Bile Acids Are Important Contributors To AKI Associated With Liver Disease: CON

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When a clear answer to a scientific question is elusive, it is sometimes valuable to take a contrarian position, to question your original assumptions. If you had asked whether we believed bile cast nephropathy was"real" before writing this article, we would have answered "probably."However, three factors suggest that interpreting bile cast nephropathy as a simple obstructive disease leading to tubular damage is incomplete:(1)absence of clear dose-response associations between hyperbilirubinemia and kidney injury;(2) lack of convincing experimental modeling implicating bilirubin in the pathogenesis of kidney damage; and (3) the bio-energetic interactions of bile acids suggest alternate (and potentially more complicated) explanations for these histologic observations are more likely.

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Renal excretion of bilirubin breakdown products is a well-described phenomenon. Normally, bilirubin is metabolized into colorless urobilinogen, which is really excreted. However, in situations where serum levels are elevated, bilirubin is also excreted in the urine, generating its dark hue. There is no doubt that bilirubin-stained casts can be detected histologically in patients with cirrhosis and AKI, as has been described in multiple autopsy series(1,2). However, there is a lack of convincing epidemiologic associations between this pathologic finding and clinical AKI to support its role as a causative mechanism of injury. To start, hyper-bilirubinemia is incredibly common in decompensated cirrhosis, yet reports of bile cast nephropathy exist on the case report level (3,4). Because AKIis are also so prevalent in this setting (5), one would expect at least some dose-response relationship between serum bilirubin, bilirubin excretion, and kidney function to be present at a population level, but such data are lacking. Further, the few extant case reports of bile cast nephropathy tend to draw from autopsy series, which are subject to selection bias of unusual or severe phenotypic pre-sensations of disease and may be compromised by post-mortem preparation artifacts.

Because of the risk of bleeding, there are only a few case series of native kidney biopsies in cirrhotic populations. The largest series of native kidney biopsies in liver transplant candidates, performed by Wadeiet al., examined the renal parenchyma of 128patients who were cirrhotic and did not report bile casts as a prominent histologic feature(6). In fact, elevated serum bilirubin was associated with less tubulointerstitial damage overall. The correlation between serum bilirubin and kidney function in noncirrhotic populations is mixed at best, with multiple studies even reporting that higher serum bilirubin is positively correlated with eGFR (7-9). It is possible this association is secondary to bilirubin scavenging superoxide, resulting in increased renal bio-availability of nitric oxide and enhancement of renal blood flow(10).

If bilirubin and bile casts were truly nephrotoxic, there should be some experimental models that support a mechanistic link between excess bile products and kidney injury. What is needed is evidence of AKI associated solely with a rise in bilirubin, uncomplicated by acute or chronic liver failure, and their own concordant multifactorial renal insults. However, this too is absent from the literature. We know of no animal studies where an infusion of exogenous bilirubin or bile acids results in the presence of bilirubin casts, histologic tubular damage, and increased renal excretion of such products to tie together a convincing mechanistic narrative. On the contrary, multiple studies have found elevated serum bilirubin levels, achieved either through genetic manipulation or intravenous or intraperitoneal infusion, to be protective against AKI. Heme oxygenase-1 is induced as an adaptive and protective response to tissue injury. One of the mechanisms by which this protection is postulated to occur is through the degradation by heme oxygenase-1 of heme into biliverdin and subsequently to bilirubin, known to have powerful anti-apoptotic and antioxidant effects. Adin et al. utilized a bilirubin flush before a 20-minute period of warm ischemia in an isolated, perfused rat kidney model to examine the potential of bilirubin for neuroprotection(11). Bilirubin treatment as compared with control resulted in significant improvements in renal vascular resistance, urine output, GFR, tubular function, and mitochondrial integrity after ischemia-reperfusion injury (IRI), and the beneficial effects were found to be dose-dependent, providing strong evidence of causality. A subsequent study found intravenous infusion of bilirubin before and after IRI in Sprague-Dawley rats resulted in a dose-dependent improvement in post-IRI serum creatinine and a trend toward the preservation of cortical proximal tubules in rats receiving a higher dose of bilirubin (12).

Examining the interaction between elevated bilirubin and nephrotoxins, Oh et al.studied rats exposed to cyclosporine, with and without pretreatment with bilirubin (13). In the experimental arm, intraperitoneal infusion of bilirubin led to a 30-fold increase in the serum concentration. Compared with controls, bilirubin-treated rats had significantly lower urinary levels of kidney injury molecule-1 and a strong trend toward lower neutrophil gelatinase-associated lipocalin after exposure to cyclosporine. Histologically, bilirubin-treated rats had significantly reduced afferent arteriolopathy, tubulointerstitial fibrosis, tubular injury, and a significantly lower number of apoptotic proximal tubule cells. In cell culture, bilirubin exposure significantly reduced the production of intracellular reactive oxygen species. Gunn rats, deficient in the enzyme uridine diphosphate glucuronyl transferase, were used by Barabas et al. to investigate the in vivo effects of unconjugated hyperbilirubinemia on cisplatin nephrotoxicity(14). This model is critical because it assesses the effects of elevated bilirubin in isolation, outside any injury or other intervention. Elevated serum bilirubin was found to have a nephroprotective effect, with significantly lower BUN and creatinine in homozygous Gunn rats, when compared with control congenic Wistar rats at day 5. Histologic grading demonstrated significant preservation of the S3 segment in Gunn rats compared with controls. Although the mechanism whereby bilirubin affords nephroprotection is not fully clear, there are suggestions it relates to the pro/anti-inflammatory balance of bile acids affecting systemic oxidative stress in the vascular compartment (15).

