Nath, Karl A.

AbstractPulmonary hypertension (PH) associated with chronic kidney disease (CKD) (PH‐CKD) affects approximately 20%–40% of CKD patients and is associated with increased morbidity and mortality. PH and CKD are both pathophysiologically associated with nitric oxide (NO) deficiency. The NO pathway, an important therapeutic domain in pulmonary arterial hypertension (PAH), is an intriguing but unexplored target in PH‐CKD. We sought to improve understanding of the clinical significance of the NO pathway in patients with PH‐CKD by assessing the hemodynamic response to inhaled NO (iNO) during right heart catheterization (RHC). In this retrospective cohort study, patients with diagnosis codes of PH and stage IV/V CKD or end‐stage renal disease and estimated glomerular filtration rate < 60 mL/min/body surface area who underwent RHC and hemodynamic drug study between July 2011 and June 2021 were eligible. Patients with mean pulmonary artery pressure (mPAP) > 20 mmHg and pulmonary vascular resistance (PVR) > 3 Wood units were included. The final cohort included 37 patients (45.9% female, mean age 72.5 ± 9.7 years). A total of 56.7% of the cohort (21/37) had precapillary PH, while 43.2% (16/37) had combined precapillary postcapillary PH (Cpc‐PH). Median survival was 3.1 years after RHC. iNO was associated with a significant decrease in both mPAP and PVR. Hemodynamic changes in mPAP and PVR were similar in precapillary and Cpc‐PH groups. Among a small subset (n = 14) who were subsequently treated with PAH‐targeted therapy, treatment response was mixed and did not reveal significant benefit. Further studies are warranted to better define the potential role of PAH therapy in PH‐CKD.

Aging and many illnesses and injuries impair skeletal muscle mass and function, but the molecular mechanisms are not well understood. To better understand the mechanisms, we generated and studied transgenic mice with skeletal muscle–specific expression of growth arrest and DNA damage inducible α (GADD45A), a signaling protein whose expression in skeletal muscle rises during aging and a wide range of illnesses and injuries. We found that GADD45A induced several cellular changes that are characteristic of skeletal muscle atrophy, including a reduction in skeletal muscle mitochondria and oxidative capacity, selective atrophy of glycolytic muscle fibers, and paradoxical expression of oxidative myosin heavy chains despite mitochondrial loss. These cellular changes were at least partly mediated by MAP kinase kinase kinase 4, a protein kinase that is directly activated by GADD45A. By inducing these changes, GADD45A decreased the mass of muscles that are enriched in glycolytic fibers, and it impaired strength, specific force, and endurance exercise capacity. Furthermore, as predicted by data from mouse models, we found that GADD45A expression in skeletal muscle was associated with muscle weakness in humans. Collectively, these findings identify GADD45A as a mediator of mitochondrial loss, atrophy, and weakness in mouse skeletal muscle and a potential target for muscle weakness in humans.

AbstractBackgroundGastroparesis is defined by delayed gastric emptying (GE) without obstruction. Studies suggest targeting heme oxygenase‐1 (HO1) may ameliorate diabetic gastroparesis. Upregulation of HO1 expression via interleukin‐10 (IL‐10) in the gastric muscularis propria is associated with reversal of delayed GE in diabetic NOD mice. IL‐10 activates the M2 cytoprotective phenotype of macrophages and induces expression of HO1 protein. Here, we assess delivery of HO1 by recombinant adeno‐associated viruses (AAVs) in diabetic mice with delayed GE.MethodsC57BL6 diabetic delayed GE mice were injected with 1 × 1012 vg scAAV9‐cre, scAAV9‐GFP, or scAAV9‐HO1 particles. Changes to GE were assessed weekly utilizing our [13C]‐octanoic acid breath test. Stomach tissue was collected to assess the effect of scAAV9 treatment on Kit, NOS1, and HO1 expression.Key ResultsDelayed GE returned to normal within 2 weeks of treatment in 7/12 mice receiving scAAV9‐cre and in 4/5 mice that received the scAAV9‐GFP, whereas mice that received scAAV9‐HO1 did not respond in the same manner and had GE that took significantly longer to return to normal (6/7 mice at 4–6 weeks). Kit, NOS1, and HO1 protein expression in scAAV9‐GFP‐treated mice with normal GE were not significantly different compared with diabetic mice with delayed GE.Conclusions and InferencesInjection of scAAV9 into diabetic C57BL6 mice produced a biological response that resulted in acceleration of GE independently of the cargo delivered by the AAV9 vector. Further research is needed to determine whether use of AAV mediated gene transduction in the gastric muscularis propria is beneficial and warranted.

