Kidney Diseases - Volume One - Chapter 13

Pathophysiology of Ischemic Acute Renal Failure: Cytoskeletal Aspects Bruce A. Molitoris Robert Bacallao

I

schemia remains the major cause of acute renal failure (ARF) in the adult population [1]. Clinically a reduction in glomerular filtration rate (GFR) secondary to reduced renal blood flow can reflect prerenal azotemia or acute tubular necrosis (ATN). More appropriate terms for ATN are acute tubular dysfunction or acute tubular injury, as necrosis only rarely is seen in renal biopsies, and renal tubular cell injury is the hallmark of this process. Furthermore, the reduction in GFR during acute tubular dysfunction can now, in large part, be related to tubular cell injury. Ischemic ARF resulting in acute tubular dysfunction secondary to cell injury is divided into initiation, maintenance, and recovery phases. Recent studies now allow a direct connection to be drawn between these clinical phases and the cellular phases of ischemic ARF (Fig. 13-1). Thus, renal function can be directly related to the cycle of cell injury and recovery. Renal proximal tubule cells are the cells most injured during renal ischemia (Fig. 13-2) [2,3]. Proximal tubule cells normally reabsorb 70% to 80% of filtered sodium ions and water and also serve to selectively reabsorb other ions and macromolecules. This vectorial transport across the cell from lumen to blood is accomplished by having a surface membrane polarized into apical (brush border membrane) and basolateral membrane domains separated by junctional complexes (Fig. 13-3) [4]. Apical and basolateral membrane domains are biochemically and functionally different with respect to many parameters, including enzymes, ion channels, hormone receptors, electrical resistance, membrane transporters, membrane lipids, membrane fluidity, and cytoskeletal associations. This epithelial cell polarity is essential for normal cell function, as demonstrated by the vectorial transport of sodium from the lumen to the blood (see Fig. 13-3). The establishment

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and maintenance of this specialized organization is a dynamic and ATP dependent multistage process involving the formation and maintenance of cell-cell and cell-substratum attachments and the targeted delivery of plasma membrane components to the appropriate domains [5]. These processes are very dependent on the cytoskeleton, in general, and the cytoskeletal membrane interactions mediated through F-actin (see Fig. 13-2, 13-3), in particular. Ischemia in vivo and cellular ATP depletion in cell culture models (“chemical ischemia”) are known to produce characteristic surface membrane structural, biochemical, and functional abnormalities in proximal tubule cells. These alterations occur in a duration-dependent fashion and are illustrated in Figures 13-2 and 13-3 and listed in Figure 13-4. Ischemia-induced alterations in the actin cytoskeleton have been postulated to mediate many of the aforementioned surface membrane changes [2,6,7]. This possible link between ischemia-induced actin cytoskeletal alterations and surface membrane structural and functional abnormalities is suggested by several lines. First, the actin cytoskeleton is known to play fundamental roles in surface membrane formation and stability, junctional complex formation and regulation, Golgi structure and function, and cell–extracellular membrane attachment [2,4,5,8]. Second, proximal tubule cell actin cytoskeleton is extremely sensitive to ischemia and ATP depletion [9,10]. Third, there is a strong correlation between the time course of actin and surface membrane alterations during ischemia or ATP depletion [2,9,10]. Finally, many of the characteristic surface membrane changes

RELATIONSHIP BETWEEN THE CLINICAL AND CELLULAR PHASES OF ISCHEMIC ACUTE RENAL FAILURE Clinical Phases

Cellular Phases

Prerenal azotemia ↓ Initiation ↓ Maintenance ↓ Recovery

Vascular and cellular adaptation ↓ ATP depletion, cell injury ↓ Repair, migration, apoptosis, proliferation ↓ Cellular differentiation

