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. Author manuscript; available in PMC: 2009 Oct 23.
Published in final edited form as: Hypertension. 2007 Dec 24;51(2):488–493. doi: 10.1161/HYPERTENSIONAHA.107.102228

Cellular Stretch Increases Superoxide Production in the Thick Ascending Limb

Jeffrey L Garvin 1, Nancy J Hong 1
PMCID: PMC2766239  NIHMSID: NIHMS116671  PMID: 18158344

Abstract

Superoxide (O2−) is an important regulator of kidney function. We have recently shown that luminal flow stimulates O2− production in the thick ascending limb (TAL), attributable in part to mechanical factors. Stretch, pressure and shear stress all change when flow increases in the TAL. We hypothesized that stretch rather than shear stress or pressure per se stimulates O2− production by TALs. We measured O2− production in isolated perfused rat TALs using fluorescence microscopy and dihydroethidium. Tubules were perfused with a Na-free solution to eliminate the confounding effect of Na transport. Flow induced an increase in O2− production from 29±4 to 90±8 AU/s (P<0.002; n=5). The response to flow is rapidly reversible. O2− production by TALs perfused at 10 nL/min decreased from 113±6 to 25±10 AU/s (P<0.003; n=4) 15 minutes after flow was stopped. Increasing pressure and stretch in the absence of shear stress caused a significant increase in O2− production (40±6 to 118±17 AU/s; P<0.02; n=5). In contrast, eliminating shear stress had no effect (107±9 versus 108±10 AU/s; n=5). Increasing stretch by 27±2% in the presence of flow while reducing pressure stimulated O2− production from 66±7 to 84±9 AU/s (29±8%; P<0.02; n=5). Tempol inhibited this increase (n=5). We conclude that increasing stretch rather than pressure or shear stress accounts for the mechanical aspect of flow-induced O2− production in the TAL. Stretch of the TAL during hypertension, diabetes, and salt loading may contribute to renal damage.

Keywords: reactive oxygen species, free radicals, oxidative stress, mechanical stress, luminal flow, kidney

Abnormal production of reactive oxygen species, including superoxide (O2−), can lead to the development of hypertension1–3 and diabetic nephropathy.4,5 O2− is an important regulator of kidney function that favors salt retention.6–8 Increases in renal O2− have been shown to cause salt-sensitive hypertension.3,9 Medullary O2− appears to contribute more to salt retention than cortical O2−.10,11 The thick ascending limb (TAL) is the main source of medullary O2−,11–13 and O2− stimulates NaCl reabsorption in this segment.8,14 Abnormal NaCl reabsorption by this segment has also been implicated in salt-sensitive hypertension.15–17 Thus, it is important to understand the factors that regulate thick ascending limb O2− production.

Luminal flow through the TAL acutely varies over a wide range18–20 thereby causing changes in mechanical stress. We21 and others22 have recently shown that increasing luminal flow stimulates O2− production in the thick ascending limb and that at least part of this effect is attributable to increased Na delivery and consequently enhanced Na transport. However, we also showed that about 50% of the increase in O2− production induced by flow is attributable to a mechanical factor. Cellular stretch, transmural pressure, and shear stress all change when flow rate changes in the TAL. However, the specific mechanical stimulus involved in flow-induced O2− production is unknown.

Stretch has been shown to increase O2− production in several types of cells including vascular smooth muscle,23 endothelial,24 and osteoblast-like cells.25 Additionally, cyclic strain increases reactive oxygen species generation by pulmonary epithelial cells26 and fibroblasts.27 Shear stress also has been shown to induce vascular O2− production.28,29

Although transmural pressure per se has not been shown to increase O2− generation, it does activate signaling pathways that could enhance O2− production.30,31

We hypothesized that stretch rather than pressure or shear stress per se stimulates O2− production by TALs. To test this hypothesis, we systematically eliminated each of these mechanical factors one by one to observe the effect on O2− generation. Our findings indicate that cellular stretch increases O2− production by TALs and that transmural pressure and shear stress do not play a role in flow-induced O2− production.

