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Mechanotransduction is an important process of sensing physical forces in the environment of organisms, tissues and cells and transducing them into a biochemical response. Due to their position on the glomerular capillaries, podocytes are exposed to near-constant biomechanical force, which can fluctuate widely. These include shear stress and hydrostatic pressure. A pathological increase in these forces can induce morphological change to podocytes, their detachment from the glomerular basement membrane and subsequent loss into the primary urine. The ability to sense and respond to variations in mechanical force would be beneficial to a cell exposed to these conditions. It is likely podocytes have such mechanisms, however their identity are unknown. Here we investigated the hypothesis that the mechanotransducer Piezo is involved in a mechanotransduction pathway in Drosophila nephrocytes, the podocyte homologue in the fly. We find Piezo is expressed in nephrocytes and localizes to the nephrocyte diaphragm. The Piezo agonist YODA, which stimulates channel opening in the absence of mechanical force, leads to a significant increase in intracellular Ca++ upon shear stress in the nephrocyte. This leads to activation of Rho1, delineating a putative Piezo mechanotransductive pathway in these cells. Loss of function analysis revealed minor defects in nephrocyte filtration function. In contrast, we show that elevated Piezo levels resulted in constantly oscillating Ca++ signals even in the absence of shear stress, increased active Rho1 and accumulation of actin stress fibers, culminating in a severe nephrocyte filtration phenotype, suggesting that pathway hyperactivity is detrimental. We asked if this phenotype could be reversed by blocking Piezo activity pharmacologically using the tarantula toxin GsMTx4. Treatment with GsMTx4 brought levels of activated Rho1 into the normal range. This work delineates a mechanotransductive pathway in nephrocytes involving Piezo, Ca++, Rho1 and the actin-cytoskeleton, and suggest this is part of a mechanism by which nephrocytes sense and adapt to changes in mechanical force.

Peritoneal injury is an important cause of technical failure of long-term peritoneal dialysis (PD). Encapsulating peritoneal sclerosis (EPS) is a severe complication of long-term PD with potentially life threatening consequences. CD147 is a glycoprotein with diverse functions including modulation of extracellular matrix via induction of matrix metalloproteinases, cell adhesion, and regulation of immune reactions. We hypothesized that CD 147 plays a role in the peritoneal cavity. In this retrospective study, we localized CD147 by immunohistochemistry in peritoneal biopsies from uremic patients not on PD (n = 8), on PD without signs of EPS (n = 7), and in biopsies in patients with the diagnosis of EPS (n = 7). Double immunofluorescence was used to co-localize α-smooth-muscle actin (α-SMA) and CD147 in selected biopsies from each group. Expression was scored semi-quantitatively. In biopsies from uremic controls, CD147 was prominently expressed in mesothelial cells, focally between fat cells and by some perivascular cells. In patients on PD, a similar distribution was present (although mesothelium was rarely conserved), with some focal accentuation. In EPS, layers of fibroblastic cells were positive for CD147. EPS biopsies demonstrated a significantly higher score in a blinded evaluation, compared to uremic patients. Cells expressing CD147 were α-SMA positive myofibroblasts as demonstrated by double immunofluorescence. Mean CD147 scores did not differ between patients with different transporter status. This is the first study demonstrating CD147 on a major part of fibroblastic cells in EPS. Future studies need to address the role of these cells in this severe complication of long-term PD.

The kidney is the most frequently transplanted solid organ. Recruitment of inflammatory cells, ranging from diffuse to nodular accumulations with defined microarchitecture, is a hallmark of acute and chronic renal allograft injury. Lymphotoxins (LTs) mediate the communication of lymphocytes and stromal cells and play a pivotal role in chronic inflammation and formation of lymphoid tissue. The aim of this study was to assess the expression of members of the LT system in acute rejection (AR) and chronic renal allograft injury such as transplant glomerulopathy (TG) and interstitial fibrosis/tubular atrophy (IFTA). We investigated differentially regulated components in transcriptomes of human renal allograft biopsies. By microarray analysis, we found the upregulation of LTβ, LIGHT, HVEM and TNF receptors 1 and 2 in AR and IFTA in human renal allograft biopsies. In addition, there was clear evidence for the activation of the NFκB pathway, most likely a consequence of LTβ receptor stimulation. In human renal allograft biopsies with transplant glomerulopathy (TG) two distinct transcriptional patterns of LT activation were revealed. By quantitative RT-PCR robust upregulation of LTα, LTβ and LIGHT was shown in biopsies with borderline lesions and AR. Immunohistochemistry revealed expression of LTβ in tubular epithelial cells and inflammatory infiltrates in transplant biopsies with AR and IFTA. Finally, activation of LT signaling was reproduced in a murine model of renal transplantation with AR. In summary, our results indicate a potential role of the LT system in acute renal allograft rejection and chronic transplant injury. Activation of the LT system in allograft rejection in rodents indicates a species independent mechanism. The functional role of the LT system in acute renal allograft rejection and chronic injury remains to be determined.

