Genetic dissection of TLR9 reveals complex regulatory and cryptic proinflammatory roles in mouse lupus.

In lupus, Toll-like receptor 7 (TLR7) and TLR9 mediate loss of tolerance to RNA and DNA, respectively. Yet, TLR7 promotes disease, while TLR9 protects from disease, implying differences in signaling. To dissect this 'TLR paradox', we generated two TLR9 point mutants (lacking either ligand (TLR9K51E) or MyD88 (TLR9P915H) binding) in lupus-prone MRL/lpr mice. Ameliorated disease of Tlr9K51E mice compared to Tlr9-/- controls revealed a TLR9 'scaffold' protective function that is ligand and MyD88 independent. Unexpectedly, Tlr9P915H mice were more protected than both Tlr9K51E and Tlr9WT mice, suggesting that TLR9 also possesses ligand-dependent, but MyD88-independent, regulatory signaling and MyD88-mediated proinflammatory signaling. Triple-mixed bone marrow chimeras showed that TLR9-MyD88-independent regulatory roles were B cell intrinsic and restrained differentiation into pathogenic age-associated B cells and plasmablasts. These studies reveal MyD88-independent regulatory roles of TLR9, shedding light on the biology of endosomal TLRs.

Morphological characteristics of SETD2-mutated locally advanced clear cell renal cell carcinoma: Comparison with BAP1-mutated clear cell renal cell carcinoma.

SET-domain containing 2 (SETD2) and BRCA1-associated protein 1 (BAP1), both chromatin remodeling genes, are frequently mutated in clear cell renal cell carcinoma (ccRCC) and involved in tumor progression and metastasis. Herein, we studied clinicopathologic features of 7 cases of locally advanced ccRCC with single SETD2 mutation, and compared to 7 cases of locally advanced ccRCC with single BAP1 mutation. SETD2-mutated ccRCC showed high-grade transformation, comprising of enlarged tumor cells with voluminous clear cytoplasm, enlarged irregular nuclei with prominent nucleoli, eosinophilic cytoplasmic granules, arranged in various architectural patterns such as large nested, tubular, tubulopapillary and solid. 71 % (5 of 7 cases) of SETD2-mutated ccRCC showed a rhabdoid morphology. SETD2-mutated ccRCC have striking propensity for invasive growth; all cases have vascular invasion and perirenal (extracapsular) adipose tissue invasion. After nephrectomy, distant metastasis was found in 67 % (4 of 7 cases) of patients with SETD2-mutated ccRCC. The most common metastatic site was the lung (3 cases), followed by precaval lymph nodes (1 case). BAP1-mutated ccRCC also showed a similar high-grade morphology, with rhabdoid and/or sarcomatoid features. Their high-grade features mostly overlapped with those of SETD2-mutated ccRCC, which makes difficult to predict the presence of BAP1 or SETD2 mutation solely from morphology. These findings justify the use of molecular testing to detect these mutations, especially when we encounter high-grade ccRCC. Detecting SETD2 and BAP1 mutation in ccRCC is useful for risk stratification and proper therapeutic strategy.

The Role of Suprapubic Superficial Fascial System Reconstruction During Repair of Adult-Acquired Buried Penis.

