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BackgroundMeasures of kidney tubule health are risk markers for acute kidney injury (AKI) in persons with chronic kidney disease (CKD) during hypertension treatment, but their associations with other adverse events (AEs) are unknown.MethodsAmong 2377 Systolic Blood Pressure Intervention Trial (SPRINT) participants with CKD, we measured at baseline eight urine biomarkers of kidney tubule health and two serum biomarkers of mineral metabolism pathways that act on the kidney tubules. Cox proportional hazards models were used to evaluate biomarker associations with risk of a composite of pre-specified serious AEs (hypotension, syncope, electrolyte abnormalities, AKI, bradycardia and injurious falls) and outpatient AEs (hyperkalemia and hypokalemia).ResultsAt baseline, the mean age was 73 ± 9 years and mean estimated glomerular filtration rate (eGFR) was 46 ± 11 mL/min/1.73 m2. During a median follow-up of 3.8 years, 716 (30%) participants experienced the composite AE. Higher urine interleukin-18, kidney injury molecule-1, neutrophil gelatinase-associated lipocalin (NGAL) and monocyte chemoattractant protein-1 (MCP-1), lower urine uromodulin (UMOD) and higher serum fibroblast growth factor-23 were individually associated with higher risk of the composite AE outcome in multivariable-adjusted models including eGFR and albuminuria. When modeling biomarkers in combination, higher NGAL [hazard ratio (HR) = 1.08 per 2-fold higher biomarker level, 95% confidence interval (CI) 1.03-1.13], higher MCP-1 (HR = 1.11, 95% CI 1.03-1.19) and lower UMOD (HR = 0.91, 95% CI 0.85-0.97) were each associated with higher composite AE risk. Biomarker associations did not vary by intervention arm (P > 0.10 for all interactions).ConclusionsAmong persons with CKD, several kidney tubule biomarkers are associated with higher risk of AEs during hypertension treatment, independent of eGFR and albuminuria.

Chronic kidney disease (CKD) remains a major epidemiological, clinical, and biomedical challenge. During CKD, renal tubular epithelial cells (TECs) present a persistent inflammatory and profibrotic response. Fatty acid oxidation (FAO), the main source of energy for TECs, is reduced in kidney fibrosis and contributes to its pathogenesis. To determine whether gain of function in FAO (FAO-GOF) could protect from fibrosis, we generated a conditional transgenic mouse model with overexpression of the fatty acid shuttling enzyme carnitine palmitoyl-transferase 1A (CPT1A) in TECs. Cpt1a-knockin (CPT1A-KI) mice subjected to 3 models of renal fibrosis (unilateral ureteral obstruction, folic acid nephropathy [FAN], and adenine-induced nephrotoxicity) exhibited decreased expression of fibrotic markers, a blunted proinflammatory response, and reduced epithelial cell damage and macrophage influx. Protection from fibrosis was also observed when Cpt1a overexpression was induced after FAN. FAO-GOF restored oxidative metabolism and mitochondrial number and enhanced bioenergetics, increasing palmitate oxidation and ATP levels, changes that were also recapitulated in TECs exposed to profibrotic stimuli. Studies in patients showed decreased CPT1 levels and increased accumulation of shortand middle chain acylcarnitines, reflecting impaired FAO in human CKD. We propose that strategies based on FAO-GOF may constitute powerful alternatives to combat fibrosis inherent to CKD.

BACKGROUND: Untreated microangiopathic hemolytic anemia in pregnancy is associated with adverse maternal and perinatal outcomes. Accurate diagnosis is challenging owing to nonspecific clinical features and pathologic findings. Timely initiation of appropriate management is essential to optimize maternal and perinatal outcomes. CASE: A 26-year-old primiparous woman presented at 20 weeks of gestation with new-onset microangiopathic hemolytic anemia on a background of poorly controlled type 1 diabetes. She received eculizumab for presumed atypical hemolytic uremic syndrome. At 24 weeks of gestation, she developed superimposed early-onset preeclampsia; she delivered at 27 weeks of gestation after continuing eculizumab. CONCLUSION(S): Eculizumab may prolong pregnancy in early-onset preeclampsia. Additional research is needed to assess short-term and long-term maternal and newborn outcomes.Copyright © 2019 by the American College of Obstetricians and Gynecologists. Published by Wolters Kluwer Health, Inc. All rights reserved.

BACKGROUND: Untreated microangiopathic hemolytic anemia in pregnancy is associated with adverse maternal and perinatal outcomes. Accurate diagnosis is challenging owing to nonspecific clinical features and pathologic findings. Timely initiation of appropriate management is essential to optimize maternal and perinatal outcomes. CASE: A 26-year-old primiparous woman presented at 20 weeks of gestation with new-onset microangiopathic hemolytic anemia on a background of poorly controlled type 1 diabetes. She received eculizumab for presumed atypical hemolytic uremic syndrome. At 24 weeks of gestation, she developed superimposed early-onset preeclampsia; she delivered at 27 weeks of gestation after continuing eculizumab. CONCLUSION(S): Eculizumab may prolong pregnancy in early-onset preeclampsia. Additional research is needed to assess short-term and long-term maternal and newborn outcomes.Copyright © 2019 by the American College of Obstetricians and Gynecologists. Published by Wolters Kluwer Health, Inc. All rights reserved.