Background. Encapsulating peritoneal sclerosis (EPS) and simple peritoneal sclerosis are important complications of long-term peritoneal dialysis (PD). Podoplanin is expressed by mesothelial cells and lymphatic vessels, which are involved in inflammatory reactions in the peritoneal cavity. Methods. We studied 69 peritoneal biopsies from patients on PD (n = 16), patients with EPS (n = 18) and control biopsies taken at the time of hernia repair (n = 15) or appendectomy (n = 20). Immunohistochemistry was performed to localize podoplanin. Additionally, markers of endothelial cells, mesothelial cells, myofibroblasts (smooth muscle actin), proliferating cells, and double labelling for smooth muscle actin/podoplanin were used on selected biopsies. Results. Podoplanin was present on the endothelium of lymphatic vessels in the submesothelial fibrous tissue and on mesothelial cells. In patients on PD and in biopsies with appendicitis, the mesothelial cells demonstrated a cuboidal appearance and circumferential podoplanin staining, with gaps between the cells. The number of lymphatic vessels was variable, but prominent at sites of fibrosis. In patients with EPS, a diffuse infiltration of podoplanin-positive cells with a fibroblastic appearance was present in 15 out of 18 biopsies. This pattern was focally present in 3 out of 16 on PD and none in the 35 controls. The podoplanin-positive cells did not express the endothelial marker or the mesothelial marker (calretinin). Conclusions. EPS is characterized by a population of podoplanin and smooth muscle actin double-positive cells. Podoplanin might be a suitable morphological marker supporting the diagnosis and might be involved in the pathogenesis of EPS.

Blocking the CD40-CD154 pathway prevents allograft rejection and induces donor-specific tolerance in various experimental models. However, the translation to clinical studies has been hampered by unexpected thromboembolic complications of CD154-blocking antibodies. Thus, blocking CD40 instead is now considered as an alternative strategy. Here, we evaluated the role of donor CD40 in allospecific T-cell responses in vitro and in an in vivo model for renal transplantation. Fully MHC-mismatched allografts from CD40-deficient donors displayed better renal function than wild type. These functional data correlated with a lower level of apoptosis in renal tubular epithelial cells and higher expression of PD-L1, which is most probably because of a reduced Th17 response in recipients of a CD40-deficient donor. This hypothesis was supported in vitro, where donor CD40 expression was important for the induction of direct allospecific T-cell responses. Especially the induction of Th17 cells was critically dependent on donor CD40. IL-17A in conjunction with interferon-γ in turn rendered renal tubular epithelial cells to a more costimulatory state by upregulating CD40 and downregulating PD-L1 expression. In conclusion, CD40 blockade not only reduces the allospecific T-cell responses, but might also lead to protection of tubular epithelium from apoptosis and thereby preserve kidney allograft function.

DNAX accessory protein-1 (DNAM-1, CD226) is a co-stimulatory and adhesion molecule expressed mainly by natural killer cells and T cells. DNAM-1 and its two ligands CD112 and CD155 are important in graft-versus-host disease, but their role in solid organ transplantation is largely unknown. We investigated the relevance of this pathway in a mouse kidney transplantation model. CD112 and CD155 are constitutively expressed on renal tubular cells and strongly upregulated in acutely rejected renal allografts. In vitro DNAM-1 blockade during allogeneic priming reduced the allospecific T cell response but not the allospecific cytotoxicity against renal tubular epithelial cells. Accordingly, absence of DNAM-1 in recipient mice or absence of CD112 or CD155 in the kidney allograft did not significantly influence renal function and severity of rejection after transplantation, but led to a higher incidence of infarcts in CD112 and CD155 deficient kidney allografts. Thus, DNAM-1 blockade is not effective in preventing transplant rejection. Despite of being highly expressed, CD112 and CD155 do not appear to play a major immunogenic role in kidney transplantation. Considering the high incidence of renal infarcts in CD112 and CD155 deficient grafts, blocking these molecules might be detrimental.

