Kidney functional reserve and damage biomarkers in subclinical chronic kidney disease and acute kidney injury.

Significant loss of kidney function is not easily identified by serum creatinine (sCr)-based measurements. In the presence of normal sCr, decreased kidney functional reserve (KFR) may identify a significant loss of function. We evaluated KFR in experimental subclinical chronic kidney disease (sCKD) before and after brief ischemia-reperfusion injury (IRI). Using fluorescein isothiocyanate-labeled sinistrin, glomerular filtration rate (GFR) was measured transcutaneously before and after adenine-induced sCKD, and 1 and 2 wk after brief IRI, and compared with urinary kidney damage biomarkers. sCKD reduced stimulated and unstimulated GFR by ∼20% while reducing KFR by 50%. IRI reduced unstimulated GFR for 14 days, but KFR remained relatively unchanged in sCKD and transiently increased in control kidneys at 7 days. sCr increased and creatinine clearance (CrCl) decreased only immediately after IRI; sCr and CrCl correlated poorly with measured GFR except on day 1 after IRI. Heterogeneity in sCr and CrCl resulted from variation in tubular creatinine secretion. The increase in damage biomarker concentrations persisted for up to 14 days after IRI, allowing retrospective detection of sCKD before AKI by urine clusterin/urine kidney injury molecule-1 with an area under the curve of 1.0. sCr and CrCl are unreliable unless sCr is acutely elevated. Measurement of KFR and urine damage biomarker excretion detected sCKD despite normal sCr and CrCl. After IRI, the urine clusterin-to-urine kidney injury molecule-1 ratio may identify prior sCKD.NEW & NOTEWORTHY Early kidney function loss is poorly identified by serum creatinine (sCr)-based measurements. Direct kidney functional reserve (KFR) measurement before kidney injury and elevated urinary biomarkers clusterin and kidney injury molecule-1 detect subclinical chronic kidney disease (sCKD) after kidney injury despite normal range sCr and creatinine clearance. Reliance on sCr masks underlying sCKD. Acute kidney injury risk evaluation requires direct glomerular filtration rate measurement and KFR, whereas kidney damage biomarkers facilitate identification of prior subclinical injury.

Subclinical chronic kidney disease modifies the diagnosis of experimental acute kidney injury.

Extensive structural damage within the kidney must be present before serum creatinine increases. However, a subclinical phase of chronic kidney disease (CKD) usually goes undetected. Here we tested whether experimental subclinical CKD would modify functional and damage biomarker profiles of acute kidney injury (AKI). Subclinical CKD was induced in rats by adenine or aristolochic acid models but without increasing serum creatinine. After prolonged recovery (three to six weeks), AKI was induced with a subnephrotoxic dose of cisplatin. Urinary levels of kidney injury molecule-1 (KIM-1), cytochrome C, monocyte chemotactic protein-1 (MCP-1), clusterin, and interleukin-18 increased during CKD induction, without an increase in serum creatinine. After AKI in adenine-induced CKD, serum creatinine increased more rapidly, while increased urinary KIM-1, clusterin, and MCP-1 were delayed and reduced. Increased serum creatinine and biomarker excretion were associated with diffuse tubulointerstitial injury in the outer stripe of outer medulla coupled with over 50% cortical damage. Following AKI in aristolochic acid-induced CKD, increased serum creatinine, urinary KIM-1, clusterin, MCP-1, cytochrome C, and interleukin-18 concentrations and excretion were greater at day 21 than day 42 and inversely correlated with cortical injury. Subclinical CKD modified functional and damage biomarker profiles in diametrically opposite ways. Functional biomarker profiles were more sensitive, while damage biomarker diagnostic thresholds and increases were diminished and delayed. Damage biomarker concentrations and excretion were inversely linked to the extent of prior cortical damage. Thus, thresholds for AKI biomarkers may need to be lower or sampling delayed in the known presence of CKD.

Dexamethasone Modifies Cystatin C-Based Diagnosis of Acute Kidney Injury During Cisplatin-Based Chemotherapy.