Common bile duct ligation(CBDL)in rats and mice is a classic model of obstructive end-stage liver disease, where elevated serum bilirubin and abnormal kidney function parameters are noted, and AKIoccurs shortly after the onset of liver fibrosis(16). If the association between bilirubin and bile salts with AKI is explicitly causal, this should be a mechanistically parsimonious model to replicate purported bile cast nephropathy noted on autopsy. However, histologic examination of the renal parenchyma of these animals at sacrifice routinely demonstrates relatively preserved tubular structure, and again, bile casts are often absent, especially early in the model timeline, when evidence of AKIis already noted on serology or via measurement of GFR. Kronos et al. fed animals norursodeoxycholic acid (nor DCA), a conjugated bile acid thought to promote favorable bioenergetic cellular interactions(17). Although they did describe evidence of kidney parenchymal injury at 8 weeks that was ameliorated by nor DCA, the degree of histologic injury was quite mild and is confounded by the long delay between the onset of AKI(usually within 1-2 weeks of bile duct ligation) and the examination of tissue at sacrifice 8 weeks later. Their findings could alternatively be explained by systemic bioenergetic interactions of UDCA conferring a renoprotective effect, or it simply may be that after such a long period of time after an irreversible liver insult, these animals develop some degree of ischemic kidney injury. Diffuse rarefaction of the renal arterial bed has been demonstrated after CBDL in mice (Figure 1)and may be expected in and of itself to foment tubular injury and fibrosis. CBDL mice have also been noted to have interstitial nephritis, but this diagnosis was not on the basis of the actual observation of acute inflammatory cells, but rather a detection of increased local VCAM-1 and macrophage marker F4/80 expression in both the interstitium and glomeruli(18).To the extent that hyperbilirubinemia does play a role in renal dysfunction, it is possible this is not due to any direct tubular toxicity, but instead via perturbations to renal and systemic hemodynamics. Elevated bilirubin has been shown to exert negative ionotropic and chronotropic cardiac effects, sometimes referred to as "jaundiced heart." Reduced enteral bile acid concentrations contribute to increased bacterial translocation and systemic endotoxemia, engendering splanchnic and systemic vasodilation, reduced effective circulating volume, and renal hypoperfusion (19). Both these mechanisms may contribute to the development of prerenal azotemia and ischemic acute tubular necrosis(ATN), producing AKI absent any direct tubular pathogenic role for bilirubin, bile salts, or casts.

If bilirubin casts, however, are not to be implicated as causal in AKI, how then does one explain the presence of such casts in autopsy series and, more commonly, in the microscopic evaluation of urine from patients with hyperbilirubinemia and AKI? Clinical conditions associated with significant elevations in serum bilirubin levels are also frequently complicated by AKI in the form of ATN. In the setting of hyperbilirubinemia, the kidneys dramatically increase bilirubin clearance via increased secretion through an organic solute transporter. When the granular casts that are the hallmark of ATNpass through the tubules in the presence of bilirubin, they become stained. It is not surprising then that utilization of a Hall or Fouchet stain would identify "bilirubin casts" in such patients with ATN. Indeed, the greater the degree of unrelated ATN, the more likely there should be bilirubin-stained casts, both on examination of urine microscopy or via biopsy or autopsy. In addition, reduced filtrate in the setting of hypoperfusion and AKI will both increase cast formation and prolong their tubular transit time, increasing the likelihood they are seen on biopsy or autopsy(21). However, whether the ATN in these patients is due to the tubular toxicity of bilirubin and bile salts or to uniquely pathogenic bilirubin casts, or whether the staining of granular casts is merely redecorating after the damage has been done is far from clear, and the presence of such casts is by no means dispositive for pathologic culpability. The same ineluctable confusion over the chicken and the egg exists when appraising the significance of the widely acknowledged association between elevated urinary bile acids levels and AKI. Once filtered, bile acids are usually reabsorbed in the proximal convoluted tubule via a Na+-bile acid active cotransporter, apical sodium-dependent bile acid transporter, resulting in fractional excretion of only 1%-2%(22). However, in the setting of cholestasis, this is downregulated, increasing bile acid clearance (23). Critically, in the presence of proximal tubule injury, the activity of virtually all local transporters is compromised and, in severe injury associated with sloughing of the brush border, rendered virtually nonfunctional. Thus, proximal tubule injury, as is seen with ATN secondary to a myriad of causes common in liver disease, will increase urinary bile acid levels, frustrating attempts to describe the injury as being secondary to said elevations. Arguments against bile acids as important contributors to AKI associated with liver disease are summarized in Table 1.

It is important to remember clinical attempts at diagnoses should be in service of optimizing a patient's treatment. Patients with cirrhosis and AKI are medically complicated and, by definition, suffer from multisystem organ failure. Very often, more than one renal insult is present, and patients rarely fit neatly into circumscribed diagnostic categories. Much remains left to learn about the effect of bilirubin and bile acids on overall organ function and hemodynamics, and it is very possible that in some patients, bile cast nephropathy may directly contribute to a multifactorial AKI, especially in patients with severe cholestatic injury and marked elevation in bilirubin (24,25). However, the presence of casts on urine microscopy in patients with cirrhosis and AKI, whether bile stained or not, is not ex post facto evidence that a rise in creatinine is primarily due to a structural injury, nor does it speak to the presence or absence of retained tubular integrity. The treatment of patients with a preserved residual renal tubular function who suffer from hypoperfusion and hemodynamic injury (such as prerenal AKI and hepatorenal syndrome) should be focused on restoring this perfusion via volume expansion, supportive care measures, and(as appropriate) splanchnic vasoconstrictors. Although the historic neglect of hyperbilirubinemia as a contributing factor to AKT is lamentable, care must be taken not to expand the potential for mismanagement by diagnostically anchoring to a single culprit and ignoring other potentially readily treatable mechanisms of kidney injury.