Manjeri A. Venkatachalam

Acute kidney injury: a springboard for progression in chronic kidney disease

Recently published epidemiological and outcome analysis studies have brought to our attention the important role played by acute kidney injury (AKI) in the progression of chronic kidney disease (CKD) to end-stage renal disease (ESRD). AKI accelerates progression in patients with CKD; conversely, CKD predisposes patients to AKI. This research gives credence to older, well-thought-out wisdom that recovery from AKI is often not complete and is marked by residual structural damage. It also mirrors older experimental observations showing that unilateral nephrectomy, a surrogate for loss of nephrons by disease, compromises structural recovery and worsens tubulointerstitial fibrosis after ischemic AKI. Moreover, review of a substantial body of work on the relationships among reduced renal mass, hypertension, and pathology associated with these conditions suggests that impaired myogenic autoregulation of blood flow in the setting of hypertension, the arteriolosclerosis that results, and associated recurrent ischemic AKI in microscopic foci play important roles in the development of progressively increasing tubulointerstitial fibrosis. How nutrition, an additional factor that profoundly affects renal disease progression, influences these events needs reevaluation in light of information on the effects of calories vs. protein and animal vs. vegetable protein on injury and progression. Considerations based on published and emerging data suggest that a pathology that develops in regenerating tubules after AKI characterized by failure of differentiation and persistently high signaling activity is the proximate cause that drives downstream events in the interstitium: inflammation, capillary rarefaction, and fibroblast proliferation. In light of this information, we advance a comprehensive hypothesis regarding the pathophysiology of AKI as it relates to the progression of kidney disease. We discuss the implications of this pathophysiology for developing efficient therapeutic strategies to delay progression and avert ESRD.

Apoptosis: definition, mechanisms, and relevance to disease

Kerr et al (1) in 1972 coined the term “apoptosis,” an ancient Greek word used to describe the “falling off” of leaves from trees or petals from flowers, referring to the particular morphology of physiological cell death. The term apoptosis is often used synonymously with programmed cell death, the latter being a more functional definition, implying that death results from the regulated activation of a preexisting death program that is encoded in the genome. The condemned cell itself, often with the help of neighboring cells and/or humoral factors, directs the death program. Apoptosis refers to the morphologic features of programmed cell death, which is characterized by cell shrinkage, nuclear condensation, membrane blebbing, fragmentation into membrane bound apoptotic bodies, and membrane changes that eventually lead to phagocytosis of the affected cell (Figure 1). During development, cell death helps sculpt parts of the body, examples being the formation of cavities and separation of digits (Figure 2A and B). It also eliminates vestigial structures that once served a function during embryogenesis (Figure 2C). Programmed cell death plays a complementary but opposite role to cell division as a homeostatic mechanism in the regulation of animal cell populations (Figure 2A). Cell death programs are required for animal development, but they also proceed into adult life. In mature animals, cell death balances cell division, maintaining the constancy of tissue mass. Removal of cells injured by genetic defects, aging, disease, or exposure to noxious agents is made possible by apoptosis (Figure 2D). Moreover, the normal immune response requires regulated elimination of specific cell populations by this mode of cell death. Apoptosis has important biological roles in the development and homeostasis of cell populations, and in the pathogenesis and expression of disease processes. Excessive or insufficient apoptosis contributes to the pathogenesis of a wide variety of diseases related to ischemia, neurodegeneration, autoimmunity, and viral infections, and is involved in the growth and regression of tumors. Although apoptosis was described as a distinct entity nearly 3 decades ago, significant advances in our understanding of fundamental mechanisms that regulate this mode of cell death were made only recently. In large part, the recent advances in our knowledge of cell death stemmed from the identification of “death genes” a decade ago (2).

Bcl-2 prevents Bax oligomerization in the mitochondrial outer membrane.

ATP depletion results in Bax translocation from cytosol to mitochondria and release of cytochrome c from mitochondria into cytosol in cultured kidney cells. Overexpression of Bcl-2 prevents cytochrome c release, without ameliorating ATP depletion or Bax translocation, with little or no association between Bcl-2 and Bax as demonstrated by immunoprecipitation (Saikumar, P., Dong, Z., Patel, Y., Hall, K., Hopfer, U., Weinberg, J. M., and Venkatachalam, M. A. (1998) Oncogene 17, 3401–3415). Now we show that translocated Bax forms homo-oligomeric structures, stabilized as chemical adducts by bifunctional cross-linkers in ATP-depleted wild type cells, but remains monomeric in Bcl-2-overexpressing cells. The protective effects of Bcl-2 did not require Bcl-2/Bax association, at least to a degree of proximity or affinity that was stable to conditions of immunoprecipitation or adduct formation by eight cross-linkers of diverse spacer lengths and chemical reactivities. On the other hand, nonionic detergents readily induced homodimers and heterodimers of Bax and Bcl-2. Moreover, associations between translocated Bax and the voltage-dependent anion channel protein or the adenine nucleotide translocator protein could not be demonstrated by immunoprecipitation of Bax, or by using bifunctional cross-linkers. Our data suggest that the in vivo actions of Bax are at least in part dependent on the formation of homo-oligomers without requiring associations with other molecules and that Bcl-2 cytoprotection involves mechanisms that prevent Bax oligomerization.