Significance Statement Syphilis is a common worldwide sexually transmitted infection. Proteinuria may occur in patients with syphilis. Membranous nephropathy (MN) is the most common cause of proteinuria in syphilis. The target antigen of MN in syphilis is unknown. This study shows that MN in syphilis is associated with a novel target antigen called neuron-derived neurotrophic factor (NDNF). NDNF-associated MN has distinctive clinical and pathologic manifestations and NDNF appears to be the target antigen in syphilis-associated MN. Background Syphilis is a common sexually transmitted infection. Membranous nephropathy (MN) is a common cause of proteinuria in syphilis. The target antigen is not known in most cases of syphilis-associated MN. Methods We performed laser microdissection of glomeruli and mass spectrometry (MS/MS) in 250 cases (discovery cohort) of phospholipase A2 receptor–negative MN to identify novel target antigens. This was followed by immunohistochemistry/confocal microscopy to localize the target antigen along the glomerular basement membrane (GBM). Western blot analyses using IgG eluted from frozen biopsy tissue were performed to detect binding to target antigen. Results MS/MS studies of the discovery cohort revealed high total spectral counts of a novel protein, neuron-derived neurotrophic factor (NDNF), in three patients: one each with syphilis and hepatitis B, HIV (syphilis status not known), and lung tumor. Next, MS/MS studies of five cases of syphilis-MN (validation cohort) confirmed high total spectral counts of NDNF (average 45±20.4) in all (100%) cases. MS/MS of 14 cases of hepatitis B were negative for NDNF. All eight cases of NDNF-associated MN were negative for known MN antigens. Electron microscopy showed stage I MN in all cases, with superficial and hump-like deposits without GBM reaction. IgG1 was the dominant IgG subtype on MS/MS and immunofluorescence microscopy. Immunohistochemistry/confocal microscopy showed granular staining and colocalization of NDNF and IgG along GBM. Western blot analyses using eluate IgG of NDNF-MN showed binding to both nonreduced and reduced NDNF, while IgG eluate from phospholipase A2 receptor-MN showed no binding. Conclusion NDNF is a novel antigenic target in syphilis-associated MN.

Heme proteins, the stuff of life, represent an ingenious biologic strategy that capitalizes on the biochemical versatility of heme, and yet is one that avoids the inherent risks to cellular vitality posed by unfettered and promiscuously reactive heme. Heme proteins, however, may be a double-edged sword because they can damage the kidney in certain settings. Although such injury is often viewed mainly within the context of rhabdomyolysis and the nephrotoxicity of myoglobin, an increasing literature now attests to the fact that involvement of heme proteins in renal injury ranges well beyond the confines of this single disease (and its analog, hemolysis); indeed, through the release of the defining heme motif, destabilization of intracellular heme proteins may be a common pathway for acute kidney injury, in general, and irrespective of the underlying insult. This brief review outlines current understanding regarding processes underlying such heme protein-induced acute kidney injury (AKI) and chronic kidney disease (CKD). Topics covered include, among others, the basis for renal injury after the exposure of the kidney to and its incorporation of myoglobin and hemoglobin; auto-oxidation of myoglobin and hemoglobin; destabilization of heme proteins and the release of heme; heme/iron/oxidant pathways of renal injury; generation of reactive oxygen species and reactive nitrogen species by NOX, iNOS, and myeloperoxidase; and the role of circulating cell-free hemoglobin in AKI and CKD. Also covered are the characteristics of the kidney that render this organ uniquely vulnerable to injury after myolysis and hemolysis, and pathobiologic effects emanating from free, labile heme. Mechanisms that defend against the toxicity of heme proteins are discussed, and the review concludes by outlining the therapeutic strategies that have arisen from current understanding of mechanisms of renal injury caused by heme proteins and how such mechanisms may be interrupted.