Rationale & objectiveRandom assignment to intensive blood pressure (BP) lowering (systolic BP<120mmHg) compared to a less intensive BP target (systolic BP<140mmHg) in the Action to Control Cardiovascular Risk in Diabetes BP (ACCORD-BP) trial resulted in a more rapid decline in estimated glomerular filtration rate (eGFR). Whether this reflects hemodynamic effects or intrinsic kidney damage is unknown.Study designLongitudinal analysis of a subgroup of clinical trial participants.Settings & participantsA subgroup of 529 participants in ACCORD-BP.ExposuresUrine biomarkers of tubular injury (kidney injury molecule 1, interleukin 18 [IL-18]), repair (human cartilage glycoprotein 39 [YKL-40]), and inflammation (monocyte chemoattractant protein 1) at baseline and year 2.OutcomesChanges in eGFR from baseline to 2 years.Analytical approachWe compared changes in biomarker levels and eGFRs across participants treated to an intensive versus less intensive BP goal using analysis of covariance.ResultsOf 529 participants, 260 had been randomly assigned to the intensive and 269 to the standard BP arm. Mean age was 62±6.5 years and eGFR was 90mL/min/1.73m2. Baseline clinical characteristics, eGFRs, urinary albumin-creatinine ratios (ACRs), and urinary biomarker levels were similar across BP treatment groups. Compared to less intensive BP treatment, eGFR was 9.2mL/min/1.73m2 lower in the intensive BP treatment group at year 2. Despite the eGFR reduction, within this treatment group, ACR was 30% lower and 4 urinary biomarker levels were unchanged or lower at year 2. Also within this group, participants with the largest declines in eGFRs had greater reductions in urinary IL-18 and YKL-40 levels. In a subgroup analysis of participants developing incident chronic kidney disease (sustained 30% decline and eGFR<60mL/min/1.73m2; n=77), neither ACR nor 4 biomarker levels increased in the intensive treatment group, whereas the level of 1 biomarker, IL-18, increased i

In antineutrophil cytoplasmic antibody-associated vasculitis (AAV), Toll-like receptors (TLRs) may be engaged by infection-associated patterns and by endogenous danger signals, linking infection and innate inflammation with this autoimmune disease. This study examined intrarenal TLR2, TLR4, and TLR9 expression and renal injury in AAV, testing the hypothesis that increased TLR expression correlates with renal injury. Patients with AAV exhibited both glomerular and tubulointerstitial expression of TLR2, TLR4, and TLR9, with TLR4 being the most prominent in both compartments. Glomerular TLR4 expression correlated with glomerular segmental necrosis and cellular crescents, with TLR2 expression correlating with glomerular segmental necrosis. The extent and intensity of glomerular and tubulointerstitial TLR4 expression and the intensity of glomerular TLR2 expression inversely correlated with the presenting estimated glomerular filtration rate. Although myeloid cells within the kidney expressed TLR2, TLR4, and TLR9, TLR2 and TLR4 colocalized with endothelial cells and podocytes, whereas TLR9 was expressed predominantly by podocytes. The functional relevance of intrarenal TLR expression was further supported by the colocalization of TLRs with their endogenous ligands high-mobility group box 1 and fibrinogen. Therefore, in AAV, the extent of intrarenal TLR4 and TLR2 expression and their correlation with renal injury indicates that TLR4, and to a lesser degree TLR2, may be potential therapeutic targets in this disease.Copyright © 2018 American Physiological Society. All rights reserved.

Tubular epithelial cells take up and degrade plasma albumin filtered by the glomerulus. Tubular damage resulting in reduced albumin uptake or degradation has been suggested as one mechanism contributing to albuminuria in kidney disease. This study investigated whether tubular albumin uptake or degradation is altered in acute and chronic glomerular disease. Mouse models of acute glomerular injury (anti-GBM disease and LPS-induced albuminuria) and chronic disease (streptozotocin-induced diabetes and db/db mice) were examined. Mice were injected intravenously with Alexa-albumin plus DQ-albumin and killed 20 minutes later. Tubular uptake of albumin (Alexa-albumin) and albumin degradation (Dye Quenched (DQ)-albumin) was assessed in tissue sections via confocal microscopy. Tubular uptake of Alexa-albumin in the models of diabetic nephropathy was not different to normal mice. However, the fluorescence signal resulting from degradation of DQ-albumin was significantly reduced in db/db mice, and the ratio of degraded to intact albumin was reduced in both models. The ratio of degraded to intact albumin in tubules was also reduced in the anti-GBM model. In the LPS model, both tubular uptake and degradation of albumin were significantly reduced, with a substantial reduction in the ratio of degraded to intact albumin in tubules. LPS stimulation of cultured tubular epithelial cells inhibited albumin uptake, indicating a direct role for LPS in modifying tubular handling of albumin. In conclusion, reduced degradation of filtered albumin in the proximal tubule is a common feature of glomerular diseases. This may be a general mechanism whereby tubular dysfunction contributes to the development of albuminuria.Copyright © 2017 John Wiley & Sons Australia, Ltd

In antineutrophil cytoplasmic antibody-associated vasculitis (AAV), Toll-like receptors (TLRs) may be engaged by infection-associated patterns and by endogenous danger signals, linking infection and innate inflammation with this autoimmune disease. This study examined intrarenal TLR2, TLR4, and TLR9 expression and renal injury in AAV, testing the hypothesis that increased TLR expression correlates with renal injury. Patients with AAV exhibited both glomerular and tubulointerstitial expression of TLR2, TLR4, and TLR9, with TLR4 being the most prominent in both compartments. Glomerular TLR4 expression correlated with glomerular segmental necrosis and cellular crescents, with TLR2 expression correlating with glomerular segmental necrosis. The extent and intensity of glomerular and tubulointerstitial TLR4 expression and the intensity of glomerular TLR2 expression inversely correlated with the presenting estimated glomerular filtration rate. Although myeloid cells within the kidney expressed TLR2, TLR4, and TLR9, TLR2 and TLR4 colocalized with endothelial cells and podocytes, whereas TLR9 was expressed predominantly by podocytes. The functional relevance of intrarenal TLR expression was further supported by the colocalization of TLRs with their endogenous ligands high-mobility group box 1 and fibrinogen. Therefore, in AAV, the extent of intrarenal TLR4 and TLR2 expression and their correlation with renal injury indicates that TLR4, and to a lesser degree TLR2, may be potential therapeutic targets in this disease.Copyright © 2018 American Physiological Society. All rights reserved.