Role of hypoxia-induced Bax translocation and cytochrome c release in reoxygenation injury.

We investigated mechanisms of cell death during hypoxia/reoxygenation of cultured kidney cells. During glucose-free hypoxia, cell ATP levels declined steeply resulting in the translocation of Bax from cytosol to mitochondria. Concurrently, there was cytochrome c release and caspase activation. Cells that leaked cytochrome c underwent apoptosis after reoxygenation. ATP depletion induced by a mitochondrial uncoupler resulted in similar alterations even in the presence of oxygen. Moreover, inclusion of glucose during hypoxia prevented protein translocations and reoxygenation injury by maintaining intracellular ATP. Thus, ATP depletion, rather than hypoxia per se, was the cause of protein translocations. Overexpression of Bcl-2 prevented cytochrome c release and reoxygenation injury without ameliorating ATP depletion or Bax translocation. On the other hand, caspase inhibitors did not prevent protein translocations, but inhibited apoptosis during reoxygenation. Nevertheless, they could not confer long-term viability, since mitochondria had been damaged. Omission of glucose during reoxygenation resulted in continued failure of ATP production, and cell death with necrotic morphology. In contrast, cells expressing Bcl-2 had functional mitochondria and remained viable during reoxygenation even without glucose. Therefore, Bax translocation during hypoxia is a molecular trigger for cell death during reoxygenation. If ATP is available during reoxygenation, apoptosis develops; otherwise, death occurs by necrosis. By preserving mitochondrial integrity, BCL-2 prevents both forms of cell death and ensures cell viability.

Mechanisms of cell death in hypoxia/reoxygenation injury.

Investigation of death pathways during cell injury in vivo caused by ischemia and reperfusion is of clinical importance, but technically difficult. Heterogeneity of cell types, differences between organ systems, diversity of death paradigms and exacerbation of tissue damage caused by inflammation are only some of the variables that need to be taken into account. With respect to the identification of necrosis and apoptosis in affected organs, technical issues related to preparation artifacts, occurrence of internucleosomal DNA cleavage in necrotic as well as apoptotic cells and other overlaps in death pathways bear consideration. In that caspase independent as well as caspase dependent processes cause cell death and that caspase inhibitors can act as anti-inflammatory agents, evaluation of ischemic death mechanisms in parenchymal cells needs to be performed with caution. When the effects of inflammation are removed by appropriate in vitro studies using purified or cultured cells, several mitochondrial factors that lead to cell death can be studied. Substantial evidence exists for the participation of electron transport defects, mitochondrial permeability transitions (MPT) and release of cytochrome c from mitochondria, effected by pro-apoptotic proteins such as Bax. The anti-apoptotic protein Bcl-2 exerts an overriding protective role in this type of injury by preserving mitochondrial structure and function. In contrast, caspase inhibitors cannot offer long-term protection to ischemically injured parenchymal cells regardless of how effectively they can inhibit apoptotic events, if the cells have suffered permanent mitochondrial damage impairing respiration.

Association of Bax and Bak homo-oligomers in mitochondria. Bax requirement for Bak reorganization and cytochrome c release.

ATP depletion induced by hypoxia or mitochondrial inhibitors results in Bax translocation from cytosol to mitochondria and release of cytochrome c from mitochondria into cytosol in cultured rat proximal tubule cells. Translocated Bax undergoes further conformational changes to oligomerize into high molecular weight complexes (Mikhailov, V., Mikhailova, M., Pulkrabek, D. J., Dong, Z., Venkatachalam, M. A., and Saikumar, P. (2001) J. Biol. Chem. 276, 18361–18374). Here we report that following Bax translocation in ATP-depleted rat proximal tubule cells, Bak, a proapoptotic molecule that normally resides in mitochondria, also reorganizes to form homo-oligomers. Oligomerization of both Bax and Bak occurred independently of Bid cleavage and/or translocation. Western blots of chemically cross-linked membrane extracts showed nonoverlapping “ladders” of Bax and Bak complexes in multiples of ∼21 and ∼23 kDa, respectively, consistent with molecular homogeneity within each ladder. This indicated that Bax and Bak complexes were homo-oligomeric. Nevertheless, each oligomer could be co-immunoprecipitated with the other, suggesting a degree of affinity between Bax and Bak that permitted co-precipitation but not cross-linking. Furthermore, dissociation of cross-linked complexes by SDS and renaturation prior to immunoprecipitation did not prevent reassociation of the two oligomeric species. Notably, expression of Bcl-2 prevented not only the oligomerization of Bax and Bak, but also the association between these two proteins in energy-deprived cells. Using Bax-deficient HCT116 and BMK cells, we show that there is stringent Bax requirement for Bak homo-oligomerization and for cytochrome c release during energy deprivation. Using Bak-deficient BMK cells we further show that Bak deficiency is associated with delayed kinetics of Bax translocation but does not affect either the oligomerization of translocated Bax or the leakage of cytochrome c. These results suggest a degree of functional cooperation between Bax and Bak in this form of cell injury, but also demonstrate an absolute requirement of Bax for mitochondrial permeabilization.

