Antibody recognition of complement factor H reveals a flexible loop involved in atypical hemolytic uremic syndrome pathogenesis.

Atypical hemolytic uremic syndrome (aHUS) is a disease associated with dysregulation of the immune complement system, especially of the alternative pathway (AP). Complement factor H (CFH), consisting of 20 domains called complement control protein (CCP1-20), downregulates the AP as a cofactor for mediating C3 inactivation by complement factor I. However, anomalies related to CFH are known to cause excessive complement activation and cytotoxicity. In aHUS, mutations and the presence of anti-CFH autoantibodies (AAbs) have been reported as plausible causes of CFH dysfunction, and it is known that CFH-related aHUS carries a high probability of end-stage renal disease. Elucidating the detailed functions of CFH at the molecular level will help to understand aHUS pathogenesis. Herein, we used biophysical data to reveal that a heavy-chain antibody fragment, termed VHH4, recognized CFH with high affinity. Hemolytic assays also indicated that VHH4 disrupted the protective function of CFH on sheep erythrocytes. Furthermore, X-ray crystallography revealed that VHH4 recognized the Leu1181-Leu1189CCP20 loop, a known anti-CFH AAbs epitope. We next analyzed the dynamics of the C-terminal region of CFH and showed that the epitopes recognized by anti-CFH AAbs and VHH4 were the most flexible regions in CCP18-20. Finally, we conducted mutation analyses to elucidate the mechanism of VHH4 recognition of CFH and revealed that VHH4 inserts the Trp1183CCP20 residue of CFH into the pocket formed by the complementary determining region 3 loop. These results suggested that anti-CFH AAbs may adopt a similar molecular mechanism to recognize the flexible loop of Leu1181-Leu1189CCP20, leading to aHUS pathogenesis.

Decreased IFT88 expression with primary cilia shortening causes mitochondrial dysfunction in cisplatin-induced tubular injury.

The relevance of primary cilia shortening in kidney disease and its pathomechanism are largely unknown. Tubular damage in acute kidney injury (AKI) is strongly associated with mitochondrial dysfunction. Thus, we investigated the interaction between primary cilia and mitochondria in cisplatin-induced AKI mouse models. We observed that the expression of intraflagellar transport 88 (IFT88), a ciliary maintenance protein, was decreased in the renal cortex following tubular damage due to cisplatin-induced AKI. This result was consistent with the decreased IFT88 expression in cisplatin-treated RPTEC/TERT1 cells (human primary proximal tubular cells) parallel to the shortening of primary cilia, suggesting a causative link between tubular damage and IFT88-mediated cilia regulation. To address the effect of impaired primary cilia with decreased IFT88 expression on tubular function, RPTEC/TERT1 cells treated with cisplatin and knocked down for IFT88 using siRNA (IFT88-KD) were assessed for phenotypic changes and mitochondrial metabolic function. Both cisplatin and IFT88-KD caused primary cilia shortening, downregulated mitochondrial oxidative phosphorylation capacity, and had defective fatty acid oxidation and decreased ATP production. Furthermore, IFT88 overexpression enhanced mitochondrial respiration, which partially counteracted cisplatin-induced defective fatty acid oxidation. These results are indicative of the contribution of IFT88 to mitochondrial homeostasis. Our findings suggest that tubular mitochondrial dysfunction in cisplatin-induced AKI is mediated, at least in part, by a decrease in IFT88 expression with primary cilia shortening. That is, tubular mitochondrial damage followed by tubular injury in AKI may occur through alteration of IFT88 expression and subsequent ciliary shortening in tubular cells.NEW & NOTEWORTHY Here, we demonstrated organelle cross-talk between primary cilia and mitochondria of proximal tubular cells in cisplatin-induced acute kidney injury. The primary cilia-mitochondria interaction may open new avenues for the development of novel therapeutic approaches in the treatment of acute kidney injury.

Efficient Gene Transfer to Kidney Mesenchymal Cells Using a Synthetic Adeno-Associated Viral Vector.