Key Points In heme protein–induced AKI, mitochondrial functional integrity, as reflected by ATP and NAD + content and NAD + /NADH ratio, is impaired. Mitochondrial quality control is compromised as reflected by impaired biogenesis, exaggerated fission, and marked ultrastructural damage. Modern concepts regarding mitochondria and AKI apply to heme protein–induced AKI, with the possibility of novel therapeutic strategies. Background Mitochondrial injury occurs in and underlies acute kidney injury (AKI) caused by ischemia-reperfusion and other forms of renal injury. However, to date, a comprehensive analysis of this issue has not been undertaken in heme protein–induced AKI (HP-AKI). We examined key aspects of mitochondrial function, expression of proteins relevant to mitochondrial quality control, and mitochondrial ultrastructure in HP-AKI, along with responses to heme in renal proximal tubule epithelial cells. Methods The long-established murine glycerol model of HP-AKI was examined at 8 and 24 hours after HP-AKI. Indices of mitochondrial function (ATP and NAD + ), expression of proteins relevant to mitochondrial dynamics, mitochondrial ultrastructure, and relevant gene/protein expression in heme-exposed renal proximal tubule epithelial cells in vitro were examined. Results ATP and NAD + content and the NAD + /NADH ratio were all reduced in HP-AKI. Expression of relevant proteins indicate that mitochondrial biogenesis (PGC-1 α , NRF1, and TFAM) and fusion (MFN2) were impaired, as was expression of key proteins involved in the integrity of outer and inner mitochondrial membranes (VDAC, Tom20, and Tim23). Conversely, marked upregulation of proteins involved in mitochondrial fission (DRP1) occurred. Ultrastructural studies, including novel 3D imaging, indicate profound changes in mitochondrial structure, including mitochondrial fragmentation, mitochondrial swelling, and misshapen mitochondrial cristae; mitophagy was also observed. Exposure of renal proximal tubule epithelial cells to heme in vitro recapitulated suppression of PGC-1 α (mitochondrial biogenesis) and upregulation of p-DRP1 (mitochondrial fission). Conclusions Modern concepts pertaining to AKI apply to HP-AKI. This study validates the investigation of novel, clinically relevant therapies such as NAD + -boosting agents and mitoprotective agents in HP-AKI.

Introduction: Arteriovenous fistulas(AVF) have been associated with high output heart failure but little is known about their effect on patients initiating hemodialysis. We sought to describe echocardiographic changes and identify those with incident heart failure after AVF creation. Methods: This was a retrospective analysis of all patients undergoing AVF creation over a 15 year period from 2000-2014 at the Mayo Clinic in Rochester, MN. Patients undergoing echocardiogram <3 months (median 40 days) before AVF creation and >6 months (median 2.5 years) following surgery were included. Patients with independent modifiers of LV structural changes (such as revascularization or myocardial infarction) were excluded by chart review. Clinical heart failure was defined by Framingham criteria. Results: From 2000-2014, 2236 patients underwent creation of AVFs and 137 patients met inclusion criteria. There was a longitudinal decrease in systolic function as evident by a drop in ejection fraction(-2%, p=0.047) with decrease in s’ tissue Doppler velocity(-0.6 cm/s, p=0.055) and increase in HR(+5 beats/min, p=0.003). There was also an increase in the prevalence of RV dilation(20 to 40%, p=0.002) and RV dysfunction(12 to 29%, p=0.002). With initiation of dialysis and decrease in weight (-7.3kg) and estimated plasma volume (-303ml) (both p<0.0001), there was a decrease in LV end diastolic dimension (-2mm, p=0.006). Despite better volume control, left atrial volume index increased (+5ml/m2, p=0.004). On multivariate Cox regression, worsening RV dilation [HR 1.8 (1.06-3.09),p=0.03] was independently associated with mortality. Incident heart failure developed in 43%(28/65) with no pre-existing HF, of whom 75% had HFPEF and 25% HFrEF. The development of new systolic HF (but not HFPEF) was associated with increased age and sex adjusted mortality [HR 3.0(1.0-7.9), p=0.045]. Conclusions: AVF creation is associated with worsening biventricular function and left atrial and RV dilatation, despite reduction in LV size. These changes are coupled with high incidence of new, unexplained heart failure. Further research is needed to identify the hemodynamic mechanisms underlying heart failure development in patients with ESRD requiring dialysis access.