Tubular epithelial cells take up and degrade plasma albumin filtered by the glomerulus. Tubular damage resulting in reduced albumin uptake or degradation has been suggested as one mechanism contributing to albuminuria in kidney disease. This study investigated whether tubular albumin uptake or degradation is altered in acute and chronic glomerular disease. Mouse models of acute glomerular injury (anti-GBM disease and LPS-induced albuminuria) and chronic disease (streptozotocin-induced diabetes and db/db mice) were examined. Mice were injected intravenously with Alexa-albumin plus DQ-albumin and killed 20 minutes later. Tubular uptake of albumin (Alexa-albumin) and albumin degradation (Dye Quenched (DQ)-albumin) was assessed in tissue sections via confocal microscopy. Tubular uptake of Alexa-albumin in the models of diabetic nephropathy was not different to normal mice. However, the fluorescence signal resulting from degradation of DQ-albumin was significantly reduced in db/db mice, and the ratio of degraded to intact albumin was reduced in both models. The ratio of degraded to intact albumin in tubules was also reduced in the anti-GBM model. In the LPS model, both tubular uptake and degradation of albumin were significantly reduced, with a substantial reduction in the ratio of degraded to intact albumin in tubules. LPS stimulation of cultured tubular epithelial cells inhibited albumin uptake, indicating a direct role for LPS in modifying tubular handling of albumin. In conclusion, reduced degradation of filtered albumin in the proximal tubule is a common feature of glomerular diseases. This may be a general mechanism whereby tubular dysfunction contributes to the development of albuminuria.Copyright © 2017 John Wiley & Sons Australia, Ltd

Common variants in the UMOD gene encoding uromodulin, associated with risk of hypertension and CKD in the general population, increase UMOD expression and urinary excretion of uromodulin, causing salt-sensitive hypertension and renal lesions. To determine the effect of selective pressure on variant frequency, we investigated the allelic frequency of the lead UMOD variant rs4293393 in 156 human populations, in eight ancient human genomes, and in primate genomes. The T allele of rs4293393, associated with CKD risk, has high frequency in most modern populations and was the one detected in primate genomes. In contrast, we identified only the derived, C allele in Denisovan and Neanderthal genomes. The distribution of the UMOD ancestral allele did not follow the ancestral susceptibility model observed for variants associated with salt-sensitive hypertension. Instead, the global frequencies of the UMOD alleles significantly correlated with pathogen diversity (bacteria, helminths) and prevalence of antibiotic-resistant urinary tract infections (UTIs). The inverse correlation found between urinary levels of uromodulin and markers of UTIs in the general population substantiates the link between UMOD variants and protection against UTIs. These data strongly suggest that the UMOD ancestral allele, driving higher urinary excretion of uromodulin, has been kept at a high frequency because of its protective effect against UTIs.

Organogenesis involves precursor cells proliferation, differentiation along with their coordinated organization into precise multicellular arrangements by planar cell polarity (PCP) pathways. The beta-catenin independent/non-canonical type of Wnt-11 signaling has been known as a PCP modulator during development. In this thesis were analyzed the roles of Wnt-11 in heart and kidney development by using in vivo functional genomics technologies. We show that the Wnt-11 gene is important for murine ventricular myocardium development, since Wnt-11 deficiency in early cardiogenesis leads to impaired organization and maturation of mouse ventricular cardiomyocytes, causing primary cardiomyopathy with in utero lethality. Wnt-11 coordinates the co-localized expression of the cell adhesion molecules N-cadherin and β-catenin, which are critical for the spatially specific organization of cardiomyocytes. We show that Wnt-11 deficiency causes primary hypertrophic and noncompaction cardiomyopathy in adult mice, with consequences for regional myocardium function. The Wnt family of secreted signals has been implicated in kidney tubule development and tubular cystic diseases such as polycystic kidney disease. We show here that Wnt-11 is expressed in mature nephrons and is involved in late steps of nephrogenesis, since the kidney tubule organization is deregulated in Wnt-11 deficient kidneys, to enlarged lumen with increased convolution. These tubule abnormalities are associated with glomerular microcyst formation and kidney failure. Wnt-11 deficiency reduced significantly Wnt-9b expression, a critical signal for PCP-mediated kidney tubule elongation. In the cortical region this associated with reduced expression of nephron and stromal progenitor cell marker. The results in this thesis point out that Wnt-11 function is required for proper myocardium organization and maturation as well as proper morphogenesis of the kidney tubules during the embryonic and postnatal developmental, Tiivistelmä Alkion sisäelinten kehityksen aikana esisolut lisääntyvät ja erilaistuvat muodostaen tarkoin määriteltyjä monisoluisia rakenteita. Muodostuvan kudosrakenteen määrittelyssä erilaiset solusignaalit ovat keskeisessä asemassa. Yksi näistä on nk. Wnt signaali perhe. Wnt perheeen jäsen Wnt-11 tehtävät on huonosti tunnettu. Wnt-11 viestittää ilmeisesti nk. planaaristen solupolariteettireittien (PCP) avulla, joka on beeta-kateniinista riippumattoman nk. ei-kanonisen Wnt signaali. Väitöskirjatyössä selvitettiin Wnt-11:n vaikutuksia sydämen ja munuaisten kehitykseen in vivo funktionaalisten genomisten menetelmien avulla. Ihmisen synnynnäiset kardiomyopatiat ovat sydänlihaksen ensisijaisia vaurioita, joiden taustalla on sydänlihaksen kehityshäiriö. Tutkimuksessa osoitetaan, että Wnt-11-geenillä on tärkeä merkitys hiiren sydänkammion kehitykselle, koska Wnt-11-geenin puute sydämen varhaisen kehityksen vaiheessa johtaa sydänlihassolujen järjestäytymisen ja kypsymisen häiriintymiseen, jolloin seurauksena on ensisijaisesta kardiomyopatiasta johtuva sikiökuolema. Wnt-11 koordinoi kahden solukiinnitysmolekyylin, N-kadheriinin ja β-kateniinin, samanaikasta ilmentymistä. Kyseiset molekyylit ovat keskeisen tärkeitä sydänlihasssolujen spatiaalisen järjestäytymisen kannalta. Tutkimuksessa osoitetaan, että Wnt-11-puutos aiheuttaa aikuisilla hiirillä ensisijaista sydänlihaksen liikakasvua ja trabekuloivaa kardiomyopatiaa, mikä vaikuttaa sydänlihaksen toimintaan. Tuloksilla voi olla merkitystä tutkittaessa ihmisen synnynnäisiä kardiomyopatioita. Wnt-signaaliperheen on osoitettu olevan yhteydessä munuaisputken kehitykseen ja sen sairauksiin, kuten munuaisten monirakkulatautiin. Väitöstutkimuksessa osoitetaan, että Wnt-11 ilmentyy kypsissä nefroneissa ja että se osallistuu nefrogeneesiin myöhempiin vaiheisiin, koska munuaisputken kehityksen säätely on poikkeavaa niissä munuaisissa, joista Wnt-11 puuttuu. Seurauksena on laajentunut, normaalia poimuttuneempi luumen. Munuaisputken po