Materials and Methods

Chemicals and Solutions

Dihydroethidium was purchased from Molecular Probes. Tempol and collagenase were obtained from Sigma-Aldrich. The Na-free solution used to perfuse tubules contained (in mmol/L) 270 mannitol; 10 HEPES, pH 7.4 (titrated with KOH) at 37°C; 5 KOH. The physiological saline used to bathe tubules contained (in mmol/L): 130 NaCl, 4 KCl, 2.5 NaH2PO4, 1.2 MgSO4, 6 L-alanine, 1 trisodium citrate, 5.5 glucose, 2 calcium dilactate, and 10 HEPES, pH 7.4 at 37°C. Both solutions were adjusted to 290±3 mOsm/kg H2O.

Isolation and Perfusion of Rat Thick Ascending Limbs

We used male Sprague-Dawley rats weighing 120 to 150 g (Charles River Breeding Laboratories, Wilmington, Mass) and maintained on a diet containing 0.22% sodium and 1.1% potassium (Purina) for at least 5 days. The Institutional Animal Care and Use Committee (IACUC) approved all protocols. Isolation and perfusion of TALs was performed as previously described.21 We perfused tubules at 10 nL/min. Intraluminal pressure was 16 to 17 mm Hg.

Measurement of O2− Using Dihydroethidium

TALs were loaded by bathing tubules in 10 μmol/L dihydroethidium for 20 minutes and then washing in dye-free solution for 30 minutes. Excitation and emission were controlled via a system composed of a xenon arc lamp, excitation filters in a λ-10 filter wheel (Sutter Instruments), and a custom-made dichroic mirror and emission filter (Chroma Technology). Oxyethidium and dihydroethidium were excited using 490 nm and 365 nm light, respectively. Emitted fluorescence intensities were measured between 520 to 600 nm (oxyethidium) and 400 to 450 nm (dihydroethidium) from regions of interest (ROI). ROIs were set larger than the tubule to accommodate changes in tubule shape caused by increasing flow, pressure, or stretch. This technique was validated by testing whether measured fluorescence changed when either the tubule was moved in the ROI or the ROI was moved with respect to the tubule. Fluorescence was imaged digitally with an image intensifier and a charge-coupled device (CCD) camera. It was recorded using Metafluor version 7 imaging software (Universal Imaging Corporation). The rate of change of the ratio between the fluorescence attributable to oxyethidium and dihydroethidium was taken to be a measure of O2− production.32,33 O2− was measured at the beginning of each period once every 5 s for 1 minute. Regression analysis of the first 6 fluorescence ratio measurements was performed and differences in slopes were statistically evaluated.

Protocols

Protocol 1: Effect of Increasing/Decreasing Luminal Flow

In the control period TALs were mounted on perfusion pipets with the distal ends open and O2− synthesis measured in the absence of luminal flow (nonperfused). Then we increased the flow rate to 10 nL/min (perfused) and measured O2− generation again. Control experiments were performed in which tubules remained nonperfused for the second measurement period.

In separate experiments, we first measured O2− generation in the presence of flow at 10 nL/min (perfused) and then again 15 minutes after stopping flow (nonperfused). Control experiments were performed in which tubules were perfused at a flow rate of 10 nL/min for both measurement periods.

Protocol 2: Effect of Stretch and Pressure in the Absence of Shear Stress

In the control period, we pinched the distal ends of tubules closed and measured O2− production in the absence of applied luminal pressure and flow. In the second period, luminal pressure was increased so that the outer diameter was the same as the average diameter observed in Protocol 1 (thus pressure and stretch were increased in the absence of shear stress) and production measured again.

Protocol 3: Effect of Maintaining Stretch and Pressure While Eliminating Shear Stress

In the control period, TALs were perfused at 10 nL/min with the distal ends open and O2− synthesis was measured. Then we stopped flow and pinched the distal ends closed to prevent luminal flow and thus shear stress. Afterward, luminal pressure was increased so that the outer diameters were the same (as were pressure and stretch) as in the control period and O2− generation measured again. Control experiments were performed in which the effect of pinching distal ends closed on O2− production was measured in the absence of applied luminal pressure and flow. Control experiments were also performed in which distal ends were not pinched and flow was maintained.