Dysregulated signaling cascades alter energy metabolism and promote cell proliferation and cyst expansion in polycystic kidney disease (PKD). Here we tested whether metabolic reprogramming towards aerobic glycolysis ("Warburg effect") plays a pathogenic role in male heterozygous Han:SPRD rats (Cy/+), a chronic progressive model of PKD. Using microarray analysis and qPCR, we found an upregulation of genes involved in glycolysis (Hk1, Hk2, Ldha) and a downregulation of genes involved in gluconeogenesis (G6pc, Lbp1) in cystic kidneys of Cy/+ rats compared with wild-type (+/+) rats. We then tested the effect of inhibiting glycolysis with 2-deoxyglucose (2DG) on renal functional loss and cyst progression in 5-week-old male Cy/+ rats. Treatment with 2DG (500 mg/kg/day) for 5 weeks resulted in significantly lower kidney weights (-27%) and 2-kidney/total-body-weight ratios (-20%) and decreased renal cyst index (-48%) compared with vehicle treatment. Cy/+ rats treated with 2DG also showed higher clearances of creatinine (1.98±0.67 vs 1.41±0.37 ml/min), BUN (0.69±0.26 vs 0.40±0.10 ml/min) and uric acid (0.38±0.20 vs 0.21±0.10 ml/min), and reduced albuminuria. Immunoblotting analysis of kidney tissues harvested from 2DG-treated Cy/+ rats showed increased phosphorylation of AMPK-α, a negative regulator of mTOR, and restoration of ERK signaling. Assessment of Ki-67 staining indicated that 2DG limits cyst progression through inhibition of epithelial cell proliferation. Taken together, our results show that targeting the glycolytic pathway may represent a promising therapeutic strategy to control cyst growth in PKD.

Summary Apoptosis controls the adaptive immune system through regulation of central and peripheral lymphocyte deletion. Therefore, substances that selectively interact with the intrinsic apoptosis pathway in lymphocytes offer unexplored opportunities to pharmacologically modulate the immune response. Here, we present evidence that the BH3-mimetic ABT-737 suppresses allogeneic immune responses. In vitro, ABT-737 prevented allogeneic T-cell activation, proliferation, and cytotoxicity by apoptosis induction, but without impairing the physiological functions of remaining viable T cells. In vivo, ABT-737 was highly selective for lymphoid cells and inhibited allogeneic T- and B-cell responses after skin transplantation. The immunosuppressive effect of ABT-737 was markedly increased in combination with low-dose cyclosporine A, as shown by the induction of long-term skin graft survival without significant inflammatory infiltrates in 50% of the recipients in an MHC class I single antigen mismatched model. Thus, pharmacological targeting of Bcl-2 proteins represents a novel immunosuppressive approach to prevent rejection of solid organ allografts.

Background Nodular inflammatory cell infiltrates with defined microarchitecture, i.e. tertiary lymphoid organs, develop in the tubulointerstitium during chronic renal inflammation. CCR6 and the corresponding ligand CCL20 are involved in the formation of gut-associated lymphatic tissue. We hypothesized that CCR6 might be involved in the formation of nodular infiltrates in the kidney. Methods CCR6- and CD20-positive B cells were localized in renal biopsies with IgA nephropathy (n = 13), membranous nephropathy (n = 12), crescentic glomerulonephritis (cGN, n = 11) and chronic interstitial nephritis (n = 13), and in pre-implantation biopsies as controls (n = 8). The mRNA expression of CCR6 and the ligand CCL20 was quantified by real-time RT-PCR in 51 renal biopsies of the same disease entities. Results In the pre-transplant biopsies, CCR6 was expressed by endothelial cells of peritubular and glomerular capillaries. In patients with glomerulonephritis, infiltrating cells were positive particularly in areas of nodular inflammatory cell accumulations. A major part of the CCR6-positive cells were CD20-positive B cells, but a part of the CD3-positive T cells were also found to be positive. The constitutive expression of CCR6 on the endothelium of glomerular capillaries was lost in biopsies with progressive injury. Tubular epithelial cells expressed CCR6 in inflamed kidneys, most commonly on the basolateral side. Conclusions CCR6 and the corresponding ligand CCL20 might therefore be involved in the recruitment of T and B cells to organized nodular infiltrates in chronic renal inflammation. The functional role of endothelial CCR6 needs to be evaluated in further studies.