BACKGROUND: Adult-Acquired Buried Penis is a disorder associated with systemic obesity that confers increased risks of malignancy, sexual dysfunction, urinary abnormalities, and psychological distress. Surgical correction improves patient-reported functional and psychological outcomes and often requires collaboration between plastic and urologic surgeons. To improve postoperative cosmetic outcomes and decrease wound complications following adult-acquired buried penis repair, we performed an anatomic and histologic study of the superficial fascial layers providing support to the external male genitalia and describe our approach for fascial reconstruction. METHODS: We characterized the superficial fascial anatomy in three patients undergoing adult-acquired buried penis repair, including two patients with Wisconsin Type II disease and one patient with Wisconsin Type IV disease. Gross specimens were sent from two patients histologic analysis using H&E and elastin-specific stains to characterize the identity of the superficial fibrofatty tissue. RESULTS: In all three patients, the fundiform ligament overlying the suspensory ligament was identified, isolated, and transected for removal with the suprapubic specimen. We found that reapproximation of this ligament following transection at the time of escutcheonectomy provided significant lift to the penis and genitals via improved support of dartos fascia. Histologic analysis of the superficial fibrofatty tissue located beneath the dermis revealed histologic similarities with the superficial fascial system described previously in abdominal and breast tissue. CONCLUSIONS: Reapproximation of the fundiform ligament and superficial fascial tissue following suprapubic/lower abdominal fat pad removal during adult-acquired buried penis may improve postoperative cosmesis by reducing strain on the dermal closure. LEVEL OF EVIDENCE V: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors http://www.springer.com/00266 .

Malignant germ cell tumors in men aged 50 years and over are associated with adverse pathologic features and higher stage at presentation.

BACKGROUND: Germ cell tumors (GCT) are the most common malignancy in men in the third and fourth decades of life. The occurrence of malignant GCT in men aged 50 years or over is rare, and their histopathologic characteristics and outcome is insufficiently characterized in the medical literature. Hence, we report the histopathologic features and clinical outcome of malignant GCTs in men aged ≥50 years at our institution. DESIGN: We performed a retrospective search of our database from 2005 to 2021 to identify men aged 50 years or older with malignant GCT. Cases of spermatocytic tumor were excluded. Clinical and histopathologic features of the tumors were reviewed. RESULTS: Forty-seven cases were identified, showing a sharp decline in incidence over the age of 65. Thirty-nine (83 %) tumors were testicular while eight (17 %) were non-testicular in presentation. Cases included 26 (55 %) seminomas, 15 (32 %) non-seminoma/mixed malignant GCT, and 5 (11 %) regressed testicular germ cell tumors. The most common component in mixed malignant GCTs was embryonal carcinoma (77 %), followed by seminoma and yolk sac tumor (62 % each). Germ cell neoplasia in situ (GCNIS) accompanied 57 % of the cases. Aggressive pathologic features, including lymphovascular invasion, retroperitoneal/lymph node involvement and higher stage at presentation, were identified in a significant proportion of cases (36/47, 77 %). Clinical follow up showed six patients (14 %) died of disease-related causes. CONCLUSION: Our findings expand and corroborate the previously reported data on malignant GCT in older men. Unique characteristics include tendency for higher stage at presentation with adverse pathologic features and more aggressive clinical course.

Bladder infection with uropathogenic Escherichia coli increases the excitability of afferent neurons.

Urinary tract infections (UTIs) cause bladder hyperactivity and pelvic pain, but the underlying causes of these symptoms remain unknown. We investigated whether afferent sensitization contributes to the bladder overactivity and pain observed in mice suffering from experimentally induced bacterial cystitis. Inoculation of mouse bladders with the uropathogenic Escherichia coli strain UTI89 caused pelvic allodynia, increased voiding frequency, and prompted an acute inflammatory process marked by leukocytic infiltration and edema of the mucosa. Compared with controls, isolated bladder sensory neurons from UTI-treated mice exhibited a depolarized resting membrane potential, lower action potential threshold and rheobase, and increased firing in response to suprathreshold stimulation. To determine whether bacterial virulence factors can contribute to the sensitization of bladder afferents, neurons isolated from naïve mice were incubated with supernatants collected from bacterial cultures with or depleted of lipopolysaccharide (LPS). Supernatants containing LPS prompted the sensitization of bladder sensory neurons with both tetrodotoxin (TTX)-resistant and TTX-sensitive action potentials. However, bladder sensory neurons with TTX-sensitive action potentials were not affected by bacterial supernatants depleted of LPS. Unexpectedly, ultrapure LPS increased the excitability only of bladder sensory neurons with TTX-resistant action potentials, but the supplementation of supernatants depleted of LPS with ultrapure LPS resulted in the sensitization of both population of bladder sensory neurons. In summary, the results of our study indicate that multiple virulence factors released from UTI89 act on bladder sensory neurons to prompt their sensitization. These sensitized bladder sensory neurons mediate, at least in part, the bladder hyperactivity and pelvic pain seen in mice inoculated with UTI89.NEW & NOTEWORTHY Urinary tract infection (UTI) produced by uropathogenic Escherichia coli (UPEC) promotes sensitization of bladder afferent sensory neurons with tetrodotoxin-resistant and tetrodotoxin-sensitive action potentials. Lipopolysaccharide and other virulence factors produced by UPEC contribute to the sensitization of bladder afferents in UTI. In conclusion, sensitized afferents contribute to the voiding symptoms and pelvic pain present in mice bladder inoculated with UPEC.