Protocol 4: Effect of Increasing Stretch While Reducing Pressure and Shear Stress

TALs were perfused with their distal ends open at 10 nL/min, and O2− production was measured. We treated the tubules with 0.1% collagenase in the bath for 5 minutes and washed for 10 minutes to partially digest the basement membrane, and then measured O2− production again. Because luminal diameter increased 27±2% and flow rate was maintained at 10 nL/min, we enhanced stretch while reducing both pressure and shear stress. Control experiments were performed in which luminal pressure was adjusted before collagenase treatment such that the average outer diameter observed during the control period of Protocol 4 was maintained after collagenase treatment.

Statistical Analysis

Results are expressed as mean±SE. The paired Student t test was used to evaluate the data and P<0.05 was considered significant.

Results

To begin to analyze whether stretch, shear stress, or pressure mediates the mechanical aspect of flow that stimulates O2− production, we measured O2− generation in the absence and presence of luminal flow. To eliminate the reported effect that increased Na delivery has on O2− generation, these experiments were performed in the absence of luminal NaCl. Figure 1 shows that during the control period in the absence of luminal flow, TALs produced O2− at a rate of 29±4 AU/s. Fifteen minutes after luminal flow was increased to 10 nL/min, O2− generation rose significantly to 90±8 AU/s (P<0.002; n=5).

Figure 1.

Figure 1

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Flow stimulates O2− production in the thick ascending limb. Perfusion at a luminal flow rate of 10 nL/min induced an increase in the rate of O2− generation from 29±4 to 90±8 AU/s (*P<0.002; n=5).

In separate experiments the protocol was reversed (Figure 2). In the control period in the presence of luminal flow at 10 nL/min O2− synthesis was 113±6 AU/s. Fifteen minutes after luminal flow was stopped, it declined to 25±10 AU/s (P<0.003; n=4). Taken together these data indicate that: (1) enhancing luminal flow augments O2− synthesis independent of changes in ion delivery; and (2) this is a rapidly reversible process.

Figure 2.

Figure 2

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Flow-induced enhancement of O2− generation is rapidly reversible. Fifteen minutes after stopping luminal flow, O2− production declined from 113±6 to 25±10 AU/s (*P<0.003; n=4).

We next examined whether stretch and pressure could stimulate O2− synthesis in the absence of shear stress. To do this we pinched the distal ends of tubules closed and increased luminal pressure to the average diameter of the tubules perfused at 10 nL/min. As shown in Figure 3, during the control period in the absence of flow and pressure, pinched TALs produced O2− at a rate of 40±6 AU/s. Fifteen minutes after tubules were pressurized resulting in stretch of the epithelial cells, O2− production rose to 118±17 AU/s (P<0.02; n=5). These data indicate that pressure, stretch, or both can stimulate O2− generation in the absence of shear stress.

Figure 3.

Figure 3

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Pressure or stretch stimulates O2− production. TALs were mounted on pipets in the absence of flow, and the distal ends were pinched closed. During the control period conditions of no pressure and stretch (No P/Str), O2− production was 40±6 AU/s. Increasing P/Str in the absence of shear stress resulted in a rise in O2− to 118±17 AU/s (*P<0.02; n=5).

To further show that shear stress was not the mechanical factor that accounts for flow-enhanced O2− generation, we removed shear stress and maintained stretch and pressure while measuring O2− (Figure 4). During the control period, we perfused tubules at 10 nL/min with open distal ends. O2− production was 107±9 AU/s. After pinching distal ends closed and pressurizing TALs to the same outer diameter (and therefore the same pressure and stretch) as during the control period, the rate did not change significantly (108±10; n=5). These data indicate that shear stress does not play a role in flow-induced O2− production.

Figure 4.