Renal fibrosis and tubular apoptosis are common mechanisms of progressive kidney disease. In vitro studies have implicated the c-Jun amino-terminal kinase (JNK) pathway in these processes. Both of the major JNK isoforms, JNK1 and JNK2, are expressed in the kidney, but their relative contribution to JNK signaling is unknown. This study investigated the role of JNK signaling in renal fibrosis and tubular apoptosis in the unilateral ureteral obstruction model using two different approaches: (1) Mice that were deficient in either JNK1 or JNK2 and (2) a specific inhibitor of all JNK isoforms, CC-401. Western blotting and immunostaining identified a marked increase in JNK signaling in the obstructed kidney, with substantial redundancy between JNK1 and JNK2 isoforms. Administration of CC-401 blocked JNK signaling in the rat obstructed kidney and significantly inhibited renal fibrosis in terms of interstitial myofibroblast accumulation and collagen IV deposition. This effect was attributed to suppression of gene transcription for the profibrotic molecules TGF-beta1 and connective tissue growth factor. CC-401 treatment also significantly reduced tubular apoptosis in the obstructed kidney. Genetic deletion of JNK1 or JNK2 did not protect mice from renal fibrosis in the unilateral ureteral obstruction model, but JNK1 deletion did result in a significant reduction in tubular cell apoptosis. In conclusion, this is the first study to demonstrate that JNK signaling plays a pathogenic role in renal fibrosis and tubular apoptosis. Furthermore, JNK1 plays a nonredundant role in tubular cell apoptosis. These studies identify the JNK pathway as a potential therapeutic target in progressive kidney disease. Copyright © 2007 by the American Society of Nephrology.

Background. Macrophage accumulation is a prominent feature in many forms of glomerulonephritis. Local proliferation of macrophages within the kidney has been described in human and experimental glomerulonephritis and may have an important role in augmenting the inflammatory response. The current study examined the relationship between local macrophage proliferation and renal expression of macrophage colony-stimulating factor (M-CSF). Methods. A total of 118 renal biopsies of patients with a wide range of glomerulonephridities were examined for M-CSF protein and macrophage proliferation (KP1 + PCNA + cells) by single and double immunohistochemistry staining, respectively. Results. Biopsies of thin membrane disease (TMD) with histologically normal kidney showed M-CSF protein expression by 33% of cortical tubules, while glomerular M-CSF expression was limited to resident macrophages and some podocytes. Glomerular M-CSF expression increased significantly in proliferative forms of glomerulonephritis, with M-CSF staining of infiltrating macrophages, podocytes and some mesangial cells. Segmental areas of strong M-CSF expression, particularly in crescents, co-localized with KP1 + PCNA + proliferating macrophages. There was also an increase in tubular M-CSF expression in most types of glomerulonephritis. Tubular M-CSF staining was strongest in areas of tubular damage and co-localized with KP1 + macrophages, including KP1 + PCNA + proliferating macrophages. Many interstitial macrophages and alpha-smooth muscle actin-positive myofibroblasts showed strong M-CSF staining. Statistical analysis showed a highly significant correlation between M-CSF expression and local macrophage proliferation in both the glomerulus and tubulointerstitium. Glomerular and tubular M-CSF expression gave a significant correlation with renal dysfunction. Conclusions. Glomerular and tubulointerstitial M-CSF expression is up-regulated in human glomerulonephritis, being most prominent in proliferative form

