Cellular senescence in the aging and diseased kidney.

The program of cellular senescence is involved in both the G1 and G2 phase of the cell cycle, limiting G1/S and G2/M progression respectively, and resulting in prolonged cell cycle arrest. Cellular senescence is involved in normal wound healing. However, multiple organs display increased senescent cell numbers both during natural aging and after injury, suggesting that senescent cells can have beneficial as well as detrimental effects in organismal aging and disease. Also in the kidney, senescent cells accumulate in various compartments with advancing age and renal disease. In experimental studies, forced apoptosis induction through the clearance of senescent cells leads to better preservation of kidney function during aging. Recent groundbreaking studies demonstrate that senescent cell depletion through INK-ATTAC transgene-mediated or cell-penetrating FOXO4-DRI peptide induced forced apoptosis, reduced age-associated damage and dysfunction in multiple organs, in particular the kidney, and increased performance and lifespan. Senescence is also involved in oncology and therapeutic depletion of senescent cells by senolytic drugs has been studied in experimental and human cancers. Although studies with senolytic drugs in models of kidney injury are lacking, their dose limiting side effects on other organs suggest that targeted delivery might be needed for successful application of senolytic drugs for treatment of kidney disease. In this review, we discuss (i) current understanding of the mechanisms and associated pathways of senescence, (ii) evidence of senescence occurrence and causality with organ injury, and (iii) therapeutic strategies for senescence depletion (senotherapy) including targeting, all in the context of renal aging and disease.

Stimulus-secretion coupling in beta-cells of transplantable human islets of Langerhans. Evidence for a critical role for Ca2+ entry.

With human islets isolated for transplantation, we examined the applicability to humans of a metabolic fuel hypothesis of glucose transduction and a Ca2+ hypothesis of depolarization-secretion coupling, both previously proposed for rodent islet beta-cells. We report that several features of human beta-cell physiology are well accounted for by these hypotheses. With whole-islet perifusion, we demonstrated that insulin secretion induced by glucose, tolbutamide, or elevated K+ is dependent on extracellular Ca2+. Insulin release induced by these secretagogues is enhanced by the dihydropyridine Ca2+ channel agonist BAYk8644 and depressed by the dihydropyridine Ca(2+)-channel antagonist nifedipine. All of the aforementioned secretagogues provoke increases in cytosolic free Ca2+, which are dependent on extracellular Ca2+ and are altered by the dihydropyridine drugs. Individual beta-cells in the islet display diminished resting membrane conductance, graded depolarization, and complex electrical patterns, including bursts of action potentials in response to stimulatory concentrations of glucose or tolbutamide. Individual islet beta-cells display voltage-dependent Ca2+ currents that are activated at membrane potentials traversed during the excursion of the action potential. In most cells, the Ca2+ currents are enhanced by BAYk8644 and depressed by nifedipine at concentrations that have parallel effects on secretagogue-induced increases in cytosolic Ca2+ and insulin secretion. These survey studies should provide the basis for more detailed investigations of the relationship of voltage-dependent ionic currents to electrical activity patterns and of electrical activity patterns to granule exocytosis in single human beta-cells.

Effects of metabolic inhibition by sodium azide on stimulus-secretion coupling in B cells of human islets of Langerhans.

Sodium azide (NaN3), a reversible inhibitor of mitochondrial respiration, blocks glucose-induced electrical activity and insulin secretion in human pancreatic islet B cells. Here we show that brief (10-15 min) application followed by removal of 3 mM NaN3 results in transient overshoot of electrical activity and insulin secretion even at substimulatory levels of glucose (3-5 mM). In addition, application of NaN3, even at very low [Ca2+]o, reversibly increases cytosolic Ca2+ to levels usually associated with substantial insulin release. These results suggest that (i) metabolic inhibition may reset B cell stimulus-secretion coupling and (ii) a rise in free cytosolic Ca2+, by itself, is not sufficient to trigger insulin secretion.

Effects of sulfonamides on a metabolite-regulated ATPi-sensitive K+ channel in rat pancreatic B-cells.

