Renal Stress Response Research Articles

Catalytic iron mediated renal stress responses during experimental cardiorenal syndrome 1 (“CRS-1”)

Cardiorenal syndrome I (CRS-1) denotes a state in which acute kidney injury occurs in the setting of acute heart failure (AHF). Isoproterenol (Iso) administration is widly used as an AHF model by transiently inducing extreme tachycardia, hypotension, and myocyte apoptosis and/or necrosis. To gain potential insights into renal manifestations of CRS-1, mice were subjected to the Iso-AHF model (50 mg Iso/kg), followed by renal functional and renal cortical assessments over 4 hours Iso induced acute azotemia (doubling of BUN, plasma creatinine) and significantly reduced renal plasma flow (prolonged plasma para-amino-hippurate clearance). Although no morphologic tubular injury was identified, marked increases in renal cortical 'stress markers' (NGAL, HO-1, IL-6, MCP-1 mRNAs) and oxidant stress (decreased glutathione, increased malondialdehyde) were observed. These changes were catalytic Fe dependent, given that the iron chelator desferrioxamine (DFO) significantly blunted, or completely reversed, these renal cortical abnormalities. Despite these acute changes, no lasting renal injury was observed (assessed over 3 days). To determine whether Iso directly impacts tubular cell integrity, cultured proximal tubule (HK-2) cells were exposed to Iso. Substantial Fe dependent cell injury (decreased MTT uptake), and Fe independent increases in HO-1/IL-6 mRNA expression were observed. We conclude that Iso-induced AHF is a useful reversible model of CRS-1. Despite its largely hemodynamic ('pre-renal') nature, Fe-mediated oxidative stress and pro-inflammatory reactions are induced. These arise, at least in part, from direct Iso- induced tubular cell toxicity, rather than simply being secondary to Iso-mediated hemodynamic events. Finally, Iso-triggered renal cytokine production can potentially contribute to 'organ cross talk' and a systemic pro-inflammatory state.

Renal Stress Responses To Work In The Heat Comparing Different Hydration Regimens

Workplace safety organizations recommend that workers predicted to incur heavy sweat loss should consume a ‘sport drink’ during work. These sugar-sweetened beverages (SSB) often include fructose, which can lead to acute kidney injury (AKI), especially when combined with hypohydration. PURPOSE: Investigate the effect of SSB on renal stress in response to simulated industrial work in the heat, while maintaining euhydration. METHODS: Twenty male participants (24 ± 2y, 179 ± 6cm, 24.7 ± 9.0% body fat) completed two randomized, matched trials of simulated industrial work (2-hr total; two 45-min work and two 15-min rest bouts) in the heat (30oC, 55% RH). Equal amounts of SSB or placebo were provided during rest and within 2-hr of completing work. Urine specific gravity (USG), heart rate (HR), and rectal temperature (Trec) were monitored throughout trials. Serum [Na+], hemoglobin, and hematocrit levels were measured at baseline, pre- and post-work, and 16-hr post-work. Urinary kidney injury molecule-1 (uKIM-1) and urinary neutrophil gelatinase-associated lipocalin (uNGAL) were measured pre- and post-work, 3-hr and 16-hr post-work. Total urine volume was measured for 16-hrs following work. Change in plasma volume (ΔPV) was calculated using the Dill & Costill equation. RESULTS: There was no significant trial difference in USG (p=.277) or HR (p=.209), but post-work (p≤.011) and 16-hr post-work USG were elevated compared to baseline (p=.043) and 3-hr post-work (p=.001). There was no difference in maximum Trec achieved between trials (SSB 38.84 ± 0.52oC; placebo 38.68 ± 0.40oC; p=.330). Serum [Na+] was elevated post-work compared to baseline and 16-hr post-work (p=.006), but there was no trial difference (p=.612). There was no trial difference in hemoglobin (p=.650) or hematocrit (p=.637). uKIM-1 was elevated post-work and 16-hr post-work compared to baseline (p≤.028) and 3-hr post-work (p≤.009), but showed no trial difference (p=.126). uNGAL 3-hr post-work was significantly lower than baseline (p=.011), but showed no trial difference (p=.992). There was no significant trial difference in total urine volume (p=.277) or ΔPV (p=.098). CONCLUSION: These findings indicate that beverage type did not affect renal stress biomarkers following simulated industrial work in the heat when euhydration was maintained.