CD100 participates in adaptive immune responses and is important in neural cell migration. To determine the role of endogenous CD100 in severe glomerular inflammation, we induced experimental crescentic glomerulonephritis by planting a foreign antigen in glomeruli of sensitized normal and CD100-deficient (CD100-\-) mice. Fewer CD100-\- glomeruli exhibited crescent formation or severe histological changes. Antigen-specific immune responses were reduced in CD100-\- mice. There was less interferon (IFN)-gamma and interleukin (IL)-4 production by splenocytes and fewer activated T and B cells were present in lymph nodes of immunized CD100 -\- mice. Serum antigen-specific immunoglobulin (IgG) levels were also decreased. Glomerular macrophage and CD4+ cell infiltration, and IgG and C3 deposition were attenuated. Normal kidneys expressed mRNA for CD100 and plexin-B1 (the tissue receptor of CD100). Direct immunofluorescence showed that renal-CD100 protein was predominantly in tubules, while plexin-B1 was present in both glomeruli and tubules. To determine whether glomerular plexin-B1 mediates leucocyte recruitment via leucocyte CD100, recruitment was studied after passive transfer of heterologous antibody (attracting neutrophils) or isologous antibody (attracting macrophages). Glomerular macrophages were reduced in CD100-\- mice, but neutrophil recruitment was equivalent, consistent with CD100 expression on macrophages, but not neutrophils. CD100 promotes severe nephritogenic immune responses and leucocyte CD100-glomerular plexin-B1 interactions enhance macrophage recruitment to glomeruli. © 2009 Blackwell Publishing Ltd.

The aim of this study was to examine the contribution of local proliferation in the development of macrophage accumulation and macrophage-mediated injury in anti-GBM glomerulonephritis. Using double immunohistochemistry staining of monocyte/macrophages plus the proliferating cell nuclear antigen (PCNA) or bromodeoxyuridine (BrdU) incorporation, we found that the initial accumulation of ED1+ macrophages in the kidney on day 1 of disease was due to an influx of circulating monocytes. However, large numbers of proliferating macrophages (ED1+PCNA+ cells), including mitotic macrophages, were present within the glomerulus and interstitium during disease progression (days 7 to 21), accounting for up to 62% of the total macrophage population and giving an excellent correlation with total macrophage accumulation (glomerulus, r = 0.92; interstitium, r = 0.94; both P < 0.001). These proliferating cells had a monocyte phenotype (ED1+ED2-ED3-), but this marked proliferative activity was restricted to the diseased kidney since no PCNA expression or BrdU incorporation was evident within circulating blood monocytes. Proliferating macrophages were almost exclusively localized in areas of severe tissue damage and they correlated significantly with glomerular and tubulointerstitial lesions (P < 0.001), proteinuria (P < 0.001) and creatinine clearance (P < 0.01). In marked contrast, glomerular PCNA- macrophages failed to correlate with these parameters. In conclusion, this study has demonstrated that local macrophage proliferation is the major mechanism of macrophage accumulation during the progression of rat anti-GBM glomerulonephritis, Furthermore, it suggests that proliferating macrophages are potent local effector cells in the mediation of progressive renal injury in this disease.

CD44 is an adhesion molecule involved in a wide range of cell-cell and cell-matrix interactions. The standard form of CD44 (CD44S) is a 85-90-kD glycoprotein, but alternative splicing of RNA encoding 10 variable exons (V1-V10) can give rise to many different CD44 variant protein isoforms of higher molecular weight. CD44 isoforms containing the V6 exon play a crucial role in tumour metastasis and lymphocyte activation. However, the role of CD44V6 in the kidney is unknown. The aim of this study was to examined renal CD44V6 expression in health, disease and in vitro. Immunohistochemistry staining with the V6-specific 1.1ASML antibody identified constitutive CD44V6 expression by occasional cortical tubular epithelial cells and medullary tubules in normal rat kidney. In immune-induced kidney disease (rat anti-glomerular basement membrane glomerulonephritis), there was a marked increase in CD44V6 expression by cortical tubules, particularly in areas of tubulointerstitial damage, which was associated with focal macrophage infiltration. There was also a marked increase in CD44V6 expression by damaged tubules in a model of non-immune kidney disease (unilateral ureteric obstruction). Reverse transcription-polymerase chain reaction revealed a complex pattern of CD44V6-containing mRNA isoforms in normal rat kidney. This pattern of CD44V6 splicing was essentially unaltered in disease. The NRK52E normal rat kidney tubular epithelial cell line expresses both CD44S and CD44V6. Stimulation of NRK52E cells with IL-1 or transforming growth factor-beta 1 induced a two-to-five-fold increase in the expression of both CD44S and CD44V6. Furthermore, triggering of NRK52E cells by antibodies to CD44S or CD44V6, but not isotype control antibodies, induced secretion of monocyte chemoattractant protein-1. In conclusion, this study has identified expression of the tumour-associated marker CD44V6 in tubular epithelial cells in normal and diseased rat kidney, and suggests that signalling through t

Diabetic nephropathy involves a renal inflammatory response induced by the diabetic milieu. Macrophages accumulate in diabetic kidneys in association with the local upregulation of monocyte chemoattractant protein-1 (MCP-1); however, the contribution of macrophages to renal injury and the importance of MCP-1 to their accrual are unclear. Therefore, we examined the progression of streptozotocin (STZ)-induced diabetic nephropathy in mice deficient in MCP-1 in order to explore the role of MCP-1-mediated macrophage accumulation in the development of diabetic kidney damage. Renal pathology was examined at 2, 8, 12 and 18 weeks after STZ treatment in MCP-1 intact (+/+) and deficient (-/-) mice with equivalent blood glucose and hemoglobin A1c levels. In MCP-1(+/+) mice, the development of diabetic nephropathy was associated with increased kidney MCP-1 production, which occurred mostly in tubules, consistent with our in vitro finding that elements of the diabetic milieu (high glucose and advanced glycation end products) directly stimulate tubular MCP-1 secretion. Diabetes of 18 weeks resulted in albuminuria and elevated plasma creatinine in MCP-1(+/+) mice, but these aspects of renal injury were largely suppressed in MCP-1(-/-) mice. Protection from nephropathy in diabetic MCP-1(-/-) mice was associated with marked reductions in glomerular and interstitial macrophage accumulation, histological damage and renal fibrosis. Diabetic MCP-1(-/-) mice also had a smaller proportion of kidney macrophages expressing markers of activation (inducible nitric oxide synthase or sialoadhesin) compared to diabetic MCP-1(+/+) mice. In conclusion, our study demonstrates that MCP-1-mediated macrophage accumulation and activation plays a critical role in the development of STZ-induced mouse diabetic nephropathy. © 2006 International Society of Nephrology.

