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This review focuses on the potential mediation in the acute kidney injury (AKI)-to-chronic kidney disease (CKD) transition by lymphocytes. We highlight evidence that lymphocytes, particularly Th17 cells, modulate the severity of both acute injury and chronic kidney disease. Th17 cells are strongly influenced by the activity of the store-operated Ca2+channel Orai1, which is upregulated on lymphocytes in animal models of AKI. Inhibition of this channel attenuates both acute and chronic kidney injury in rodent models. In addition, Oria1+ cells are increased in peripheral blood of patients with AKI. Similarly, peripheral blood cells manifest an early and sustained increase in Orai1 expression in a rat model of ischemia/reperfusion, suggesting that blood cell Orai1 may represent a marker informing potential Th17 activity in the setting of AKI or the AKI-to-CKD transition.

PURPOSE OF THE REVIEW: Hypertension has been demonstrated to be a chief contributor to morbidity and mortality throughout the world. Though the etiology of hypertension is multifactorial, emerging evidence, obtained in experimental studies, as well as observational studies in humans, points to the role of inflammation and immunity. Many aspects of immune function have now been implicated in hypertension and end-organ injury; this review will focus upon the recently-described role of Th17 cells in this pathophysiological response. RECENT FINDINGS: Studies in animal models and human genetic studies point to a role in the adaptive immune system as playing a contributory role in hypertension and renal tissue damage. Th17 cells, which produce the cytokine IL17, are strongly pro-inflammatory cells which may contribute to tissue damage if expressed in chronic disease conditions. The activity of these cells may be enhanced by physiological factors associated with hypertension such as dietary salt or Ang II. This activity may culminate in the increased sodium retaining activity and exacerbation of inflammation and renal fibrosis via multiple cellular mechanisms. SUMMARY: Th17 cells are a distinct component of the adaptive immune system that may strongly enhance pathways leading to increased sodium reabsorption, elevated vascular tone and end-organ damage. Moreover, this pathway may lend itself toward specific targeting for treatment of kidney disease and hypertension.

The pathophysiology of AKI during tumor lysis syndrome (TLS) is not well understood due to the paucity of data. We aimed to decipher crystal-dependent and crystal-independent mechanisms of TLS-induced AKI.Crystalluria, plasma cytokine levels, and extracellular histones levels were measured in two cohorts of patients with TLS. We developed a model of TLS in syngeneic mice with acute myeloid leukemia, and analyzed ultrastructural changes in kidneys and endothelial permeability using intravital confocal microscopy. In parallel, we studied the endothelial toxicity of extracellular histonesThis study sheds new light on the pathophysiology of TLS-induced AKI and suggests that extracellular histones may constitute a novel target for therapeutic intervention in TLS when endothelial dysfunction occurs.

Wet bulb temperatures (T(wet)) during extreme heat events are commonly 31°C. Recent predictions indicate that T(wet) will approach or exceed 34°C. Epidemiological data indicate that exposure to extreme heat events increases kidney injury risk. We tested the hypothesis that kidney injury risk is elevated to a greater extent during prolonged exposure to T(wet) = 34°C compared with T(wet) = 31°C. Fifteen healthy men rested for 8 h in T(wet) = 31 (0)°C and T(wet) = 34 (0)°C. Insulin-like growth factor-binding protein 7 (IGFBP7), tissue inhibitor of metalloproteinase 2 (TIMP-2), and thioredoxin 1 (TRX-1) were measured from urine samples. The primary outcome was the product of IGFBP7 and TIMP-2 ([IGFBP7·TIMP-2]), which provided an index of kidney injury risk. Plasma interleukin-17a (IL-17a) was also measured. Data are presented at preexposure and after 8 h of exposure and as mean (SD) change from preexposure. The increase in [IGFBP7·TIMP-2] was markedly greater at 8 h in the 34°C [+26.9 (27.1) (ng/mL)(2)/1,000) compared with the 31°C [+6.2 (6.5) (ng/mL)(2)/1,000] trial (P < 0.01). Urine TRX-1, a marker of renal oxidative stress, was higher at 8 h in the 34°C [+77.6 (47.5) ng/min] compared with the 31°C [+16.2 (25.1) ng/min] trial (P < 0.01). Plasma IL-17a, an inflammatory marker, was elevated at 8 h in the 34°C [+199.3 (90.0) fg/dL; P < 0.01] compared with the 31°C [+9.0 (95.7) fg/dL] trial. Kidney injury risk is exacerbated during prolonged resting exposures to T(wet) experienced during future extreme heat events (34°C) compared with that experienced currently (31°C), likely because of oxidative stress and inflammatory processes. NEW AND NOTEWORTHY We have demonstrated that kidney injury risk is increased when men are exposed over an 8-h period to a wet bulb temperature of 31°C and exacerbated at a wet bulb temperature of 34°C. Importantly, these heat stress conditions parallel those that are encountered during current (31°C) and future (34°C) extreme heat events. The kidney injury biomarker analyses indicate both the proximal and distal tubules as the locations of potential renal injury and that the injury is likely due to oxidative stress and inflammation.

Over the last 20 years, there has been an increased appreciation of the long-term sequelae of acute kidney injury (AKI) and the potential development of chronic kidney disease (CKD). Several pathophysiological features have been proposed to mediate the AKI to CKD progression including maladaptive alterations in tubular, interstitial, inflammatory and vascular cells. These alterations likely interact to culminate in the progression to CKD. In this article we focus primarily on evidence of vascular rarefaction secondary to AKI, and the potential mechanisms by which rarefaction occurs in relation to other alterations in tubular and interstitial compartments. We further focus on the potential that rarefaction contributes to renal hypoxia. Consideration the role of hypoxia in the AKI to CKD transition focuses on experimental evidence of persistent renal hypoxia following AKI and experimental maneuvers to evaluate the influence of hypoxia, per se, in progressive disease. Finally, consideration of methods to evaluate hypoxia in patients is provided with the suggestion that non-invasive measurement of renal hypoxia may provide insight into progression in post AKI patients.

Multiple clinical and preclinical studies have demonstrated that plasma levels of asymmetric dimethylarginine (ADMA) are strongly associated with hypertension, diabetes, and cardiovascular and renal disease. Genetic studies in rodents have provided evidence that ADMA metabolizing dimethylarginine dimethylaminohydrolase (DDAH)-1 plays a role in hypertension and cardiovascular disease. However, it remains to be established whether ADMA is a bystander, biomarker, or sufficient contributor to the pathogenesis of hypertension and cardiovascular and renal disease. The goal of the present investigation was to develop a pharmacological molecule to specifically lower ADMA and determine the physiologic consequences of ADMA lowering in animal models. Further, we sought to determine whether ADMA lowering will produce therapeutic benefits in vascular disease in which high ADMA levels are produced. A novel long-acting recombinant DDAH (M-DDAH) was produced by post-translational modification, which effectively lowered ADMA in vitro and in vivo. Treatment with M-DDAH improved endothelial function as measured by increase in cGMP and in vitro angiogenesis. In a rat model of hypertension, M-DDAH significantly reduced blood pressure (vehicle: 187 ± 19 mm Hg vs. M-DDAH: 157 ± 23 mm Hg; P < 0.05). Similarly, in a rat model of ischemia-reperfusion injury, M-DDAH significantly improved renal function as measured by reduction in serum creatinine (vehicle: 3.14 ± 0.74 mg/dl vs. M-DDAH: 1.1 ± 0.75 mg/dl; P < 0.01), inflammation, and injured tubules (vehicle: 73.1 ± 11.1% vs. M-DDAH: 22.1 ± 18.4%; P < 0.001). These pharmacological studies have provided direct evidence for a pathologic role of ADMA and the therapeutic benefits of ADMA lowering in preclinical models of endothelial dysfunction, hypertension, and ischemia-reperfusion injury. SIGNIFICANCE STATEMENT: High levels of ADMA occur in patients with cardiovascular and renal disease. A novel modified dimethylarginine dimethylaminohydrolase by PEGylation effectively lowers ADMA, improves endothelial function, reduces blood pressure and protects from ischemia-reperfusion renal injury.

