Urinary soluble HLA-DR is a potential biomarker for acute renal transplant rejection

BACKGROUND.: Urine is a potentially rich source of biomarkers for monitoring kidney dysfunction. In this study, we have investigated the potential of soluble human leukocyte antigen (sHLA)-DR in the urine for noninvasive monitoring of renal transplant patients. METHODS.: Urinary soluble HLA-DR levels were measured by sandwich enzyme-linked immunosorbent assay in 103 patients with renal diseases or after renal transplantation. sHLA-DR in urine was characterized by Western blotting and mass spectrometry. RESULTS.: Acute graft rejection was associated with a significantly elevated level of urinary sHLA-DR (P<0.0001), compared with recipients with stable graft function or healthy individuals. A receiver operating characteristic curve analysis showed the area under the curve to be 0.88 (P<0.001). At a selected threshold, the sensitivity was 80% and specificity was 98% for detection of acute renal transplant rejection. sHLA-DR was not exosomally associated and was of lower molecular weight compared with the HLA-DR expressed as heterodimer on the plasma membrane of antigen-presenting cells. CONCLUSIONS.: sHLA-DR excreted into urine is a promising indicator of renal transplant rejection.

Dialysis and Renal Transplantation in HIV-Infected Patients: a European Survey

OBJECTIVES:: To determine prevalence and characteristics of end-stage renal diseases (ESRD) [dialysis and renal transplantation (RT)] among European HIV-infected patients. METHODS:: Cross-sectional multicenter survey of EuroSIDA clinics during 2008. RESULTS:: Prevalence of ESRD was 0.5%. Of 122 patients with ESRD 96 were on dialysis and 26 had received a RT. Median age was 47 years, 73% were males and 43% were black. Median duration of HIV infection was 11 years. Thirty-three percent had prior AIDS; 91% were receiving antiretrovirals; and 88% had undetectable viral load. Median CD4T-cell count was 341 cells per cubic millimetre; 20.5% had hepatitis C coinfection. Most frequent causes of ESRD were HIV-associated nephropathy (46%) and other glomerulonephritis (28%). Hemodialysis (93%) was the most common dialysis modality; 34% of patients were on the RT waiting list. A poor HIV control was the reason for exclusion from RT waiting list in 22.4% of cases. All the RT recipients were all alive at the time of the survey. Acute rejection was reported in 8 patients (30%). Functioning graft was present in 21 (80%). CONCLUSIONS:: This is the first multinational cross-sectional study of ESRD among European HIV population. Low prevalence of ESRD was found. Two-thirds of patients were excluded from RT for non-HIV/AIDS-related pathologies. Most patients had a functioning graft despite a high acute rejection rate.

Involvement of drug-specific T cells in acute drug-induced interstitial nephritis

Drug-induced interstitial nephritis can be caused by a plethora of drugs and is characterized by a sudden impairment of renal function, mild proteinuria, and sterile pyuria. For investigation of the possible pathomechanism of this disease, drug-specific T cells were analyzed, their function was characterized, and these in vitro findings were correlated to histopathologic changes that were observed in kidney biopsy specimens. Peripheral blood mononuclear cells from three patients showed a proliferative response to only one of the administered drugs, namely flucloxacillin, penicillin G, and disulfiram, respectively. The in vitro analysis of the flucloxacillin-reactive cells showed an oligoclonal immune response with an outgrowth of T cells bearing the T cell receptor Vbeta9 and Vbeta21.3. Moreover, flucloxacillin-specific T cell clones could be generated from peripheral blood, they expressed CD4 and the alphabeta-T cell receptor, and showed a heterogeneous cytokine secretion pattern with no clear commitment to either a Th1- or Th2-type response. The immunohistochemistry of kidney biopsies of these patients revealed cell infiltrations that consisted mostly of T cells (CD4+ and/or CD8+). An augmented presence of IL-5, eosinophils, neutrophils, CD68+ cells, and IL-12 was observed. In agreement with negative cytotoxicity assays, no cytotoxicity-related molecules such as Fas and perforin were detected by immunohistochemistry. The data indicate that drug-specific T cells are activated locally and orchestrate a local inflammation via secretion of various cytokines, the type of which depends on the cytokine pattern secreted and which probably is responsible for the renal damage.

