pressure.

Diverse Receptor Tyrosine Kinase Phosphorylation in Urine-Derived Tubular Epithelial Cells from Autosomal Dominant Polycystic Kidney Disease Patients.

BACKGROUNDS: The clinical features of autosomal dominant polycystic kidney disease (ADPKD) differ among patients even if they have the same gene mutation in PKD1 or PKD2. This suggests that there is diversity in the expression of other modifier genes or in the underlying molecular mechanisms of ADPKD, but these are not well understood. METHODS: We primarily cultured solute carrier family 12 member 3 (SLC12A3)-positive urine-derived distal tubular epithelial cells from 6 ADPKD patients and 4 healthy volunteers and established immortalized cell lines. The diversity in receptor tyrosine kinase (RTK) phosphorylation by phospho-RTK array in immortalized tubular epithelial cells was analyzed. RESULTS: We noted diversity in the activation of several molecules, including Met, a receptor of hepatocyte growth factor (HGF). Administration of golvatinib, a selective Met inhibitor, or transfection of small interfering RNA for Met suppressed cell proliferation and downstream signaling only in the cell lines in which hyperphosphorylation of Met was observed. In three-dimensional culture of Madin-Darby canine kidney (MDCK) cells as a cyst formation model of ADPKD, HGF activated Met, resulting in an increased total cyst number and total cyst volume. Administration of golvatinib inhibited these phenotypes in MDCK cells. CONCLUSION: Analysis of urine-derived tubular epithelial cells demonstrated diverse RTK phosphorylation in ADPKD, and Met phosphorylation was noted in some patients. Considering the difference in the effects of golvatinib on immortalized tubular epithelial cells among patients, this analysis may aid in selecting suitable drugs for individual ADPKD patients.

Intratubular epithelial-mesenchymal transition and tubular atrophy after kidney injury in mice.

Tubular atrophy is a common pathological feature of kidney fibrosis. Although fibroblasts play a predominant role in tissue fibrosis, the role of repairing tubular epithelia in tubular atrophy is unclear. We demonstrated the essential role of focal adhesion kinase (FAK)-mediated intratubular epithelial-mesenchymal transition (EMT) in the pathogenesis of tubular atrophy after severe ischemia-reperfusion injury (IRI). Actively proliferating tubular epithelia undergoing intratubular EMT were noted in the acute phase of severe IRI, resulting in tubular atrophy in the chronic phase, reflecting failed tubular repair. Furthermore, FAK was phosphorylated in the tubular epithelia in the acute phase of severe IRI, and its inhibition ameliorated both tubular atrophy and interstitial fibrosis in the chronic phase after injury. In vivo clonal analysis of single-labeled proximal tubular epithelial cells after IRI using proximal tubule reporter mice revealed substantial clonal expansion after IRI, reflecting active epithelial proliferation during repair. The majority of these proliferating epithelia were located in atrophic and nonfunctional tubules, and FAK inhibition was sufficient to prevent tubular atrophy. In vitro, transforming growth factor-ß induced FAK phosphorylation and an EMT phenotype, which was also prevented by FAK inhibition. In an in vitro tubular epithelia gel contraction assay, transforming growth factor-ß treatment accelerated gel contraction, which was suppressed by FAK inhibition. In conclusion, injury-induced intratubular EMT is closely related to tubular atrophy in a FAK-dependent manner.

Pharmacological inhibition of ataxia-telangiectasia mutated exacerbates acute kidney injury by activating p53 signaling in mice.

The DNA damage response after kidney injury induces cell cycle arrest in renal tubular epithelial cells, resulting in the secretion of pro-fibrotic cytokines, thereby promoting interstitial fibrosis in a paracrine manner. Phosphorylation of ataxia-telangiectasia mutated (ATM) is the initial step in the DNA damage response and subsequent cell cycle arrest; however, the effects of ATM inhibition on the injured kidney have not been explored. Pharmacological ATM inhibition by KU55933 in cisplatin-treated mice did not ameliorate, but instead exacerbated cisplatin-induced DNA damage and tubular injury, thereby increasing mortality. Analysis of isolated tubular epithelia by FACS from bigenic SLC34a1-CreERt2; R26tdTomato proximal tubular-specific reporter mice revealed that KU55933 upregulated p53 and subsequent pro-apoptotic signaling in tubular epithelia of cisplatin-treated mice, leading to marked mitochondrial injury and apoptosis. In addition, KU55933 attenuated several DNA repair processes after cisplatin treatment, including single-strand DNA repair and Fanconi anemia pathways, suggesting that DNA repair after dual treatment of cisplatin and KU55933 was not sufficient to prevent the cisplatin-induced tubular injury. Our study suggested that ATM inhibition does not increase DNA repair after cisplatin-induced DNA damage and exacerbates tubular injury through the upregulation of p53-dependent pro-apoptotic signaling. Acute kidney injury must be carefully monitored when ATM inhibitors become available in clinical practice in the future.

Comprehensive renoprotective effects of ipragliflozin on early diabetic nephropathy in mice.

Clinical and experimental studies have shown that sodium glucose co-transporter 2 inhibitors (SGLT2i) contribute to the prevention of diabetic kidney disease progression. In order to clarify its pharmacological effects on the molecular mechanisms underlying the development of diabetic kidney disease, we administered different doses of the SGLT2i, ipragliflozin, to type 2 diabetic mice. A high-dose ipragliflozin treatment for 8 weeks lowered blood glucose levels and reduced urinary albumin excretion. High- and low-dose ipragliflozin both inhibited renal and glomerular hypertrophy, and reduced NADPH oxidase 4 expression and subsequent oxidative stress. Analysis of glomerular phenotypes using glomeruli isolation demonstrated that ipragliflozin preserved podocyte integrity and reduced oxidative stress. Regarding renal tissue hypoxia, a short-term ipragliflozin treatment improved oxygen tension in the kidney cortex, in which SGLT2 is predominantly expressed. We then administered ipragliflozin to type 1 diabetic mice and found that high- and low-dose ipragliflozin both reduced urinary albumin excretion. In conclusion, we confirmed dose-dependent differences in the effects of ipragliflozin on early diabetic nephropathy in vivo. Even low-dose ipragliflozin reduced renal cortical hypoxia and abnormal hemodynamics in early diabetic nephropathy. In addition to these effects, high-dose ipragliflozin exerted renoprotective effects by reducing oxidative stress in tubular epithelia and glomerular podocytes.

Trans-collateral angioplasty in vascular access intervention therapy for subacute occluded vessel.

We describe the first reported case of successful trans-collateral angioplasty (TCA) in vascular access intervention therapy (VAIVT) for a subacute occluded lesion in the vascular access route. TCA is a technique which has been developed in the field of endovascular therapy for peripheral arterial disease and is usually applied for a long chronic total occluded lesion with no available distal puncture site. Because such lesion characteristics suitable for being applied with TCA are not usually seen in the patients who receive VAIVT, there is little opportunity when TCA is performed in VAIVT. The present patient showed subacute occlusion in the vascular access route with well-developed collateral blood vessels. Because antegrade wiring resulted in subintimal tracking, we failed to antegradely introduce the guidewire to the vascular true lumen. Moreover, no puncture site in the venous side was anatomically available. Therefore, we adopted the strategy of TCA and successfully completed the procedure. Although we rarely encounter the situation in which TCA is necessary for VAIVT, the strategy of TCA is a promising procedure if the condition permits.