BACKGROUND/AIMS: Plasma cystatin C (pCysC) may be superior to serum creatinine (sCr) as a surrogate of GFR. However, the performance of pCysC for diagnosing acute kidney injury (AKI) after cisplatin-based chemotherapy is potentially affected by accompanying corticosteroid anti-emetic therapy and hydration. METHODS: In a prospective observational study pCysC, sCr, urinary kidney injury molecule-1 (KIM-1), and urinary clusterin were measured over 2 weeks in 27 patients given first-cycle chemotherapy. The same variables were measured over 2 weeks in Sprague-Dawley rats given a single intraperitoneal injection of dexamethasone, cisplatin, or both, and in controls. RESULTS: In patients, pCysC increases were greater than sCr 41% vs. 16%, mean paired difference 25% (95% CI: 16-34%)], relative increases were ≥ 50% in 9 patients (35%) for pCysC compared with 2 (8%) for sCr (p = 0.04) and increases in sCr were accompanied by increased KIM-1 and clusterin excretion, but increases in pCysC alone were not. In rats, dexamethasone administration produced dose-dependent increases in pCysC (and augmented cisplatin-induced increases in pCysC), but did not augment histological injury, increases in sCr, or KIM-1 and clusterin excretion. CONCLUSIONS: In the presence of dexamethasone, elevation of pCysC does not reliably diagnose AKI after cisplatin-based chemotherapy.

Disruption of E-cadherin by matrix metalloproteinase directly mediates epithelial-mesenchymal transition downstream of transforming growth factor-beta1 in renal tubular epithelial cells.

Epithelial-mesenchymal transition (EMT) plays an important role in organ fibrosis, including that of the kidney. Loss of E-cadherin expression is a hallmark of EMT; however, whether the loss of E-cadherin is a consequence or a cause of EMT remains unknown, especially in the renal system. In this study, we show that transforming growth factor (TGF)-beta1-induced EMT in renal tubular epithelial cells is dependent on proteolysis. Matrix metalloproteinase-mediated E-cadherin disruption led directly to tubular epithelial cell EMT via Slug. TGF-beta1 induced the proteolytic shedding of E-cadherin, which caused the nuclear translocation of beta-catenin, the transcriptional induction of Slug, and the repression of E-cadherin transcription in tubular epithelial cells. These findings reveal a direct role for E-cadherin and for matrix metalloproteinases in causing EMT downstream of TGF-beta1 in fibrotic disease. Specific inhibition rather than activation of matrix metalloproteinases may offer a novel approach for treatment of fibrotic disease.

Selection and validation of reference genes for normalisation of gene expression in ischaemic and toxicological studies in kidney disease.

Normalisation to standard reference gene(s) is essential for quantitative real-time polymerase chain reaction (RT-qPCR) to obtain reproducible and comparable results of a gene of interest (GOI) between subjects and under varying experimental conditions. There is limited evidence to support selection of the commonly used reference genes in rat ischaemic and toxicological kidney models. Employing these models, we determined the most stable reference genes by comparing 4 standard methods (NormFinder, qBase+, BestKeeper and comparative ΔCq) and developed a new 3-way linear mixed-effects model for evaluation of reference gene stability. This new technique utilises the intra-class correlation coefficient as the stability measure for multiple continuous and categorical covariates when determining the optimum normalisation factor. The model also determines confidence intervals for each candidate normalisation gene to facilitate selection and allow sample size calculation for designing experiments to identify reference genes. Of the 10 candidate reference genes tested, the geometric mean of polyadenylate-binding nuclear protein 1 (PABPN1) and beta-actin (ACTB) was the most stable reference combination. In contrast, commonly used ribosomal 18S and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were the most unstable. We compared the use of PABPN1×ACTB and 2 commonly used genes 18S and GAPDH on the expression of 4 genes of interest know to vary after renal injury and expressed by different kidney cell types (KIM-1, HIF1α, TGFß1 and PECAM1). The less stable reference genes gave varying patterns of GOI expression in contrast to the use of the least unstable reference PABPN1×ACTB combination; this improved detection of differences in gene expression between experimental groups. Reduced within-group variation of the now more accurately normalised GOI may allow for reduced experimental group size particularly for comparison between various models. This objective selection of stable reference genes increased the reliability of comparisons within and between experimental groups.

Formation of tight junctions between neighboring podocytes is an early ultrastructural feature in experimental crescentic glomerulonephritis.