Effect of donor-specific transfusions on the outcome of renal allografts in the cyclosporine era

Despite the introduction of new immunosuppressive agents, a steady decline of functioning renal allografts after living donation is observed. Thus nonpharmacological strategies to prevent graft loss have to be reconsidered, including donor-specific transfusions (DST). We introduced a cyclosporine-based DST protocol for renal allograft recipients from living-related/unrelated donation. From 1993 to 2003, 200 ml of whole blood, or the respective mononuclear cells from the potential living donor were administered twice to all of our 61 recipient candidates. The transplanted subjects were compared with three groups of patients without DST from the Collaborative Transplant Study (Heidelberg, Germany) during a 6-year period. Six patients were sensitized without delay for a subsequent cadaveric kidney. DST patients had less often treatment for rejection and graft survival was superior compared with subjects from the other Swiss transplant centers (n = 513) or from Western Europe (n = 7024). To diminish the probability that superior results reflect patient selection rather than effects of DST, a 'matched-pair' analysis controlling for relevant factors of transplant outcome was performed. Again, this analysis indicated that recipients with DST had better outcome. Thus, our observation suggests that DST improve the outcome of living kidney transplants even when modern immunosuppressive drugs are prescribed.

Characterization of the regulation of renal Na+/H+ exchanger NHE3 by insulin

Insulin receptors are widely distributed in the kidney and affect multiple aspects of renal function. In the proximal tubule, insulin regulates volume and acid-base regulation through stimulation of the Na(+)/H(+) exchanger NHE3. This paper characterizes the signaling pathway by which insulin stimulates NHE3 in a cell culture model [opossum kidney (OK) cell]. Insulin has two distinct phases of action on NHE3. Chronic insulin (24 h) activates NHE3 through the classic phosphatidylinositol 3-kinase-serum- and glucocorticoid-dependent kinase 1 (PI3K-SGK1) pathway as insulin stimulates SGK1 phosphorylation and the insulin effect can be blocked by the PI3K inhibitor wortmannin or a dominant-negative SGK1. We showed that SGK1 transcript and protein are expressed in rat proximal tubule and OK cells. We previously showed that glucocorticoids augment the effect of insulin on NHE3 (Klisic J, Hu MC, Nief V, Reyes L, Fuster D, Moe OW, Ambuhl PM. Am J Physiol Renal Physiol 283: F532-F539, 2002). Part of this can be mediated via induction of SGK1 by glucocorticoids, and indeed the insulin effect on NHE3 can also be amplified by overexpression of SGK1. We next addressed the acute effect of insulin (1-2 h) on NHE3 by systematically examining the candidate signaling cascades and activation mechanisms of NHE3. We ruled out the PI3K-SGK1-Akt and TC10 pathways, increased surface NHE3, NHE3 phosphorylation, NHE3 association with calcineurin homologous protein 1 or megalin as mechanisms of acute activation of NHE3 by insulin. In summary, insulin stimulates NHE3 acutely via yet undefined pathways and mechanisms. The chronic effect of insulin is mediated by the classic PI3K-SGK1 route.

Origin of renal myofibroblasts in the model of unilateral ureter obstruction in the rat

Tubulo-interstitial fibrosis is a constant feature of chronic renal failure and it is suspected to contribute importantly to the deterioration of renal function. In the fibrotic kidney there exists, besides normal fibroblasts, a large population of myofibroblasts, which are supposedly responsible for the increased production of intercellular matrix. It has been proposed that myofibroblasts in chronic renal failure originate from the transformation of tubular cells via epithelial-mesenchymal transition (EMT) or from infiltration by bone marrow-derived precursors. Little attention has been paid to the possibility of a transformation of resident fibroblasts into myofibroblasts in renal fibrosis. Therefore we examined the fate of resident fibroblasts in the initial phase of renal fibrosis in the classical model of unilateral ureter obstruction (UUO) in the rat. Rats were perfusion-fixed on days 1, 2, 3 and 4 after ligature of the right ureter. Starting from 1 day of UUO an increasing expression of alpha-smooth muscle actin (alphaSMA) in resident fibroblasts was revealed by immunofluorescence and confirmed by the observation of bundles of microfilaments and webs of intermediate filaments in the electron microscope. Inversely, there was a decreased expression of 5'-nucleotidase (5'NT), a marker of renal cortical fibroblasts. The RER became more voluminous, suggesting an increased synthesis of matrix. Intercellular junctions, a characteristic feature of myofibroblasts, became more frequent. The mitotic activity in fibroblasts was strongly increased. Renal tubules underwent severe regressive changes but the cells retained their epithelial characteristics and there was no sign of EMT. In conclusion, after ureter ligature, resident peritubular fibroblasts proliferated and they showed progressive alterations, suggesting a transformation in myofibroblasts. Thus the resident fibroblasts likely play a central role in fibrosis in that model.

