PHYSIOLOGIC HALLMARKS OF ACUTE RENAL FAILURE

Ischemia/reperfusion and cisplatin predictably lower glomerular filtration rate (GFR), the latter in a clear dose-dependent manner¹ after a single drug exposure. Early proteinuria is mild (<500 mg/day), as is glycosuria. In a few patients, significant urinary electrolyte wasting may be provoked by cisplatin², including severe sodium, divalent action, and phosphate wasting, seen primarily with high-dose therapy. Most common is the gradual onset of nonoliguric renal failure, with an accompanying defect in concentrating ability Figure 1³. In micropuncture experiments, the site of altered water reabsorption is beyond the late distal tubule, probably in the apparently morphologically intact collecting duct³. In rats, the cause of the fall in glomerular filtration is afferent vasoconstriction and possibly an altered ultrafiltration coefficient before evidence of tubule obstruction¹. The precise pathogenesis for these changes in segmental water transport and vascular resistance remains unknown, but molecular studies are beginning to yield insights into these events (see below).

Figure 1.

Maximum concentrating ability in control and cisplatin (CDDP)-treated animals following a 24-hour period of water depreviation and 1000 mU of ADH. 

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MORPHOLOGY OF ARF

At least three cellular fates can be identified in acute renal failure (ARF): cells may die either by frank necrosis or by apoptosis; they may replicate and divide; or they may appear indifferent to the stress Figure 2. Frank necrosis, as is often seen experimentally, is not prominent in the vast majority of human cases. Necrosis is usually patchy, involving individual cells or small clusters of cells, sometimes resulting in small areas of denuded basement membrane. Less obvious injury is more often noted, including loss of brush borders, flattening of the epithelium, detachment of cells, intratubular cast formation, and dilatation of the lumen. Although proximal tubules show many of theses changes, injury to the distal nephron can also be demonstrated when human biopsy material is closely examined, but experimentally, the thick ascending limb is spared from necrosis.

Figure 2.

Radiohistogram of outer stripe of outer medulla of rat kidney taken from animal 5 days after cisplatin 5mg/kg BW. Note three cell fates: necrosis (ND) of cells lining injured S3 segment; apparent indifference of thick ascending limb (TAL) and collecting duct (CD) epithelial cells; cells of the proximal tubule (PT) undergoing DNA synthesis (arrow). Condensed nuclear debris may also be seen in such section indicating apoptotic bodies (not shown).

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Apoptosis has been noted in ischemic and nephrotoxic forms of ARF. This form of cell death differs from frank necrosis in that it requires the activation of a regulated program that leads to DNA fragmentation, cytoplasmic condensation, and cell loss without precipitating an inflammatory response. In contradistinction to necrosis, the principal site of apoptotic cell death is the distal nephron.

THE RENAL STRESS RESPONSE

Cellular stress, including that induced by oxidants and DNA-damaging agents similar to cisplatin, activates two related parts of the mitogen-activated protein kinase signaling pathway, or MAPKs—the extracellular-regulated protein kinases, or ERKs, and the stress-activated protein kinase (SAPK), also know as Jun N-terminal kinase (JNK)⁴. A balance between the activation of the ERKs and the SAPKs seems to exist in determining cell fate during the stress applied by ischemia/reperfusion. Observations in vivo revealed activation of ERK was restricted to surviving thick ascending limb cells during ischemia/reperfusion, while JNK activation was demonstrated in both proximal and distal tubule cells⁵. Furthermore, inhibition of JNK in vivo ameliorated ischemic/reperfusion–induced ARF⁶. By manipulating the balance between ERK and JNK, we showed that oxidant injury–induced necrosis could be ameliorated by either up-regulation of endogenous ERK, or inhibition of JNK⁷. These studies confirmed the crucial role played by the MAPK pathway in determining outcome.

CELL CYCLE EVENTS IN ACUTE RENAL FAILURE

The kidney also commits to DNA synthesis rapidly after injury, and the commitment coincides temporally with the emergence of the morphologic and functional derangements of ARF. The rapid appearance of mRNA for the "immediate-early" genes including proliferating cell nuclear antigen (PCNA), mark the commitment to the cell cycle¹⁰.

