Systematic analysis of a novel human renal glomerulus-enriched gene expression dataset.

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

Expression of the chemokine receptor CCR6 in human renal inflammation

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 studie

Podoplanin-positive cells are a hallmark of encapsulating peritoneal sclerosis

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 EP

Expansion-enhanced super-resolution radial fluctuations enable nanoscale molecular profiling of pathology specimens

Expansion microscopy physically enlarges biological specimens to achieve nanoscale resolution using diffraction-limited microscopy systems1. However, optimal performance is usually reached using laser-based systems (for example, confocal microscopy), restricting its broad applicability in clinical pathology, as most centres have access only to light-emitting diode (LED)-based widefield systems. As a possible alternative, a computational method for image resolution enhancement, namely, super-resolution radial fluctuations (SRRF)2,3, has recently been developed. However, this method has not been explored in pathology specimens to date, because on its own, it does not achieve sufficient resolution for routine clinical use. Here, we report expansion-enhanced super-resolution radial fluctuations (ExSRRF), a simple, robust, scalable and accessible workflow that provides a resolution of up to 25 nm using LED-based widefield microscopy. ExSRRF enables molecular profiling of subcellular structures from archival formalin-fixed paraffin-embedded tissues in complex clinical and experimental specimens, including ischaemic, degenerative, neoplastic, genetic and immune-mediated disorders. Furthermore, as examples of its potential application to experimental and clinical pathology, we show that ExSRRF can be used to identify and quantify classical features of endoplasmic reticulum stress in the murine ischaemic kidney and diagnostic ultrastructural features in human kidney biopsies.</p

Lymphotoxin expression in human and murine renal allografts

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 LT beta, 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 NF kappa B pathway, most likely a consequence of LT beta 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 LTa, LT beta and LIGHT was shown in biopsies with borderline lesions and AR. Immunohistochemistry revealed expression of LT beta 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

Inhibition of Aerobic Glycolysis Attenuates Disease Progression in Polycystic Kidney Disease

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

Effect of Sodium-Glucose Cotransport Inhibition on Polycystic Kidney Disease Progression in PCK Rats.

The sodium-glucose-cotransporter-2 (SGLT2) inhibitor dapagliflozin (DAPA) induces glucosuria and osmotic diuresis via inhibition of renal glucose reabsorption. Since increased diuresis retards the progression of polycystic kidney disease (PKD), we investigated the effect of DAPA in the PCK rat model of PKD. DAPA (10 mg/kg/d) or vehicle was administered by gavage to 6 week old male PCK rats (n=9 per group). Renal function, albuminuria, kidney weight and cyst volume were assessed after 6 weeks of treatment. Treatment with DAPA markedly increased glucose excretion (23.6 ± 4.3 vs 0.3 ± 0.1 mmol/d) and urine output (57.3 ± 6.8 vs 19.3 ± 0.8 ml/d). DAPA-treated PCK rats had higher clearances for creatinine (3.1 ± 0.1 vs 2.6 ± 0.2 ml/min) and BUN (1.7 ± 0.1 vs 1.2 ± 0.1 ml/min) after 3 weeks, and developed a 4-fold increase in albuminuria. Ultrasound imaging and histological analysis revealed a higher cyst volume and a 23% higher total kidney weight after 6 weeks of DAPA treatment. At week 6 the renal cAMP content was similar between DAPA and vehicle, and staining for Ki67 did not reveal an increase in cell proliferation. In conclusion, the inhibition of glucose reabsorption with the SGLT2-specific inhibitor DAPA caused osmotic diuresis, hyperfiltration, albuminuria and an increase in cyst volume in PCK rats. The mechanisms which link glucosuria to hyperfiltration, albuminuria and enhanced cyst volume in PCK rats remain to be elucidated

Inhibition of Sodium-GlucoseCotransporter 2 with dapagliflozin in Han: SPRD rats with polycystic kidney disease

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

CD147 expression in peritoneal injury

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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

Image3_A high-throughput drug discovery pipeline to optimize kidney normothermic machine perfusion.TIFF

Kidney transplantation is the only definitive therapy for end-stage kidney disease. The shortage of organs for transplantation is the main limitation of this life-saving treatment. Normothermic machine perfusion (NMP) is a novel preservation technique with the potential to increase the number of transplantable kidneys through reducing delayed graft function and organ evaluation under physiological conditions. To date, the cellular effects and possible pharmacological interventions during machine perfusion are incompletely understood. A major limitation is the technically complex, time-consuming, and small-scale replication of NMP in rodent models. To overcome this, we developed a 3D-printed, high throughput ex-vivo mouse kidney slice incubator (KSI) mimicking mouse kidney NMP by working under closely resembling conditions. KSI significantly reduced the time per experiment and increased the sample throughput (theoretical: 54 incubations with n = 500/day). The model recapitulated the cellular responses during NMP, namely increased endoplasmic reticulum stress (ER stress). Using KSI, five pharmacological interventions against ER stress taken from the literature were tested. While four were ineffective and excluded, one, β-Nicotinamide-adenine-dinucleotide (NADH), ameliorated ER stress significantly during KSI. The test of NADH in mouse kidney NMP replicated the positive effects against ER stress. This suggests that testing the addition of NADH during clinical kidney NMP might be warranted.</p