A simplified method for isolation of large numbers of defined nephron segments

Schafer, James A.; Watkins, Mary L.; Li, L.; Herter, Peter; Haxelmans, Sabine; Schlatter, Eberhard

doi:10.1152/ajprenal.1997.273.4.f650

Cited by 72 publications

(84 citation statements)

References 24 publications

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“…Isolation of mouse primary tubular epithelial cells was performed under a modified protocol described by Schafer et al 30 Briefly, kidneys were harvested after cardiac perfusion with 0.025% collagenase (Worthington) in M199 Hank's solution (Lonza). Renal cortex was isolated, minced, and incubated at 37°C in the collagenase solution while 5% CO 2 balanced with room air gas was injected into the solution for 40 minutes.…”

Section: Isolation Of Primary Tubular Epithelial Cellsmentioning

confidence: 99%

Distinct Macrophage Phenotypes Contribute to Kidney Injury and Repair

Lee¹,

Huen²,

Nishio³

et al. 2011

Journal of the American Society of Nephrology

756

796

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The ischemically injured kidney undergoes tubular cell necrosis and apoptosis, accompanied by an interstitial inflammatory cell infiltrate. In this study, we show that iNos-positive proinflammatory (M1) macrophages are recruited into the kidney in the first 48 hours after ischemia/reperfusion injury, whereas arginase 1-and mannose receptor-positive, noninflammatory (M2) macrophages predominate at later time points. Furthermore, depletion of macrophages before ischemia/reperfusion diminishes kidney injury, whereas depletion at 3 to 5 days after injury slows tubular cell proliferation and repair. Infusion of Ifn␥-stimulated, bone marrowderived macrophages into macrophage-depleted mice at the time of kidney reperfusion restored injury to the level seen without macrophage depletion, suggesting that proinflammatory macrophages worsen kidney damage. In contrast, the appearance of macrophages with the M2 phenotype correlated with the proliferative phase of kidney repair. In vitro studies showed that IFN␥-stimulated, proinflammatory macrophages begin to express markers of M2 macrophages when cocultured with renal tubular cells. Moreover, IL-4 -stimulated macrophages with an M2 phenotype, but not IFN␥-stimulated proinflammatory macrophages, promoted renal tubular cell proliferation. Finally, tracking fluorescently labeled, IFN␥-stimulated macrophages that were injected after injury showed that inflammatory macrophages can switch to an M2 phenotype in the kidney at the onset of kidney repair. Taken together, these studies show that macrophages undergo a switch from a proinflammatory to a trophic phenotype that supports the transition from tubule injury to tubule repair.

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Section: Isolation Of Primary Tubular Epithelial Cellsmentioning

confidence: 99%

Distinct Macrophage Phenotypes Contribute to Kidney Injury and Repair

Lee¹,

Huen²,

Nishio³

et al. 2011

Journal of the American Society of Nephrology

756

796

View full text Add to dashboard Cite

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“…18,19 Forty glomeruli and 40 mm of tubules were collected and dissolved in TRIzol solution for RNA extraction. Separated groups of freshly dissected PTs (80 mm) were sonicated in 10 L of HEPES buffer (25 mmol/L sodium HEPES, 1 mmol/L EDTA, and 0.1 mmol/L PMSF) and incubated with Hcys to measure the enzymatic activities of the transsulfuration pathway using HPLC analysis.…”

Section: Microdissection Of Nephron Segments From Rat Kidneymentioning

confidence: 99%

Hyperhomocysteinemia Associated With Decreased Renal Transsulfuration Activity in Dahl S Rats