GABARAP deficiency modulates expression of NaPi-IIa in renal brush-border membranes

Renal reabsorption of inorganic phosphate (P(i)) is mainly mediated by the Na(+)-dependent P(i)-cotransporter NaPi-IIa that is expressed in the brush-border membrane (BBM) of renal proximal tubules. Regulation and apical expression of NaPi-IIa are known to depend on a network of interacting proteins. Most of the interacting partners identified so far associate with the COOH-terminal PDZ-binding motif (TRL) of NaPi-IIa. In this study GABA(A) receptor-associated protein (GABARAP) was identified as a novel interacting partner of NaPi-IIa applying a membrane yeast-two-hybrid system (MYTH 2.0) to screen a mouse kidney library with the TRL-truncated cotransporter as bait. GABARAP mRNA and protein are present in renal tubules, and the interaction of NaPi-IIa and GABARAP was confirmed by using glutathione S-transferase pulldowns from BBM and coimmunoprecipitations from transfected HEK293 cells. Amino acids 36-68 of GABARAP were identified as the determinant for the described interaction. The in vivo effects of this interaction were studied in a murine model. GABARAP(-/-) mice have reduced urinary excretion of P(i), higher Na(+)-dependent (32)P(i) uptake in BBM vesicles, and increased expression of NaPi-IIa in renal BBM compared with GABARAP(+/+) mice. The expression of Na(+)/H(+) exchanger regulatory factor (NHERF)1, an important scaffold for the apical expression of NaPi-IIa, is also increased in GABARAP(-/-) mice. The absence of GABARAP does not interfere with the regulation of the cotransporter by either parathyroid hormone or acute changes of dietary P(i) content.

Occurrence of spontaneous tumours of the renal proximal tubules in oscars Astronotus ocellatus

This report describes the occurrence of renal papillary cystic adenomas and adenocarcinomas in oscars Astronotus ocellatus Cuvier, 1829. Samples from 5 oscars with abdominal swelling were collected between 1996 and 2004 and compared to a published case from the USA. Macroscopically, all cases revealed a large, well-demarcated, greyish-brown nodular mass in a retroperitoneal position within the body cavity, and originating from the posterior kidney. Histologically, these neoplasms were composed of epithelial cells, which were arranged in papillary cystic tubular structures and partly covered by cilia. In this study, microscopic and ultrastructural examination confirmed that the origin of the neoplasm was the proximal tubules of the kidney.

Expression of the neural stem cell markers NG2 and L1 in human angiomyolipoma: are angiomyolipomas neoplasms of stem cells?

Angiomyolipomas are benign tumors of the kidney which express phenotypes of smooth muscle, fat, and melanocytes. These tumors appear with increased frequency in the autosomal dominant disorder tuberous sclerosis and are the leading cause of morbidity in adults with tuberous sclerosis. While benign, these tumors are capable of provoking life threatening hemorrhage and replacement of the kidney parenchyma, resulting in renal failure. The histogenesis of these tumors is currently unclear, although currently, we believe these tumors arise from "perivascular epithelioid cells" of which no normal counterpart has been convincingly demonstrated. Recently, stem cell precursors have been recognized that can give rise to smooth muscle and melanocytes. These precursors have been shown to express the neural stem cell marker NG2 and L1. In order to determine whether angiomyolipomas, which exhibit smooth muscle and melanocytic phenotypes, express NG2 and L1, we performed immunocytochemistry on a cell line derived from a human angiomyolipoma, and found that these cells are uniformly positive. Immunohistochemistry of human angiomyolipoma specimens revealed uniform staining of tumor cells, while renal cell carcinomas revealed positivity only of angiogenic vessels. These results support a novel histogenesis of angiomyolipoma as a defect in differentiation of stem cell precursors.