BACKGROUND: After injury, mesenchymal progenitors in the kidney interstitium differentiate into myofibroblasts, cells that have a critical role in kidney fibrogenesis. The ability to deliver genetic material to myofibroblast progenitors could allow new therapeutic approaches to treat kidney fibrosis. Preclinical and clinical studies show that adeno-associated viruses (AAVs) efficiently and safely transduce various tissue targets in vivo; however, protocols for transduction of kidney mesenchymal cells have not been established. METHODS: We evaluated the transduction profiles of various pseudotyped AAV vectors expressing either GFP or Cre recombinase reporters in mouse kidney and human kidney organoids. RESULTS: Of the six AAVs tested, a synthetic AAV called Anc80 showed specific and high-efficiency transduction of kidney stroma and mesangial cells. We characterized the cell specificity, dose dependence, and expression kinetics and showed the efficacy of this approach by knocking out Gli2 from kidney mesenchymal cells by injection of Anc80-Cre virus into either homozygous or heterozygous Gli2-floxed mice. After unilateral ureteral obstruction, the homozygous Gli2-floxed mice had less fibrosis than the Gli2 heterozygotes had. We observed the same antifibrotic effect in ß-catenin-floxed mice injected with Anc80-Cre virus before obstructive injury, strongly supporting a central role for canonical Wnt signaling in kidney myofibroblast activation. Finally, we showed that the Anc80 synthetic virus can transduce the mesenchymal lineage in human kidney organoids. CONCLUSIONS: These studies establish a novel method for inducible knockout of floxed genes in mouse mesangium, pericytes, and perivascular fibroblasts and are the foundation for future gene therapy approaches to treat kidney fibrosis.

The reduced expression of proximal tubular transporters in acquired Fanconi syndrome with κ light chain deposition.

In a case of acquired Fanconi syndrome associated with smoldering myeloma, we confirmed the deposition of protease-resistant κ light chain proteins in a proximal tubular injury and found the decreased expression of apical tubular transporters including sodium glucose co-transporter, sodium phosphate co-transporter, uric acid transporter 1, and a decrease of Na(+)/K(+)-ATPase in the basolateral membrane. The protease-resistant kappa light chain has a pathological role in the expression of tubular transporters in the proximal tubule and causes Fanconi syndrome associated with smoldering myeloma.

Eculizumab for adult patients with atypical haemolytic-uraemic syndrome: full dataset analysis of Japanese post-marketing surveillance.

BACKGROUND: Eculizumab has been approved for atypical haemolytic-uraemic syndrome (aHUS) in Japan since 2013. Post-marketing surveillance enrolled patients with aHUS who received ≥ 1 dose of eculizumab to assess eculizumab safety and effectiveness. METHODS: We evaluated serious adverse events and effectiveness endpoints, i.e., haematologic normalization, a decrease of ≥ 25% in serum creatinine (sCr) levels, and complete thrombotic microangiopathy (TMA) response in adult patients with aHUS without other underlying diseases. In addition, the difference of baseline characteristics between patients who did and did not meet effectiveness endpoints was examined. RESULTS: In this safety and effectiveness analysis, 79 adult patients were included; median age was 54.0 years, median treatment duration was 30 weeks. Total exposure time of eculizumab was 75.51 patient-years, and 94 serious adverse events were reported in 39 patients. No unexpected safety signals were identified in this population. Mean platelet count, lactate dehydrogenase and estimated glomerular filtration rate significantly improved after 7 days of treatment. Complete TMA response, haematologic normalization and the improvement of sCr levels were met by 35.3%, 40.4% and 51.3% of patients, respectively. Median treatment duration was shorter in patients who did not achieve complete TMA response (6 weeks) than in patients who did (114 weeks). Multivariate analysis suggested that the time from the most recent TMA episode to start of eculizumab treatment was negatively associated with kidney function improvement. CONCLUSIONS: No unexpected safety signals of eculizumab were identified in Japanese patients with aHUS in a real-world setting. Renal outcomes were negatively associated with the time from the most recent TMA episode to the initiation of eculizumab treatment.

A case of malignant hypertension as a presentation of atypical hemolytic uremic syndrome.

INTRODUCTION: Malignant hypertension (mHTN) damages multiple target organs, including the kidneys. mHTN has been regarded as one of the causes of secondary thrombotic microangiopathy (TMA); however, a high prevalence of complement gene abnormalities was recently reported in cohorts of mHTN. CASE REPORT: We herein describe a 47-year-old male who presented with severe hypertension, renal failure (serum creatinine (sCr): 11.6 mg/dL), heart failure, retinal hemorrhage, hemolytic anemia, and thrombocytopenia. Renal biopsy findings were consistent with acute hypertensive nephrosclerosis. The patient was diagnosed with secondary TMA associated with mHTN. However, his previous medical history of TMA of unknown origin and family history of atypical hemolytic uremic syndrome (aHUS) suggested as aHUS presenting mHTN, and genetic testing revealed a pathogenic C3 mutation (p.I1157T). The patient required plasma exchange and hemodialysis for 2 weeks and was able to withdraw from dialysis by antihypertensive therapy without eculizumab. Renal function gradually improved to a sCr level of 2.7 mg/dL under antihypertensive therapy for 2 years after the event. There was no recurrence, and renal function was preserved throughout a 3-year follow-up. DISCUSSION: mHTN is a common presentation of aHUS. In cases of mHTN, abnormalities in complement-related genes may be involved in the development of the disease.