induced by ischemia can be mimicked by F-actin disassembly mediated by cytochalasin D [11]. Although these correlations are highly suggestive of a central role for actin alterations in the pathophysiology of ischemia-induced surface membrane damage they fall short in providing mechanistic data that directly relate actin cytoskeletal changes to cell injury. Proximal tubule cell injury during ischemia is also known to be principally responsible for the reduction in GFR. Figure 13-5 illustrates the three known pathophysiologic mechanisms that relate proximal tubule cell injury to a reduction in GFR. Particularly important is the role of the cytoskeleton in mediating these three mechanisms of reduced GFR. First, loss of apical membrane into the lumen and detachment of PTC result in substrate for cast formation. Both events have been related to actin cytoskeletal and integrin polarity alterations [12–15]. Cell detachment and the loss of integrin polarity are felt to play a central role in tubular obstruction (Fig. 13-6). Actin cytoskeletalmediated tight junction opening during ischemia occurs and results in back-leak of glomerular filtrate into the blood. This results in ineffective glomerular filtration (Fig. 13-7). Finally, abnormal proximal sodium ion reabsorption results in large distal tubule sodium delivery and a reduction in GFR via tubuloglomerular feedback mechanisms [2,16,17]. In summary, ischemia-induced alterations in proximal tubule cell surface membrane structure and function are in large part responsible for cell and organ dysfunction. Actin cytoskeletal dysregulation during ischemia has been shown to be responsible for much of the surface membrane structural damage. FIGURE 13-1 Relationship between the clinical and cellular phases of ischemic acute renal failure. Prerenal azotemia results from reduced renal blood flow and is associated with reduced organ function (decreased glomerular filtration rate), but cellular integrity is maintained through vascular and cellular adaptive responses. The initiation phase occurs when renal blood flow decreases to a level that results in severe cellular ATP depletion that, in turn, leads to acute cell injury. Severe cellular ATP depletion causes a constellation of cellular alterations culminating in proximal tubule cell injury, cell death, and organ dysfunction [2]. During the clinical phase known as maintenance, cells undergo repair, migration, apoptosis, and proliferation in an attempt to re-establish and maintain cell and tubule integrity [3]. This cellular repair and reorganization phase results in slowly improving cell and organ function. During the recovery phase, cell differentiation continues, cells mature, and normal cell and organ function return [18].

Pathophysiology of Ischemic Acute Renal Failure: Cytoskeletal Aspects

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FIGURE 13-2 Ischemic acute renal failure in the rat kidney. Light A, B, transmission electron, C, D, and immunofluorescence E, F, microscopy of control renal cortical sections, A, C, E, and after moderate ischemia induced by 25 minutes of renal artery occlusion, B, D, F. Note the extensive loss of apical membrane structure, B, D, in proximal (PT) but not distal tubule cells. This has been shown to correlate with extensive alterations in F-actin as shown by FITC-phalloidin labeling, E, F. G, Drawing of a proximal tubule cell under physiologic conditions. Note the orderly arrangement of the actin cytoskeleton and its extensive interaction with the surface membrane at the zonula occludens (ZO, tight junction) zonula adherens (ZA, occludens junction), interactions with ankyrin to mediate Na+, K+-ATPase [2] stabilization and cell adhesion molecule attachment [5,8]. The actin cytoskeleton also mediates attachment to the extracellular matrix (ECM) via integrins [12,15]. Microtubules (MT) are involved in the polarized delivery of endocytic and exocytic vesicles to the surface membrane. Finally, F-actin filaments bundle together via actin-bundling proteins [19] to mediate amplification of the apical surface membrane via microvilli (MV). The actin bundle attaches to the surface membrane by the actin-binding proteins myosin I and ezrin [19,20].

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Acute Renal Failure

Proximal tubule cell

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ISCHEMIA INDUCED PROXIMAL TUBULE CELL ALTERATIONS Alterations Surface Membrane Alterations 1. Microvilli fusion, internalization, fragmentation and luminal shedding resulting in loss of surface membrane area and tubular obstruction 2. Loss of surface membrane polarity for lipids and proteins 3. Junctional complex dissociation with unregulated paracellular permeability (backleak) 4. Reduced PTC vectorial transport Actin Cytoskeletal Alterations 1. Polymerization of actin throughout the cell cytosol 2. Disruption and delocalization of F-actin structures including stress fibers, cortical actin and the junctional ring 3. Accumulation of intracellular F-actin aggregates containing surface membrane proteins—myosin I, the tight junction proteins ZO-1, ZO-2, cingulin 4. Disruption and dissociation of the spectrin cytoskeleton 5. Disruption of microtubules during early reflow in vivo 6. The cytoskeleton of proximal tubule cells, as compared to distal tubule cells, is more sensitive to ischemia in vivo and ATP depletion in vitro

Necrosis

References [21] [2,22,23] [6,24–27] [28] [6,16,29] [2,7,16] [20,30] [31,32] [33] [6,16,34]

FIGURE 13-3 Fate of an injured proximal tubule cell. The fate of a proximal tubule cell after an ischemic episode depends on the extent and duration of the ischemia. Cell death can occur immediately via necrosis or in a more programmed fashion (apoptosis) hours to days after the injury. Fortunately, most cells recover either in a direct fashion or via an intermediate undifferentiated cellular pathway. Again, the severity of the injury determines the route taken by a particular cell. Adjacent cells are often injured to varying degrees, especially during mild to moderate ischemia. It is believed that the rate of organ functional recovery relates directly to the severity of cell injury during the initiation phase. ECM—extracellular membrane; Na+—sodium ion; K+—potassium ion; P1—phosphate.

FIGURE 13-4 Ischemia induced proximal tubule cell alterations.