Non-canonical Wnt/calcium signaling is protective against podocyte injury and glomerulosclerosis.

Activation of canonical Wnt signaling has been implicated in podocyte injury and proteinuria. As Wnts are secreted proteins, whether Wnts derived from podocytes are obligatory for promoting proteinuria remains unknown. To address this, we generated conditional knockout mice where Wntless, a cargo receptor protein required for Wnt secretion, was specifically deleted in glomerular podocytes. Mice with podocyte-specific ablation of Wntless (Podo-Wntless-/-) were phenotypically normal. However, after inducing kidney damage with Adriamycin for six days, Podo-Wntless-/- mice developed more severe podocyte injury and albuminuria than their control littermates. Surprisingly, ablation of Wntless resulted in upregulation of ß-catenin, accompanied by reduction of nephrin, podocin, podocalyxin, and Wilms tumor 1 proteins. In chronic injury induced by Adriamycin, increased albuminuria, aggravated podocyte lesions and extracellular matrix deposition were evident in Podo-Wntlessl-/- mice, compared to wild type mice. Mechanistically, specific ablation of Wntless in podocytes caused down-regulation of the nuclear factor of activated T cell 1 (NFAT1) and Nemo-like kinase (NLK), key downstream mediators of non-canonical Wnt/calcium signaling. In vitro, knockdown of either NFAT1 or NLK induced ß-catenin activation while overexpression of NLK significantly repressed ß-catenin induction and largely preserved nephrin in glomerular podocytes. Thus, our results indicate that podocyte-derived Wnts play an important role in protecting podocytes from injury by repressing ß-catenin via activating non-canonical Wnt/calcium signaling.

Calyceal diverticula: Clinical, radiological and histopathological findings of an uncommon entity with presumed congenital origin.

Calyceal diverticula (CD) are relatively uncommon urologic conditions that generally follow an asymptomatic course and rarely require medical intervention. CD are thought to have a congenital origin due to abnormalities during the process of ureteral bud formation. Clinically and radiologically, they can mimic multiple neoplastic and non-neoplastic renal processes, with potentially relevant differences in the management of these patients. Symptoms are usually associated with the presence of stones, obstruction to the drainage of the diverticulum, large size, or secondary infection. In chronic cases, surgery might become necessary, creating an opportunity to examine the histopathological characteristics of this condition. Although these are benign in the majority of patients, some rare instances of malignancy arising from the CD have been reported. In this series, we addressed the clinical, radiological, and histopathological findings of CD.

Genetic or pharmacologic Nrf2 activation increases proteinuria in chronic kidney disease in mice.