Immunohistochemistry May Not Replace Immunofluorescence in Paraffin-embedded Tissue for Detecting Masked Monoclonal Immunoglobulin Deposits.

An 84-year-old man developed a membranoproliferative glomerulonephritis pattern of injury, and the most likely cause detected during a workup was monoclonal IgG-λ in the urine and serum. Predominant IgG and λ light chain deposition was confirmed only by immunofluorescence using formalin-fixed, paraffin-embedded tissue and not by immunohistochemistry. A smaller and non-linear dynamic range of immunohistochemistry makes it less quantitative than immunofluorescence staining and may explain why immunohistochemistry failed to detect the light chain restriction. This case suggests that immunohistochemistry may not serve as a substitute for immunofluorescence on formalin-fixed, paraffin-embedded tissue in detecting masked monoclonal immunoglobulin deposits, although further research is warranted.

Immunoelectron microscopy findings in a patient with C3 glomerulonephritis.

We performed a kidney biopsy in a 36-year-old man to evaluate microscopic hematuria and proteinuria. Light microscopy showed increased mesangial matrix and partial swelling of the glomerular basement membrane (GBM), and immunofluorescence showed positive staining only for C3. Immunoelectron microscopy showed that gold particle-labeled C3 was localized in the electron-dense and moderately electron-dense deposits shown by electron microscopy in the mesangium, the thickened GBM near the paramesangium, and the thickened distal portion of the GBM but was not localized in the non-thickened GBM. Gold-labeled immunoglobulin G, κ, and λ were not seen. C3 glomerulonephritis was more evident in gold-labeled electron microscopy, which further clarified the localization of C3 deposition.

Glyoxalase I retards renal senescence.

Although kidney functions deteriorate with age, little is known about the general morphological alterations and mechanisms of renal senescence. We hypothesized that carbonyl stress causes senescence and investigated the possible role of glyoxalase I (GLO1), which detoxifies precursors of advanced glycation end products in the aging process of the kidney. We observed amelioration of senescence in GLO1-transgenic aged rats (assessed by expression levels of senescence markers such as p53, p21(WAF1/CIP1), and p16(INK4A)) and a positive rate of senescence-associated ß-galactosidase (SABG) staining, associated with reduction of renal advanced glycation end product accumulation (estimated by the amount of carboxyethyl lysine). GLO1-transgenic rats showed amelioration of interstitial thickening (observed as an age-related presentation in human renal biopsy specimens) and were protected against age-dependent decline of renal functions. We used GLO1 overexpression or knockdown in primary renal proximal tubular epithelial cells to investigate the effect of GLO1 on cellular senescence. Senescence markers were significantly up-regulated in renal proximal tubular epithelial cells at late passage and in those treated with etoposide, a chemical inducer of senescence. GLO1 cellular overexpression ameliorated and knockdown enhanced the cellular senescence phenotypes. Furthermore, we confirmed the association of decreased GLO1 enzymatic activity and age-dependent deterioration of renal function in aged humans with GLO1 mutation. These findings indicate that GLO1 ameliorates carbonyl stress to retard renal senescence.

A case of de novo focal segmental glomerulosclerosis occurred one and half years after kidney transplantation supposed to be caused by calcineurin inhibitor.