Identification of a novel tumor suppressor gene on human chromosome 3p14-p12 involved in renal cell carcinoma

Nonpapillary renal cell carcinoma (RCC) is an adult cancer of the kidney which occurs both in familial and sporadic forms. The familial form of RCC is associated with translocations involving chromosome 3 with a breakpoint at 3p14-p13. Studies focused on sporadic RCC have shown two commonly deleted regions at 3p14.3-p13 and 3p21.3. In addition, a more distal region mapping to 3p26-p25 has been linked to the Von Hippel Lindau (VHL) disease gene. A large proportion of VHL patients develop RCC. The short arm of human chromosome 3 can, therefore, be dissected into three distinct regions which could encode tumor suppressor genes for RCC. Loss or inactivation of one or more of these loci may be an important step in the genesis of RCC.^ I have used the technique of microcell-mediated chromosome transfer to introduce an intact, normal human chromosome 3 and defined fragments of 3p, dominantly marked with pSV2neo, into the highly malignant RCC cell line SN12C.19. The introduction of chromosome 3 and of a centric fragment of 3p, encompassing 3p14-q11, into SN12C.19 resulted in dramatic suppression of tumor growth in nude mice. Another defined deletion hybrid contained the region 3p12-q24 of the introduced human chromosome and failed to suppress tumorigenicity. These data define the region 3p14-p12, the most proximal region of high frequency allele loss in sporadic RCC as well as the region containing the translocation breakpoint in familial RCC, to contain a novel tumor suppressor locus involved in RCC. We have designated this locus nonpapillary renal cell carcinoma-1 (NRC-1). Furthermore, we have functional evidence that NRC-1 controls the growth of RCC cells by inducing rapid cell death in vivo. ^

Regulation of sodium/glucose cotransporter expression in LLC-PK$\sb1$ cells

Expression of the Na$\sp+$/glucose cotransporter (SGLT1), a differentiated function of the pig kidney epithelial cell line LLC-PK$\sb1$ derived from proximal tubule, was further investigated. The differentiation inducer hexamethylene bisacetamide (HMBA) and IBMX, an inhibitor of cAMP phosphodiesterase, each stimulated a significant increase in Na$\sp+$/glucose cotransport activity, levels of the 75 kD cotransporter subunit and steady-state levels of the SGLT1 message. The action of HMBA is associated with involvement of polyamines and protein kinase C, and is synergistic with cAMP. We provide evidence that cAMP-elevating agents increase Na$\sp+$/glucose cotransporter expression, at least in part, via a post-transcriptional mechanism. Two molecular species of SGLT1 mRNA (3.9 kb and 2.2 kb) are transcribed from the same gene in LLC-PK$\sb1$ cells and differ only in the length of the 3$\sp\prime$ untranslated region (3$\sp\prime$ UTR). cAMP elevation differentially stabilized the 3.9 kb SGLT1 transcript from degradation but not the 22 kb species. UV-cross-linking and label transfer experiments indicated that cyclic AMP elevation was associated with formation of a 48 kD protein complex with a specific domain within the 3$\sp\prime$ UTR of SGLT1 mRNA. The binding was competitively inhibited by poly (U) and other U-rich RNA species such as c-fos ARE, and modulated by a protein kinase A-mediated phosphorylation/dephosphorylation mechanism. The binding site was mapped to a 120-nucleotide 3$\sp\prime$ UTR sequence which contains a uridine-rich region (URE). Our study provides the first demonstration that renal SGLT1 is post-transcriptionally regulated by a phosphorylation/dephosphorylation mechanism, and provides a deeper insight into gene regulation of this physiologically important cotransporter. ^

