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T cells mediate organ injury and repair. A proportion of unconventional kidney T cells called double-negative (DN) T cells (TCR + CD4 - CD8 - ), with anti-inflammatory properties, were previously demonstrated to protect from early injury in moderate experimental acute kidney injury (AKI). However, their role in repair after AKI has not been studied. We hypothesized that DN T cells mediate repair after severe AKI. C57B6 mice underwent severe (40 min) unilateral ischemia-reperfusion injury (IRI). Kidney DN T cells were studied by flow cytometry and compared with gold-standard anti-inflammatory CD4 + regulatory T cells (Tregs). In vitro effects of DN T cells and Tregs on renal tubular epithelial cell (RTEC) repair after injury were quantified with live-cell analysis. DN T cells, Tregs, CD4, or vehicle were adoptively transferred after severe AKI. Glomerular filtration rate (GFR) was measured using fluorescein isothiocyanate (FITC)-sinistrin. Fibrosis was assessed with Masson's trichrome staining. Profibrotic genes were measured with qRT-PCR. Percentages and the numbers of DN T cells substantially decreased during repair phase after severe AKI, as well as their activation and proliferation. Both DN T cells and Tregs accelerated RTEC cell repair in vitro. Post-AKI transfer of DN T cells reduced kidney fibrosis and improved GFR, as did Treg transfer. DN T cell transfer lowered transforming growth factor (TGF)β1 and α-smooth muscle actin (αSMA) expression. DN T cells reduced effector-memory CD4 + T cells and IL-17 expression. DN T cells undergo quantitative and phenotypical changes after severe AKI, accelerate RTEC repair in vitro as well as improve GFR and renal fibrosis in vivo. DN T cells have potential as immunotherapy to accelerate repair after AKI. NEW & NOTEWORTHY Double-negative (DN) T cells (CD4 - CD8 - ) are unconventional kidney T cells with regulatory abilities. Their role in repair from acute kidney injury (AKI) is unknown. Kidney DN T cell population decreased during repair after ischemic AKI, in contrast to regulatory T cells (Tregs) which increased. DN T cell administration accelerated tubular repair in vitro, while after severe in vivo ischemic injury reduced kidney fibrosis and increased glomerular filtration rate (GFR). DN T cell infusion is a potential therapeutic agent to improve outcome from severe AKI.

Acute kidney injury (AKI) due to cisplatin is a significant problem that limits its use as an effective chemotherapeutic agent. T cell receptor + CD4 - CD8 - double negative (DN) T cells constitute the major T cell population in the human and mouse kidney, express programmed cell death protein (PD)-1, and protect from ischemic AKI. However, the pathophysiological roles of DN T cells in cisplatin-induced AKI is unknown. In this study, wild-type mice were treated with cisplatin (30 mg/kg) or vehicle, and the effects on kidney DN T cell numbers and function were measured. In vitro experiments evaluated effects of kidney DN T cells on cisplatin-induced apoptosis and PD ligand 1 (PD-L1) in renal epithelial cells. Adoptive transfer experiments assessed the therapeutic potential of DN T cells during cisplatin-induced AKI. Our results show that kidney DN T cell population increased at 24 h and declined by 72 h after cisplatin treatment. Cisplatin treatment increased kidney DN T cell proliferation, apoptosis, CD69, and IL-10 expression, whereas CD62L, CD44, IL-17A, interferon-γ, and TNF-α were downregulated. Cisplatin treatment decreased both PD-1 and natural killer 1.1 subsets of kidney DN T cells with a pronounced effect on the PD-1 subset. In vitro kidney DN T cell coculture decreased cisplatin-induced apoptosis in kidney proximal tubular epithelial cells, increased Bcl-2, and decreased cleaved caspase 3 expression. Cisplatin-induced expression of PD ligand 1 was reduced in proximal tubular epithelial cells cocultured with DN T cells. Adoptive transfer of DN T cells attenuated kidney dysfunction and structural damage from cisplatin-induced AKI. These results demonstrate that kidney DN T cells respond rapidly and play a protective role during cisplatin-induced AKI.