Exacerbation of adverse cardiovascular effects of aircraft noise in an animal model of arterial hypertension

Arterial hypertension is the most important risk factor for the development of cardiovascular disease. Recently, aircraft noise has been shown to be associated with elevated blood pressure, endothelial dysfunction, and oxidative stress. Here, we investigated the potential exacerbated cardiovascular effects of aircraft noise in combination with experimental arterial hypertension. C57BL/6J mice were infused with 0.5 mg/kg/d of angiotensin II for 7 days, exposed to aircraft noise for 7 days at a maximum sound pressure level of 85 dB(A) and a mean sound pressure level of 72 dB(A), or subjected to both stressors. Noise and angiotensin II increased blood pressure, endothelial dysfunction, oxidative stress and inflammation in aortic, cardiac and/or cerebral tissues in single exposure models. In mice subjected to both stressors, most of these risk factors showed potentiated adverse changes. We also found that mice exposed to both noise and ATII had increased phagocytic NADPH oxidase (NOX-2)-mediated superoxide formation, immune cell infiltration (monocytes, neutrophils and T cells) in the aortic wall, astrocyte activation in the brain, enhanced cytokine signaling, and subsequent vascular and cerebral oxidative stress. Exaggerated renal stress response was also observed. In summary, our results show an enhanced adverse cardiovascular effect between environmental noise exposure and arterial hypertension, which is mainly triggered by vascular inflammation and oxidative stress. Mechanistically, noise potentiates neuroinflammation and cerebral oxidative stress, which may be a potential link between both risk factors. The results indicate that a combination of classical (arterial hypertension) and novel (noise exposure) risk factors may be deleterious for cardiovascular health.

Pharmacological and genetic inhibition of downstream targets of p38 MAPK in experimental nephrotic syndrome.

Nie X, Chanley MA, Pengal R, Thomas DB, Agrawal S, Smoyer WE. Pharmacological and genetic inhibition of downstream targets of p38 MAPK in experimental nephrotic syndrome. Am J Physiol Renal Physiol 314: F602-F613, 2018. First published November 29, 2017; doi: 10.1152/ajprenal.00207.2017 .-The p38 MAPK pathway plays a crucial role in various glomerulopathies, with activation being associated with disease and inhibition being associated with disease amelioration. We hypothesized that the downstream targets of p38 MAPK, MAPK-activated protein kinase 2 and/or 3 (MK2 and/or MK3), play an important role in mediating injury in experimental nephrotic syndrome via their actions on their downstream substrates heat shock protein B1 (HSPB1) and cyclooxygenase-2 (COX-2). To test this hypothesis, the effects of both pharmacological and genetic inhibition of MK2 and MK3 were examined in mouse adriamycin (ADR) and rat puromycin aminonucleoside (PAN) nephropathy models. MK2-/-, MK3-/-, and MK2-/-MK3-/- mice were generated in the Sv129 background and subjected to ADR-induced nephropathy. MK2 and MK3 protein expression was completely abrogated in the respective knockout genotypes, and massive proteinuria and renal histopathological changes developed after ADR treatment. Furthermore, renal cortical HSPB1 was induced in all four genotypes by day 21, but HSPB1 was activated only in the wild-type and MK3-/- mice. Expression of the stress proteins HSPB8 and glucose-regulated protein 78(GRP78) remained unaltered across all genotypes. Finally, while MK2 and/or MK3-knockout downregulated the proinflammatory enzyme COX-2, ADR significantly induced renal cortical COX-2 only in MK2-/- mice. Additionally, pharmacological MK2 inhibition with PF-318 during PAN-induced nephropathy did not result in significant proteinuria reduction in rats. Together, these data suggest that while the inhibition of MK2 and/or MK3 regulates the renal stress response, our currently available approaches are not yet able to safely and effectively reduce proteinuria in experimental nephrotic syndrome and that other p38MAPK downstream targets should also be considered to improve the future treatment of glomerular disease.