Both acute and chronic kidney disease have a strong underlying inflammatory component. This review focuses primarily on T helper 17 (Th17) cells as mediators of inflammation and their potential to modulate acute and chronic kidney disease. We provide updated information on factors and signaling pathways that promote Th17 cell differentiation with specific reference to kidney disease. We highlight numerous clinical studies that have investigated Th17 cells in the setting of human kidney disease and provide updated summaries from various experimental animal models of kidney disease indicating an important role for Th17 cells in renal fibrosis and hypertension. We focus on the pleiotropic effects of Th17 cells in different renal cell types as potentially relevant to the patho genesis of kidney disease. Finally, we highlight studies that present contrasting roles for Th17 cells in kidney disease progression.

To investigate T helper type 17 (Th17) cells in the setting of acute kidney injury, the gene encoding the master regulator of Th17 cell differentiation, that is, RAR-related orphan receptor-γ (RORγT), was mutated in Lewis rats using CRISPR/Cas9 technology. In response to 40 min of bilateral renal ischemia-reperfusion (I/R), RAR-related orphan receptor C ( Rorc)−/− rats were resistant to injury relative to wild-type Rorc+/+ rats. This protection was associated with inhibition of IL-17 expression and reduced infiltration of CD4+ cells, CD8+ cells, B cells, and macrophages. To evaluate the effect of Th17 cells on repair, ischemia was increased to 50 min in Rorc−/− rats. This maneuver equalized the initial level of injury in Rorc−/− and Rorc+/+ rats 1 to 2 days post-I/R based on serum creatinine values. However, Rorc−/− rats, but not Rorc+/+ rats, failed to successfully recover renal function and had high mortality by 4 days post-I/R. Histological assessment of kidney tubules showed evidence of repair by day 4 post-I/R in Rorc+/+ rats but persistent necrosis and elevated cell proliferation in Rorc−/− rats. Adoptive transfer of CD4+ cells from the spleen of Rorc+/+ rats or supplementation of exogenous rIL-17 by an osmotic minipump improved renal function and survival of Rorc−/− rats following 50 min of I/R. This was associated with a relative decrease in the number of M1-type macrophages and a relative increase in the percentage of T regulatory cells. Taken together, these data suggest that Th17 cells have both a deleterious and a beneficial role in kidney injury and recovery, contributing to early postischemic injury and inflammation but also possibly being critical in the resolution of inflammation during kidney repair.

Ischemic preconditioning confers organ-wide protection against subsequent ischemic stress. A substantial body of evidence underscores the importance of mitochondria adaptation as a critical component of cell protection from ischemia. To identify changes in mitochondria protein expression in response to ischemic preconditioning, we isolated mitochondria from ischemic preconditioned kidneys and sham-treated kidneys as a basis for comparison. The proteomic screen identified highly upregulated proteins, including NADP+-dependent isocitrate dehydrogenase 2 (IDH2), and we confirmed the ability of this protein to confer cellular protection from injury in murine S3 proximal tubule cells subjected to hypoxia. To further evaluate the role of IDH2 in cell protection, we performed detailed analysis of the effects of Idh2 gene delivery on kidney susceptibility to ischemia-reperfusion injury. Gene delivery of IDH2 before injury attenuated the injury-induced rise in serum creatinine (P

Damage to endothelial cells contributes to acute kidney injury (AKI) by leading to impaired perfusion. Endothelial colony-forming cells (ECFC) are endothelial precursor cells with high proliferative capacity, pro-angiogenic activity, and in vivo vessel forming potential. We hypothesized that ECFC may ameliorate the degree of AKI and/or promote repair of the renal vasculature following ischemia-reperfusion (I/R). Rat pulmonary microvascular endothelial cells (PMVEC) with high proliferative potential were compared with pulmonary artery endothelial cells (PAEC) with low proliferative potential in rats subjected to renal I/R. PMVEC administration reduced renal injury and hastened recovery as indicated by serum creatinine and tubular injury scores, while PAEC did not. Vehicle-treated control animals showed consistent reductions in renal medullary blood flow (MBF) within 2 h of reperfusion, while PMVEC protected against loss in MBF as measured by laser Doppler. Interestingly, PMVEC mediated protection occurred in the absence of homing to the kidney. Conditioned medium (CM) from human cultured cord blood ECFC also conveyed beneficial effects against I/R injury and loss of MBF. Moreover, ECFC-CM significantly reduced the expression of ICAM-1 and decreased the number of differentiated lymphocytes typically recruited into the kidney following renal ischemia. Taken together, these data suggest that ECFC secrete factors that preserve renal function post ischemia, in part, by preserving microvascular function.

Highly aerobic organs like the kidney are innately susceptible to ischemia-reperfusion (I/R) injury, which can originate from sources including myocardial infarction, renal trauma, and transplant. Therapy is mainly supportive and depends on the cause(s) of damage. In the absence of hypervolemia, intravenous fluid delivery is frequently the first course of treatment but does not reverse established AKI. Evidence suggests that disrupting leukocyte adhesion may prevent the impairment of renal microvascular perfusion and the heightened inflammatory response that exacerbate ischemic renal injury. We investigated the therapeutic potential of hydrodynamic isotonic fluid delivery (HIFD) to the left renal vein 24 hours after inducing moderate-to-severe unilateral IRI in rats. HIFD significantly increased hydrostatic pressure within the renal vein. When conducted after established AKI, 24 hours after I/R injury, HIFD produced substantial and statistically significant decreases in serum creatinine levels compared with levels in animals given an equivalent volume of saline via peripheral infusion (P

Tubulointerstitial fibrosis has long been recognized as the most significant structural lesion associated with the development and progression of CKD. Since the pioneering work of Border and Ruoslahti,[1][1] the pleiotropic factor TGF- β has assumed a central role in the genesis of

We hypothesized that the store-operated calcium entry (SOCE) channel, Orai1, participates in the activation of Th17 cells and influences renal injury. In rats, following renal ischemia/reperfusion (I/R), there was a rapid and sustained influx of Orai1+ CD4 T cells and IL-17 expression was restricted to Orai1+ cells. When kidney CD4+ cells of post-acute kidney injury (post-AKI) rats were stimulated with angiotensin II and elevated Na+ (10-7 M/170 mM) in vitro, there was an enhanced response in intracellular Ca2+ and IL-17 expression, which was blocked by SOCE inhibitors 2APB, YM58483/BTP2, or AnCoA4. In vivo, YM58483/BTP2 (1 mg/kg) attenuated IL-17+ cell activation, inflammation, and severity of AKI following either I/R or intramuscular glycerol injection. Rats treated with high-salt diet (5-9 weeks after I/R) manifested progressive disease indicated by enhanced inflammation, fibrosis, and impaired renal function. These responses were significantly attenuated by YM58483/BTP2. In peripheral blood of critically ill patients, Orai1+ cells were significantly elevated by approximately 10-fold and Th17 cells were elevated by approximately 4-fold in AKI versus non-AKI patients. Further, in vitro stimulation of CD4+ cells from AKI patients increased IL-17, which was blocked by SOCE inhibitors. These data suggest that Orai1 SOCE is a potential therapeutic target in AKI and CKD progression.