Expression of spermine/spermidine-N1-acetyltransferase (SSAT), the rate-limiting enzyme of polyamine backconversion cascade, increases after ischemia-reperfusion injuries (IRI). We hypothesized that SSAT plays an important role in the mediation of IRI. To test our hypothesis, wild-type (SSAT-wt) and SSAT-deficient (SSAT-ko) mice were subjected to liver or kidney IRI by ligation of hepatic or renal arteries. The liver and kidney content of putrescine (Put), a downstream by-product of SSAT activity, increased in SSAT-wt animals but not in SSAT-ko animals after IRI, indicating that polyamine backconversion is not functional in SSAT-deficient mice. When subjected to hepatic IRI, SSAT-ko mice were significantly protected against liver damage compared with SSAT-wt mice. Similarly, SSAT-ko animals subjected to renal IRI showed significantly greater protection against damage to kidney tubules than SSAT-wt mice. These studies indicate that SSAT-deficient animals are protected against IRI and suggest that SSAT is an important mediator of the tissue damage in IRI.

Branching morphogenesis of the metanephric kidney is critically dependent on the delicate orchestration of diverse cellular processes including proliferation, apoptosis, migration, and differentiation. Sphingosine-1-phosphate (S1P) is a potent lipid mediator influencing many of these cellular events. We report increased expression and activity of both sphingosine kinases and S1P phosphatases during development of the mouse metanephric kidney from induction at embryonic day 11.5 to maturity. Sphingosine kinase activity exceeded S1P phosphatase activity in embryonic kidneys, resulting in a net accumulation of S1P, while kinase and phosphatase activities were similar in adult tissue, resulting in reduced S1P content. Sphingosine kinase expression was greater in the metanephric mesenchyme than in the ureteric bud, while the S1P phosphatase SPP2 was expressed at greater levels in the ureteric bud. Treatment of cultured embryonic kidneys with sphingosine kinase inhibitors resulted in a dose-dependent reduction of ureteric bud tip numbers and increased apoptosis. Exogenous S1P rescued kidneys from apoptosis induced by kinase inhibitors. Ureteric bud tip number was unaffected by exogenous S1P in kidneys treated with N,N-dimethylsphingosine, although tip number increased in those treated with d,l-threo-dihydrosphingosine. S1P1 and S1P2 were the predominant S1P receptors expressed in the embryonic kidney. S1P1 expression increased during renal development while expression of S1P2 decreased, and both receptors were expressed predominantly in the metanephric mesenchyme. These results demonstrate dynamic regulation of S1P homeostasis during renal morphogenesis and suggest that differential expression of S1P metabolic enzymes and receptors provides a novel mechanism contributing to the regulation of kidney development.

There is growing evidence that sphingosine 1-phosphate (S1P) plays an important role in regulating the development, morphology, and function of the cardiovascular system. There is little data, however, regarding the relative contribution of endogenous S1P and its cognate receptors (referred to as S1P(1-5)) to cardiovascular homeostasis. We used S1P(2) receptor knockout mice (S1P(2)(-/-)) to evaluate the role of S1P(2) in heart and vascular function. There were no significant differences in blood pressure between wild-type and S1P(2)(-/-) mice, measured in awake mice. Cardiac function, evaluated in situ by using a Millar catheter, was also not different in S1P(2)(-/-) mice under baseline or stimulated conditions. In vivo analysis of vascular function by flowmetry revealed decreases in mesenteric and renal resistance in S1P(2)(-/-) mice, especially during vasoconstriction with phenylephrine. In intact aortic rings, the concentration-force relations for both KCl and phenylephrine were right shifted in S1P(2)(-/-) mice, whereas the maximal isometric forces were not different. By contrast, in deendothelialized rings the concentration-force relations were not different but the maximal force was significantly greater in S1P(2)(-/-) aorta. Histologically, there were no apparent differences in vascular morphology. These data suggest that the S1P(2) receptor plays an important role in the function of the vasculature and is an important mediator of normal hemodynamics. This is mediated, at least in part, through an effect on the endothelium, but direct effects on vascular smooth muscle cannot be ruled out and require further investigation.