Figure 4

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Eliminating shear stress (SS) does not affect O2− generation. During the control period, TALs with open distal ends and perfused at 10 nL/min produced O2− at a rate of 107±9 AU/s. When we stopped flow, pinched the distal ends, and adjusted pressure/stretch (P/Str) to the same as during the control period, O2− generation did not change (108±10 AU/s; n=5).

To investigate whether stretch or pressure is responsible for flow-induced O2− production, we perfused TALs before and after collagenase treatment. Figure 5 shows that during the control period O2− production was 66±7 AU/s when tubules were perfused at 10 nL/min. After collagenase treatment O2− generation increased to 84±9 AU/s (29±8%; P<0.02; n=5), whereas the outer diameter of the tubules, and therefore stretch, increased 27±2%. Because diameter increased and flow rate was constant, pressure declined greater than 50% in these experiments. O2− production did not significantly change after collagenase treatment in separate experiments in which diameter was maintained at the control value. These data indicate that stretch rather than pressure stimulates O2− production in the TAL.

Figure 5.

Figure 5

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Increasing stretch while reducing pressure enhances O2− production. Treatment of TALs with collagenase (collag) causes an increase in stretch (27±2% increase in tubule diameter) which results in a 29±8% increase in O2− production from 66±7 to 84±9 AU/s (*P<0.02; n=5). The luminal flow rate was 10 nL/min throughout the experiment.

To demonstrate that the changes in fluorescence ratio measurements are attributable to O2− generation, we added the O2− scavenger tempol to the bath at the beginning of the experiment. As shown in Figure 6, in the presence of 100 μmol/L tempol perfused TALs produced O2− at an expected reduced rate of 31±5 AU/s. Treatment with collagenase did not result in a significant difference in O2− generation (25±6 AU/s; n=5). In control experiments, O2− production did not differ over time in the presence of tempol when tubules were perfused at 10 nL/min. These data confirm that the stretch-induced increases in fluorescence ratios are attributable to increases in O2− generation.

Figure 6.

Figure 6

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The O2− scavenger tempol blocks the effect of stretch on O2− production. In the presence of 100 μmol/L tempol, O2− production in TALs perfused at a rate of 10 nL/min did not significantly change after treating the tubules with collagenase to increase stretch (31±5 versus 25±6 AU/s; n=5).

Discussion

We have demonstrated that increasing luminal flow enhances O2− production in the TAL independent of ion delivery and that this process is rapidly reversible. We found that increasing pressure and stretch in the absence of shear stress augments O2− generation, whereas removing shear stress while maintaining pressure and stretch has no effect on flow-induced O2− production. We found that using collagenase to increase stretch while reducing pressure and shear stress augments O2− production, and that tempol could block this effect. Therefore it is stretch rather than pressure or shear stress that accounts for the mechanical aspect of flow-induced O2− production in the TAL.

To our knowledge, this is the first study to demonstrate that stretch enhances O2− generation in the TAL. Abe et al22 showed that enhanced ion delivery and transport associated with increased flow augmented O2− in the TAL. However, they also acknowledged the possible contribution of mechanical factors to this effect. We recently showed that NaCl absorption attributable to Na/K/2Cl cotransport and flow-associated mechanical factors contribute equally to flow-induced stimulation of O2− production.21 The present study is the first to elucidate the specific mechanical component of flow that affects O2− production in the TAL.

The results from our flow versus no flow experiments, which demonstrated that the response to mechanical stimulation induced by a flow rate of 10 nL/min is rapidly reversible, provided a basis for our experiments examining specific mechanical stimuli. To distinguish the roles of each mechanical factor, we had to mimic in occluded tubules the mechanical parameters associated with free flow. We pinched tubules closed to prevent flow and then applied pressure to dilate the tubules. It may be argued that the filling of the tubule creates a transient shear stress that could elicit a flow-induced response. However, the volume of a typical TAL used in our experiments is 0.4 nL. Assuming that tubules are filled over 1 minute, filling rate is only 4% of the 10 nL/min used in free-flow experiments. The contribution is even less if one considers that flow falls to zero once tubules are filled, and we waited 15 minutes before measuring a response. Other investigators have also found a negligible effect of a transient flow impulse in studies of flow and stretch in the cortical collecting duct.34