In the kidney, hypoxia contributes to tubulointerstitial fibrosis, but little is known about its implications for glomerular damage and glomerulosclerosis. Chronic hypoxia was hypothesized to be involved in nephrosclerosis (NSC) or "hypertensive nephropathy." In the present study genome-wide expression data from microdissected glomeruli were studied to examine the role of hypoxia in glomerulosclerosis of human NSC. Functional annotation analysis revealed prominent regulation of hypoxia-associated biological processes in NSC, including angiogenesis, fibrosis, and inflammation. Glomerular expression levels of a majority of genes regulated by the hypoxia-inducible factors (HIFs) were significantly altered in NSC. Among these HIF targets, chemokine C-X-C motif receptor 4 (CXCR4) was prominently induced. Glomerular CXCR4 mRNA induction was confirmed by quantitative RT-PCR in an independent cohort with NSC but not in those with other glomerulopathies. By immunohistological analysis, CXCR4 showed enhanced positivity in podocytes in NSC biopsy specimens. This CXCR4 positivity was associated with nuclear localization of HIF1alpha only in podocytes of NSC, indicating transcriptional activity of HIF. As the CXCR4 ligand CXCL12/SDF-1 is constitutively expressed in podocytes, autocrine signaling may contribute to NSC. In addition, a blocking CXCR4 antibody caused significant inhibition of wound closure by podocytes in an in vitro scratch assay. These data support a role for CXCR4/CXCL12 in human NSC and indicate that hypoxia not only is involved in tubulointerstitial fibrosis but also contributes to glomerular damage in NSC.

Accumulation of inflammatory cells in different renal compartments is a hallmark of progressive kidney diseases including glomerulonephritis (GN). Lymphotoxin β receptor (LTβR) signaling is crucial for the formation of lymphoid tissue, and inhibition of LTβR signaling has ameliorated several non-renal inflammatory models. Therefore, we tested whether LTβR signaling could also have a role in renal injury. Renal biopsies from patients with GN were found to express both LTα and LTβ ligands, as well as LTβR. The LTβR protein and mRNA were localized to tubular epithelial cells, parietal epithelial cells, crescents, and cells of the glomerular tuft, whereas LTβ was found on lymphocytes and tubular epithelial cells. Human tubular epithelial cells, mesangial cells, and mouse parietal epithelial cells expressed both LTα and LTβ mRNA upon stimulation with TNF in vitro. Several chemokine mRNAs and proteins were expressed in response to LTβR signaling. Importantly, in a murine lupus model, LTβR blockade improved renal function without the reduction of serum autoantibody titers or glomerular immune complex deposition. Thus, a preclinical mouse model and human studies strongly suggest that LTβR signaling is involved in renal injury and may be a suitable therapeutic target in renal diseases.