Studies were performed to investigate the regulatory mechanism of bladder cyclooxygenase-2 (COX-2) expression after outlet obstruction. In situ hybridization of murine bladder tissues using COX-2-specific riboprobes demonstrated that COX-2 expression was induced predominantly in the bladder smooth muscle cells after outlet obstruction. To study the effect of increased mechanical stretch on COX isoform expression, cultured rat bladder smooth muscle cells were grown on silicone elastomer-bottomed plates coated with collagen type I and were subjected to continuous cycles of stretch/relaxation for variable duration. COX-1 mRNA levels did not change with stretch. COX-2 expression increased in a time-dependent manner after stretch, with maximal mRNA and protein levels occurring after 4 h. PGE2 levels increased more than 40-fold in the culture media after stretch, consistent with increased COX activity, and this was reduced to near completion in the presence of a COX-2 inhibitor, NS-398. Exposure to stretch over a 48-h period induced a 4.7 +/- 0.6-fold increase in tritiated thymidine incorporation rate. This increase in DNA synthesis was markedly suppressed when the cells were stretched in the presence of NS-398. We conclude that in bladder obstruction COX-2 activation occurs predominantly in the smooth muscle cells in response to mechanical stretch. Our findings also suggest that stretch-activated COX-2 expression may participate in bladder smooth muscle cell proliferation and thereby play a role in pathological bladder wall thickening after obstruction.

Grb7, a signaling protein whose physiological function is unknown, binds receptor tyrosine kinases important for normal kidney development. By investigating and correlating Grb7 gene expression with that reported for Grb7-binding receptors, we provide clues to Grb7 function(s). RT-PCR and immunoblot were used to demonstrate Grb7 gene and protein expression in the mature kidney. Additional RT-PCR studies detected gene expression in all microdissected adult nephron segments examined, except glomeruli, and in the mouse metanephric kidney from embryonic day 11 (E11) through to day 17 (E17). In situ hybridization at E14 demonstrated the following cellular pattern of localization: Grb7 mRNA in metanephric epithelia of mesenchymal and ureteric bud origin; no expression in the undifferentiated mesenchyme; and little expression in podocyte-destined cells or primitive glomeruli. Grb7 mRNA was also present in the epithelia of the lung and gut at E14. Thus Grb7 may have a basic function in growth factor signaling in terminally differentiated epithelia along the nephron and in developing epithelia in the kidney, lung, and gut. It is localized in a pattern permissive for a role in Her2 and Ret receptor signaling.

Studies were undertaken to assess expression of inducible cyclooxygenase (COX)-2 in bladder during fetal development and COX-1 and COX-2 expression after outlet obstruction. Bladder tissue or bladder progenitor tissue was harvested from CD-1 murine embryos at embryonic days 11.5 (E11.5), E14.5, E17.5, E20.5 (newborn), and from adult. Bladder obstruction was created in adult female mice by ligating the urethra, and bladders were harvested after 3-24 h of obstruction. Gene expression was assessed by semiquantitative reverse transcription-polymerase chain reaction and Western blotting. COX-2 was highly expressed at the early stages of bladder development and declined progressively throughout gestation. In adult bladder, both COX-1 and COX-2 were detectable at low levels under basal conditions. An approximately 30-fold increase in COX-2 mRNA was seen after 24 h of obstruction. In contrast, COX-1 did not change with obstruction. COX-2 mRNA levels peaked at 6 h of obstruction. In regional bladder-distention models, COX-2 induction was confined to the area of distention. Bladder outlet obstruction stimulates COX-2 expression dramatically, reactivating a gene that is highly expressed during fetal development.