Failed Tubule Recovery, AKI-CKD Transition, and Kidney Disease Progression

The transition of AKI to CKD has major clinical significance. As reviewed here, recent studies show that a subpopulation of dedifferentiated, proliferating tubules recovering from AKI undergo pathologic growth arrest, fail to redifferentiate, and become atrophic. These abnormal tubules exhibit persistent, unregulated, and progressively increasing profibrotic signaling along multiple pathways. Paracrine products derived therefrom perturb normal interactions between peritubular capillary endothelium and pericyte-like fibroblasts, leading to myofibroblast transformation, proliferation, and fibrosis as well as capillary disintegration and rarefaction. Although signals from injured endothelium and inflammatory/immune cells also contribute, tubule injury alone is sufficient to produce the interstitial pathology required for fibrosis. Localized hypoxia produced by microvascular pathology may also prevent tubule recovery. However, fibrosis is not intrinsically progressive, and microvascular pathology develops strictly around damaged tubules; thus, additional deterioration of kidney structure after the transition of AKI to CKD requires new acute injury or other mechanisms of progression. Indeed, experiments using an acute-on-chronic injury model suggest that additional loss of parenchyma caused by failed repair of AKI in kidneys with prior renal mass reduction triggers hemodynamically mediated processes that damage glomeruli to cause progression. Continued investigation of these pathologic mechanisms should reveal options for preventing renal disease progression after AKI.

Role of apoptosis in hypoxic/ischemic damage in the kidney

Cell death by hypoxia/ischemia may occur by apoptosis as well as necrosis in experimental models of renal injury both in vivo and in vitro. Necrosis can occur during hypoxia/ischemia as a result of widespread cellular degradation, and during reoxygenation/reperfusion as a consequence of the development of the mitochondrial permeability transition pore (PTP). In vitro models of hypoxia/reoxygenation suggest that apoptotic cell death may occur during reoxygenation as a consequence of mitochondrial release of cytochrome c (Cyt c) during hypoxia. In hypoxic renal cells, Bax and Bak, 2 pro-apoptotic proteins of the Bcl-2 family, collaborate to permeabilize the mitochondrial outer membrane to intermembrane proteins such as Cyt c, although Bax, per se, appears to play the dominant role. Cyt c then acts to trigger the downstream apoptotic cascade. Caspase inhibitors suppress these downstream events, but not Cyt c release. However, the anti-apoptotic Bcl-2 prevents mitochondrial permeabilization and maintains viability. Inflammation is known to play a major role in exacerbating parenchymal damage during reperfusion. Recent studies suggest that the apoptosis-related mechanisms contribute to the inflammatory process. By inhibiting tubular cell apoptosis, by suppressing an apoptotic chain reaction in accumulating inflammatory cells, and by inhibiting caspase-1 processing in injured tissue, caspase inhibitors may reduce inflammation, and thereby reduce the cascading parenchymal injury that is associated with inflammation.

Tolerance of the Human Kidney to Isolated Controlled Ischemia

Tolerance of the human kidney to ischemia is controversial. Here, we prospectively studied the renal response to clamp ischemia and reperfusion in humans, including changes in putative biomarkers of AKI. We performed renal biopsies before, during, and after surgically induced renal clamp ischemia in 40 patients undergoing partial nephrectomy. Ischemia duration was >30 minutes in 82.5% of patients. There was a mild, transient increase in serum creatinine, but serum cystatin C remained stable. Renal functional changes did not correlate with ischemia duration. Renal structural changes were much less severe than observed in animal models that used similar durations of ischemia. Other biomarkers were only mildly elevated and did not correlate with renal function or ischemia duration. In summary, these data suggest that human kidneys can safely tolerate 30-60 minutes of controlled clamp ischemia with only mild structural changes and no acute functional loss.

Glomerular permeability of macromolecules. Effect of molecular configuration on the fractional clearance of uncharged dextran and neutral horseradish peroxidase in the rat.

Molecular parameters other than size and charge are likely to influence the filtration of macromolecules across the glomerular filter. We have studies, therefore, the glomerular permeability of macromolecules with widely different molecular configuration such as horseradish peroxidase, a plant glycoprotein with an isoelectric point in the physiologic pH range, and dextran, an uncharged sugar polymer of D-glucopyranose. Simultaneous fractional clearances were determined for both test macromolecules in five Wistar-Furth rats. The results indicate that for a molecular radius of 28.45 A, as measured by gel filtration, the sugar polymer has a fractional clearance of 0.483 on the average, exceeding that of the protein tracer, with a value of 0.068, by a factor less than 7. We conclude that other molecular parameters such as shape, flexibility, and deformability play important roles in the transport of macromolecules across the extracellular matrix that constitutes the glomerular filter.