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Pathophysiology of Ischemic Acute Renal Failure: Cytoskeletal Aspects

Efferent arteriole

Glomerular plasma flow

Glomerular hydrostatic pressure

Glomerular filtration

Intratubular pressure

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Glomerular pressure

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Obstructing cast

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FIGURE 13-5 Mechanisms of proximal tubule cell—mediated reductions in glomerular filtration rate (GFR) following ischemic injury. A, GFR depends on four factors: 1) adequate blood flow to the glomerulus; 2) an adequate glomerular capillary pressure as determined by afferent and efferent arteriolar resistance; 3) glomerular permeability; and 4) low intratubular pressure. B, Afferent arteriolar constriction diminishes GFR by reducing blood flow—and, therefore, glomerular capillary pressure. This occurs in response to a high distal sodium delivery and is mediated by tubular glomerular feedback. C, Obstruction of the tubular lumen by cast formation increases tubular pressure and, when it exceeds glomerular capillary pressure, a marked decrease or no filtration occurs. D, Back-leak occurs when the paracellular space between cells is open for the flux of glomerular filtrate to leak back into the extracellular space and into the blood stream. This is believed to occur through open tight junctions.

D RG

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FIGURE 13-6 Overview of potential therapeutic effects of cyclic integrin-binding peptides. A, During ischemic injury, tubular obstruction occurs as a result of loss of apical membrane, cell contents, and detached cells released into the lumen. B, Also, basolateral integrins diffuse to the apical region of the cell. Biotinylated cyclic peptides containing the sequence cRGDDFV bind to desquamated cells in the ascending limb of the loop of Henle and in proximal tubule cells in ischemic rat kidneys. The desquamated cells can adhere to injured cells or aggregate, causing tubule obstruction. (Continued on next page)

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Acute Renal Failure

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FIGURE 13-6 (Continued) C, When cyclic peptides that contain the RGD canonical binding site of integrins are perfused intra-arterially, the peptides ameliorate the extent of acute renal failure, as demonstrated by a higher glomerular filtration rate (GFR) in rats receiving peptide containing the RGD sequence. B, Proposed mechanism of renal protection by cyclic RGD peptides. By adhering to the RGD binding sites of the integrins located on the apical plasma membrane or distributed randomly on desquamated cells, the cyclic peptide blocks cellular aggregation and tubular obstruction [12–15]. (Courtesy of MS Goligorski, MD.)

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FIGURE 13-7 Functional and morphologic changes in tight junction integrity associated with ischemic injury or intracellular ATP depletion. A and B, Ruthenium red paracellular permeability in rat proximal tubules. A, In control kidneys, note the electron-dense staining of the brush border, which cuts off at the tight junctions (tj, arrows). B, Sections from a perfusion-fixed kidney after 20 minutes of renal artery crossclamp [35]. The electron-dense staining can be seen at cell contact sites beyond the tight junction (arrows). The paracellular pathway is no longer sealed by the tight junction, permitting backleak of the electron-dense ruthenium red. C, Changes in the transepithelial resistance (TER) versus time during ATP depletion and ATP repletion [36]. Paracellular resistance to electron movement (Continued on next page)

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Pathophysiology of Ischemic Acute Renal Failure: Cytoskeletal Aspects

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FIGURE 13-7 (Continued) (the TER falls to zero with ATP depletion). The cellular junctional complex that controls the TER is the tight junction. When the TER falls to zero, this suggests that tight junction structural integrity has been compromised. D and E, Staining of renal epithelial cells with antibodies that bind to a component of the tight junction, ZO-1 [37]. D, ZO-1 staining in untreated Mardin-Darby carnine kidney (MDCK) cells. ZO-1 is located at the periphery of cells at cell contact sites, forming a continuous linear contour. E, In ATP–depleted cells the staining pattern is discontinuous. F and G, Ultrastructural analysis of the tight junction in MDCK cells. In untreated MDCK cells, electron micrographs of the tight junction shows a continuous ridge like structure in freeze fracture preparations [38]. In ATP depleted cells the strands are disrupted, forming aggregates (arrows). Note that the continuous strands are no longer present and large gaps are observable.

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Acknowledgment These studies were in part supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grants DK 41126 (BAM) and DK4683 (RB) and by an American Heart

Association Established Investigator Award (BAM), a VA Merrit Review Grant (BAM), and a NKF Clinical Scientist Award (RB).

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2.

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3.

Thadhani R, Pascual M, Bonventre JV: Acute renal failure. N Engl J Med 1996, 334:1448–1457.

4.

Drubin DG, Nelson WJ: Origins of cell polarity. Cell 1996, 84:335–344.

5.

Mays RW, Nelson WJ, Marrs JA: Generation of epithelial cell polarity: Roles for protein trafficking, membrane-cytoskeleton, and E-cadherin–mediated cell adhesion. Cold Spring Harbor Symposia on Quantitative Biol 1995, 60:763–773.