Chen²,

Muh³

et al. 2006

Hypertension

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Abstract-Elevated plasma homocysteine (Hcys) has been reported to participate in the development of arterial and glomerular sclerosis in Dahl salt-sensitive hypertensive (SS) rats. The mechanism resulting in hyperhomocysteinemia in these animals remains unknown. Disposal of Hcys in the kidneys plays an important role in regulating the plasma Hcys level. We, therefore, examined the activities and expressions of the enzymes involved in the metabolism of Hcys in the kidneys of SS rats, compared with that of Brown Norway rats and SSBN13 rats, a consomic subcolony of SS rats that carries a substituted chromosome 13 from Brown Norway rats. High-performance liquid chromatography analysis demonstrated that plasma Hcys levels were significantly higher in SS rats. The conversion of S-adenosylhomocysteine into Hcys via S-adenosylhomocysteine hydrolase by renal tissue was not different among these 3 rat strains. However, the metabolic rate of Hcys into cysteine was markedly reduced in the SS rat kidneys. The mRNA and protein levels of cystathionine ␤-synthase (CBS), one of the key enzymes in the transsulfuration pathway in the kidneys, were significantly lower in SS rats. In microdissected nephron segments, CBS mRNA was shown to be mainly present in renal proximal tubules (PTs). The mRNA levels of CBS in the PTs were also significantly decreased in SS rats, accompanied by a reduced CBS activity in PTs. We conclude that hyperhomocysteinemia is associated with a decreased activity and expression of CBS in renal PTs because of the defect of chromosome 13 in SS rats. (Hypertension. 2006; 47:1094-1100.)

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“…Isolation of human nephron segments was performed from unaltered cortices of patients (with their consent) undergoing tumor nephrectomy (25,26). Proximal tubules of a total length of 200 m or IHKE-1 cells were lysed in a 4 M guanidinium chloride buffer and total RNA was isolated and incubated with 10 U DNase I (Promega, Heidelberg, Germany) at 37°C for 60 min to digest traces of genomic DNA.…”

Section: Polymerase Chain Reactionmentioning

confidence: 99%

Up-Regulation of Early Growth Response Gene-1 Via the CXCR3 Receptor Induces Reactive Oxygen Species and Inhibits Na+/K+-ATPase Activity in an Immortalized Human Proximal Tubule Cell Line

Bek

Reinhardt

Fischer

et al. 2003

The Journal of Immunology

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The CXCR3 chemokine receptor, a member of the CXCR family, has been linked to a pathological role in autoimmune disease, inflammatory disease, allograft rejection, and ischemia. In the kidney, expression of the CXCR3 receptor and its ligands is up-regulated in states of glomerulonephritis and in allograft rejection, but little is known about the expression and functional role the CXCR3 receptor might play. Here, we study the function of the CXCR3 chemokine receptor in an immortalized human proximal tubular cell line (IHKE-1). Stimulation of the CXCR3 receptor by its selective agonist monokine induced by IFN-γ leads via a Ca2+-dependent mechanism to an up-regulation of early growth response gene (EGR)-1. Overexpression of EGR-1 induces down-regulation of copper-zinc superoxide dismutase and manganese superoxide dismutase and stimulates the generation of reactive oxygen species (ROS) via the NADH/NADPH-oxidase system. EGR-1 overexpression or treatment with monokine induced by IFN-γ resulted in a ROS-dependent inhibition of basolateral Na+/K+-ATPase activity, compromising sodium transport in these cells. Thus, activation of the CXCR3 receptor in proximal tubular cells might disturb natriuresis during inflammatory and ischemic kidney disease via EGR-1-mediated imbalance of ROS.

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A simplified method for isolation of large numbers of defined nephron segments

Cited by 72 publications

References 24 publications

Distinct Macrophage Phenotypes Contribute to Kidney Injury and Repair

Distinct Macrophage Phenotypes Contribute to Kidney Injury and Repair

Hyperhomocysteinemia Associated With Decreased Renal Transsulfuration Activity in Dahl S Rats

Up-Regulation of Early Growth Response Gene-1 Via the CXCR3 Receptor Induces Reactive Oxygen Species and Inhibits Na+/K+-ATPase Activity in an Immortalized Human Proximal Tubule Cell Line

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