However, coincident with this increased activity, we have shown that the p21^{WAF1/CIP1/SDI1} gene is activated in murine kidney cells in all experimental models of ARF¹⁰. This protein binds to and inhibits cyclin-dependent protein kinases, whose cyclic oscillations coincide with the progression of cells through consecutive stages of the cell cycle. The sites of p21 mRNA over-expression were localized by in situ hybridization to the cells of the thick ascending limbs and distal convoluted tubule cells in the cortex that do not participate in the DNA synthetic response to ARF. Experiments in mice whose p21 gene has been knocked out demonstrate a protective role of the p21 gene product in ARF¹⁰.

MOLECULAR RESPONSES TO RENAL INJURY

The MAPK pathway activates and represses gene expression by phosphorylating constitutively expressed transcription factors. These include the activator protein-1 (AP-1) transcription factor components, c-fos and c-Jun. These factors in combination with other factors activate and repress large groups of genes in response to cellular stress⁸. We found that prominent members of these stress-related genes were activated following ischemia- or cisplatin-induced ARF Figure 3. Inhibition of new protein synthesis superinduced these genes during ischemia, proving that, like other situations of stress, the kidney is able to mount a typical immediate early gene response. Other immediate early genes were activated in the kidney as a consequence of ischemia and cisplatin, including the chemokines interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1), which are potent leukocyte chemotactic agents⁸. Subsequently increased expression of several cytokines and chemokines has been demonstrated in various models of ARF, including tumor necrosis factor- (TNF-), transforming growth factor- (TGF-), RANTES, and MIP-2⁹, some of which were found to play important roles in the pathogenesis of ARF. Thus, the kidney expresses a stress response, including activation of the MAPKs and subsequent activation of proto-oncogene and chemokines, which are crucial to the outcome of ischemia- and cisplatin-induced ARF.

Figure 3.

Northern analysis of kidney c-fos and c-jun mRNA in control and cisplatin-induced acute renal failure. 

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ANALYSIS OF THE RENAL TRANSCRIPTOSOME DURING CISPLATIN-INDUCED ACUTE RENAL FAILURE

Given the complex nature of the reaction of the kidney to injury, including differences in segment-specific cell fate and altered renal function, and the observation that activation of signal transduction and differential gene regulation occurs well before obvious changes in renal function and morphology take place, we adopted the use of gene microarrays to analyze changes in the renal transcriptosome more broadly over time. We used the model of cisplatin-induced renal failure because unlike the renal ischemia/reperfusion model, the course of renal failure is predictable and slowly evolving after a single exposure to the drug. Using the 129/SV strain of mouse receiving 20 mg/kg cisplatin intraperitoneally, we isolated whole kidney mRNA from control and cisplatin-exposed animals one and three days after cisplatin. Hybridization of cRNA to an Affymetrix HU95A microarray chip was performed and hybridization monitored using Affymetrix software (Affymetrix; Santa Clara, CA, USA). Differences in gene expression were analyzed for significance by two-way analysis of variance (ANOVA). Further analysis included selection for changes in gene expression that exceeded control levels by threefold. These genes were further analyzed by comparing the expression of these genes to the expression profile of p21 and prepro epidermal growth factor (EGF) with a 99% correlation coefficient because these genes are known to be up- and down-regulated, respectively, during cisplatin- and ischemia/reperfusion injury^8,10. Only annotated genes were included in the analysis. This analysis yielded 303 genes. The heat map Figure 4 shows that a set of 77 genes increased monotonically from low to high (green through black to red) after cisplatin, while the remainder shows depression of gene expression through the observation period (red-green).

Figure 4.

Heat map of the changes in mouse kidney gene expression after cisplatin injection. Using changes in gene expression of 3-fold or greater and applying hierarchic clustering, the expression profile of each of these genes that correlated with the expression profile of preproEGF by 0.99 or greater, and ordered by z-score.

