Human Transplant Kidneys on Normothermic Machine Perfusion Display Endocrine Activity

Background. Normothermic machine perfusion (NMP) is an alternative to hypothermic machine perfusion (HMP) for donor kidney preservation before transplantation. Contrary to HMP, NMP allows for functional assessment of donor kidneys because normothermic conditions allow for metabolic activity. The kidneys are key producers of hormones. Yet, it remains unknown whether donor kidneys during NMP display endocrine functions. Methods. Fifteen donor kidneys were subjected to HMP followed by 2 h of NMP before transplantation. NMP perfusate was collected at 3 time points (0, 1, 2 h) for the measurements of prorenin/renin, erythropoietin (EPO), and vitamin D, and urine samples were collected at 1 h and 2 h for urodilatin measurement. Fifteen HMP perfusate samples were collected for the same measurements. Results. Kidneys on NMP secreted significantly more prorenin, renin, EPO, and active vitamin D than during HMP. EPO and vitamin D secretion remained stable during 2 h of NMP, whereas the prorenin release rate increased and renin release rate decreased after 1 h. Donation after brain death kidneys secreted more vitamin D and less EPO during NMP than donation after circulatory death kidneys. Twelve donor kidneys produced urine during NMP and released detectable levels of urodilatin. Kidneys exhibited a large variation in hormone release rates. No significant differences were found in hormone release capacity between delayed graft function (DGF) and non-DGF kidneys, and no significant correlations were found between hormone release rates and the duration of DGF or 1-mo posttransplant serum creatinine levels. Conclusions. Human transplant kidneys display endocrine activity during NMP. To explore whether correlations exist between hormone release rates and posttransplant kidney function, large numbers of kidneys are required.</p

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

SARS-CoV-2 infects the human kidney and drives fibrosis in kidney organoids

Kidney failure is frequently observed during and after COVID-19, but it remains elusive whether this is a direct effect of the virus. Here, we report that SARS-CoV-2 directly infects kidney cells and is associated with increased tubule-interstitial kidney fibrosis in patient autopsy samples. To study direct effects of the virus on the kidney independent of systemic effects of COVID-19, we infected human induced pluripotent stem cell-derived kidney organoids with SARS-CoV-2. Single cell RNA-sequencing indicated injury and dedifferentiation of infected cells with activation of pro-fibrotic signaling pathways. Importantly, SARS-CoV-2 infection also led to increased collagen 1 protein expression in organoids. A SARS-CoV-2 protease inhibitor was able to ameliorate the infection of kidney cells by SARS-CoV-2. Our results suggest that SARS-CoV-2 can directly infect kidney cells and induce cell injury with subsequent fibrosis. These data could explain both acute kidney injury in COVID-19 patients and the development of chronic kidney disease in Long-COVID

Dietary potassium and the renal control of salt balance and blood pressure

Dietary potassium (K(+)) intake has antihypertensive effects, prevents strokes, and improves cardiovascular outcomes. The underlying mechanism for these beneficial effects of high K(+) diets may include vasodilation, enhanced urine flow, reduced renal renin release, and negative sodium (Na(+)) balance. Indeed, several studies demonstrate that dietary K(+) intake induces renal Na(+) loss despite elevated plasma aldosterone. This review briefly highlights the epidemiological and experimental evidences for the effects of dietary K(+) on arterial blood pressure. It discusses the pivotal role of the renal distal tubule for the regulation of urinary K(+) and Na(+) excretion and blood pressure and highlights that it depends on the coordinated interaction of different nephron portions, epithelial cell types, and various ion channels, transporters, and ATPases. Moreover, we discuss the relevance of aldosterone and aldosterone-independent factors in mediating the effects of an altered K(+) intake on renal K(+) and Na(+) handling. Particular focus is given to findings suggesting that an aldosterone-independent downregulation of the thiazide-sensitive NaCl cotransporter significantly contributes to the natriuretic and antihypertensive effect of a K(+)-rich diet. Last but not least, we refer to the complex signaling pathways enabling the kidney to adapt its function to the homeostatic needs in response to an altered K(+) intake. Future work will have to further address the underlying cellular and molecular mechanism and to elucidate, among others, how an altered dietary K(+) intake is sensed and how this signal is transmitted to the different epithelial cells lining the distal tubule

The mineralocorticoid receptor (MR) regulates ENaC but not NCC in mice with random MR deletion

Aldosterone binds to the mineralocorticoid receptor (MR) and increases renal Na+ reabsorption via up-regulation of the epithelial Na+ channel (ENaC) and the Na+-K+- ATPase in the collecting system (CS) and possibly also via the NaCl cotransporter (NCC) in the distal convoluted tubule (DCT). However, whether aldosterone directly regulates NCC via MR, or indirectly through systemic alterations remains controversial. We used mice with deletion of MR in ~20% of renal tubule cells (MR/X mice), in which MR-positive (MRwt) and -negative (MRko) cells can be studied sideby- side in the same physiological context. Adult MR/X mice showed similar mRNA and protein levels of renal ion transport proteins to control mice. In MR/X mice, no differences in NCC abundance and phosphorylation was seen between MRwt and MRko cells and dietary Na+ restriction up-regulated NCC to similar extent in both groups of cells. In contrast, MRko cells in the CS did not show any detectable alpha- ENaC abundance or apical targeting of ENaC neither on control diet nor in response to dietary Na+ restriction. Furthermore, Na+-K+-ATPase expression was unaffected in MRko cells of the DCT, while it was lost in MRko cells of the CS. In conclusion, MR is crucial for ENaC and Na+-K+-ATPase regulation in the CS, but is dispensable for NCC and Na+-K+-ATPase regulation in the DCT