The nuclear factor erythroid 2-related factor 2 (Nrf2) pathway upregulates key cellular defenses. Clinical trials are utilizing pharmacologic Nrf2 inducers such as bardoxolone methyl to treat chronic kidney disease, but Nrf2 activation has been linked to a paradoxical increase in proteinuria. To understand this effect, we examined genetically engineered mice with elevated Nrf2 signaling due to reduced expression of the Nrf2 inhibitor, Kelch-like ECH-associated protein 1 (Keap1). These Keap1FA/FA mice lacked baseline proteinuria but exhibited increased proteinuria in experimental models evoked by adriamycin, angiotensin II, or protein overload. After injury, Keap1FA/FA mice had increased glomerulosclerosis, nephrin disruption and shedding, podocyte injury, foot process effacement, and interstitial fibrosis. Keap1FA/FA mice also had higher daytime blood pressures and lower heart rates measured by radiotelemetry. Conversely, Nrf2 knockout mice were protected from proteinuria. We also examined the pharmacologic Nrf2 inducer CDDO-Im. Compared to angiotensin II alone, the combination of angiotensin II and CDDO-Im significantly increased proteinuria, a phenomenon not observed in Nrf2 knockout mice. This effect was not accompanied by additional increases in blood pressure. Finally, Nrf2 was found to be upregulated in the glomeruli of patients with focal segmental glomerulosclerosis, diabetic nephropathy, fibrillary glomerulonephritis, and membranous nephropathy. Thus, our studies demonstrate that Nrf2 induction in mice may exacerbate proteinuria in chronic kidney disease.

Absolute quantitative proteomics using the total protein approach to identify novel clinical immunohistochemical markers in renal neoplasms.

BACKGROUND: Renal neoplasms encompass a variety of malignant and benign tumors, including many with shared characteristics. The diagnosis of these renal neoplasms remains challenging with currently available tools. In this work, we demonstrate the total protein approach (TPA) based on high-resolution mass spectrometry (MS) as a tool to improve the accuracy of renal neoplasm diagnosis. METHODS: Frozen tissue biopsies of human renal tissues [clear cell renal cell carcinoma (n = 7), papillary renal cell carcinoma (n = 5), chromophobe renal cell carcinoma (n = 5), and renal oncocytoma (n = 5)] were collected for proteome analysis. Normal adjacent renal tissue (NAT, n = 5) was used as a control. Proteins were extracted and digested using trypsin, and the digested proteomes were analyzed by label-free high-resolution MS (nanoLC-ESI-HR-MS/MS). Quantitative analysis was performed by comparison between protein abundances of tumors and NAT specimens, and the label-free and standard-free TPA was used to obtain absolute protein concentrations. RESULTS: A total of 205 differentially expressed proteins with the potential to distinguish the renal neoplasms were found. Of these proteins, a TPA-based panel of 24, including known and new biomarkers, was selected as the best candidates to differentiate the neoplasms. As proof of concept, the diagnostic potential of PLIN2, TUBB3, LAMP1, and HK1 was validated using semi-quantitative immunohistochemistry with a total of 128 samples assessed on tissue micro-arrays. CONCLUSIONS: We demonstrate the utility of combining high-resolution MS and the TPA as potential new diagnostic tool in the pathology of renal neoplasms. A similar TPA approach may be implemented in any cancer study with solid biopsies.

Early activation of fibroblasts is required for kidney repair and regeneration after injury.

Acute kidney injury (AKI) is a devastating condition with high morbidity and mortality. AKI is characterized by tubular injury, inflammation, and vascular impairment. However, the role of interstitial fibroblasts in the pathogenesis of AKI is largely unknown. Here, we show that fibroblasts were activated, as defined by vimentin expression, at 1 h after AKI triggered by ischemia-reperfusion injury (IRI). They rapidly entered the cell cycle with Ki-67-positive staining, which started at 1 h and peaked at 12 h after IRI, whereas tubular cell proliferation peaked at 3 d. The trigger for such an early activation of fibroblasts was identified as sonic hedgehog (Shh), which was rapidly induced in renal tubules and could target interstitial fibroblasts. Tubule-specific knockout of Shh in mice inhibited fibroblast activation and aggravated kidney injury and functional decline after IRI. Likewise, pharmacologic inhibition of Shh signaling with cyclopamine also hindered fibroblast activation and exacerbated kidney damage. These studies uncover that tubule-derived Shh triggers the early activation of fibroblasts, which is required for kidney repair and regeneration. Our findings for the first time illustrate a previously unrecognized importance of interstitial fibroblasts in conferring renal protection in AKI.-Zhou, D., Fu, H., Liu, S., Zhang, L., Xiao, L., Bastacky, S. I., Liu, Y. Early activation of fibroblasts is required for kidney repair and regeneration after injury.