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Table 1 summarizes the general findings of this analysis. First, individual genes whose expression was known to change during cisplatin-induced ARF were also identified by this approach, and have been classified in broad categories as before. Thus, changes in gene expression denoting regeneration, apoptosis, and survival, as well as those denoting inflammation and cell adhesion were confirmed. Another theme validated by this approach involves what appears to be a loss of the mature phenotype of the kidney. Many of the genes whose expression is down-regulated during cisplatin-induced ARF are those genes that express at high levels only when the kidney achieves full maturity of function. These include the prepro EGF and Tamm-Horsfall protein genes, whose functions are unknown in the kidney, but also include the down-regulation of important membrane transporter genes, such as aquaporin-2, and sodium proton exchanger 3 (NHE3). The loss of these latter proteins may be responsible, in part, for the tubular reabsorptive defects typical of this and other forms of ARF.

Table 1 - Molecular responses to renal ischemia.

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We interrogated this dataset for changes relevant to metabolism because it has been proposed that an early target of cisplatin, as well as ischemia-induced renal failure, is the mitochondria's ability to generate ATP. The mRNA for pyruvate dehydrogenase kinase, which phosphorylates and inhibits pyruvate dehydrogenase, a rate-limiting enzyme for mitochondrial oxidation of pyruvate, and implies inhibition of aerobic glycolysis. Phosphoenolpyruvate carboxykinase is markedly up-regulated by cisplatin, as well, suggesting increased utilization of pyruvate to generate glucose, as this enzyme is the key regulator of gluconeogenesis. This notion is further supported by the increase in fructose-1,6-biphosphatase, which, like PEPCK, is a key enzyme in the gluconeogenic pathway. We are also able to propose that these changes in gene regulation could be a consequence of cisplatin-induced acidification, as we found reduced expression of acid extruder and base loader mRNA in our analysis. We have incorporated these predictions in Figure 5, and are actively pursuing these predictions both in vivo and vitro with promising results. We believe that this information will lead to important new insights into the mechanisms responsible for ARF.

Figure 5.

Predicted impact of cisplatin on renal metabolism of glucose and intracellular pH. 

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CONCLUSION

Cell death, survival, and repair are intimately interrelated after renal injury. The stress response characterized by transduction pathways and gene transcription that serve both positive and negative aspects of cell survival is intimately involved in the outcome of ischemic and nephrotoxic damage. The cell cycle and its regulation are key components of the life and death of the stressed cells throughout the kidney. Some cells participating in this response will survive and repair, whereas others will die. What determines whether a cell will recover from such injury or undergo cell death by necrosis or apoptosis is probably a function of the severity of the stress, the specific changes in gene regulation that the cell is capable of mounting, and the availability of survival factors in the cell's external milieu.

Manipulation of the signal transduction and molecular pathways provoked by toxic injury may provide an opportunity in the future to influence ARF Figure 6. We have shown already that up-regulation of the ERK pathway and down-regulation of JNK improves survival to oxidant stress. Similarly, p21 over-expression protects cells from ischemic and toxic damage, and may be reasonable focus of future intervention. Inhibition of cytokine pathways also ameliorates ARF, and may do so by bringing a more positive balance in these pathways promoting survival. We propose that intervening in the metabolic consequences of the molecular response, such as the inhibition of pyruvate oxidation, may also be amenable to manipulation. Additional targets will almost certainly be provided by the application and analysis of high-throughput gene expression and proteomics just now being applied to this interesting and pertinent field of study.

Figure 6.

Survival factors may influence the signal transduction and transcriptional programs initiated by stress. The balance between the prosurvival or prodeath aspects of the stress response determines the ultimate fate of the cells. Both the cyclin-kinase inhibitor p21 protein and extracellular-regulated kinase (ERK) protect renal cells from oxidant injury.

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References

References

Acknowledgments

I want to thank my colleagues Drs. Peter Price, Judit Megyesi, and Istvan Arany at UAMS, and Drs. David Gorenstein and Bruce Luxon at UTMB Galveston, who have contributed directly to my studies of acute renal failure. I also want to acknowledge Dr. Thomas Andreoli, who provided me with the opportunity to extend these studies here at UAMS, and who published, as Editor in Chief of KI, my first paper on the molecular responses to ARF, and who provided the encouraging words of an editor so sought after by a young and struggling clinical scientist desperate to continue working in the laboratory.