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Bacallao R, Garfinkel A, Monke S, et al.: ATP depletion: A novel method to study junctional properties in epithelial tissues. I. Rearrangement of the actin cytoskeleton. J Cell Sci 1994, 107:3301–3313. Kroshian VM, Sheridan AM, Lieberthal W: Functional and cytoskeletal changes induced by sublethal injury in proximal tubular epithelial cells. Am J Physiol 1994, F21–F30. Fish EM, Molitoris BA: Alterations in epithelial polarity and the pathogenesis of disease states. N Engl J Med 1994, 330:1580–1588.

9.

Glaumann B, Glauman H, Berezesky IK, et al.: Studies on the cellular recovery from injury II. Ultrastructural studies on the recovery of the pars convoluta of the proximal tubule of the rat kidney from temporary ischemia. Virchows Arch B 1977, 24:1–18. 10. Kellerman PS, Norenberg SL, Jones GM: Early recovery of the actin cytoskeleton during renal ischemic injury in vivo. Am J Kidney Dis 1996, 16:33–42.

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20. Wagner MC, Molitoris BA: ATP depletion alters myosin Ib cellular location in LLC-PK1 cells. Am J Physiol 1997, 272:C1680–C1690. 21. Venkatachalam MA, Jones DB, Rennke HG, et al.: Mechanism of proximal tubule brush border loss and regeneration following mild ischemia. Lab Invest 1981, 45:355–365. 22. Ritter D, Dean AD, Guan ZH, et al.: Polarized distribution of renal natriuretic peptide receptors in normal physiology and ischemia. Am J Physiol 1995, 269:F918–F925. 23. Alejandro VSJ, Nelson WJ, Huie P, et al.: Postischemic injury, delayed function and Na+/K+-ATPase distribution in the transplanted kidney. Kidney Int 1995, 48:1308–1315. 24. Donohoe JF, Venkatachalam MA, Benard DB, et al.: Tubular leakage and obstruction after renal ischemia: Structural-functional correlations. Kidney Int 1978, 13:208–222. 25. Molitoris BA, Falk SA, Dahl RH: Ischemic-induced loss of epithelial polarity. Role of the tight junction. J Clin Invest 1989, 84:1334–1339. 26. Mandel LJ, Bacallao R, Zampighi G: Uncoupling of the molecular fence and paracellular gate functions in epithelial tight junctions. Nature 1993, 361:552–555. 27. Kwon O, Nelson J, Sibley RK, et al.: Backleak, tight junctions and cell-cell adhesion in postischemic injury to the renal allograft (Abstract). J Am Soc Nephrol 1996, 7:A2907. 28. Molitoris BA. Na+-K+-ATPase that redistributes to apical membrane during ATP depletion remains functional. Am J Physiol 1993, 265:F693–F597. 29. Kellerman PS: Exogenous adenosine triphosphate (ATP) proximal tubule microfilament structure and function in vivo in a maleic acid model of ATP depletion. J Clin Invest 1993, 92:1940–1949. 30. Tsukamoto T, Nigam SK: ATP depletion causes tight junction proteins to form large, insoluble complexes with cytoskeletal proteins in renal epithelial cells. J Biol Chem 1997, 273:F463–F472. 31. Molitoris BA, Dahl R, Hosford M: Cellular ATP depletion induces disruption of the spectrin cytoskeletal network. Am J Physiol 1996, 271:F790–F798. 32. Edelstein CL, Ling H, Schrier RW: The nature of renal cell injury. Kidney Int 1997, 51:1341–1351. 33. Abbate M, Bonventre JV, Brown D: The microtubule network of renal epithelial cells is disrupted by ischemia and reperfusion. Am J Physiol 1994, 267:F971–F978. 34. Sheridan AM, Schwartz JH, Kroshian VM, et al.: Renal mouse proximal tubular cells are more susceptible than MDCK cells to chemical anoxia. Am J Physiol 1993, 265:F342–F350. 35. Molitoris BA, Falk SA, Dahl RH: Ischemia-induced loss of epithelial polarity. Role of the tight junction. J Clin Invest 1989, 84:1334–1339. 36. Doctor RB, Bacallao R, Mandel LJ: Method for recovering ATP content and mitochondrial function after chemical anoxia in renal cell cultures. Am J Physiol 1994, 266:C1803–C1811. 37. Stevenson BR, Siliciano JD, Mooseker MS, et al.: Identification of ZO-1: A high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia. J Cell Biol 1986, 103:755–766. 38. Mandel LJ, Bacallao R, Zampighi G: Uncoupling of the molecular ‘fence’ and paracellular ‘gate’ functions in epithelial tight junctions. Nature 1993, 361:552–555.