2-Methoxyestradiol Attenuates Angiotensin II-Induced Hypertension, Cardiovascular Remodeling, and Renal Injury.

Estradiol may antagonize the adverse cardiovascular effects of angiotensin II (Ang II). We investigated the effects of 2-methoxyestradiol (2-ME), a nonestrogenic estradiol metabolite, on Ang II-induced cardiovascular and renal injury in male rats. First, we determined the effects of 2-ME on Ang II-induced acute changes in blood pressure, renal hemodynamics, and excretory function. Next, we investigated the effects of 2-ME and 2-hydroxyestardiol (2-HE) on hypertension and cardiovascular and renal injury induced by chronic infusion of Ang II. Furthermore, the effects of 2-ME on blood pressure and cardiovascular remodeling in the constricted aorta (CA) rat model and on isoproterenol-induced (ISO) cardiac hypertrophy and fibrosis were examined. 2-ME had no effects on Ang II-induced acute changes in blood pressure, renal hemodynamics, or glomerular filtration rate. Both 2-ME and 2-HE reduced hypertension, cardiac hypertrophy, proteinuria, and mesangial expansion induced by chronic Ang II infusions. In CA rats, 2-ME attenuated cardiac hypertrophy and fibrosis and reduced elevated blood pressure above the constriction. Notably, 2-ME reduced both pressure-dependent (above constriction) and pressure-independent (below constriction) vascular remodeling. 2-ME had no effects on ISO-induced renin release yet reduced ISO-induced cardiac hypertrophy and fibrosis. This study shows that 2-ME protects against cardiovascular and renal injury due to chronic activation of the renin-angiotensin system. This study reports for the first time that in vivo 2-ME reduces trophic (pressure-independent) effects of Ang II and related cardiac and vascular remodeling.

Captopril Attenuates Cardiovascular and Renal Disease in a Rat Model of Heart Failure With Preserved Ejection Fraction.

Heart failure with preserved ejection fraction (HFpEF), a prevalent form of heart failure, is frequently accompanied by the metabolic syndrome and kidney disease. Because current treatment options of HFpEF are limited, evaluation of therapies in experimental models of HFpEF with the metabolic syndrome and kidney disease is needed. In this study, we evaluated the effects of captopril, furosemide, and their combination in aged, obese ZSF1 rats, an animal model of HFpEF with the metabolic syndrome and chronic kidney disease as comorbidities. Captopril (100 mg/kg), furosemide (50 mg/kg), or their combination was administered orally to obese ZSF1 rats aged 20 to 44 weeks. Untreated ZSF1 rats served as controls. After 24 weeks of treatment, captopril significantly lowered systemic blood pressure and attenuated HFpEF as evidenced by significantly reduced left ventricular end diastolic pressures (10.5 ± 1.4 vs. 4.9 ± 1.3 mm Hg in Control vs. Captopril, respectively) and significantly lower left ventricular relaxation time constants (28.1 ± 2.9 vs. 18.3 ± 3.1 ms in Control vs. Captopril, respectively). The captopril-induced improvement in left ventricular function was associated with reduced cardiac hypertrophy, ischemia, necrosis, and vasculitis. Captopril also increased renal blood flow and glomerular filtration rate, reduced renal vascular resistance and proteinuria, and improved renal histology (ie, reduced renal hypertrophy, glomerulosclerosis, and tubular atrophy/dilation). Furosemide alone provided little benefit; moreover, furosemide did not augment the therapeutic benefits of captopril. This study suggests that chronic administration of captopril, but not furosemide, could be beneficial in patients with HFpEF, particularly in those with comorbidities such as obesity, diabetes, and dyslipidemias.