To determine the effect of stretch per se, we used collagenase to partially digest the basement membrane of the tubules. Treatment of TALs with collagenase causes a 27% increase in the diameter of the tubule. This increase in stretch was achieved without an increase in pressure, because we kept flow rate constant. Because volume is proportional to the square of the radius, a 27% increase in the radius corresponds to a 60% increase in volume. At a constant flow rate, an increase in volume results in a proportional decrease in pressure.

The greater increase in O2− production (about 80 AU/s) observed in Figure 3 compared with that observed in Figure 5 (about 20 AU/s) is expected because of the different amounts of stretch. Although it is difficult to quantify the degree of stretch that cells undergo in Figure 3, it is reasonable to infer that cells undergo a much larger increase in stretch in Figure 3 than in Figure 5. In the absence of pressure/stretch (Figure 3), cells begin essentially at “zero” stretch. The lumen appears to have infoldings attributable to the columnar shape of the TAL cells in the absence of stretch. When pressure is applied, the cells stretch and the inner (luminal) diameter increases dramatically. In the absence of pressure and stretch, the inner diameter is approximately 3 μm and increases to 12 μm as pressure and stretch are applied. Therefore, cell length increases from 1.9 to 7.5 μm. In contrast, the cells in Figure 5 are already stretched because of flow and undergo only a 27% increase in length after collagenase treatment. As expected the 29% increase in O2− production in Figure 5 is not as large as that observed in Figure 3.

Mechanical strain-induced O2− production has been widely studied in endothelial cells.24,28,35,36 O2− generation in response to mechanical stress has also been described in vascular smooth muscle cells,23 cardiac myocytes,37 and pulmonary epithelial cells.26 Although numerous studies have revealed an important role of mechanical strain in reactive oxygen species production in the vasculature, little is known about the role of O2− as a mediator of mechanotransduction in renal tubules. The TAL is one of the most compliant nephron segments,18 and therefore the pressure that accompanies an increase in luminal flow can stretch TAL cells and thereby trigger O2− production.

The mechanism involved in stretch-induced generation of O2− in the TAL is unknown. However, several different signaling pathways triggered by mechanical stimuli can also lead to increased O2− production. Pathways that mediate O2− production include PKC,4,38,39 Src,40 phospholipase A2,41 and Akt.42 Stretch has been shown to stimulate intracellular Ca34 in cortical collecting ducts, which in turn can activate PKC. A PKC-dependent activation of S6 kinase by cyclic stretch in mesangial cells has also been demonstrated.43 Stretch also activates Src,44 which in turn can trigger O2− production.40

Mechanically-sensitive increases in O2− may contribute to some of the pathological conditions associated with oxidative stress. Increased O2− production can lead to inappropriate NaCl retention7 and various types of hypertension, including renovascular,45 angiotensin-induced,7 and salt-sensitive hypertension.1 Increased flow in the TAL may occur chronically because of extensive renal ablation,46 high salt intake,47 hypertension,48 or diabetes.49 Although other factors that increase O2− may be involved, the mechanical stimuli related to increased flow in the TAL may play a role in the O2−-associated nephropathy observed in diseases such as hypertension and diabetes.

In summary, we found that stretch is the mechanical component involved in flow-induced O2− production in the TAL. Enhanced O2− in the TAL may be important in the pathogenesis of hypertension and other diseases associated with abnormal production of reactive oxygen species.

Perspectives

Renal O2− contributes to hypertension and diabetic nephropathy. Luminal flow through the nephron increases chronically because of diabetes, salt loading, and hypertension. High flow through the TAL and the resultant increased stretch of the tubule may contribute to the renal damage associated with these pathological conditions.

Sources of Funding

This work was supported by grants from the National Institutes of Health (HL 28982, HL 70985) to J.L. Garvin.

Footnotes

Disclosures

None.

References

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