Acute kidney injury (AKI) is a strong independent predictor of mortality and often results in incomplete recovery of renal function, leading to progressive chronic kidney disease (CKD). Many clinical trials have been conducted on the basis of promising preclinical data, but no therapeutic interventions have been shown to improve long-term outcomes after AKI. This is partly due to the failure of preclinical studies to accurately model clinically relevant injury and long-term outcomes on CKD progression. Here, we evaluated the long-term effects of AKI on CKD progression in three animal models reflecting diverse etiologies of AKI: repeat-dose cisplatin, rhabdomyolysis, and ischemia-reperfusion injury. Using transdermal measurement of glomerular filtration rate as a clinically relevant measure of kidney function and quantification of peritubular capillary density to measure capillary rarefaction, we showed that repeat-dose cisplatin caused capillary rarefaction and decreased renal function in mice without a significant increase in interstitial fibrosis, whereas rhabdomyolysis-induced AKI led to severe interstitial fibrosis, but renal function and peritubular capillary density were preserved. Furthermore, long-term experiments in mice with unilateral ischemia-reperfusion injury showed that restoration of renal function 12 wk after a contralateral nephrectomy was associated with increasing fibrosis, but a reversal of capillary rarefaction was seen at 4 wk. These data demonstrate that clear dissociation between kidney function and fibrosis in these models of AKI to CKD progression and suggest that peritubular capillary rarefaction is more strongly associated with CKD progression than renal fibrosis.

Acute kidney injury (AKI) is associated with high mortality rates and predisposes development of chronic kidney disease (CKD). Distant organ damage, particularly in the lung, may contribute to mortality in AKI patients. Animal models of AKI demonstrate an increase in pulmonary infiltration of lymphocytes and reveal an acute compromise of lung function, but the chronic effects of AKI on pulmonary inflammation are unknown. We hypothesized that in response to renal ischemia/reperfusion (I/R), there is a persistent systemic increase in Th17 cells with potential effects on pulmonary structure and function. Renal I/R injury was performed on rats, and CKD progression was hastened by unilateral nephrectomy and exposure to 4.0% sodium diet between 35 and 63 days post-I/R. Th17 cells in peripheral blood showed a progressive increase up to 63 days after recovery from I/R injury. Infiltration of leukocytes including Th17 cells was also elevated in bronchiolar lavage (BAL) fluid 7 days after I/R and remained elevated for up to 63 days. Lung histology demonstrated an increase in alveolar cellularity and a significant increase in picrosirius red staining. Suppression of lymphocytes with mycophenolate mofetil (MMF) or an IL-17 antagonist significantly reduced Th17 cell infiltration and fibrosis in lung. In addition, tracheal smooth muscle contraction to acetylcholine was significantly enhanced 63-days after I/R relative to sham-operated controls. These data suggest that AKI is associated with a persistent increase in circulating and lung Th17 cells which may promote pulmonary fibrosis and the potential alteration in airway contractility.

Acute kidney injury is associated with alterations in vascular tone that contribute to an overall reduction in GFR. Studies in animal models indicate that ischemia triggers alterations in endothelial function that contribute significantly to the overall degree and severity of a kidney injury. Putative mediators of vasoconstriction that may contribute to the initial loss of renal blood flow and GFR are highlighted. In addition, there is discussion of how intrinsic damage to the endothelium impairs homeostatic responses in vascular tone as well as promotes leukocyte adhesion and exacerbating the reduction in renal blood flow. The timing of potential therapies in animal models as they relate to the evolution of AKI, as well as the limitations of such approaches in the clinical setting are discussed. Finally, we discuss how acute kidney injury induces permanent alterations in renal vascular structure. We posit that the cause of the sustained impairment in kidney capillary density results from impaired endothelial growth responses and suggest that this limitation is a primary contributing feature underlying progression of chronic kidney disease.

Gene therapy has been proposed as a novel alternative to treat kidney disease. This goal has been hindered by the inability to reliably deliver transgenes to target cells throughout the kidney, while minimizing injury. Since hydrodynamic forces have previously shown promising results, we optimized this approach and designed a method that utilizes retrograde renal vein injections to facilitate transgene expression in rat kidneys. We show, using intravital fluorescence two-photon microscopy, that fluorescent albumin and dextrans injected into the renal vein under defined conditions of hydrodynamic pressure distribute broadly throughout the kidney in live animals. We found injection parameters that result in no kidney injury as determined by intravital microscopy, histology, and serum creatinine measurements. Plasmids, baculovirus, and adenovirus vectors, designed to express EGFP, EGFP-actin, EGFP-occludin, EGFP-tubulin, tdTomato-H2B, or RFP-actin fusion proteins, were introduced into live kidneys in a similar fashion. Gene expression was then observed in live and ex vivo kidneys using two-photon imaging and confocal laser scanning microscopy. We recorded widespread fluorescent protein expression lasting more than 1 mo after introduction of transgenes. Plasmid and adenovirus vectors provided gene transfer efficiencies ranging from 50 to 90%, compared with 10–50% using baculovirus. Using plasmids and adenovirus, fluorescent protein expression was observed 1) in proximal and distal tubule epithelial cells; 2) within glomeruli; and 3) within the peritubular interstitium. In isolated kidneys, fluorescent protein expression was observed from the cortex to the papilla. These results provide a robust approach for gene delivery and the study of protein function in live mammal kidneys.

T cells have been implicated in the pathogenesis of acute kidney injury (AKI) and its progression to chronic kidney disease (CKD). Previous studies suggest that Th17 cells participate during the AKI-to-CKD transition, and inhibition of T cell activity by mycophenolate mofetil (MMF) or losartan attenuates the development of fibrosis following AKI. We hypothesized that T cell-deficient rats may have reduced levels of IL-17 cytokine leading to decreased fibrosis following AKI. Renal ischemis-reperfusion (I/R) was performed on T cell-deficient athymic rats (Foxn1rnu−/rnu−) and control euthymic rats (Foxn1rnu−/+), and CKD progression was hastened by unilateral nephrectomy at day 33 and subsequent exposure to 4.0% sodium diet. Renal fibrosis developed in euthymic rats and was reduced by MMF treatment. Athymic rats exhibited a similar degree of fibrosis, but this was unaffected by MMF treatment. FACS analysis demonstrated that the number of IL-17+ cells was similar between postischemic athymic vs. euthymic rats. The source of IL-17 production in euthymic rats was predominately from conventional T cells (CD3+/CD161−). In the absence of conventional T cells in athymic rats, a compensatory pathway involving natural killer cells (CD3−/CD161+) was the primary source of IL-17. Blockade of IL-17 activity using IL-17Rc receptor significantly decreased fibrosis and neutrophil recruitment in both euthymic and athymic rats compared with vehicle-treated controls. Taken together, these data suggest that IL-17 secretion participates in the pathogenesis of AKI-induced fibrosis possibly via the recruitment of neutrophils and that the source of IL-17 may be from either conventional T cells or NK cells.