Tubule-Derived Wnts Are Required for Fibroblast Activation and Kidney Fibrosis.

Cell-cell communication via Wnt ligands is necessary in regulating embryonic development and has been implicated in CKD. Because Wnt ligands are ubiquitously expressed, the exact cellular source of the Wnts involved in CKD remains undefined. To address this issue, we generated two conditional knockout mouse lines in which Wntless (Wls), a dedicated cargo receptor that is obligatory for Wnt secretion, was selectively ablated in tubular epithelial cells or interstitial fibroblasts. Blockade of Wnt secretion by genetic deletion of Wls in renal tubules markedly inhibited myofibroblast activation and reduced renal fibrosis after unilateral ureteral obstruction. This effect associated with decreased activation of ß-catenin and downstream gene expression and preserved tubular epithelial integrity. In contrast, fibroblast-specific deletion of Wls exhibited little effect on the severity of renal fibrosis after obstructive or ischemia-reperfusion injury. In vitro, incubation of normal rat kidney fibroblasts with tubule-derived Wnts promoted fibroblast proliferation and activation. Furthermore, compared with kidney specimens from patients without CKD, biopsy specimens from patients with CKD also displayed increased expression of multiple Wnt proteins, predominantly in renal tubular epithelium. These results illustrate that tubule-derived Wnts have an essential role in promoting fibroblast activation and kidney fibrosis via epithelial-mesenchymal communication.

Muc1 enhances the ß-catenin protective pathway during ischemia-reperfusion injury.

The hypoxia-inducible factor (HIF)-1 and ß-catenin protective pathways represent the two most significant cellular responses that are activated in response to acute kidney injury. We previously reported that murine mucin (Muc)1 protects kidney function and morphology in a mouse model of ischemia-reperfusion injury (IRI) by stabilizing HIF-1α, enhancing HIF-1 downstream signaling, and thereby preventing metabolic stress (Pastor-Soler et al. Muc1 is protective during kidney ischemia-reperfusion injury. Am J Physiol Renal Physiol 308: F1452-F1462, 2015). We asked if Muc1 regulates the ß-catenin protective pathway during IRI as 1) ß-catenin nuclear targeting is MUC1 dependent in cultured human cells, 2) ß-catenin is found in coimmunoprecipitates with human MUC1 in extracts of both cultured cells and tissues, and 3) MUC1 prevents ß-catenin phosphorylation by glycogen synthase kinase (GSK)3ß and thereby ß-catenin degradation. Using the same mouse model of IRI, we found that levels of active GSK3ß were significantly lower in kidneys of control mice compared with Muc1 knockout (KO) mice. Consequently, ß-catenin was significantly upregulated at 24 and 72 h of recovery and appeared in the nuclear fraction at 72 h in control mouse kidneys. Both ß-catenin induction and nuclear targeting were absent in Muc1 KO mice. We also found downstream induction of ß-catenin prosurvival factors (activated Akt, survivin, transcription factor T cell factor 4 (TCF4), and its downstream target cyclin D1) and repression of proapoptotic factors (p53, active Bax, and cleaved caspase-3) in control mouse kidneys that were absent or aberrant in kidneys of Muc1 KO mice. Altogether, the data clearly indicate that Muc1 protection during acute kidney injury proceeds by enhancing both the HIF-1 and ß-catenin protective pathways.

Extracellular Superoxide Dismutase Protects against Proteinuric Kidney Disease.