PURPOSE: In crescentic glomerulonephritis (CGN), the development of cellular bridges between podocytes and parietal epithelial cells (PECs) triggers glomerular crescent formation. However, the sequential changes in glomerular ultrastructure in CGN are not well defined. This study investigated the time course of glomerular ultrastructure in experimental CGN. METHODS: Transmission electron microscopy (TEM) was performed using kidney samples from rats with nephrotoxic serum nephritis (NSN) from day 1 to day 14. Morphometric analysis was conducted on randomly selected glomeruli captured on TEM digital images. RESULTS: On day 1 of NSN, there was widespread formation of focal contacts between the cell bodies of neighboring podocytes, and tight junctions were evident at the site of cell-to-cell contact. This was confirmed by the increased expression of the tight junction molecule, zonula occludens-1 (ZO-1), which localized to the points of podocyte cell-cell body contact. On day 2, the interpodocyte distance decreased and the glomerular basement membrane thickness increased. Foot process effacement (FPE) was segmental on day 3 and diffuse by day 5, accompanied by the formation of podocyte cellular bridges with Bowman's capsule, as confirmed by a decrease in podocyte-to-PEC distance. Fibrinoid necrosis and cellular crescents were evident in all glomeruli by days 7 and 14. In vitro, the exposure of podocytes to macrophage-conditioned media altered cellular morphology and caused an intracellular redistribution of ZO-1. CONCLUSION: The formation of tight junctions between podocytes is an early ultrastructural abnormality in CGN, preceding FPE and podocyte bridge formation and occurring in response to inflammatory injury. Podocyte-to-podocyte tight junction formation may be a compensatory mechanism to maintain the integrity of the glomerular filtration barrier following severe endocapillary injury.

Fab fragments of ovine antibody to colchicine enhance its clearance in the rat.

CONTEXT: Colchicine is an anti-inflammatory alkaloid used for the treatment of acute gout, but has a narrow therapeutic index. Colchicine overdoses are relatively rare, but have high mortality requiring rapid treatment. OBJECTIVE: To evaluate the ability of a newly available ovine fragment antigen-binding (Fab) antibody to colchicine (ColchiFab(™)) to protect rats against renal and other injury 24 h after colchicine ingestion. MATERIALS AND METHODS: Rats were gavaged with colchicine (5 mg/kg), then 2 h later injected intraperitoneally with 5 ml of sterile saline, or Fab anti-colchicine, a newly available ovine antibody to colchicine. Samples of blood were taken at 1, 2, 5 and 24 h after gavage, and urine was collected from 5 to 24 h after gavage. Concentrations of colchicine in tissue, blood and urine were measured by liquid chromatography/mass spectrometry, concentrations of Fab anti-colchicine, urinary neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 or KIM-1 by enzyme-linked immunosorbent assay or ELISA, while concentrations of creatine kinase and creatinine (Cr) were measured enzymatically. RESULTS: Colchicine equilibrated rapidly throughout the body and increased serum creatine kinase. Fab anti-colchicine also rapidly redistributed to the blood and remained at high concentrations over 24 h. Fab anti-colchicine caused a rapid 7.1-fold increase in serum colchicine level, followed by excretion of both colchicine and Fab anti-colchicine through the urine. This was associated with the accumulation of colchicine in the kidney, a reversal of colchicine-induced diarrhoea, and increasing urinary NGAL level; from 168 ± 48 to 477 ± 255 ng/mmol Cr [mean ± standard deviation or SD]. DISCUSSION: Fab anti-colchicine greatly increased the clearance of colchicine, although increasing NGAL level suggested the presence of mild kidney damage. CONCLUSION: These data suggest clinical utility for Fab anti-colchicine in the treatment of colchicine overdose.

Induction monotherapy with sirolimus has selected beneficial effects on glomerular and tubulointersititial injury in nephrotoxic serum nephritis.

BACKGROUND: The study aimed to test the hypothesis that therapeutic treatment with a mammalian target of rapamycin complex 1 inhibitor reduces renal cell proliferation and attenuates glomerular and tubulointerstitial injury in the early phase of nephrotoxic serum nephritis (NSN) in rats. METHODS: Male Wistar-Kyoto rats received a single tail-vein injection of sheep anti-rat glomerular basement membrane serum (day 0) and were treated with vehicle or sirolimus (0.25 mg/kg/day by subcutaneous injection) from day 1 until day 14. RESULTS: Treatment with sirolimus attenuated kidney enlargement by 41% (P<0.05), improved endogenous creatinine clearance by 50% (P<0.05), and reduced glomerular and tubulointerstitial cell proliferation by 53% and 70%, respectively, (P<0.05 compared to vehicle) in rats with NSN. In glomeruli, sirolimus reduced segmental fibrinoid necrosis by 69%, autologous rat immunoglobulin G deposition, glomerular capillary tuft enlargement, and periglomerular myofibroblast (α-smooth muscle actin-positive cells) accumulation (all P<0.05) but did not significantly affect glomerular crescent formation (P=0.15), macrophage accumulation (P=0.25), or the progression of proteinuria. In contrast, sirolimus preserved tubulointerstitial structure and attenuated all markers of injury (interstitial ED-1- and α-smooth muscle actin-positive cells and tubular vimentin expression; all P<0.05). By immunohistochemistry and Western blot analysis, sirolimus reduced the glomerular and tubulointerstitial expression of phosphorylated (Ser 235/236) S6-ribosomal protein (P<0.05). CONCLUSION: Induction monotherapy with sirolimus suppressed target of rapamycin complex 1 activation, renal cell proliferation, and injury during the early stages of rodent NSN, but the degree of histological protection was more consistent in the tubulointerstitium than the glomerular compartment.