Crucial Roles of the Protein Kinases MK2 and MK3 in a Mouse Model of Glomerulonephritis

Elevated mitogen-activated protein kinase p38 (p38 MAPK) signaling has been implicated in various experimental and human glomerulopathies, and its inhibition has proven beneficial in animal models of these diseases. p38 MAPK signaling is partially mediated through MK2 and MK3, two phylogenetically related protein kinases that are its direct substrates. The current study was designed to determine the specific roles of MK2 and MK3 in a mouse model of acute proliferative glomerulonephritis, using mice with disrupted MK2 and/or MK3 genes. We found that the absence of MK3 alone worsened the disease course and increased mortality slightly compared to wild-type mice, whereas the absence of MK2 alone exhibited no significant effect. However, in an MK3-free background, the disease course depended on the presence of MK2 in a gene dosage-dependent manner, with double knock-out mice being most susceptible to disease induction. Histological and renal functional analyses confirmed kidney damage following disease induction. Because the renal stress response plays a crucial role in kidney physiology and disease, we analyzed the stress response pattern in this disease model. We found that renal cortices of diseased mice exhibited a pronounced and specific pattern of expression and/or phosphorylation of stress proteins and other indicators of the stress response (HSPB1, HSPB6, HSPB8, CHOP, eIF2α), partially in a MK2/MK3 genotype-specific manner, and without induction of a general stress response. Similarly, the expression and activation patterns of other protein kinases downstream of p38 MAPK (MNK1, MSK1) depended partially on the MK2/MK3 genotype in this disease model. In conclusion, MK2 and MK3 together play crucial roles in the regulation of the renal stress response and in the development of glomerulonephritis, which can potentially be exploited to develop novel therapeutic approaches to treat glomerular disease.

Role of Mitofusin 2 in the Renal Stress Response

The role of mitofusin 2 (MFN2), a key regulator of mitochondrial morphology and function in the renal stress response is unknown. To assess its role, the MFN2 floxed gene was conditionally deleted in the kidney of mice (MFN2 cKO) by Pax2 promoter driven Cre expression (Pax2Cre). MFN2 cKO caused severe mitochondrial fragmentation in renal epithelial cells that are critical for normal kidney tubular function. However, despite a small (20%) decrease in nephron number, newborn cKO pups had organ or tubular function that did not differ from littermate Cre-negative pups. MFN2 deficiency in proximal tubule epithelial cells in primary culture induced mitochondrial fragmentation but did not significantly alter ATP turnover, maximal mitochondrial oxidative reserve capacity, or the low level of oxygen consumption during cyanide exposure. MFN2 deficiency also did not increase apoptosis of tubule epithelial cells under non-stress conditions. In contrast, metabolic stress caused by ATP depletion exacerbated mitochondrial outer membrane injury and increased apoptosis by 80% in MFN2 deficient vs. control cells. Despite similar stress-induced Bax 6A7 epitope exposure in MFN2 deficient and control cells, MFN2 deficiency significantly increased mitochondrial Bax accumulation and was associated with greater release of both apoptosis inducing factor and cytochrome c. In conclusion, MFN2 deficiency in the kidney causes mitochondrial fragmentation but does not affect kidney or tubular function during development or under non-stress conditions. However, MFN2 deficiency exacerbates renal epithelial cell injury by promoting Bax-mediated mitochondrial outer membrane injury and apoptosis.

Uremia induces proximal tubular cytoresistance and heme oxygenase-1 expression in the absence of acute kidney injury

Acute kidney injury (AKI) induces adaptive responses within proximal tubular (PT) cells that serve to protect them from further ischemic or toxic damage. However, it is not known whether uremia, a potential consequence of AKI, independently alters susceptibility to tubular injury. To address this issue, we subjected CD-1 mice to bilateral ureteral transection (BUTx), which produces uremia (blood urea nitrogen approximately 150 mg/dl) in the absence of direct renal damage. PT segments were then isolated from BUTx and control mice and subjected to in vitro hypoxic injury. Additionally, "in vitro uremia" was modeled in isolated tubules or in cultured PT (HK-2) cells by addition of 1) peritoneal dialysate (obtained from mice with bilateral ureteral obstruction), 2) peritoneal fluid (from BUTx mice), or 3) normal human urine (pH 7.4, with and without boiling). Effects on injury severity (lactate dehydrogenase release) were assessed. Finally, because uremia is a prooxidant state, it was hypothesized that BUTx would increase renal lipid peroxidation (malondialdehyde) and induce heme oxygenase-1 (HO-1), a redox-sensitive cytoprotective protein. BUTx conferred striking protection against hypoxic damage. This could be partially modeled in tubules and HK-2 cells by induction of in vitro uremia. Urine's protective action was heat labile (largely destroyed by boiling). BUTx caused a tripling of renal malondialdehyde and HO-1 protein levels. Increased HO-1 transcription was likely involved, as indicated by a tripling of HO-1 mRNA and RNA polymerase II binding along the HO-1 gene (chromatin immunoprecipitation assay). "Gene-activating" histone modifications [H3K4 trimethylation (H3K4m3) and histone 2 variant (H2A.Z)] at HO-1 gene loci were also observed. Uremia, per se, can contribute to the AKI-induced cytoresistance. Low-molecular-weight, heat-labile, cytoprotective factor(s) and uremia-induced renal stress responses (e.g., HO-1 gene activation) are likely involved. Finally, renal HO-1 induction following AKI may reflect direct cell injury effects and adaptations to uremia.