The mouse isolated perfused kidney technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy. This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney's preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout

Dietary potassium and the renal control of salt balance and blood pressure

Dietary potassium (K(+)) intake has antihypertensive effects, prevents strokes, and improves cardiovascular outcomes. The underlying mechanism for these beneficial effects of high K(+) diets may include vasodilation, enhanced urine flow, reduced renal renin release, and negative sodium (Na(+)) balance. Indeed, several studies demonstrate that dietary K(+) intake induces renal Na(+) loss despite elevated plasma aldosterone. This review briefly highlights the epidemiological and experimental evidences for the effects of dietary K(+) on arterial blood pressure. It discusses the pivotal role of the renal distal tubule for the regulation of urinary K(+) and Na(+) excretion and blood pressure and highlights that it depends on the coordinated interaction of different nephron portions, epithelial cell types, and various ion channels, transporters, and ATPases. Moreover, we discuss the relevance of aldosterone and aldosterone-independent factors in mediating the effects of an altered K(+) intake on renal K(+) and Na(+) handling. Particular focus is given to findings suggesting that an aldosterone-independent downregulation of the thiazide-sensitive NaCl cotransporter significantly contributes to the natriuretic and antihypertensive effect of a K(+)-rich diet. Last but not least, we refer to the complex signaling pathways enabling the kidney to adapt its function to the homeostatic needs in response to an altered K(+) intake. Future work will have to further address the underlying cellular and molecular mechanism and to elucidate, among others, how an altered dietary K(+) intake is sensed and how this signal is transmitted to the different epithelial cells lining the distal tubule

Aldosterone controls primary cilium length and cell size in renal collecting duct principal cells

Primary cilia are nonmotile sensory organelles found on the surface of almost all kidney tubule epithelial cells. Being exposed to the tubular lumen, primary cilia are thought to be chemo- and mechanosensors of luminal composition and flux, respectively. We hypothesized that, Na+ transport and primary cilia exist in a sensory functional connection in mature renal tubule epithelial cells. Our results demonstrate that primary cilium length is reduced in mineralocorticoid receptor (MR) knockout (KO) mice in a cell autonomous manner along the aldosterone-sensitive distal nephron (ADSN) compared with wild type (as µm ± SEM; 3.1 ± 0.2 vs 4.0 ± 0.1). In mouse cortical collecting duct (mCCD)cl1 cells, which are a model of collecting duct (CD) principal cells, changes in Na+ transport intensity were found to mediate primary cilium length in response to aldosterone (as µm ± SEM: control: 2.7 ± 0.9 vs aldosterone treated: 3.8 ± 0.8). Cilium length was positively correlated with the availability of IFT88, a major intraflagellar anterograde transport complex B component, which is stabilized in response to exposure to aldosterone treatment. This suggests that the abundance of IFT88 is a regulated, rate limiting factor in the elongation of primary cilia. As previously observed in vivo, aldosterone treatment increased cell volume of cultured CD principal cells. Knockdown of IFT88 prevents ciliogenesis and inhibits the adaptive increase in cell size that was observed in response to aldosterone treatment. In conclusion, our results reveal a functional connection between Na+ transport, primary cilia, and cell size, which may play a key role in the morphological and functional adaptation of the CD to sustained changes in active Na+ reabsorption due to variations in aldosterone secretion

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

Multiorgan tropism of SARS-CoV-2 lineage B.1.1.7

Due to the development of novel functionalities, distinct SARS-CoV-2 variants such as B.1.1.7 fuel the current pandemic. B.1.1.7 is not only more transmissible, but may also cause an increased mortality compared to previous SARS-CoV-2 variants. Human tissue analysis of the SARS-CoV-2 lineage B.1.1.7 is urgently needed, and we here present autopsy data from 7 consecutive SARS-CoV-2 B.1.1.7 cases. The initial RT-qPCR analyses from nasopharyngeal swabs taken post mortem included typing assays for B.1.1.7. We quantitated SARS-CoV-2 B.1.1.7 viral load in autopsy tissue of multiple organs. Highest levels of SARS-CoV-2 B.1.1.7 copies normalized to ß-globin were detected in the respiratory system (lung and pharynx), followed by the liver and heart. Importantly, SARS-CoV-2 lineage B.1.1.7 was found in 100% of cases in the lungs and in 85.7% in pharynx tissue. Detection also in the kidney and brain highlighting a pronounced organ tropism. Comparison of the given results to a former cohort of SARS-CoV-2 deaths during the first wave in spring 2020 showed resembling organ tropism. Our results indicate that also SARS-CoV-2 B.1.1.7 has a relevant organ tropism beyond the respiratory tract. We speculate that B.1.1.7 spike protein's affinity to human ACE2 facilitates transmission, organ tropism, and ultimately morbidity and mortality. Further studies and larger cohorts are obligatory to proof this link