A 64-yr-old man with end-stage kidney disease caused by hypertensive nephrosclerosis underwent living-unrelated ABO-identical kidney transplantation (KTx) at the age of 60 yr from his 60-yr-old wife. Maintenance trough concentration of cyclosporine A (CsA) was 100 ± 30 ng/mL. Five months after KTx, proteinuria gradually increased to around 1 g/d. TRBx at eight months after KTx revealed the new-onset alteration of mild arteriolosclerosis with intimal hyalinosis, which might reflect calcineurin inhibitor (CNI)-associated arteriopathy (CAA). Nearly one and half years after KTx, urinary protein excretion became nearly 2 g/d. TRBx revealed the advanced CAA and findings of focal segmental glomerulosclerosis (FSGS). Then, CNI was switched from CsA to tacrolimus (TAC). TRBx at two and half years after KTx revealed progressed arteriolar transmural thickening and striped fibrosis, which were supposed to be induced by an increase in serum TAC concentration because of acute enterocolitis. Then, TAC dose was reduced to serum trough concentration 5-8 ng/mL, but urinary protein excretion was increased up to 10 g/d. Reduction of TAC to trough concentration 2.0 ± 0.5 ng/mL reduced urinary protein excretion. Attempts to elevate TAC trough concentration within normal range (4-8 ng/mL) reproducibly induced the recurrence of an increase in sCr or urinary protein excretion. All these findings supported the etiology of graft dysfunction, and proteinuria of this case was FSGS.

Pathogenesis of Atypical Hemolytic Uremic Syndrome.

Atypical hemolytic uremic syndrome (aHUS) is a type of thrombotic microangiopathy (TMA) defined by thrombocytopenia, microangiopathic hemolytic anemia, and renal failure. aHUS is caused by uncontrolled complement activation in the alternative pathway (AP). A variety of genetic defects in complement-related factors or acquired autoantibodies to the complement regulators have been found in 50 to 60% of all cases. Recently, however, the classification and diagnosis of aHUS are becoming more complicated. One reason for this is that some factors, which have not been regarded as complement-related factors, have been reported as predisposing factors for phenotypic aHUS. Given that genotype is highly correlated with the phenotype of aHUS, careful analysis of underlying genetic abnormalities or acquired factors is needed to predict the prognosis or to decide an optimal treatment for the disease. Another reason is that complement dysregulation in the AP have also been found in a part of other types of TMA such as transplantation-related TMA and pregnancy-related complication. Based on these findings, it is now time to redefine aHUS according to the genetic or acquired background of abnormalities.Here, we review the pathogeneses and the corresponding phenotypes of aHUS and complement-related TMAs.

Wnt signaling in kidney tubulointerstitium during disease.

The evolutionary conserved Wnt signaling transduction pathway plays essential roles in a wide array of biologic processes including embryonic development, branching morphogenesis, proliferation and carcinogenesis. Over the past ten years it has become increasingly clear that Wnt signaling also regulates the response of adult organs to disease processes, including kidney disease. This review will focus on the growing literature implicating important roles for Wnt signaling during disease in two separate kidney compartments: the tubular epithelium and the interstitium.

Pharmacological GLI2 inhibition prevents myofibroblast cell-cycle progression and reduces kidney fibrosis.

Chronic kidney disease is characterized by interstitial fibrosis and proliferation of scar-secreting myofibroblasts, ultimately leading to end-stage renal disease. The hedgehog (Hh) pathway transcriptional effectors GLI1 and GLI2 are expressed in myofibroblast progenitors; however, the role of these effectors during fibrogenesis is poorly understood. Here, we demonstrated that GLI2, but not GLI1, drives myofibroblast cell-cycle progression in cultured mesenchymal stem cell-like progenitors. In animals exposed to unilateral ureteral obstruction, Hh pathway suppression by expression of the GLI3 repressor in GLI1+ myofibroblast progenitors limited kidney fibrosis. Myofibroblast-specific deletion of Gli2, but not Gli1, also limited kidney fibrosis, and induction of myofibroblast-specific cell-cycle arrest mediated this inhibition. Pharmacologic targeting of this pathway with darinaparsin, an arsenical in clinical trials, reduced fibrosis through reduction of GLI2 protein levels and subsequent cell-cycle arrest in myofibroblasts. GLI2 overexpression rescued the cell-cycle effect of darinaparsin in vitro. While darinaparsin ameliorated fibrosis in WT and Gli1-KO mice, it was not effective in conditional Gli2-KO mice, supporting GLI2 as a direct darinaparsin target. The GLI inhibitor GANT61 also reduced fibrosis in mice. Finally, GLI1 and GLI2 were upregulated in the kidneys of patients with high-grade fibrosis. Together, these data indicate that GLI inhibition has potential as a therapeutic strategy to limit myofibroblast proliferation in kidney fibrosis.

[Serum calcium concentration and the safety of vitamin D therapy].