A Gene–Environment Interaction Model of Stress-Induced Hypertension

The case for a gene-environment interaction model of stress-induced hypertension is detailed in this paper. We hypothesize that repeated exposure to stress in combination with an environmentally and/or genetically mediated susceptibility may lead to the development of essential hypertension. Previously, we reviewed the evidence for a genetic influence on the two major intermediate phenotypes of our model: cardiovascular reactivity to psychological stress and stress-induced sodium retention, representing the cardiovascular and renal stress response, respectively. Here we first describe how genes underlying the physiological systems mediating the stress response of heart, vasculature, and kidney (i.e., the sympathetic nervous system, renin-angiotensin- aldosterone system and sodium reabsorption, and the endothelial system) may increase vulnerability to stress and confer susceptibility to development of essential hypertension. Next, we extend our model and review genes underlying three additional systems that may mediate the influence of stress on the development of essential hypertension: the parasympathetic nervous system, the serotonergic system, and the hypothamamus-pituitary-adrenal axis. The elucidation of our gene-environment interaction model of stress-induced essential hypertension will improve the understanding of the contribution of stress to the development of essential hypertension. This knowledge may lead to more effective primary and secondary prevention programs involving lifestyle interventions in which the role of stress, both acute and chronic, will be taken into account, particularly for individuals at increased genetic risk of essential hypertension.

Urinary heat shock protein-72 excretion in clinical and experimental renal ischemia.

Renal ischemia not only causes injury but also induces repair mechanisms, such as the cellular induction of the 72-kilodalton heat shock protein HSP-72. The aim of this study was to determine whether HSP-72 is excreted in urine after ischemic renal injury. The first urine of six pediatric allograft recipients was examined for proteinuria and urinary HSP-72 excretion. Sprague-Dawley rats were treated with renal ischemia or hyperthermia and renal cortex and urinary HSP-72 levels were determined. HSP-72 was excreted in the first urine of renal allografts. In rats, renal HSP-72 was induced both by renal ischemia or hyperthermia. However, only renal ischemia resulted in urinary excretion of HSP-72. Urinary excretion of HSP-72 indicates an increased renal stress response and loss of tubular cell integrity after clinical and experimental renal ischemia.

Dissecting the genetic architecture of the cardiovascular and renal stress response

We review the evidence for a genetic basis of the cardiovascular and renal stress response. A bio-behavioral model of stress-induced hypertension is presented that explains how repeated exposure to stress in combination with genetic susceptibility might lead to the development of hypertension. In this model, we focus on three underlying physiological systems that mediate the stress response of the heart, vasculature and kidney: the sympathetic nervous system (SNS), the renin-angiotensin-aldosterone system (RAAS) and the endothelial system (ES). We then review the evidence for a genetic influence on cardiovascular reactivity to psychological stress and stress-induced sodium retention using data from twin and family studies and a limited number of candidate gene studies. Finally, by describing the underlying physiological systems of our model and their genetic underpinning we emphasize the importance of inclusion of genetic measurements in any future studies testing the reactivity hypothesis.

Renal Cholesterol Accumulation: A Durable Response after Acute and Subacute Renal Insults