Investigators approaching the problem of renal organogenesis have been hampered by a paucity of suitable molecular markers that specify distinct developmental phenotypes. To identify such markers, differential display-polymerase chain reaction (DD-PCR) was used to survey the temporal pattern of gene expression in mouse kidney at 11.5, 13.5, 15.5, and 17.5 days after conception and in the adult kidney. Twenty-two differentially expressed amplification products were identified, isolated, and sequenced. Seventeen clones showed no significant similarity with previously reported nucleotide sequences: two were similar to two housekeeping gene products, and three were similar to human or rat expressed sequence tags. To confirm the differential expression patterns observed by DD-PCR, semiquantitative reverse transcription-PCR was performed using sequence-specific oligonucleotide primers. Nineteen of 22 clones were differentially expressed during kidney development [mouse embryonic renal marker (MERM) sequences 1-19]. The value of MERMs as developmental markers was further assessed in mouse metanephric organ culture, where the pattern of MERM transcript expression mimicked that observed in vivo. Therefore, the DD-PCR method permitted development of a panel of marker sequences that can be used to characterize renal developmental processes and that may allow the identification of novel, functionally relevant gene products.

We have previously reported that in several renal cell types, adenosine receptor agonists inhibit adenylyl cyclase and activate phospholipase C via a pertussis toxin-sensitive G protein. In the present study, in 28A cells, both of these adenosine receptor-mediated responses were inhibited by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a highly selective A1 adenosine receptor antagonist. The binding characteristics of the adenosine A1 receptor in the 28A renal cell line were studied using the radiolabeled antagonist [3H]DPCPX to determine whether two separate binding sites could account for these responses. Saturation binding of [3H]DPCPX to 28A cell membranes revealed a single class of A1 binding sites with an apparent Kd value of 1.4 nM and maximal binding capacity of 64 fmol/mg protein. Competition experiments with a variety of adenosine agonists gave biphasic displacement curves with a pharmacological profile characteristic of A1 receptors. Comparison of [3H]DPCPX competition binding data from 28A cell membranes with rabbit brain membranes, a tissue with well-characterized A1 receptors, reveals that the A1 receptor population in 28A cells has similar agonist binding affinities to the receptor population in brain but has a considerably lower density. Addition of guanosine 5'-triphosphate (100 microM) to 28A cell membranes caused the competition curves to shift from biphasic to monophasic, indicating that the A1 receptors exist in two interconvertible affinity states because of their coupling to G proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine has been reported to produce a biphasic renal blood flow (RBF) response (vasoconstriction followed by a return of flow to control level) and a decrease in glomerular filtration rate (GFR) when infused into the kidney. Intrarenal adenosine infusion also leads to a decrease in renin release. By altering the hemodynamic response to adenosine, we sought to determine whether the decrease in renin release depends on vascular or filtration-induced events. In nine dogs with nonfiltering kidneys, adenosine infusion (3 X 10(-7) mol/min) resulted in a biphasic RBF response and an inhibition of renin release (309 +/- 53 vs. 71 +/- 26 ng ANG I/min). In 11 dogs treated with verapamil (10 micrograms X kg-1 X min-1) no vasoconstriction or decrease in GFR occurred; however, renin release was inhibited by adenosine (1,300 +/- 159 vs. 534 +/- 225 ng ANG I/min). In a third group of nine dogs whose ureteral pressure was raised to 80 cmH2O, adenosine infusion produced a sustained vasoconstriction and an inhibition of renin release (3,086 +/- 1,144 vs. 328 +/- 130 ng ANG I/min). These experiments, in which the renin release effects of adenosine are dissociated from the hemodynamic effects, lead us to conclude that the inhibition of renin release produced by adenosine does not depend either on the vascular or filtration-induced effects of adenosine.