Damage to endothelial cells contributes to acute kidney injury (AKI) by causing impaired perfusion, while the permanent loss of the capillary network following AKI has been suggested to promote chronic kidney disease. Therefore, strategies to protect renal vasculature may impact both short‐term recovery and long‐term functional preservation post‐AKI. Human adipose stromal cells (hASCs) possess pro‐angiogenic and anti‐inflammatory properties and therefore have been tested as a therapeutic agent to treat ischaemic conditions. This study evaluated hASC potential to facilitate recovery from AKI with specific attention to capillary preservation and inflammation. Male Sprague Dawley rats were subjected to bilateral ischaemia/reperfusion and allowed to recover for either two or seven days. At the time of reperfusion, hASCs or vehicle was injected into the suprarenal abdominal aorta. hASC‐treated rats had significantly greater survival compared to vehicle‐treated rats (88.7% versus 69.3%). hASC treatment showed hastened recovery as demonstrated by lower creatinine levels at 48 hrs, while tubular damage was significantly reduced at 48 hrs. hASC treatment resulted in a significant decrease in total T cell and Th17 cell infiltration into injured kidneys at 2 days post‐AKI, but an increase in accumulation of regulatory T cells. By day 7, hASC‐treated rats showed significantly attenuated capillary rarefaction in the cortex (15% versus 5%) and outer medulla (36% versus 18%) compared to vehicle‐treated rats as well as reduced accumulation of interstitial alpha‐smooth muscle actin‐positive myofibroblasts. These results suggest for the first time that hASCs improve recovery from I/R‐induced injury by mechanisms that contribute to decrease in inflammation and preservation of peritubular capillaries.

To facilitate its role in maintaining plasma homeostasis, the kidney relies on a complex vascular architecture to carry out multiple functions. Given that the plasma volume is filtered approximately 60 times per day and that approximately 99% of the filtrate is reabsorbed and returned to the plasma

Brown Norway rats (BN, BN/NHsdMcwi) are profoundly resistant to developing acute kidney injury (AKI) following ischemia reperfusion. To help define the genetic basis for this resistance, we used consomic rats, in which individual chromosomes from BN rats were placed into the genetic background of Dahl SS rats (SS, SS/JrHsdMcwi) to determine which chromosomes contain alleles contributing to protection from AKI. The parental strains had dramatically different sensitivity to ischemia reperfusion with plasma creatinine levels following 45 min of ischemia and 24 h reperfusion of 4.1 and 1.3 mg/dl in SS and BN, respectively. No consomic strain showed protection similar to the parental BN strain. Nine consomic strains (SS-7(BN), SS-X(BN), SS-8(BN), SS-4(BN), SS-15(BN), SS-3(BN), SS-10(BN), SS-6(BN), and SS-5(BN)) showed partial protection (plasma creatinine about 2.5-3.0 mg/dl), suggesting that multiple alleles contribute to the severity of AKI. In silico analysis was performed using disease ontology database terms and renal function quantitative trait loci from the Rat Genome Database on the BN chromosomes giving partial protection from AKI. This tactic identified at least 36 candidate genes, with several previously linked to the pathophysiology of AKI. Thus, natural variants of these alleles or yet-to-be identified alleles on these chromosomes provide protection against AKI. These alleles may be potential modulators of AKI in susceptible patient populations.

Objective Chronic kidney disease (CKD) is histologically characterized by interstitial fibrosis, which may be driven by peritubular capillary dropout and hypoxia. Surprisingly, peritubular capillaries have little repair capacity. We sought to establish long-term cultures of rat kidney endothelial cells to investigate their growth regulatory properties.

Chronic kidney disease pathogenesis involves both tubular and vascular injuries. Despite abundant investigations to identify the risk factors, the involvement of chronic endothelial dysfunction in developing nephropathies is insufficiently explored. Previously, soluble thrombomodulin (sTM), a cofactor in the activation of protein C, has been shown to protect endothelial function in models of acute kidney injury. In this study, the role for sTM in treating chronic kidney disease was explored by employing a mouse model of chronic vascular activation using endothelial-specific TNF-α-expressing (tie2-TNF) mice. Analysis of kidneys from these mice after 3 mo showed no apparent phenotype, whereas 6-mo-old mice demonstrated infiltration of CD45-positive leukocytes accompanied by upregulated gene expression of inflammatory chemokines, markers of kidney injury, and albuminuria. Intervention with murine sTM with biweekly subcutaneous injections during this window of disease development between months 3 and 6 prevented the development of kidney pathology. To better understand the mechanisms of these findings, we determined whether sTM could also prevent chronic endothelial cell activation in vitro. Indeed, treatment with sTM normalized increased chemokines, adhesion molecule expression, and reduced transmigration of monocytes in continuously activated TNF-expressing endothelial cells. Our results suggest that vascular inflammation associated with vulnerable endothelium can contribute to loss in renal function as suggested by the tie2-TNF mice, a unique model for studying the role of vascular activation and inflammation in chronic kidney disease. Furthermore, the ability to restore the endothelial balance by exogenous administration of sTM via downregulation of specific adhesion molecules and chemokines suggests a potential for therapeutic intervention in kidney disease associated with chronic inflammation.

Acute kidney injury induces the loss of renal microvessels, but the fate of endothelial cells and the mechanism of potential vascular endothelial growth factor (VEGF)-mediated protection is unknown. Cumulative cell proliferation was analyzed in the kidney of Sprague-Dawley rats following ischemia-reperfusion (I/R) injury by repetitive administration of BrdU (twice daily) and colocalization in endothelial cells with CD31 or cablin. Proliferating endothelial cells were undetectable for up to 2 days following I/R and accounted for only ∼1% of BrdU-positive cells after 7 days. VEGF-121 preserved vascular loss following I/R but did not affect proliferation of endothelial, perivascular cells or tubular cells. Endothelial mesenchymal transition states were identified by localizing endothelial markers (CD31, cablin, or infused tomato lectin) with the fibroblast marker S100A4. Such structures were prominent within 6 h and sustained for at least 7 days following I/R. A Tie-2-cre transgenic crossed with a yellow fluorescent protein (YFP) reporter mouse was used to trace the fate of endothelial cells and demonstrated interstititial expansion of YFP-positive cells colocalizing with S100A4 and smooth muscle actin following I/R. The interstitial expansion of YFP cells was attenuated by VEGF-121. Multiphoton imaging of transgenic mice revealed the alteration of YFP-positive vascular cells associated with blood vessels characterized by limited perfusion in vivo. Taken together, these data indicate that vascular dropout post-AKI results from endothelial phenotypic transition combined with an impaired regenerative capacity, which may contribute to progressive chronic kidney disease.

