Optimized protocol for the multi-omics processing of cryopreserved human kidney tissue.

Biobanking of tissue from clinically obtained kidney biopsies for later use with multi-omic and imaging techniques is an inevitable step to overcome the need of disease model systems and towards translational medicine. Hence, collection protocols ensuring integration into daily clinical routines using preservation media not requiring liquid nitrogen but instantly preserving kidney tissue for clinical and scientific analyses are of paramount importance. Thus, we modified a robust single nucleus dissociation protocol for kidney tissue stored snap frozen or in the preservation media RNAlaterand CellCover. Using porcine kidney tissue as surrogate for human kidney tissue, we conducted single nucleus RNA sequencing with the Chromium 10X Genomics platform. The resulting data sets from each storage condition were analyzed to identify any potential variations in transcriptomic profiles. Furthermore, we assessed the suitability of the preservation media for additional analysis techniques (proteomics, metabolomics) and the preservation of tissue architecture for histopathological examination including immunofluorescence staining. In this study, we show that in daily clinical routines the RNAlater facilitates the collection of highly preserved human kidney biopsies and enables further analysis with cutting-edge techniques like single nucleus RNA sequencing, proteomics, and histopathological evaluation. Only metabolome analysis is currently restricted to snap frozen tissue. This work will contribute to build tissue biobanks with well-defined cohorts of the respective kidney disease that can be deeply molecularly characterized, opening new horizons for the identification of unique cells, pathways and biomarkers for the prevention, early identification, and targeted therapy of kidney diseases.

Transcription regulates HIF-1α expression in CD4(+) T cells.

The transcription factor hypoxia inducible factor-1α (HIF-1α) mediates the metabolic adaptation of cells to hypoxia and T-helper cell fate. However, HIF-1α regulation in CD4(+) T cells (T cells) remains elusive. Here we observed that depletion of oxygen (O2⩽2%) alone was not sufficient to induce HIF-1α expression in T cells. However, when hypoxic T cells were stimulated, HIF-1α was expressed and this was dependent on nuclear factor-κB- and nuclear factor of activated T cell (NFAT)-mediated transcriptional upregulation of Hif-1α mRNA. HIF-1α upregulation could be blocked by drugs inhibiting NF-κB, NFAT or mammalian target of rapamycin precluding CD4(+) T-cell stimulation or translation in T cells, as well as by blocking transcription. CD3, CD28, phorbol-12-myristat-13-acetat (PMA) or ionomycin-stimulated T cells did not express HIF-1α under normoxic conditions. In conclusion, regulation of HIF-1α expression in CD4(+) T cells in hypoxia gravely relies on its transcriptional upregulation and subsequent enhanced protein stabilization.

Single-cell transcriptomics reveals a mechanosensitive injury signaling pathway in early diabetic nephropathy.

BACKGROUND: Diabetic nephropathy (DN) is the leading cause of end-stage renal disease, and histopathologic glomerular lesions are among the earliest structural alterations of DN. However, the signaling pathways that initiate these glomerular alterations are incompletely understood. METHODS: To delineate the cellular and molecular basis for DN initiation, we performed single-cell and bulk RNA sequencing of renal cells from type 2 diabetes mice (BTBR ob/ob) at the early stage of DN. RESULTS: Analysis of differentially expressed genes revealed glucose-independent responses in glomerular cell types. The gene regulatory network upstream of glomerular cell programs suggested the activation of mechanosensitive transcriptional pathway MRTF-SRF predominantly taking place in mesangial cells. Importantly, activation of MRTF-SRF transcriptional pathway was also identified in DN glomeruli in independent patient cohort datasets. Furthermore, ex vivo kidney perfusion suggested that the regulation of MRTF-SRF is a common mechanism in response to glomerular hyperfiltration. CONCLUSIONS: Overall, our study presents a comprehensive single-cell transcriptomic landscape of early DN, highlighting mechanosensitive signaling pathways as novel targets of diabetic glomerulopathy.

Collapsing Focal Segmental Glomerulosclerosis in Viral Infections.

Collapsing glomerulopathy represents a special variant of the proteinuric kidney disease focal segmental glomerulosclerosis (FSGS). Histologically, the collapsing form of FSGS (cFSGS) is characterized by segmental or global condensation and obliteration of glomerular capillaries, the appearance of hyperplastic and hypertrophic podocytes and severe tubulointerstitial damage. Clinically, cFSGS patients present with acute kidney injury, nephrotic-range proteinuria and are at a high risk of rapid progression to irreversible kidney failure. cFSGS can be attributed to numerous etiologies, namely, viral infections like HIV, cytomegalovirus, Epstein-Barr-Virus, and parvovirus B19 and also drugs and severe ischemia. Risk variants of the APOL1 gene, predominantly found in people of African descent, increase the risk of developing cFSGS. Patients infected with the new Corona-Virus SARS-CoV-2 display an increased rate of acute kidney injury (AKI) in severe cases of COVID-19. Besides hemodynamic instability, cytokine mediated injury and direct viral entry and infection of renal epithelial cells contributing to AKI, there are emerging reports of cFSGS associated with SARS-CoV-2 infection in patients of mainly African ethnicity. The pathogenesis of cFSGS is proposed to be linked with direct viral infection of podocytes, as described for HIV-associated glomerulopathy. Nevertheless, there is growing evidence that the systemic inflammatory cascade, activated in acute viral infections like COVID-19, is a major contributor to the impairment of basic cellular functions in podocytes. This mini review will summarize the current knowledge on cFSGS associated with viral infections with a special focus on the influence of systemic immune responses and potential mechanisms propagating the development of cFSGS.

HIF-1α- and hypoxia-dependent immune responses in human CD4+CD25high T cells and T helper 17 cells.

The central oxygen sensitive transcription factor HIF-1α has been implicated in the differentiation of n(T(reg)) and Th17 cells and to orchestrate metabolic changes of activated T cells. However, data on the functional relevance of HIF-1α and Hox, in general, for nT(reg)-suppressive activity and T cell function in primary human cells are still missing. Therefore, we analyzed the effect of Hox and HIF-1α on human T(res), n(Treg), and Th17 cells. Under Hox, nT(reg)-mediated suppression of T(res) proliferation, CD25 expression, and secretion of IFN-γ were significantly reduced, whereas expression levels of VEGF, TNF-α, and IL-10 were significantly increased. In contrast to observations in mice, Th17 lineage commitment, as determined by RORγt expression, was not affected by activation or inhibition of HIF-1α expression using DMOG or YC-1 treatment, respectively. Nevertheless, the secretion of IL-17A was increased by DMOG and reduced by YC-1 under Th17-skewing conditions in a dose- dependent manner. In conclusion, Hox and HIF-1α substantially influence human T cell-mediated immune responses by modulation of nT(reg)-suppressive function and IL-17A secretion by Th17 cells.