Aim: A number of growth factors have been shown to induce proliferation of renal cell types in animal models of kidney disease. In vitro studies suggest that many such growth factors induce renal cell proliferation through the extracellular signal-regulated kinase (ERK) pathway. The aim of this study was to determine the functional role of ERK signalling in cell proliferation in the obstructed kidney. Method(s): Unilateral ureteric obstruction was induced in C57BL/6J mice which then received an ERK inhibitor drug (U0126 100 mg/kg t.i.d.), vehicle (DMSO) or no treatment, starting at day 2 after unilateral ureteric obstruction surgery and continuing until animals were killed on day 5. Cell proliferation was assessed by uptake of bromodeoxyuridine (BrdU). Result(s): In normal mice, phosphorylation (activation) of ERK (p-ERK) was restricted to collecting ducts. Western blotting identified a marked increase in p-ERK in the obstructed kidney in the no-treatment and vehicle-treated groups. Immunostaining showed strong p-ERK staining in many tubules and in interstitial cells. U0126 treatment inhibited ERK phosphorylation as assessed by western blot and immunostaining. The number of BrdU+ cortical tubular cells was reduced by vehicle treatment but was not further changed by U0126 treatment. In contrast, interstitial cell proliferation in the obstructed kidney was unaltered by vehicle treatment, but this was significantly inhibited by U0126. This was associated with a reduction in interstitial macrophage accumulation, but no effect was seen upon interstitial accumulation of alpha-SMA+ myofibroblasts. Renal fibrosis, as assessed by collagen deposition, was unaffected by U0126 or vehicle treatment. Conclusion(s): These studies show that accumulation of interstitial macrophages in the obstructed kidney is, in part, dependent upon the ERK signalling pathway. © 2008 The Authors.

Renal fibrosis and tubular apoptosis are common mechanisms of progressive kidney disease. In vitro studies have implicated the c-Jun amino-terminal kinase (JNK) pathway in these processes. Both of the major JNK isoforms, JNK1 and JNK2, are expressed in the kidney, but their relative contribution to JNK signaling is unknown. This study investigated the role of JNK signaling in renal fibrosis and tubular apoptosis in the unilateral ureteral obstruction model using two different approaches: (1) Mice that were deficient in either JNK1 or JNK2 and (2) a specific inhibitor of all JNK isoforms, CC-401. Western blotting and immunostaining identified a marked increase in JNK signaling in the obstructed kidney, with substantial redundancy between JNK1 and JNK2 isoforms. Administration of CC-401 blocked JNK signaling in the rat obstructed kidney and significantly inhibited renal fibrosis in terms of interstitial myofibroblast accumulation and collagen IV deposition. This effect was attributed to suppression of gene transcription for the profibrotic molecules TGF-beta1 and connective tissue growth factor. CC-401 treatment also significantly reduced tubular apoptosis in the obstructed kidney. Genetic deletion of JNK1 or JNK2 did not protect mice from renal fibrosis in the unilateral ureteral obstruction model, but JNK1 deletion did result in a significant reduction in tubular cell apoptosis. In conclusion, this is the first study to demonstrate that JNK signaling plays a pathogenic role in renal fibrosis and tubular apoptosis. Furthermore, JNK1 plays a nonredundant role in tubular cell apoptosis. These studies identify the JNK pathway as a potential therapeutic target in progressive kidney disease. Copyright © 2007 by the American Society of Nephrology.

Background. Macrophage accumulation is a prominent feature in many forms of glomerulonephritis. Local proliferation of macrophages within the kidney has been described in human and experimental glomerulonephritis and may have an important role in augmenting the inflammatory response. The current study examined the relationship between local macrophage proliferation and renal expression of macrophage colony-stimulating factor (M-CSF). Methods. A total of 118 renal biopsies of patients with a wide range of glomerulonephridities were examined for M-CSF protein and macrophage proliferation (KP1 + PCNA + cells) by single and double immunohistochemistry staining, respectively. Results. Biopsies of thin membrane disease (TMD) with histologically normal kidney showed M-CSF protein expression by 33% of cortical tubules, while glomerular M-CSF expression was limited to resident macrophages and some podocytes. Glomerular M-CSF expression increased significantly in proliferative forms of glomerulonephritis, with M-CSF staining of infiltrating macrophages, podocytes and some mesangial cells. Segmental areas of strong M-CSF expression, particularly in crescents, co-localized with KP1 + PCNA + proliferating macrophages. There was also an increase in tubular M-CSF expression in most types of glomerulonephritis. Tubular M-CSF staining was strongest in areas of tubular damage and co-localized with KP1 + macrophages, including KP1 + PCNA + proliferating macrophages. Many interstitial macrophages and alpha-smooth muscle actin-positive myofibroblasts showed strong M-CSF staining. Statistical analysis showed a highly significant correlation between M-CSF expression and local macrophage proliferation in both the glomerulus and tubulointerstitium. Glomerular and tubular M-CSF expression gave a significant correlation with renal dysfunction. Conclusions. Glomerular and tubulointerstitial M-CSF expression is up-regulated in human glomerulonephritis, being most prominent in proliferative form