Intracellular ATP (ATPi)-sensitive K+ [K+(ATP)] channels are now a recognized site of action of clinically useful hypoglycemic and hyperglycemic sulfonamides. We have further examined the action of these agents on single K+ channels in rat pancreatic B-cells 1) Tolbutamide and glyburide, two hypoglycemic sulfonylureas which decrease K+(ATP) channel activity in the cell-attached patch, affect the kinetics of K+(ATP) channel in a manner similar to glucose. They shorten the duration of the "burst," or cluster of open channel events, while lengthening the intervals between bursts. 2) The hyperglycemic vasodilator diazoxide increases mean K+(ATP) channel activity in the cell-attached patch as well as in the inside-out excised patch exposed to ATPi. It appears to lengthen channel bursts and shorten the intervals between them. Two structurally similar diuretics, hydrochlorothiazide and furosemide, which have mild hyperglycemic effects, do not increase K+(ATP) channel activity even at clinically toxic concentrations. 3) Neither the sulfonylureas nor diazoxide directly affect the activity of single delayed rectifier K+ channels or single calcium and voltage-activated K+ channels in normal B-cells.

'Perforated patch recording' allows long-term monitoring of metabolite-induced electrical activity and voltage-dependent Ca2+ currents in pancreatic islet B cells.

We describe the application of 'perforated patch recording' using the pore-forming antibiotic nystatin, to monitor the electrical activity and underlying ionic currents of rat and human pancreatic islet B cells. We demonstrate that glucose-induced electrical activity is seen even in single B cells during current-clamp recordings lasting hours 'L-type' Ca2+-channel currents can also be monitored over this period of time. This technique may prove useful in examining hormone and neurotransmitter modulation of electrical activity in B cells, while minimizing the effects of cytoplasmic 'wash-out'.

Activity of ion channels during volume regulation by clonal N1E115 neuroblastoma cells.

When exposed to a hypotonic bathing solution, clonal N1E115 neuroblastoma cells initially swell and then undergo a regulatory volume decrease (RVD). Using cell-attached patch-clamp recording, we have found that the activity of a stretch-sensitive, nonselective cation [C+(SA)] channel increases shortly after the onset of osmotically induced cell swelling; this depolarizes the cells as much as 30 mV. Shortly thereafter, and roughly coincident with the onset of RVD, two types of voltage-dependent channels open at the new resting potential; these are (i) a delayed-rectifier type K+ [K+(DR)] channel and (ii) a large-conductance anion channel. We suggest that opening of the C+(SA) channel may contribute to the volume "sensor" mechanism, while the depolarization-induced opening of the K+(DR) and anion channels may constitute a significant K+ salt exit pathway, operating in RVD.

Metabolite-regulated ATP-sensitive K+ channel in human pancreatic islet cells.

In patch-clamped surface cells of human islets, we identified an inwardly rectifying, voltage-independent K+ channel that may be a crucial link between substrate metabolism and depolarization-induced insulin secretion. It is the major channel open at rest. It closes on exposure of the cell to secretagogue concentrations of glucose or other metabolic fuels and oral hypoglycemic sulfonylureas but reopens on addition of either a metabolic inhibitor that prevents substrate utilization or the hyperglycemic sulfonamide diazoxide. Onset of electrical activity coincides with channel closure by the secretagogues. In excised patches, the activity of this channel is inhibited at its cytoplasmic surface by ATP. These results suggest that in humans, as in rodents, 1) rises in cytoplasmic ATP levels during substrate metabolism trigger K+-channel closure and cell depolarization and 2) clinically useful sulfonamides modulate glucose-induced insulin secretion, in part by affecting a readily identifiable resting conductance pathway for K+.

A stretch-activated anion channel in tobacco protoplasts.

Stretch-activated ion channels have been described in animal cells, where they might serve as mechanoreceptors, baroreceptors or osmoreceptors, as well as in yeast and bacteria, where osmoregulatory functions have been suggested. Here we report a large conductance, stretch-activated, anion-selective channel in protoplasts of a higher plant, tobacco, and discuss its possible role in osmoregulation.