Incomplete Distal Renal Tubular Acidosis from a Heterozygous Mutation of the V-ATPase B1 Subunit

Congenital distal renal tubular acidosis (dRTA) from mutations of the B1 subunit of the V-ATPase is considered an autosomal recessive disease. We analyzed a dRTA kindred with a truncation-mutation of B1 (p.Phe468fsX487) previously shown to have failure of assembly into the V1 domain of the V-ATPase. All heterozygous carriers in this kindred have normal plasma bicarbonate concentrations, thus evaded the diagnosis of RTA. However, inappropriately high urine pH, hypocitraturia, and hypercalciuria are present either individually or in combination in the heterozygotes at baseline. Two of the heterozygotes studied also have inappropriate urinary acidification with acute ammonium chloride loading and impaired urine-blood pCO2 gradient during bicarbonaturia indicating presence of H+ gradient and flux defects. In normal human renal papillae, wild type B1 is located primarily on the plasma membrane but papilla from one of the heterozygote who had kidney stones had renal tissue secured from surgery showed B1 in both plasma membrane as well as a diffuse intracellular staining. Titrating increasing amounts of the mutant B1 subunit did not exhibit negative dominance over the expression, cellular distribution, or H+-pump activity of the wild type B1 in mammalian HEK293 cells and in V-ATPase-deficient S. cerevisiae. This is the first demonstration of renal acidification defects and nephrolithiasis in heterozygous carriers of mutant B1 subunit; which cannot be attributable to negative dominance. We propose that heterozygosity may lead to mild real acidification defects due to haploinsufficiency. B1 heterozygosity should be considered in patients with calcium nephrolithiasis and urinary abnormalities such as alkalinuria or hypocitraturia.

Lindane induced cellular dysfunction in MDCK cells: The role of the peripheral benzodiazepine receptor

The central nervous system GABAA/Benzodiazepine (GABAA/BZD) receptors are targets for many pharmaceutical agents and several classes of pesticides. Lindane is an organochlorine pesticide, although banned from production in the U.S. since 1977, still imported for use as an insecticide and pharmaceutically to control ectoparasites (ATSDR, 1994). Lindane functions as a GABA/BZD receptor antagonist within the central nervous system (CNS). Outside of the CNS, peripheral BZD receptors have been localized to the distal tubule of the kidney. Previous research in our laboratory has shown that incubation of renal cortical slices with lindane can produce an increase in kallikrein leakage, suggesting a distal tubular effect. In this study, Madin Darby Canine Kidney (MDCK) cells were used as an in vitro system to assess the toxicity of lindane. This purpose of this study was to determine if interactions between a renal distal tubular BZD-like receptor and lindane could lead to perturbations in renal distal cellular chloride (Cl−) transport and mitochondrial dysfunction and ultimately, cellular death. ^ Pertubations in renal chloride transport were measured indirectly by determining if lindane altered cell function responsiveness following osmotic stress. MDCK cells pre-treated with lindane and then subjected to osmotic stress remained swollen for up to 12 hours post-stress. Lindane-induced dysfunction was assessed through stress protein induction measured by Western Blot analysis. Lindane pretreatment delayed Heat Shock Protein 72 (HSP72) induction by 36 hours in osmotically stressed cells. Pretreatment with 1 × 10 −5 M LIN followed by osmotic stress elevated p38 and Stress Activated Protein Kinase (SAPK/JNK) at 15 minutes which declined at 30 minutes. Lindane appeared to have no effect on Endoplasmic Reticulum Related Kinase (ERK) induction. Lindane did not effect osmotically stressed LLC-PKI cells, a control cell line. ^ Lindane-treated MDCK cells did not exhibit necrosis. Instead, apoptosis was observed in lindane-treated MDCK cells in both time- and dose-dependent manners. LLC-PKI cells were not affected by LIN treatment. ^ To better understand the mechanism of lindane-induced apoptosis, mitochondrial function was measured. No changes in cytochrome c release or mitochondrial membrane potential were observed suggesting the mitochondrial pathway was not involved in lindane-induced apoptosis. ^ Further research will need to be conducted to determine the mechanism of lindane-induced adverse cellular effects. ^