CD100 participates in adaptive immune responses and is important in neural cell migration. To determine the role of endogenous CD100 in severe glomerular inflammation, we induced experimental crescentic glomerulonephritis by planting a foreign antigen in glomeruli of sensitized normal and CD100-deficient (CD100-\-) mice. Fewer CD100-\- glomeruli exhibited crescent formation or severe histological changes. Antigen-specific immune responses were reduced in CD100-\- mice. There was less interferon (IFN)-gamma and interleukin (IL)-4 production by splenocytes and fewer activated T and B cells were present in lymph nodes of immunized CD100 -\- mice. Serum antigen-specific immunoglobulin (IgG) levels were also decreased. Glomerular macrophage and CD4+ cell infiltration, and IgG and C3 deposition were attenuated. Normal kidneys expressed mRNA for CD100 and plexin-B1 (the tissue receptor of CD100). Direct immunofluorescence showed that renal-CD100 protein was predominantly in tubules, while plexin-B1 was present in both glomeruli and tubules. To determine whether glomerular plexin-B1 mediates leucocyte recruitment via leucocyte CD100, recruitment was studied after passive transfer of heterologous antibody (attracting neutrophils) or isologous antibody (attracting macrophages). Glomerular macrophages were reduced in CD100-\- mice, but neutrophil recruitment was equivalent, consistent with CD100 expression on macrophages, but not neutrophils. CD100 promotes severe nephritogenic immune responses and leucocyte CD100-glomerular plexin-B1 interactions enhance macrophage recruitment to glomeruli. © 2009 Blackwell Publishing Ltd.

The aim of this study was to examine the contribution of local proliferation in the development of macrophage accumulation and macrophage-mediated injury in anti-GBM glomerulonephritis. Using double immunohistochemistry staining of monocyte/macrophages plus the proliferating cell nuclear antigen (PCNA) or bromodeoxyuridine (BrdU) incorporation, we found that the initial accumulation of ED1+ macrophages in the kidney on day 1 of disease was due to an influx of circulating monocytes. However, large numbers of proliferating macrophages (ED1+PCNA+ cells), including mitotic macrophages, were present within the glomerulus and interstitium during disease progression (days 7 to 21), accounting for up to 62% of the total macrophage population and giving an excellent correlation with total macrophage accumulation (glomerulus, r = 0.92; interstitium, r = 0.94; both P < 0.001). These proliferating cells had a monocyte phenotype (ED1+ED2-ED3-), but this marked proliferative activity was restricted to the diseased kidney since no PCNA expression or BrdU incorporation was evident within circulating blood monocytes. Proliferating macrophages were almost exclusively localized in areas of severe tissue damage and they correlated significantly with glomerular and tubulointerstitial lesions (P < 0.001), proteinuria (P < 0.001) and creatinine clearance (P < 0.01). In marked contrast, glomerular PCNA- macrophages failed to correlate with these parameters. In conclusion, this study has demonstrated that local macrophage proliferation is the major mechanism of macrophage accumulation during the progression of rat anti-GBM glomerulonephritis, Furthermore, it suggests that proliferating macrophages are potent local effector cells in the mediation of progressive renal injury in this disease.

CD44 is an adhesion molecule involved in a wide range of cell-cell and cell-matrix interactions. The standard form of CD44 (CD44S) is a 85-90-kD glycoprotein, but alternative splicing of RNA encoding 10 variable exons (V1-V10) can give rise to many different CD44 variant protein isoforms of higher molecular weight. CD44 isoforms containing the V6 exon play a crucial role in tumour metastasis and lymphocyte activation. However, the role of CD44V6 in the kidney is unknown. The aim of this study was to examined renal CD44V6 expression in health, disease and in vitro. Immunohistochemistry staining with the V6-specific 1.1ASML antibody identified constitutive CD44V6 expression by occasional cortical tubular epithelial cells and medullary tubules in normal rat kidney. In immune-induced kidney disease (rat anti-glomerular basement membrane glomerulonephritis), there was a marked increase in CD44V6 expression by cortical tubules, particularly in areas of tubulointerstitial damage, which was associated with focal macrophage infiltration. There was also a marked increase in CD44V6 expression by damaged tubules in a model of non-immune kidney disease (unilateral ureteric obstruction). Reverse transcription-polymerase chain reaction revealed a complex pattern of CD44V6-containing mRNA isoforms in normal rat kidney. This pattern of CD44V6 splicing was essentially unaltered in disease. The NRK52E normal rat kidney tubular epithelial cell line expresses both CD44S and CD44V6. Stimulation of NRK52E cells with IL-1 or transforming growth factor-beta 1 induced a two-to-five-fold increase in the expression of both CD44S and CD44V6. Furthermore, triggering of NRK52E cells by antibodies to CD44S or CD44V6, but not isotype control antibodies, induced secretion of monocyte chemoattractant protein-1. In conclusion, this study has identified expression of the tumour-associated marker CD44V6 in tubular epithelial cells in normal and diseased rat kidney, and suggests that signalling through t

Diabetic nephropathy involves a renal inflammatory response induced by the diabetic milieu. Macrophages accumulate in diabetic kidneys in association with the local upregulation of monocyte chemoattractant protein-1 (MCP-1); however, the contribution of macrophages to renal injury and the importance of MCP-1 to their accrual are unclear. Therefore, we examined the progression of streptozotocin (STZ)-induced diabetic nephropathy in mice deficient in MCP-1 in order to explore the role of MCP-1-mediated macrophage accumulation in the development of diabetic kidney damage. Renal pathology was examined at 2, 8, 12 and 18 weeks after STZ treatment in MCP-1 intact (+/+) and deficient (-/-) mice with equivalent blood glucose and hemoglobin A1c levels. In MCP-1(+/+) mice, the development of diabetic nephropathy was associated with increased kidney MCP-1 production, which occurred mostly in tubules, consistent with our in vitro finding that elements of the diabetic milieu (high glucose and advanced glycation end products) directly stimulate tubular MCP-1 secretion. Diabetes of 18 weeks resulted in albuminuria and elevated plasma creatinine in MCP-1(+/+) mice, but these aspects of renal injury were largely suppressed in MCP-1(-/-) mice. Protection from nephropathy in diabetic MCP-1(-/-) mice was associated with marked reductions in glomerular and interstitial macrophage accumulation, histological damage and renal fibrosis. Diabetic MCP-1(-/-) mice also had a smaller proportion of kidney macrophages expressing markers of activation (inducible nitric oxide synthase or sialoadhesin) compared to diabetic MCP-1(+/+) mice. In conclusion, our study demonstrates that MCP-1-mediated macrophage accumulation and activation plays a critical role in the development of STZ-induced mouse diabetic nephropathy. © 2006 International Society of Nephrology.