Background/Aims: Dapagliflozin (DAPA) is a selective inhibitor of the sodium-glucose cotransporter 2 (SGLT2) which induces glucosuria and osmotic diuresis. The therapeutic effect of DAPA in progressing stages of polycystic kidney disease (PKD) has not been studied. Methods: We examined the effect of DAPA in the Han: SPRD rat model of PKD. DAPA (10 mg/kg/day) or vehicle (VEH) was administered orally via gavage to 5 week old male Han: SPRD (Cy/+) or control (+/+) rats (n = 8-9 per group) for 5 weeks. Blood and urine were collected at baseline and after 2.5 and 5 weeks of treatment to assess renal function and albuminuria. At the end of the treatment, rats were sacrificed and kidneys were excised for histological analysis. Results: After 5 weeks of treatment, DAPA-treated Cy/+ and +/+ rats exhibited significantly higher glucosuria, water intake and urine output than VEH-treated rats. DAPA-treated Cy/+ rats also exhibited significantly higher clearances for creatinine and BUN and less albuminuria than VEH-treated Cy/+ rats. DAPA treatment for 5 weeks resulted in a significant increase of the kidney weight in Cy/+ rats but no change in cyst growth. The degree of tubular epithelial cell proliferation, macrophage infiltration and interstitial fibrosis was also similar in DAPA-and VEH-treated Cy/+ rats. Conclusion: The induction of glucosuria with the SGLT2-specific inhibitor DAPA was associated with improved renal function and decreased albuminuria, but had no effect on cyst growth in Cy/+ rats. Overall the beneficial effects of DAPA in this PKD model were weaker than the previously described effects of the combined SGLT1/2 inhibitor phlorizin.

Introduction: Simple peritoneal fibrosis and encapsulating peritoneal sclerosis (EPS) are important lesions in the peritoneum of patients on peritoneal dialysis (PD). We have previously described a population of podoplanin-positive myofibroblasts in peritoneal biopsies from patients with EPS. Platelet-derived growth factor receptor-β (PDGFRβ) is a marker of pericytes, and PDGFs might be involved in the fibrotic response of the peritoneum. This study aimed to describe PDGFRβ in the human peritoneum. Methods: In this retrospective analysis, we localized PDGFRβ in peritoneal biopsies from patients with EPS (n = 6) and patients on PD without signs of EPS (n = 5), and compared them with normal peritoneum (n = 4) and peritoneum from uremic patients (n = 5). Consecutive sections were stained for smooth-muscle actin (SMA) and podoplanin. Slides were scored semiquantitatively by 2 observers blinded to the diagnosis. Results: PDGFRβ was expressed by cells of arterial walls in all biopsies. A prominent population of PDGFRβ-positive cells was present in the normal peritoneum, which were SMA negative on consecutive sections. In patients on PD, a high number of PDGFRβ were also positive for SMA. In EPS, the majority of podoplanin-positive cells were positive for PDGFRβ. In peritoneal biopsies from normal and uremic patients, the expression of SMA was mainly restricted to cells of arterial walls. Podoplanin expression was restricted to lymphatic vessels in normal peritoneum, in uremic patients, and in patients on PD without EPS. Conclusions: As podoplanin-positive myofibroblasts express PDGFRβ, these cells might be related to pericytes (rather than other sources of fibroblasts). PDGFRβ might turn out to be a therapeutic target in EPS.

The B-cell survival factors APRIL and BLyS are important for B-cell maturation and activation and contribute to human autoimmune diseases. Interference with B-cell function by targeting these molecules is currently being investigated in large clinical trials for systemic lupus erythematosus. The local expression patterns of APRIL and BLyS have not been investigated in detail in kidneys with lupus nephritis. We studied the mRNA expression of APRIL, BLyS, and the corresponding receptors BCMA, TACI, and BAFF-R in microdissected human biopsies with proliferative lupus nephritis (n=25) and compared it with pretransplant biopsies of living donors (n=9). APRIL and BLyS mRNA levels were significantly higher in glomeruli of patients with proliferative lupus nephritis (12- and 30-fold, respectively). Tubulointerstitial expression of APRIL, BLyS, BCMA, and TACI was also significantly elevated. To localize the respective proteins in the kidney, APRIL, BLyS, and BAFF-R were studied by immunohistochemistry in renal biopsies with proliferative (n=21) or membranous (n=8) lupus nephritis. APRIL was prominently expressed in glomeruli with proliferative, but not membranous, lupus nephritis. The staining pattern was consistent with mesangial cells. A prominent accumulation of CD68-positive cells was present in glomeruli in association with APRIL expression. APRIL, BLyS, and BAFF-R were also expressed in interstitial inflammatory cell accumulation. This is the first study, which details local expression of APRIL and BLyS in glomeruli and tubulointerstitium of human proliferative lupus nephritis. This information might help define intrarenal effects of APRIL and BLyS inhibition in human lupus nephritis.