Role of organic cation transporters in the renal secretion of nucleoside analogs

The mammalian kidney maintains homeostasis of the extracellular environment and eliminates toxic substances from the body, in part via secretion by the organic cation transporters (OCT). Some nucleosides are also secreted by the kidney. Previous work indicated that the deoxyadenosine analog, 2′ -deoxytubercidin (dTub), is secreted by mouse kidney through the OCTs. This study examines the role of OCTs in the renal secretion of dTub and other nucleoside analogs. ^ Using the Xenopus laevis oocyte expression system, the basolateral type rat organic cation transporter rOCT1 was shown to transport dTub and other nucleosides. The positive charged form of dTub (dTub +) appears to be the substrate for rOCT1. Tetraethylammonium (TEA) and dTub competitively inhibit the other's uptake by rOCT1 in a manner consistent with their interaction at a common site. Although 67% homologous with rOCT1, rOCT2 does not mediate the uptake of these nucleosides. Kinetic studies demonstrated the difference in substrate specificity between rOCT1 and rOCT2 to be largely due to a poor affinity of rOCT2 for dTub+. This difference in affinity is located within transmembrane domains 2–7 as determined by chimeric constructs. ^ OCT1 knockout mice were used to evaluate the role of OCT1 in the renal secretion of dTub. No significant difference in tissue distribution and urinary excretion of dTub was observed between the knockout and wild-type mice, indicating that OCT1 is not necessary for the renal secretion of dTub. Apical transporters are postulated to participate in its active secretion. To characterize a possible apical transporter, we screened several renal cell lines for a nucleoside-sensitive OCT. American opossum kidney proximal tubule cells (OK) express a TEA efflux transporter that is inhibited by dTub and other nucleoside analogs. This carrier is metabolic-dependent and distinct from the cloned OCTs to date, i.e. it is sodium- and proton-independent. In conclusion, dTub is a good substrate for OCT1; however, this OCT is not necessary for its renal secretion in mice. The novel TEA efflux transporter identified in OK cells is likely to participate in the renal secretion of dTub and perhaps other nucleoside analogs. ^

Evaluation of oxidative damage and renal distal tubule cell stress response following exposure to lindane

Lindane, or γ-hexachlorocyclohexane, is a chlorinated hydrocarbon pesticide that was banned from U.S. production in 1976, but until recently continued to be imported and applied for occupational and domestic purposes. Lindane is known to cause central nervous system (CNS), immune, cardiovascular, reproductive, liver, and kidney toxicity. The mechanism for which lindane interacts with the CNS has been elucidated, and involves antagonism of the γ-aminobutyric acid/benzodiazepine (GABAA/BZD) receptor. Antagonism of this receptor results in the inhibition of Cl- channel flux, with subsequent convulsions, seizures, and paralysis. This response makes lindane a desirable defense against arthropod pests in agriculture and the home. However, formulation and application of this compound can contribute to human toxicity. In conjunction with this exposure scenario, workers may be subject to both heat and physical stress that may increase their susceptibility to pesticide toxicity by altering their cellular stress response. The kidneys are responsible for maintaining osmotic homeostasis, and are exposed to agents that undergo urinary excretion. The mechanistic action of lindane on the kidneys is not well understood. Lindane, in other organ systems, has been shown to cause cellular damage by generation of free radicals and oxidative stress. Previous research in our laboratory has shown that lindane causes apoptosis in distal tubule cells, and delays renal stress response under hypertonic stress. Characterizing the mechanism of action of lindane under conditions of physiologic stress is necessary to understand the potential hazard cyclodiene pesticides and other organochlorine compounds pose to exposed individuals under baseline conditions, as well as under conditions of physiologic stress. We demonstrated that exposure to lindane results in oxidative damage and dysregulation of glutathione response in renal distal tubule (MDCK) cells. We showed that under conditions of hypertonic stress, lindane-induced oxidative stress resulted in early onset apoptosis and corresponding down-regulated expression of the anti-apoptotic protein, Bcl-xL. Thus, the interaction of lindane with renal peripheral benzodiazepine receptors (PBR) is associated with attenuation of cellular protective proteins, making the cell more susceptible to injury or death. ^