To investigate the cellular mechanisms underlying the epithelial actions of adenosine, we studied adenosine receptor-effector coupling in cultured rabbit cortical collecting tubule (RCCT) cells. We previously reported, in RCCT cells isolated by immunodissection, that a potent A2 adenosine analogue [5'-N-ethylcarboxamideadenosine (NECA)] stimulates cAMP production [effective concentration 50% (EC50) = 1 microM], and potent A1 analogues [N6-cyclohexyladenosine (CHA) and R-N6-phenylisopropyladenosine (PIA)] inhibit basal and AVP-stimulated cAMP production (EC50 = 5 nM). The present study was undertaken to determine whether adenosine receptors in RCCT cells are also coupled to a signal transduction system leading to the mobilization of intracellular free calcium. RCCT cells were loaded with the fluorescent calcium indicator, fura-2, and were treated with the adenosine analogues NECA, CHA, and PIA. All three adenosine analogues produced dose-dependent (1 nM-0.1 mM), transient increases in intracellular calcium concentration with equal potency (EC50 = 0.5 microM). Chelation of extracellular calcium with ethyleneglycol-bis(beta-aminoethyl ether)N,N,N',N' tetraacetic acid (EGTA) did not abolish the increase in calcium. The adenosine receptor antagonists, 1,3-diethyl-8-propylxanthine and 8-cyclopentyl-1,3-dipropylxanthine, and pretreatment of RCCT cells with pertussis toxin blocked the increase in calcium. These results demonstrate that RCCT cells have, in addition to adenosine receptors associated with the stimulation and inhibition of cAMP, a pertussis-toxin sensitive receptor system that leads to the mobilization of intracellular calcium.

To aid in characterizing adenosine receptors in renal cells, primary cultures of rabbit cortical collecting tubule (RCCT) cells were infected with an adenovirus 12-simian virus 40 hybrid, resulting in a continuous cell line. The cells, designated RCCT-28A, retained their epithelial morphology and reacted with a monoclonal antibody specific for rabbit collecting tubule. Adenosine 3',5'-cyclic monophosphate (cAMP) accumulation was stimulated by vasopressin (AVP), isoproterenol, prostaglandin E2 (PGE2), calcitonin, parathyroid hormone, and a potent adenosine A1- and A2-receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA). A more selective adenosine A1-receptor agonist, N6-cyclohexyl adenosine (CHA) inhibited basal and AVP-stimulated cAMP accumulation. Cytosolic free calcium was transiently elevated by bradykinin, PGE2, NECA, and CHA. To examine the mechanism by which adenosine analogues increase intracellular free calcium, phosphoinositide (PI) turnover was assessed in the 28A cells after labeling with myo-[3H]inositol. NECA and CHA increased [3H]inositol phosphate formation with an approximate half-maximal effective concentration of 0.1 microM for both analogues. The increase in PI turnover was blocked by the selective adenosine A1-receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine and pretreatment of the 28A cells with pertussis toxin. These results suggest that adenosine analogues increase cytosolic free calcium by stimulating PI turnover.

A mouse monoclonal antibody designated IgG3(rct-30) has been prepared that reacts specifically with an antigen on the surface of all cells comprising the cortical and medullary rabbit renal collecting tubule including the arcades. Plastic culture dishes coated with IgG3(rct-30) were used to isolate collecting tubule cells from collagenase dispersions of rabbit renal cortical cells by immunoadsorption. Typically, 10(6) rabbit cortical collecting tubule (RCCT) cells were obtained from 5 g of renal cortex (2 kidneys). Initial purity was greater than 96% based on immunocytofluorescent staining with three different anti-collecting tubule antibodies. Between 20 and 30% of the RCCT cells were reactive with peanut lectin suggesting that RCCT cells are a mixture of principal and intercalated cells. Approximately 10(7) RCCT cells were obtained after 4 to 5 days in primary culture. Moreover, RCCT cells continued to proliferate after passaging with a doubling time of approximately 32 h. RCCT cells passaged once and then cultured 4-5 days were found 1) to synthesize cAMP in response to arginine vasopressin (AVP), prostaglandin E2 (PGE2), isoproterenol, and parathyroid hormone, but not calcitonin, prostaglandin D2, or prostaglandin I, and 2) to release PGE2 in response to bradykinin but not arginine vasopressin or isoproterenol. Our results indicate that cultured RCCT cells retain many of the hormonal, histochemical, and morphological properties expected for a mixture of principal and intercalated rabbit cortical collecting tubule epithelia. RCCT cells should prove useful both for studying hormonal interactions in the cortical collecting tubule and as a starting population for isolating intercalated collecting tubule epithelia.