Proximal tubular cholesterol levels rise within 18 hours of diverse forms of acute renal tubular injury (eg, myoglobinuria, ischemia/reperfusion, urinary tract obstruction). These increments serve to protect against further bouts of tubular attack (so-called "acquired cytoresistance"). Whether these cholesterol increments are merely transitory, or persist into the maintenance phase of acute renal failure (ARF), has not been previously defined. Furthermore, whether subacute/insidious tubular injury [eg, cyclosporine A (CSA), tacrolimus toxicity], nontubular injury (eg, acute glomerulonephritis), or physiological stress (eg, mild dehydration) impact renal cholesterol homeostasis have not been addressed. This study sought to resolve these issues. Male CD-1 mice were subjected to glycerol-induced ARF. Renal cortical-free cholesterol (FC) and cholesterol ester (CE) levels were determined 3, 5, 7, or 14 days later, and the values contrasted to prevailing blood-urea nitrogen concentrations. The impact of 40 minutes of unilateral renal ischemia plus reflow (3 to 6 days) on mouse cortical FC/CE content was also assessed. Additionally, FC/CE levels were measured in rat renal cortex either 10 days after CSA or tacrolimus therapy, or 48 hours after induction of nephrotoxic serum nephritis. Finally, the impact of overnight dehydration on mouse renal cortical/medullary FC/CE profiles was determined. Compared to sham-treated animals, glycerol, CSA, tacrolimus, ischemia-reperfusion, and nephrotoxic serum each induced dramatic CE +/- FC elevations, rising as much as 10x control values. In the glycerol model, striking correlations (r </= 0.99) between FC/CE and blood-urea nitrogen levels were observed. The FC/CE increases were specific to damaged kidney (glycerol did not raise hepatic FC/CE; unilateral renal ischemia did not alter contralateral renal FC/CE levels). Overnight dehydration raised renal CE levels, most notably in the medulla. FC/CE accumulation is a hallmark of the maintenance phase of ischemic and nephrotoxic ARF, and can reflect its severity. That cholesterol accumulation can result from glomerular injury and dehydration suggests that it is a generic renal stress response, with potential relevance extending beyond just the phenomenon of acquired cytoresistance.

Cell Cycle Events During Renal Injury

Recovery from ischemic and nephrotoxic acute renal failure requires replacement of damaged tubule cells in order to restore the morphological and functional integrity of the renal epithelium. A more thorough understanding of the renal stress response and its molecular interactions with the cell cycle machinery will be important areas of biomarker research in nephrotoxicity. Many of these molecular targets should be detectable hopefully without resorting the invasive techniques.

Stress Response in a Leporine Renal Cell Model

It is well established that renal proximal tubule (RPT) cells grown under standard in vitro conditions attenuate many of their in vivo properties and functions. Thus, the study of renal stress response mechanisms requires an appropriate cell culture model. In the present study, we compared the heat stress (10 min, 45°C) response of freshly isolated RPT cells with that of RPT cells grown in vitro for 6 days under two different culture conditions: (1) SHAKE conditions, where oxygen levels and physiological functions are maintained via continuous media motion [Nowak G, Schnellmann RG: Am J Physiol 1996;271:C2072–2080] and (2) STILL conditions, involving standard cell culture which leads to partial hypoxia and a marked reduction in physiological functions. The freshly isolated RPT cells progressively synthesized heat shock proteins (HSPs) and stress glycoproteins (SGs) during a 3-hour culture period in vitro. Under these conditions, heat stress did not further increase HSP and SG synthesis. In RPT cells grown under SHAKE conditions, HSP70 synthesis was detected 1 h after heat stress and decreased below detection by 3 h. In contrast, the uptake of radiolabeled mannose into (glycoprotein) GP62 (M_r 62,000), GP50, and GP38 was observed in control SHAKE cultures and was not further increased after heat stress. These results are consistent with immunohistochemistry studies, where similar changes in HSP70 and GP50 expression were noted. RPT cells grown under STILL conditions showed both increased synthesis of HSP70 and increased glycosylation of GP62, GP50, and GP38 as early as 1 h after heat stress, but in contrast to SHAKE conditions, this heat-induced stress response further intensified at 3 h after heat stress. By 7 h after heating, HSP synthesis returned to control levels, while glycosylation of GP62 and GP50 remained elevated. Based on our results, we conclude that freshly isolated RPT cells exhibit a stress response that may be caused by acute cell isolation/culture stress. While this stress response unfolds, freshly isolated RPT cells appear unable to respond to additional heat stress. RPT cells grown under SHAKE and STILL conditions exhibit high rates of SG glycosylation, especially that of GP62, possibly reflecting a ‘stress’ condition associated with growth on plastic substrate. Concurrently, RPT cells from STILL cultures show a higher capacity for responding to acute heat stress than SHAKE cultures, evidenced by the transiently increased HSP synthetic rates. The interpretation of the renal stress response capacity, therefore, must be linked to a specific culture condition.