Ischemia-reperfusion (I/R)-induced acute kidney injury (AKI) results in prolonged impairment of peripheral (i.e., nonrenal) vascular function since skeletal muscle resistance arteries derived from rats 5 wk post-I/R injury, show enhanced responses to ANG II stimulation but not other constrictors. Because vascular superoxide increases ANG II sensitivity, we hypothesized that peripheral responsiveness following recovery from AKI was attributable to vascular oxidant stress. Gracilis arteries (GA) isolated from post-I/R rats (∼5 wk recovery) showed significantly greater superoxide levels relative to sham-operated controls, as detected by dihydroeithidium, which was further augmented by acute ANG II stimulation in vitro. Hydrogen peroxide measured by dichlorofluorescein was not affected by ANG II. GA derived from postischemic animals manifested significantly greater constrictor responses in vitro to ANG II than GA from sham-operated controls. The addition of the superoxide scavenging reagent Tempol (10−5 M) normalized the response to values similar to sham-operated controls. Apocynin (10−6 M) and endothelial denudation nearly abrogated all ANG II-stimulated constrictor activity in GA from post-AKI rats, suggesting an important role for an endothelial-derived source of peripheral oxidative stress. Apocynin treatment in vivo abrogated GA oxidant stress and attenuated ANG II-induced pressor responses post-AKI. Interestingly, gene expression studies in GA vessels indicated a paradoxical reduction in NADPH oxidase subunit and AT1-receptor genes and no effect on several antioxidant genes. Taken together, this study demonstrates that AKI alters peripheral vascular responses by increasing oxidant stress, likely in the endothelium, via an undefined mechanism.

The present studies evaluated intrarenal hemodynamics, pressure natriuresis, and arterial blood pressure in rats following recovery from renal ischemia-reperfusion (I/R) injury. Acute I/R injury, induced by 40 min of bilateral renal arterial occlusion, resulted in an increase in plasma creatinine that resolved within a week. Following 5 wk of recovery on a 0.4% NaCl diet, the pressure-natriuresis response was assessed in anesthetized rats in which the kidney was denervated and extrarenal hormones were administered intravenously. Increasing renal perfusion pressure (RPP) from 107 to 141 mmHg resulted in a fourfold increase in urine flow and sodium excretion in sham control rats. In comparison, pressure diuresis and natriuresis were significantly attenuated in post-I/R rats. In sham rats, glomerular filtration rate (GFR) averaged 1.6 ± 0.2 ml·min−1·g kidney weight−1and renal blood flow (RBF) averaged 7.8 ± 0.7 ml·min−1·g kidney weight−1at RPP of 129 mmHg. Renal cortical blood flow, measured by laser-Doppler flowmetry, was well autoregulated whereas medullary blood flow and renal interstitial hydrostatic pressure increased directly with elevated RPP in sham rats. In contrast, GFR and RBF were significantly reduced whereas medullary perfusion and interstitial pressure demonstrated an attenuated response to RPP in post-I/R rats. Further experiments demonstrated that conscious I/R rats develop hypertension when sodium intake is increased. The present data indicate that the pressure-natriuretic-diuretic response in I/R rats is blunted because of a decrease in GFR and RBF and the depressed pressure-dependent increase in medullary blood flow and interstitial pressure.

The RNA-binding protein human antigen R (HuR) participates in the posttranscriptional regulation of mRNAs bearing 3′ AU-rich and U-rich elements, which HuR can stabilize under conditions of cellular stress. Using the LLC-PK1proximal tubule cell line model, we recently suggested a role for HuR in protecting kidney epithelia from injury during ischemic stress (Jeyaraj S, Dakhlallah D, Hill SR, Lee BS. J Biol Chem 280: 37957–37964, 2005; Jeyaraj SC, Dakhlallah D, Hill SR, Lee BS. Am J Physiol Renal Physiol 291: F1255–F1263, 2006). Here, we have extended this work to show that small interfering RNA-mediated suppression of HuR in LLC-PK1cells increased apoptosis during energy depletion, while overexpression of HuR diminished apoptosis. Suppression of HuR also resulted in diminished levels of key cell survival proteins such as Bcl-2 and Hsp70. Furthermore, rat kidneys were subjected in vivo to transient ischemia followed by varying periods of reperfusion. Ischemia and reperfusion (I/R) affected intensity and distribution of HuR in a nephron segment-specific manner. Cells of the proximal tubule, which are most sensitive to I/R injury, demonstrated a transient shift of HuR to the cytoplasm immediately following ischemia. Over a 14-day period following the onset of reperfusion, nuclear and total HuR protein gradually increased in cortical and medullary proximal tubules, but not in non-proximal tubule cells. HuR mRNA was expressed in two forms with alternate transcriptional start sites that increased over a 14-day I/R period, and in vitro studies suggest selective translatability of these two mRNAs. Baseline and I/R-stimulated levels of HuR mRNA did not parallel those of HuR protein, suggesting translational control of HuR expression, particularly in medullary proximal tubules. These findings suggest that alterations in distribution and expression of the antiaptotic protein HuR specifically in cells of the proximal tubule effect a protective mechanism during and following I/R injury in kidney.

Reductions in vascular density occur following acute ischemia-reperfusion (I/R) injury that may predispose the development of chronic kidney disease. The mechanisms mediating vascular loss are not clear but may relate to the lack of effective vascular repair responses. To determine the regulation of the VEGF/VEGFR pathway following I/R injury, male Sprague-Dawley rats were subjected to bilateral renal ischemia (45 min) and allowed to recover for 1, 3, 7, and 35 days. VEGF mRNA expression was repressed by greater than 50% of control values up to 3 days postischemia, while VEGF protein was repressed for up to 7 days postischemia. The renal mRNA expression of receptors was not altered postischemia; however, VEGFR1 (flt-1) protein was transiently reduced in kidney while soluble flt-1 was elevated in plasma at 7 days following injury. Microarray analysis of angiogenesis-related genes identified the enhanced expression of a number of genes, among these was ADAMTS-1 (a disintegrin and metalloproteinase with thrombospondin motif-1), a secreted VEGF inhibitor. The altered expression of ADAMTS-1 was confirmed using RT-PCR and Western blot analysis; immunofluorescence localized its expression to proximal tubules following I/R injury. Other genes identified using microarray included aminopeptidase N, Smad-1, and Id-3 and their localization was also examined using immunohistochemistry. In summary, the data indicate no clear pattern of anti-angiogenic gene expression following renal I/R injury. However, the studies do suggest an overall inhibition of the VEGF pathway during the early injury and repair phase of renal ischemia that may contribute to an overall reduction in renal microvascular density.