Glomerular diseases account for the majority of cases with chronic renal failure. Several genes have been identified with key relevance for glomerular function. Quite a few of these genes show a specific or preferential mRNA expression in the renal glomerulus. To identify additional candidate genes involved in glomerular function in humans we generated a human renal glomerulus-enriched gene expression dataset (REGGED) by comparing gene expression profiles from human glomeruli and tubulointerstitium obtained from six transplant living donors using Affymetrix HG-U133A arrays. This analysis resulted in 677 genes with prominent overrepresentation in the glomerulus. Genes with 'a priori' known prominent glomerular expression served for validation and were all found in the novel dataset (e.g. CDKN1, DAG1, DDN, EHD3, MYH9, NES, NPHS1, NPHS2, PDPN, PLA2R1, PLCE1, PODXL, PTPRO, SYNPO, TCF21, TJP1, WT1). The mRNA expression of several novel glomerulus-enriched genes in REGGED was validated by qRT-PCR. Gene ontology and pathway analysis identified biological processes previously not reported to be of relevance in glomeruli of healthy human adult kidneys including among others axon guidance. This finding was further validated by assessing the expression of the axon guidance molecules neuritin (NRN1) and roundabout receptor ROBO1 and -2. In diabetic nephropathy, a prevalent glomerulopathy, differential regulation of glomerular ROBO2 mRNA was found.In summary, novel transcripts with predominant expression in the human glomerulus could be identified using a comparative strategy on microdissected nephrons. A systematic analysis of this glomerulus-specific gene expression dataset allows the detection of target molecules and biological processes involved in glomerular biology and renal disease.

Encapsulating peritoneal sclerosis (EPS) is a life threatening complication of peritoneal dialysis (PD). Podoplanin is a glycoprotein expressed by mesothelial cells, lymphatic endothelial cells, and myofibroblasts in peritoneal biopsies from patients with EPS. To evaluate podoplanin as a marker of EPS we measured podoplanin mRNA and described the morphological patterns of podoplanin-positive cells in EPS. Included were 20 peritoneal biopsies from patients with the diagnosis of EPS (n = 5), patients on PD without signs of EPS (n = 5), and control patients (uremic patients not on PD, n = 5, non-uremic patients n = 5). EPS patient biopsies revealed significantly elevated levels of podoplanin mRNA (p

Functional consequences of integrin-linked kinase activation in podocyte damage . Background The delicate foot process architecture of glomerular podocytes critically depends on integrin mediated cell-glomerular basement membrane (GBM) interaction. Integrin signaling via the integrin-linked kinase (ILK) is activated in podocyte damage and associated with considerable podocyte phenotype alterations. ILK has been shown to regulate cell fate via nuclear interaction of β-catenin with lymphoid enhancer factor (LEF-1) transcription factors. The aim of this study was to elucidate the molecular mechanisms of ILK dependant phenotype regulation in podocytes. Methods ILK function was evaluated in conditionally immortalized murine glomerular epithelial cells using overexpression of ILK and a small molecule ILK inhibitor in puromycin/adriamycin-induced podocyte damage in vitro and in vivo . Results Kinase active, but not mutant ILK induced translocation of β-catenin to the cell nucleus, de novo expression of LEF-1, and nuclear colocalization of β-catenin and LEF-1. The role of ILK signaling in podocyte damage was evaluated using puromycin, an agent known to cause selective proteinuria and to increase ILK activity. The small molecular ILK inhibitor MC-5 blocked puromycin-induced nuclear translocation of β-catenin, podocyte detachment, cell proliferation, and repression of the slit membrane molecules P-cadherin and CD2ap. In vivo activation of the β-catenin pathway could be shown by nuclear colocalization of β-catenin with WT-1 in adriamycin nephropathy. Conclusion ILK regulates podocyte cell matrix interaction, proliferation, and slit membrane gene expression in podocyte damage. As this pathway is amendable to pharmacologic intervention, further detailed studies of in vivo ILK function in glomerular disease appear justified.

SPECIFIC AIMWe sought to identify and characterize novel molecular pathways activated in glomerular filtration barrier failure. An expression screening approach on glomeruli from children with cong...