Extracellular superoxide dismutase (EC-SOD), also known as SOD3, is an antioxidant expressed at high levels in normal adult kidneys. Because oxidative stress contributes to a variety of kidney injuries, we hypothesized that EC-SOD may be protective in CKD progression. To study this hypothesis, we used a murine model of ADR nephropathy characterized by albuminuria and renal dysfunction. We found that levels of EC-SOD diminished throughout the course of disease progression and were associated with increased levels of NADPH oxidase and oxidative stress markers. EC-SOD null mice were sensitized to ADR injury, as evidenced by increases in albuminuria, serum creatinine, histologic damage, and oxidative stress. The absence of EC-SOD led to increased levels of NADPH oxidase and an increase in ß-catenin signaling, which has been shown to be pathologic in a variety of kidney injuries. Exposure of EC-SOD null mice to either chronic angiotensin II infusion or to daily albumin injections also caused increased proteinuria. In contrast, EC-SOD null mice subjected to nonproteinuric CKD induced by unilateral ureteral obstruction exhibited no differences compared with wild-type mice. Finally, we also found a decrease in EC-SOD in human CKD biopsy samples, similar to our findings in mice. Therefore, we conclude that EC-SOD is protective in CKDs characterized by proteinuria.

Increased urothelial paracellular transport promotes cystitis.

Changes in the urothelial barrier are observed in patients with cystitis, but whether this leads to inflammation or occurs in response to it is currently unknown. To determine whether urothelial barrier dysfunction is sufficient to promote cystitis, we employed in situ adenoviral transduction to selectively overexpress the pore-forming tight junction-associated protein claudin-2 (CLDN-2). As expected, the expression of CLDN-2 in the umbrella cells increased the permeability of the paracellular route toward ions, but not to large organic molecules. In vivo studies of bladder function revealed higher intravesical basal pressures, reduced compliance, and increased voiding frequency in rats transduced with CLDN-2 vs. controls transduced with green fluorescent protein. While the integrity of the urothelial barrier was preserved in the rats transduced with CLDN-2, we found that the expression of this protein in the umbrella cells initiated an inflammatory process in the urinary bladder characterized by edema and the presence of a lymphocytic infiltrate. Taken together, these results are consistent with the notion that urothelial barrier dysfunction may be sufficient to trigger bladder inflammation and to alter bladder function.

Muc1 is protective during kidney ischemia-reperfusion injury.

Ischemia-reperfusion injury (IRI) due to hypotension is a common cause of human acute kidney injury (AKI). Hypoxia-inducible transcription factors (HIFs) orchestrate a protective response in renal endothelial and epithelial cells in AKI models. As human mucin 1 (MUC1) is induced by hypoxia and enhances HIF-1 activity in cultured epithelial cells, we asked whether mouse mucin 1 (Muc1) regulates HIF-1 activity in kidney tissue during IRI. Whereas Muc1 was localized on the apical surface of the thick ascending limb, distal convoluted tubule, and collecting duct in the kidneys of sham-treated mice, Muc1 appeared in the cytoplasm and nucleus of all tubular epithelia during IRI. Muc1 was induced during IRI, and Muc1 transcripts and protein were also present in recovering proximal tubule cells. Kidney damage was worse and recovery was blocked during IRI in Muc1 knockout mice compared with congenic control mice. Muc1 knockout mice had reduced levels of HIF-1α, reduced or aberrant induction of HIF-1 target genes involved in the shift of glucose metabolism to glycolysis, and prolonged activation of AMP-activated protein kinase, indicating metabolic stress. Muc1 clearly plays a significant role in enhancing the HIF protective pathway during ischemic insult and recovery in kidney epithelia, providing a new target for developing therapies to treat AKI. Moreover, our data support a role specifically for HIF-1 in epithelial protection of the kidney during IRI as Muc1 is present only in tubule epithelial cells.

D-Amino Acid Substitution of Peptide-Mediated NF-κB Suppression in mdx Mice Preserves Therapeutic Benefit in Skeletal Muscle, but Causes Kidney Toxicity.