The present study determined the effect of immune suppression with mycophenolate mofetil (MMF) on sodium-sensitive hypertension following recovery from ischemia reperfusion (I/R)-induced acute renal failure. Male Sprague-Dawley rats fed 0.4% NaCl chow were subjected to 40 min bilateral I/R or control sham surgery. After 35 days of recovery, when plasma creatinine levels had returned to normal, the rats were switched to 4.0% NaCl chow for 28 days and administered vehicle or MMF (20 mg·kg−1·day−1 ip). High-salt mean arterial pressure was significantly higher in I/R rats (144 ± 16 mmHg) compared with vehicle-treated sham rats (122 ± 2 mmHg). Treatment of I/R rats with MMF during the period of high salt intake prevented the salt-induced increase in arterial pressure (114 ± 3 mmHg). Conscious creatinine clearance was lower in I/R rats (0.27 ± 0.07 ml·min−1·100 g body wt−1) compared with vehicle-treated sham rats (0.58 ± 0.04 ml·min−1·100 g body wt−1); MMF treatment prevented the decrease in creatinine clearance in I/R rats (0.64 ± 0.07 ml·min−1·100 g body wt−1). I/R injury also significantly increased glomerular tissue damage and increased the presence of ED-1 positive (macrophages) and S100A4 positive cells (fibroblasts) in the renal interstitium. The I/R rats treated with MMF exhibited a significant reduction in infiltrating macrophages and fibroblasts and decreased histological damage. The present data indicate that infiltrating immune cells mediate or participate in the development of sodium-sensitive hypertension and renal damage in rats apparently recovered from renal I/R injury.

Recovery of renal function is a well-characterized feature of models of acute renal failure; however, more recent studies have reported a predisposition to chronic renal disease. This study sought to determine the susceptibility to sodium-dependent hypertension following recovery from ischemic acute renal failure. Following ischemia-reperfusion (I/R) injury, rats were allowed to recover for 35 days on a 0.4% salt diet, then were switched to 4.0% salt diet for an additional 28 days. Blood pressure was significantly increased in postischemic rats switched to high-sodium diet at day 35 (19 ± 9 mmHg) compared with postischemic rats maintained on low-sodium diet. Plasma renin activity and creatinine clearance were not affected by I/R injury. The ischemic injury combined with transfer to 4.0% salt diet resulted in marked renal hypertrophy characterized by interstitial cellular deposition, tubular dilation, and enhanced rates of albumin excretion. Glomerular structure was altered in post-I/R rats switched to high-sodium diet but not in those maintained on low-sodium diets. When rats were acclimated to high-sodium diet before I/R injury, the early injury was similar to that observed in animals acclimated to low-sodium diet, and these animals progressed rapidly toward chronic kidney disease, as evidenced by advancement of albuminuria. These data suggest that the recovery from acute I/R injury is not complete, compromises Na homeostasis, and predisposes hypertension and secondary renal disease.

Background: Despite poor sensitivity in acutely ill patients, serum creatinine (and estimated glomerular filtration rate [eGFR SCR ]) remains the sole means of risk-stratifying patients for acute kidney injury (AKI) prior to contrast-enhanced CT imaging (CECT). Hypothesis: We hypothesized that an acute phase marker of renal dysfunction, cystatin-C (expressed as eGFR CYS ), would more accurately predict contrast-induced nephropathy (CIN) than eGFR SCR . Given the risk of arterial vascular events subsequent to AKI, we also evaluated eGFR CYS in risk-stratifying patients for major adverse events (MAE) within 1 year of CECT. Methods: We followed 462 consecutive adults, without end-stage renal disease, undergoing CECT (any indication) in the outpatient, emergency care setting for CIN and 1-year MAE: death, renal failure, myocardial infarction, stroke, and/or peripheral vascular event requiring intervention (blinded, adjudicated outcome). We excluded patients with life-threatening CECT indications and collected serum for eGFR SCR and eGFR CYS prior to CECT. Predictive accuracy was defined as the area under the receiver operating characteristic curve (AUROC) and likelihood ratios (LR+ and LR-). A threshold of ≤60 ml/min/m 2 defined an abnormal eGFR SCR or eGFR CYS . Results: CIN occurred in 14% and a MAE in 17% (low observer variability, κ>0.9) of our heterogeneous population: mean age 50 yrs (±16 yrs), 51% discharged after CECT, 16% with diabetes mellitus (DM), and only 16% with eGFR SCR ≤60ml/min/m 2 . CIN was associated with 1-year MAE: RR 2.4 (1.5-4.0) after adjusting for age and existing co-morbidities (active malignancy, CHF, DM, and CAD). The AUROC, LR+ and LR- for eGFR SCR were 0.55 (0.47-0.63), 0.9 (0.4-2.1) and 1.0 (0.9-1.1). In comparison, the AUROC, LR+, and LR- for eGFR CYS were 0.79 (0.62-0.96), 5.5 (3.9-7.6) and 0.43 (0.31-0.57), respectively. The MAE rate did not differ in patients with normal (13%) or abnormal (15%, p=0.5) pre-CECT eGFR SCR . Whereas, an abnormal eGFR SCR was associated with a 29% (p Conclusions: In patients undergoing CECT in the outpatient setting, eGFR CYS more accurately predicted CIN and more effectively risk-stratified patients for 1-year MAE than eGFR SCR . These findings warrant prospective validation.

In addition to the long-term renal complications, previous studies suggested that after acute renal failure (ARF), rats manifest an increased pressor response to an overnight infusion of ANG II. The present study tested whether recovery from ARF results in alterations in sensitivity to the peripheral vasculature. ARF was induced in Sprague-Dawley rats by 45 min of bilateral renal ischemia and reperfusion. Animals were allowed to recover renal structure and function for 5–8 wk, after which the acute pressor responses to ANG II were evaluated either in vivo in in situ skeletal muscle arterioles or in isolated gracilis muscle arteries in vitro. Baseline arterial pressure was not different in ARF rats vs. sham-operated controls, although ARF rats exhibited an enhanced pressor response to bolus ANG II infusion compared with control rats. Steady-state plasma ANG II concentration and plasma renin activity were similar between ARF and control rats. Constrictor reactivity of in situ cremasteric arterioles from ARF rats was enhanced in response to increasing concentrations of ANG II; however, no difference was observed in arteriolar responses to elevated Po2, norepinephrine, acetylcholine, or sodium nitroprusside. Isolated gracilis muscle arteries from ARF rats also showed increased vasoconstriction in response to ANG II but not norepinephrine. In conclusion, recovery from ischemic ARF is not associated with hypertension but is associated with increased arteriolar constrictor reactivity to ANG II. Although the mechanisms of this altered responsiveness are unclear, such changes may relate, in part, to cardiovascular complications in patients with ARF and/or after renal transplant.