A comparison of the ability of levels of urinary biomarker proteins and exosomal mRNA to predict outcomes after renal transplantation.

BACKGROUND: mRNA for biomarkers of kidney injury extracted from urinary exosomes may reflect or predict levels of the corresponding protein after transplantation and clinical outcomes. METHODS: Urinary exosomes were isolated from patients following renal transplantation, from healthy controls, and patients with CKD. Expression of exosomal mRNA for the injury biomarkers neutrophil gelatinase-associated lipocalin (NGAL), interleukin-18 (IL-18), kidney injury molecule-1 (KIM-1), and cystatin C was compared with the concentrations of corresponding urinary proteins, 18S RNA and serum creatinine. RESULTS: All biomarker protein concentrations increased after transplantation, and urinary NGAL and IL-18 at 24 and 168 h correlated with the day 7 creatinine reduction ratio (CRR). Exosomal18S RNA increased after transplantation, but exosomal mRNA for NGAL, IL-18 and cystatin C did not correlate with the day 7 CRR, or urinary biomarker concentrations at any time after transplantation. Exosomal NGAL mRNA was lower 4 h after transplantation than in control exosomes. In contrast, exosomal mRNA for cystatin C was unchanged after transplantation and in CKD, although urinary cystatin C temporarily increased following transplantation. Urinary KIM-1 increased after transplantation, but exosomal mRNA for KIM-1 remained undetectable. In CKD 18S RNA was raised, and exosomal mRNA for NGAL, IL-18 and cystatin C was detected in all patients. While urinary NGAL was greater in CKD than control subjects, exosomal NGAL mRNA was unchanged. Exosomal IL-18 mRNA was increased in CKD, but not IL-18 protein. CONCLUSIONS: After renal transplantation, urinary NGAL and IL-18 levels reflect the day 7 CRR. However, while mRNA for these biomarkers is present in exosomes, their levels do not reflect or predict urinary biomarker levels or the CRR. This likely reflects the fact that packaging of mRNA in exosomes is selective, and is not necessarily representative of mRNA in the parent cells responsible for biomarker production.

Therapeutic role of sirolimus in non-transplant kidney disease.

Sirolimus is a member of a novel class of immunosuppressant drug that potently suppresses T cell proliferation and expansion by inhibition of the Target of Rapamycin Complex 1 (TORC1) protein kinase. Sirolimus also has anti-proliferative effects on intrinsic cells of the kidney, and increasing evidence suggests that it may have a therapeutic role in non-transplant renal diseases. In the normal kidney, sirolimus is considered to be non-nephrotoxic. In the diseased kidney, sirolimus may be beneficial or detrimental, depending on the type of renal injury. In polycystic kidney disease, TORC1 activation mediates renal tubular epithelial cell (TEC) proliferation and cyst growth in animals, and Phase III clinical trials are underway to determine the effect of sirolimus in attenuating disease progression in humans. In contrast, in acute kidney injury, sirolimus transiently impairs proximal TEC regeneration and delays renal recovery. In animal models of lupus nephritis and diabetic kidney disease, sirolimus prevents disease progression. However, the efficacy of sirolimus in human glomerulonephritis as well as in diabetic chronic kidney disease remains unclear, as it paradoxically exacerbates renal dysfunction when the baseline glomerular filtration rate is low (< 40 ml/min/1.73 m(2)) and there is heavy proteinuria (> 300 mg/day). This may, in part, be due to inhibition of compensatory glomerular capillary repair through the suppression of endothelial cell proliferation and angiogenic growth factor production by podocytes. Therefore, at present, polycystic kidney disease is the most promising therapeutic application for sirolimus in non-transplant renal diseases, and further studies are needed to clarify its role in other situations.