Aim: A number of growth factors have been shown to induce proliferation of renal cell types in animal models of kidney disease. In vitro studies suggest that many such growth factors induce renal cell proliferation through the extracellular signal-regulated kinase (ERK) pathway. The aim of this study was to determine the functional role of ERK signalling in cell proliferation in the obstructed kidney. Method(s): Unilateral ureteric obstruction was induced in C57BL/6J mice which then received an ERK inhibitor drug (U0126 100 mg/kg t.i.d.), vehicle (DMSO) or no treatment, starting at day 2 after unilateral ureteric obstruction surgery and continuing until animals were killed on day 5. Cell proliferation was assessed by uptake of bromodeoxyuridine (BrdU). Result(s): In normal mice, phosphorylation (activation) of ERK (p-ERK) was restricted to collecting ducts. Western blotting identified a marked increase in p-ERK in the obstructed kidney in the no-treatment and vehicle-treated groups. Immunostaining showed strong p-ERK staining in many tubules and in interstitial cells. U0126 treatment inhibited ERK phosphorylation as assessed by western blot and immunostaining. The number of BrdU+ cortical tubular cells was reduced by vehicle treatment but was not further changed by U0126 treatment. In contrast, interstitial cell proliferation in the obstructed kidney was unaltered by vehicle treatment, but this was significantly inhibited by U0126. This was associated with a reduction in interstitial macrophage accumulation, but no effect was seen upon interstitial accumulation of alpha-SMA+ myofibroblasts. Renal fibrosis, as assessed by collagen deposition, was unaffected by U0126 or vehicle treatment. Conclusion(s): These studies show that accumulation of interstitial macrophages in the obstructed kidney is, in part, dependent upon the ERK signalling pathway. © 2008 The Authors.

Tubulointerstitial localization of major histocompatibility complex (MHC) class II (Ia) antigens and immune-activated mononuclear cells (IL-2R+) and its suppression by prednisolone (PSL) were studied in a progressive model of focal glomerulosclerosis (FGS) induced by aminonucleoside (AN) and protamine sulfate (PS) given as 4 four day courses in rats. Tissues were prepared for the quantification of leucocytes on days 24, 52 and 80. The numbers of total leucocytes (CD45+), Ia+ cells, monocytes/macrophages, T lymphocytes (CD5+) and IL-2R+ cells (CD25+) were enumerated in immunoperoxidase sections using monoclonal antibodies: OX1, OX6, ED1, OX19 and ART18, respectively. The effects of PSL at a dose of 3 mg/kg body weight, which was begun from day 30, 12 days after the second series of injections, and continued for 50 days, were studied on day 80. There was no significant increase of glomerular T cells or IL-2R+ cells throughout the study. By contrast, each cell group in the interstitium increased in number throughout the disease evolution. Specifically IL-2R+ cells, which were rarely seen before AN + PS injection, were expressed by 5% of total leucocytes on day 80. Ia antigens also increased time-dependently and were found in proximal tubular cells on day 80. PSL treatment suppressed IL-2R and Ia expression in proportion to the decrease of total leucocytes and T cells. These results imply that delayed type hypersensitivity related to the appearance of immune activated T cells (IL-2R+) and MHC class II antigens may play a role in the progression of interstitial lesions and consequent renal dysfunction in a nonimmune-initiated FGS model.

Abnormal sodium (Na+) and water handling by the kidney may be the cause of hypertension in rats of the Milan hypertensive strain (MHS). In addition, the plasma cell membrane Ca-ATPase at Vmax is lower in MHS than in normotensive controls (MNS). The isolated kidney preparation was used to explore the role of extracellular free calcium (Ca2+) concentrations (1.25, 1.00 and 0.75 mmol/l) in tubular Na+ transport of pre-hypertensive MHS rats. At the Ca2+ concentration of 1.0 mmol/l, the following parameters were significantly higher (P less than 0.01) in MHS: glomerular filtration rate (GFR) and urinary volume (UV), +104.8 and +147.2%, respectively; urinary Na+ excretion (UNa+), +318.0%; tubular Na+ reabsorption (TNa+), +91.0% and oxygen consumption Q O2, +42.9%. Raising the Ca2+ concentration to 1.25 mmol/l increased UV (+167.0%) and UNa+ (+231.0%) in MHS without substantially affecting MNS. The difference between the two strains was therefore considerably larger for these parameters, but disappeared for TNa+ and Q O2. The overall kidney function of MHS was poor as compared wit renal function at Ca2+ 1.0 mmol/l. The differences between the two strains virtually disappeared at the lowest Ca2+ concentration (0.75 mmol/l); only GFR and TNa+ remained slightly greater in MHS mainly because of the poor viability of MNS kidneys in this experimental condition. It thus appears that the lower activity of Ca2+-ATPase of MHS compared with MNS plasma cell membranes influences the differences in Na+ and water handling in the two strains