Recovery from ischemic acute renal failure (ARF) involves a well-described regenerative process; however, recovery from ARF also results in a predisposition to a progressive renal disease that is not well understood. This study sought to identify alterations in renal gene expression in postischemic, recovered animals that might play important roles in this progressive disorder. RNA isolated from sham-operated control rats or rats 35 days after recovery from bilateral ischemia-reperfusion (I/R) injury was compared using a cDNA microarray containing ∼2,000 known rat genes. A reference hybridization strategy was utilized to define a 99.9% interval and to identify 16 genes that were persistently altered after recovery from I/R injury (12 were upregulated and 4 were downregulated). Real-time PCR verified the altered expression of six of eight genes that had been positively identified. Several genes that were identified had not previously been evaluated within the context of ARF. S100A4, a specific marker of fibroblasts, was identified in a population of interstitial cells that were present postischemic injury. S100A4-positive cells were also identified in tubular cells at earlier time points postischemia. Genes associated with calcification, including osteopontin and matrix Gla protein, were also enhanced postischemic injury. Several proinflammatory genes were identified, including complement C4, were enhanced in postischemic tissues. Conversely, renal kallikrein expression was specifically reduced in the postischemic kidney. In summary, genes with known inflammatory, remodeling, and vasoactive activities were identified in rat kidneys after recovery from ARF, some of which may play a role in altering long-term renal function after recovery from ARF.

Transforming growth factor (TGF)-β1and a number of TGF-β-responsive genes are transiently enhanced following induction of ischemic acute renal failure (ARF) in the rat. The mRNA and protein expression of TGF-β receptors were analyzed in postischemic rat kidneys by ribonuclease protection, in situ hybridization, and immunohistochemistry. TGF-βRI and -RII were enhanced within 3 days of ischemia-reperfusion (I/R) injury and remained elevated for up 7 days post-I/R; TGF-β receptor expression was localized primarily in regenerating tubules within the outer medulla. A neutralizing TGF-β antibody exacerbated cellular proliferation observed on day 3 postischemia but had no effect on day 1 or 2. TGF-β antibody treatment had no measurable effect on loss of renal function or the restoration of renal function during the recovery response for up to 35 days postsurgery. However, ischemic injury resulted in modest renal hypertrophy that is due, in part, to in an increase in the number of interstitial cells in the postischemic kidney. Immunohistochemistry showed that several of these cells stained positively for the fibroblast-specific marker, S100A4 positive. Anti-TGF-β treatment substantially attenuated the renal hypertrophy, interstitial cellularity, and S100A4-positive cells present at 35 days post-I/R. Finally, TGF-β immunoneutralization attenuated the loss of renal vascular density following recovery from I/R injury. These data suggest that the TGF-β/TβR system is enhanced in the postischemic kidney. However, the current study failed to identify a prominent role for this system in the repair of proximal tubules following ARF. In contrast, the activation of this system may play an important role in the long-term structure of the postischemic kidney by influencing microvascular structure and interstitial cellularity.

The Kidney Research National Dialogue, supported by the National Institute of Diabetes and Digestive and Kidney Diseases, asked the scientific community to formulate and prioritize research objectives that would improve our understanding of kidney function and disease. High-priority objectives for AKI were identified, including enhancing training and collaborative opportunities, improving phenotyping and development of clinical tools, expanding our understanding of the pathophysiology and interaction between injury and reparative processes, and identifying determinants of long-term outcomes. Research in animal models must be translated to improve diagnosis and treatment of patients. The objectives identified by the Kidney Research National Dialogue provide the research community with future opportunities for improving the prevention, diagnosis, and treatment of people with AKI.

Resistance to ischemic acute renal failure in the Brown Norway rat: A new model to study cytoprotection. Background An in vivo model of intrinsic resistance to ischemia could be invaluable to define how specific pathways to injury or putative protectors from injury affect the severity of acute renal failure (ARF). The purpose of this study was to determine whether separate rat strains had differential sensitivity to renal ischemia, characterize the extent of protection, and begin to define differences in gene expression that might impact on the severity of ARF. Methods The sensitivity to 45 minutes of renal ischemia in Sprague-Dawley rat (SD) was compared with 2 lines of Brown-Norway rats (BN/Mcw, BN/Hsd). Constitutive and inducible stress protein expression was compared between strains. Results At 24hours' reperfusion, SD rats had higher creatinine (3.4mg/dL), elevated Na and water excretion, and proximal tubule necrosis. Both strains of BN rats were resistant to loss of renal function (Scr = 0.9mg/dL at 24hours' reflow) and had preserved renal morphology. BN rats had no redistribution of Na,K-ATPase into detergent-soluble cortical extracts found early (15 minutes) after ischemia in SD rats. Hsc73 expression did not differ between strains and was not induced by ischemia. Compared with SD, induction of Hsp25 and 72 by renal ischemia was blunted in both BN strains. Constitutive Hsp25 was higher in both BN-Mcw and BN-Hsd compared with SD rat kidney. Constitutive Hsp72 was significantly higher only in BN-Mcw kidneys. Immunohistochemistry showed baseline Hsp72 and 25 expression was increased in proximal tubules of BN-Mcw versus SD. Conclusion BN rat kidney is resistant to ischemic injury and provides a new model for studying cytoprotective mechanisms. Initial study of strain-specific gene expression suggests particular stress proteins are among the potential mechanisms contributing to protection against ARF.

Ischemic acute renal failure (ARF) results in the permanent loss of peritubular capillaries and predisposes the progression of chronic renal failure. The present study was undertaken to determine whether renal hypoxia, which may represent an important mediator in disease progression, is persistently exacerbated after recovery from ARF. Rats were subjected to ischemia-reperfusion injury and allowed to recover for 5 or 20 wk. Immunohistochemistry of the hypoxia-sensitive marker 2-pimonidizole at 5 wk revealed an overall increase in incorporation in the outer medullary region after recovery from ARF compared with sham-operated controls. Unilateral nephrectomy, in combination with ischemia-reperfusion injury resulted in greater 2-pimonidizole staining than that observed in the bilateral injury model. In addition, in the unilateral ischemia-nephrectomy model, proteinuria, interstitial fibrosis, and renal functional loss developed significantly faster than in the bilateral model of ARF when animals were allowed to recover for 20 wk. l-Arginine in the drinking water (∼0.5 g/day) increased total renal blood flow ∼30%, decreased pimonidizole staining, and attenuated manifestations of chronic renal disease. These data suggest that a reduction in the peritubular capillary density after ARF results in a persistent reduction in renal Po2and that hypoxia may play an important role in progression of chronic renal disease after ARF.

Kidney pericytes are progenitors of scar-forming interstitial myofibroblasts that appear after injury. The function of kidney pericytes as microvascular cells and how these cells detach from peritubular capillaries and migrate to the interstitial space, however, are poorly understood. Here, we used an unbiased approach to identify genes in kidney pericytes relevant to detachment and differentiation in response to injury in vivo, with a particular focus on genes regulating proteolytic activity and angiogenesis. Kidney pericytes rapidly activated expression of a disintegrin and metalloprotease with thrombospondin motifs-1 (ADAMTS1) and downregulated its inhibitor, tissue inhibitor of metalloproteinase 3 (TIMP3) in response to injury. Similarly to brain pericytes, kidney pericytes bound to and stabilized capillary tube networks in three-dimensional gels and inhibited metalloproteolytic activity and angiogenic signaling in endothelial cells. In contrast, myofibroblasts did not have these vascular stabilizing functions despite their derivation from kidney pericytes. Pericyte-derived TIMP3 stabilized and ADAMTS1 destabilized the capillary tubular networks. Furthermore, mice deficient in Timp3 had a spontaneous microvascular phenotype in the kidney resulting from overactivated pericytes and were more susceptible to injury-stimulated microvascular rarefaction with an exuberant fibrotic response. Taken together, these data support functions for kidney pericytes in microvascular stability, highlight central roles for regulators of extracellular proteolytic activity in capillary homoeostasis, and identify ADAMTS1 as a marker of activation of kidney pericytes.