Vitamin D and its analogues are administered to patients with secondary hyperparathyroidism (SHPT) in chronic renal failure (CRF). Hypercalcemia is one of the complications of this therapy, and is thought to be not rare, though its frequency is not clearly documented. There is a need to monitor serum corrected (ionized) calcium concentration and parathyroid hormone (PTH) in order to administer vitamin D and its analogues safely. Intravenous administration of vitamin D provides advantages over oral administration, though no definite evidence could show which is better. Calcitriol and maxacalcitol are administered intravenously, and the latter is more likely to increase serum calcium concentration than the former. It must be very careful to administer vitamin D to patients with predialysis CRF because hypercalcemia can accelerate progression of renal dysfunction.

Glyoxalase I reduces glycative and oxidative stress and prevents age-related endothelial dysfunction through modulation of endothelial nitric oxide synthase phosphorylation.

Endothelial dysfunction is a major contributor to cardiovascular disease (CVD), particularly in elderly people. Studies have demonstrated the role of glycation in endothelial dysfunction in nonphysiological models, but the physiological role of glycation in age-related endothelial dysfunction has been poorly addressed. Here, to investigate how vascular glycation affects age-related endothelial function, we employed rats systemically overexpressing glyoxalase I (GLO1), which detoxifies methylglyoxal (MG), a representative precursor of glycation. Four groups of rats were examined, namely young (13 weeks old), mid-age (53 weeks old) wild-type, and GLO1 transgenic (WT/GLO1 Tg) rats. Age-related acceleration in glycation was attenuated in GLO1 Tg rats, together with lower aortic carboxymethyllysine (CML) and urinary 8-hydroxydeoxyguanosine (8-OHdG) levels. Age-related impairment of endothelium-dependent vasorelaxation was attenuated in GLO1 Tg rats, whereas endothelium-independent vasorelaxation was not different between WT and GLO1 Tg rats. Nitric oxide (NO) production was decreased in mid-age WT rats, but not in mid-age GLO1 Tg rats. Age-related inactivation of endothelial NO synthase (eNOS) due to phosphorylation of eNOS on Thr495 and dephosphorylation on Ser1177 was ameliorated in GLO1 Tg rats. In vitro, MG increased phosphorylation of eNOS (Thr495) in primary human aortic endothelial cells (HAECs), and overexpression of GLO1 decreased glycative stress and phosphorylation of eNOS (Thr495). Together, GLO1 reduced age-related endothelial glycative and oxidative stress, altered phohphorylation of eNOS, and attenuated endothelial dysfunction. As a molecular mechanism, GLO1 lessened inhibitory phosphorylation of eNOS (Thr495) by reducing glycative stress. Our study demonstrates that blunting glycative stress prevents the long-term impact of endothelial dysfunction on vascular aging.

Downregulation of miR-205 modulates cell susceptibility to oxidative and endoplasmic reticulum stresses in renal tubular cells.

BACKGROUND: Oxidative stress and endoplasmic reticulum (ER) stress play a crucial role in tubular damage in both acute kidney injury (AKI) and chronic kidney disease (CKD). While the pathophysiological contribution of microRNAs (miRNA) to renal damage has also been highlighted, the effect of miRNA on renal damage under oxidative and ER stresses conditions remains elusive. METHODS: We assessed changes in miRNA expression in the cultured renal tubular cell line HK-2 under hypoxia-reoxygenation-induced oxidative stress or ER stress using miRNA microarray assay and real-time RT-PCR. The pathophysiological effect of miRNA was evaluated by cell survival rate, intracellular reactive oxygen species (ROS) level, and anti-oxidant enzyme expression in miRNA-inhibited HK-2 or miRNA-overexpressed HK-2 under these stress conditions. The target gene of miRNA was identified by 3'-UTR-luciferase assay. RESULTS: We identified 8 and 10 miRNAs whose expression was significantly altered by oxidative and ER stresses, respectively. Among these, expression of miR-205 was markedly decreased in both stress conditions. Functional analysis revealed that decreased miR-205 led to an increase in cell susceptibility to oxidative and ER stresses, and that this increase was associated with the induction of intracellular ROS and suppression of anti-oxidant enzymes. While increased miR-205 by itself made no change in cell growth or morphology, cell viability under oxidative or ER stress conditions was partially restored. Further, miR-205 bound to the 3'-UTR of the prolyl hydroxylase 1 (PHD1/EGLN2) gene and suppressed the transcription level of EGLN2, which modulates both intracellular ROS level and ER stress state. CONCLUSIONS: miR-205 serves a protective role against both oxidative and ER stresses via the suppression of EGLN2 and subsequent decrease in intracellular ROS. miR-205 may represent a novel therapeutic target in AKI and CKD associated with oxidative or ER stress in tubules.