In Duchenne muscular dystrophy (DMD) patients and the mdx mouse model of DMD, chronic activation of the classical nuclear factor-κB (NF-κB) pathway contributes to the pathogenesis that causes degeneration of muscle fibers, inflammation and fibrosis. Prior studies demonstrate that inhibition of inhibitor of κB kinase (IKK)-mediated NF-κB activation using L-isomer NF-κB essential modulator (NEMO)-binding domain (NBD) peptide-based approaches reduce muscle pathology in the mdx mouse. For our studies, the NBD peptide is synthesized as a fusion peptide with an eight-lysine (8K) protein transduction domain to facilitate intracellular delivery. We hypothesized that the d-isoform peptide could have a greater effect than the naturally occurring L-isoform peptide due to the longer persistence of the D-isoform peptide in vivo. In this study, we compared systemic treatment with low (1 mg/kg) and high (10 mg/kg) doses of L- and D-isomer 8K-wild-type-NBD peptide in mdx mice. Treatment with both L- or D-isoform 8K-wild-type-NBD peptide resulted in decreased activation of NF-κB and improved histology in skeletal muscle of the mdx mouse. However, we observed kidney toxicity (characterized by proteinuria), increased serum creatinine, activation of NF-κB and pathological changes in kidney cortex that were most severe with treatment with the D-isoform of 8K-wild-type-NBD peptide. The observed toxicity was also seen in normal mice.

MicroRNA-17~92 is required for nephrogenesis and renal function.

Deletion of all microRNAs (miRNAs) in nephron progenitors leads to premature loss of these cells, but the roles of specific miRNAs in progenitors have not been identified. Deletions in the MIR17HG cluster (miR-17~92 in mice), detected in a subset of patients with Feingold syndrome, represent the first miRNA mutations to be associated with a developmental defect in humans. Although MIR17HG is expressed in the developing kidney, and patients with Feingold syndrome caused by MYCN mutations have renal anomalies, it remains unclear to what extent MIR17HG contributes to renal development and function. To define the role of miR-17~92, we generated mice with a conditional deletion of miR-17~92 in nephron progenitors and their derivatives. The nephron progenitor population was preserved in these mice; however, this deletion impaired progenitor cell proliferation and reduced the number of developing nephrons. Postnatally, mutant mice developed signs of renal disease, including albuminuria by 6 weeks and focal podocyte foot process effacement and glomerulosclerosis at 3 months. Taken together, these data support a role for this miRNA cluster in renal development, specifically in the regulation of nephron development, with subsequent consequences for renal function in adult mice.

Membranoproliferative glomerulonephritis: the role for laser microdissection and mass spectrometry.

Monoclonal gammopathy is increasingly recognized as a common cause of membranoproliferative glomerulonephritis (MPGN); however, establishing this diagnosis can be challenging. We report the case of a 58-year-old asymptomatic woman who presented with proteinuria with protein excretion of 5,000mg/d, microscopic hematuria, and normal kidney function. Kidney biopsy was consistent with MPGN pattern of injury. Immunofluorescence studies were positive for nonspecific segmental immunoglobulin M (IgM) and C3 staining. Electron microscopy showed subendothelial, subepithelial, and mesangial electron-dense deposits. The workup excluded an infectious or autoimmune disease, but IgG κ monoclonal protein was detected in serum at a concentration of 0.4mg/dL. Because there was a mismatch between the serum monoclonal protein (IgG κ) and immunofluorescence staining pattern (nonspecific IgM, no light chain restriction), laser microdissection and mass spectrometry were performed on the kidney biopsy tissue. This identified the deposits as monoclonal IgG κ, thereby leading to the diagnosis of monoclonal gammopathy-associated MPGN. Our case emphasizes the importance of searching for an underlying cause of MPGN, reviews the technique of laser microdissection-mass spectrometry, and highlights its application as a pathology tool for the evaluation of monoclonal gammopathy-related glomerulonephritis.