Acute kidney injury (AKI) represents a significant clinical concern that is associated with high mortality rates and also represents a significant risk factor for the development of chronic kidney disease (CKD). This article will consider alterations in renal endothelial function in the setting of AKI that may underlie impairment in renal perfusion and how inefficient vascular repair may manifest post-AKI and contribute to the potential transition to CKD. We provide updated terminology for cells previously classified as “endothelial progenitor” that may mediate vascular repair such as pro-angiogenic cells and endothelial colony forming cells. We consider how endothelial repair may be mediated by these different cell types following vascular injury, particularly in models of AKI. We further summarize the potential ability of these different cells to mitigate the severity of AKI, improve perfusion and maintain vascular structure in pre-clinical studies.

Acute kidney dysfunction after ischemia-reperfusion injury may be a consequence of persistent intrarenal vasoconstriction. Regulators of G protein Signaling (RGS) proteins are GTPase activating proteins for heterotrimeric G proteins that can regulate vascular tone. RGS4 is expressed in vascular smooth muscle cells in the kidney. However, RGS4 protein levels are low in many tissues as a consequence of N-end rule-mediated polyubiquination and proteasomal degradation. In this work, the role of RGS4 in the renal response to ischemia/reperfusion injury (IRI) was investigated. A murine model of IRI was employed and RGS4-null mice (R4KO) were highly sensitized to the development of renal dysfunction after IRI. Furthermore, R4KO mice exhibited reduced renal blood flow after IRI. Kidneys were studied for intrinsic RGS4 function by ex vivo isolation. R4KO kidneys exhibited increased renal vasoconstriction in response to endothelin-1 infusion. The intrinsic renal activity of RGS4 was examined in an in vivo model of syngeneic renal transplantation. Transplanted R4KO kidneys exhibited significantly reduced reperfusion blood flow and increased renal cell death. To increase RGS4 activity, wild type mice were administered the proteasomal inhibitor MG-132 and this resulted in increased renal RGS4 protein. Furthermore, MG-132 treatment inhibited the development of renal dysfunction after IRI in wild type - but not R4KO - mice. These results demonstrate that RGS4 antagonizes the development of renal dysfunction in response to IRI.

We performed a pilot study to assess whether renal shock wave lithotripsy influences metabolic syndrome onset and severity.Three-month-old juvenile female Ossabaw miniature pigs were treated with shock wave lithotripsy (2,000 shock waves at 24 kV with 120 shock waves per minute in 2) or sham shock wave lithotripsy (no shock waves in 2). Shock waves were targeted to the upper pole of the left kidney to model treatment that would also expose the pancreatic tail to shock waves. Pigs were then instrumented to directly measure arterial blood pressure via an implanted radiotelemetry device. They later received a hypercaloric atherogenic diet for about 7 months. Metabolic syndrome development was assessed by the intravenous glucose tolerance test.Metabolic syndrome progression and severity were similar in the sham treated and lithotripsy groups. The only exception arterial blood pressure, which remained relatively constant in sham treated pigs but began to increase at about 2 months towards hypertensive levels in lithotripsy treated pigs. Metabolic data on the 2 groups were pooled to provide a more complete assessment of metabolic syndrome development and progression in this juvenile pig model. The intravenous glucose tolerance test revealed substantial insulin resistance with impaired glucose tolerance within 2 months on the hypercaloric atherogenic diet with signs of further metabolic impairment at 7 months.These preliminary results suggest that renal shock wave lithotripsy is not a risk factor for worsening glucose tolerance or diabetes mellitus onset. However, it appears to be a risk factor for early onset hypertension in metabolic syndrome.

Ischemic preconditioning is well known to afford protection against subsequent episodes of ischemia/reperfusion (I/R) in multiple organs, including the kidney, but the mechanisms involved are not fully understood. We proposed that ischemic preconditioning results in an altered mitochondrial proteome that confers resistance to subsequent I/R injury. We subjected rats to moderate ischemic injury and used liquid chromatography – mass spectroscopy (LC/MS) to analyze the proteome of kidney cortical mitochondria isolated following 14 days of recovery from I/R. The expression of several proteins was elevated following preconditioning, and two were selected for further analysis: Isocitrate dehydrogenase 2 (IDH2) and a member of the sulfotransferase family (SULT1A1). The effect of increased expression of these proteins was evaluated by using hydrodynamic gene delivery of plasmid vectors to overexpress each protein. Strikingly, hydrodynamic delivery of IDH2 and SULT1A1 genes attenuated the peak in serum creatinine ...

First published August 9, 2001; 10.1152/ajprenal.00050.2001.—Acute episodes of severe renal ischemia result in acute renal failure (ARF). These episodes are followed by a characteristic recovery and repair response, whereby tubular morphology and renal function appear completely restored within ∼1 mo. However, the chronic effects of such an injury have not been well studied. Male rats were subjected to 60-min bilateral ischemia followed by reperfusion, yielding a characteristic injury. Postischemic animals manifested severe diuresis, peaking at 1 wk postinjury (volume: >45 ml/day, ARF vs. 18 ml/day, sham; P < 0.05). Urine flow subsequently declined but remained significantly elevated vs. sham animals for a 40-wk period. The prolonged alteration in urinary concentrating ability was attributable, in part, to a diminished capacity to generate a hypertonic medullary interstitium. By week 16, proteinuria developed in the post-ARF group and progressed for the duration of the study. Histological examination revealed essentially normal tubular morphology at 4 and 8 wk postinjury but the development of tubulointerstitial fibrosis at 40 wk. Transforming growth factor (TGF)-β1 expression was elevated at 40 wk, but not at 4 and 8 wk postinjury. Microfil analysis revealed an ∼30–50% reduction in peritubular capillary density in the inner stripe of the outer medulla at 4, 8, and 40 wk in post-ARF groups vs. sham animals. In addition, post-ARF rats manifested a significant pressor response to a low dose of ANG II (15 ng · kg−1· min−1). We hypothesize that severe ischemic injury results in a permanent alteration of renal capillary density, contributing to a urinary concentrating defect and the predisposition toward the development of renal fibrosis.

Acute kidney injury (AKI) remains a significant health care concern. Many reports describe high mortality rates that largely have remained unchanged for several decades (1,2). With such a poor prognostic outlook, scientists have sought to develop novel therapies that are based on information gleaned from clinical studies as well as in vitro and in vivo models of renal injury. These approaches have been geared toward understanding molecular, cellular, and physiologic responses of renal injury and have led to a focus on pathways that are related to metabolism, oxidant stress, hemodynamics, inflammation, and growth factors, to name a few, as potential targets of therapy (3). Will such scientific approaches lead to therapies that bear promise and ultimately affect outcome? Thus far, they have not. It has become clear that a reevaluation of approaches in the study of AKI is at hand, including the incorporation of novel therapeutic approaches, better and more clinically relevant model systems, and the application of better prognostic indicators as a means to establish treatment.