Dependence on multivalent cations of quantal release of transmitter induced by black widow spider venom.

Application of alpha-latrotoxin (alpha-LT), the active component of black widow spider venom (BWSV), to a vertebrate neuromuscular junction, in the presence of millimolar bath concentrations of Ca2+ or Mg2+, greatly increases the frequency of miniature end-plate potentials (Fmepp). We have further characterized the cation dependence of alpha-LT action at the frog cutaneous pectoris neuromuscular junction. The divalent cations, Ca, Sr, Ba at less than or equal to 50 microM, Zn, Mn, Cd at greater than or equal to 50-100 microM, and Mg at greater than or equal to 1.0 mM, as well as the trivalent cation La at greater than or equal to 15 microM, all increase Fmepp exponentially to greater than or equal to 100-200 s-1 over several minutes time. The exponential rate of rise is graded with extracellular cation concentration and can be reduced by increasing [K+] of the bath from 2 to 25-40 mM. Long-term exposure to alpha-LT in the presence of Sr2+ or Mn2+ results in the exhaustion of the releasable quantal store of transmitter, which in the case of Mn2+ correlates well with depletion of synaptic vesicles. These data support the hypothesis that BWSV promotes an increase in Fmepp by increasing nerve terminal permeability to multivalent cations that enter the nerve terminal down their electrochemical gradients and then may bind to quantal release activating sites or displace Ca2+ from intracellular stores.

Cation dependence of posttetanic potentiation of neuromuscular transmission.

We have investigated the possibility that much of posttetanic potentiation (PTP) of quantal release of neurotransmitter at the frog neuromuscular junction may be due to posttetanic accumulation of [Ca2+]i, via a plasmalemmal Cao2+-Nai+ exchanger that is powered by an increase in Nai+ during the tetanus. Our new findings on the cationic dependence of PTP are consistent with this hypothesis. 1) Several manuevers that decrease Na+-K+ pump activity, (decreasing [K+]o, replacing K+o with Rb+o or Li+o, or adding acetylstrophanthidin to Ringers), all increase the intratetanic rise and prolong the posttetanic decay of epp quantal content (m) and miniature epp frequency (fmepp). 2) Increasing [Ca2+]i or [Sr2+]o, but not [Mg2+]o, increases posttetanic fmepp in a graded fashion. 3) PTP of fmepp is still present after addition of Mn2+o, which blocks voltage dependent Ca2+ entry.

A metabolite-regulated potassium channel in rat pancreatic B cells.

In B cells from dispersed rat islet of Langerhans we have identified an inward rectifying voltage-independent K+ channel whose behavior parallels the metabolically regulated potassium permeability (PK) found in tracer flux and microelectrode recording studies. In cell-attached patches of membrane, the channel is closed when any one of several substrates (glucose, mannose, leucine, or glyceraldehyde) is added to the cell's bathing solution but is reopened on addition of an appropriate metabolic inhibitor, which prevents utilization of that substrate. In inside-out excised patches, a K+ channel with nearly identical kinetic features is closed by addition of micromolar concentrations of ATP to the "cytoplasmic" solution. The ATP sensitivity of channel activity is modified by addition of ADP, suggesting competition at a nucleotide binding site. These results suggest the presence of a metabolically regulated K+ channel gated by intracellular concentrations of ATP or the ratio of ATP/ADP concentrations.

Bristow-Latarjet repair for recurrent anterior shoulder instability; an eight-year study.

A follow-up study was made of 44 patients who underwent 47 should operations as described by Bristow and Latarjet. The group consisted of 32 recurrent shoulder dislocations and 15 so-called spontaneous shoulder instabilities. The average follow-up was 3.7 years. No significant complications occurred either per- or postoperatively and relapse of luxation was not seen. Only one patient had objectively confirmed shoulder instability after the operation. The average limitation of external rotation at 90 degrees abduction was 12 degrees.